Gene therapy for treatment of syngap1-related intellectual disability

Abstract

SYNGAP1 -related intellectual disability is a genetic neurodevelopmental disorder caused by an insufficient level of SynGAPl. Provided herein are autologous hematopoietic stem and progenitor cells transduced with a lentiviral vector expressing a modified form of SYNGAP1, and methods of making the same, which can be useful in treating patients diagnosed with SYNGAP1 -related intellectual disability.

Description

[0001] GENE THERAPY FOR TREATMENT OF SYNGAP1-RELATED INTELLECTUAL DISABILITY

[0002] CROSS-REFERENCE TO RELATED APPLICATION(S)

[0003] This application claims priority to U.S. Provisional Application Serial No. 63 / 656,866, filed June 6, 2024. The entire content of the application referenced above is hereby incorporated by reference herein.

[0004] BACKGROUND

[0005] Synaptic Ras GTPase activating protein 1 -related intellectual disability (SRID) is a genetic neurodevel opmental disorder (NDDs) caused by an insufficient level of SynGAPl. Most cases are haploinsufficient and are due to de novo mutations which occur in the SYNGAP1 gene.1'5Reduction in wild type levels or complete loss of SynGAPl protein expression leads to Ras activation and excessive AMPA receptor (a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid) in the cellular membrane. This affects multiple neuronal functions including dendritic spine formation, synaptic development, integrity, signaling, and strength, and the long-term potentiation.6'9Due to the lack of normal levels of SynGAPl, neuronal synapses do not function properly and develop too early which presents with a wide array of clinical phenotypes including intellectual disabilities, decreased motor skills, epilepsy, speech abnormalities, and sleep and behavioral disorders.1'5Currently there is no cure for SRID and treatments are typically individualized depending on the patient-specific clinical phenotypes which are displayed.1'2These include physical and speech therapy, rehabilitation, behavioral therapy, and anti-seizure medications. Therefore, novel therapies need to be developed that allow for the delivery of functional SynGAPl to affected neurons to prevent abnormal development and disease progression.

[0006] SUMMARY

[0007] Accordingly, provided herein are novel therapies that allow for the delivery of functional SynGAPl.

[0008] Certain embodiments provide a synaptic Ras GTPase activating protein 1 (SynGAPl) protein having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NOTO, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, wherein the SynGAPl protein comprises one or more non-naturally occurring glycosylation sites.

[0009] Certain embodiments provide a polynucleotide comprising a nucleic acid sequence that encodes a synaptic Ras GTPase activating protein 1 (SynGAPl), wherein the encoded SynGAPl comprises one or more non-naturally occurring glycosylation sites. In certain embodiments, the polynucleotide further comprises a polynucleotide encoding a secretion signal. In certain embodiments, the polynucleotide further comprises a regulatory sequence, such as a promoter, that directs expression of the encoded SynGAPl.

[0010] Certain embodiments provide a vector comprising a polynucleotide as described herein.

[0011] Certain embodiments provide a population of hematopoietic stem and progenitor cells (HSPCs) that have been transduced with a vector described herein.

[0012] Certain embodiments provide a population of hematopoietic stem and progenitor cells (HSPCs) that comprise a polynucleotide described herein.

[0013] Certain embodiments provide a population of hematopoietic stem and progenitor cells (HSPCs) that comprise a protein described herein.

[0014] Certain embodiments provide a method of treating a human subject with SYNGAP1- related intellectual disability, comprising administering to a human subject in need thereof an effective amount of a vector described herein.

[0015] Certain embodiments provide the use of a vector described herein in the treatment of a SYNGAP1 -related intellectual disability.

[0016] Certain embodiments provide a method of treating a human subject with SYNGAP1- related intellectual disability, comprising administering to a human subject in need thereof an effective amount of the population of HSPCs described herein.

[0017] Certain embodiments provide the use of the population of a population of HSPCs described herein in the treatment of a SYNGAP1 -related intellectual disability.

[0018] BRIEF DESCRIPTION OF THE FIGURES

[0019] Figure 1 (A)-(C). Functionality of the SynGAPl expressing vector in SynGAPl -affected B lymphocytes. (A) a modified version of mouse SYNGAP1 was cloned under the control of an MNDU3 promoter into a self-inactivating lentiviral vector backbone, CCLc-X. (B) B lymphocytes obtained from a SynGAPl patient were left nontransduced (NT) or transduced with either the empty vector (EV) or the SynGAPl expressing vector (synl8). Total RNA was extracted and SYNGAP1 transcripts were quantified by QPCR (N=3). Data is represented as fold increase in expression with the NT cell values normalized to 1.0. Mean and standard error of the mean are shown. (C) Total protein was extracted from the cells and Western blots were performed to detect expression of SynGAPl (-135 kDa). GAPDH (-36 kDa) was used as an internal loading control.

[0020] Figure 2 (A)-(C). CFU assay and expression of SynGAPl in vector transduced human CD34+ HSPC. A CFU assay was performed with nontransduced (NT), empty vector transduced (EV), and SYNGAP1 vector transduced (synl8) human CD34+ HSPC. (A) After 14 days of culture, total GM, GEMM, and BFU-E colonies were counted (N=3). (B) Fold expansion of the cells was also calculated using the input number of cells and the final cell count after the 14-day CFU assay (N=3). Mean and standard error of the mean are shown. (C) The cells were further differentiated into macrophages in vitro. Total protein was extracted from the cells and Western blots were performed to detect expression of SynGAPl (-135 kDa). GAPDH (-35 kDA) was used as an internal loading control.

[0021] Figure 3 (A)-(F). Open field analysis of BGS mice transplanted with SYNGAP1 vector transduced human CD34+ HSPC. Eight-week-old BGS (B6-IL2rg- / ySYNGAPl+ / -) mice were left either nontransplanted (NT) (N=9) or transplanted with human CD34+ HSPC transduced with either the empty vector (EV) (N=9) or the SynGAPl expressing vector (synl8) (N=9). Nontransplanted wild type (WT) (B6-IL2rg- / ySYNGAPl+ / +) mice were used as controls (N=9). Sixteen weeks post-transplant, the mice were subjected to a 15-minute open field assay to evaluate (A) total distance traveled, (B) total horizontal activity, and (C) total vertical episodes. The data from the synl8 group was further divided into subgroups representing a high VCN (>2.0) named synl8 high (N=4) and a low VCN (<2.0) named synl8 low (N=5). (D) total distance traveled, (E) total horizontal activity, and (F) total vertical episodes were analyzed. Mean and standard error of the mean are shown. * = p<0.05 and ** = p<0.01.

[0022] Figure 4 (A)-(B). Rotorod analysis of BGS mice transplanted with SYNGAP1 vector transduced human CD34+ HSPC. Eight-week-old BGS (B6-IL2rg- / ySYNGAPl+ / -) mice were left either nontransplanted (NT) (N=9) (— ) or transplanted with human CD34+ HSPC transduced with either the empty vector (EV) (N=9) (■) or the SYNGAP1 expressing vector (synl8) (A) (N=9). Nontransplanted wild type (WT) (•) (B6-IL2rg- / ySYNGAPl+ / +) mice were used as controls (N=9). Sixteen weeks post-transplant, the mice were subjected to a rotorod assay for three consecutive days. (A) total latency time to fall was measured with a maximum time of 5 minutes. (B) The data from the synl8 group was further divided into subgroups representing a high VCN (>2.0) named synl8 high (N=4) (A) and a low VCN (<2.0) named synl8 low (N=5) (♦) and reanalyzed. Mean and standard error of the mean are shown. * = p<0.05 and ** = p<0.01.

[0023] Figure 5 (A)-(B). Immunohistochemical detection of SynGAPl in brain tissue obtained from BGS mice. Sagittal sections of brain tissue obtained from the nontransplanted wild type (WT) (N=5), nontransplanted heterozygous SYNGAP1+ / - (NT) (N=6), empty vector (EV) transduced (N=6), or SYNGAP1 expressing (synl8) vector transduced (N=9) cell transplanted mice were labeled with a polyclonal antibody for mouse SynGAPl. (A) Enumeration of positive cells was performed and compared between the groups. Mean and standard error of the mean are shown. (B) representative images of a region of the frontal lobe from each group are displayed. Scale bar in the lower left comer represents 100 pm. Arrows indicate a positive cell. * = p<0.05.

[0024] DETAILED DESCRIPTION

[0025] Currently, there is not a cure for SRID. As such, novel therapies are needed to treat SRID, e.g., which provide delivery of functional SynGAPl to affected neurons to prevent abnormal development and disease progression.

[0026] A stem cell gene therapy strategy to treat SRID has great potential in offering a therapeutic intervention for affected patients through a process called cross-correction. In this approach, autologous hematopoietic stem and progenitor cells (HSPC) are gene modified to express the therapeutic protein defective or missing in the respective disorder. Upon transplantation of the cells, the genetically modified HSPC engraft in the bone marrow and have the ability to differentiate into immune cell progeny. Myeloid cells derived from the infused HSPC can engraft in the brains of transplanted patients as microglia and deliver functional protein to affected neurons.

[0027] As described herein, a lentiviral vector expressing a modified form of mouse SYNGAP1 was developed, and the in vivo efficacy was evaluated in an immunodeficient mouse model of SRID. As described herein for what is believed to be the first time, significant rescue of SRID- related phenotypes are demonstrated, which indicates that a stem cell gene therapy approach can provide a therapeutic benefit for SRID patients.

[0028] In particular, as a novel approach toward the treatment of SRID, a lentiviral vector expressing a modified form of SynGAPl, was generated for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene modified cells were then transplanted into adult immunodeficient SYNGAP1+ / - heterozygous mice and evaluated for improvement of SRID clinical phenotypes. Expression of SynGAPl was also evaluated in the brain tissue of transplanted mice. Accordingly, provided herein is a synaptic Ras GTPase activating protein 1 (SynGAPl) protein having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, wherein the SynGAPl protein comprises one or more non-naturally occurring glycosylation sites.

[0029] In certain embodiments, the SynGAPl protein has at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0030] In certain embodiments, the SynGAPl protein has at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO:8.

[0031] In certain embodiments, the SynGAPl protein comprises from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) non-naturally occurring glycosylation sites.

[0032] Certain embodiments provide a polynucleotide comprising a nucleic acid sequence that encodes a synaptic Ras GTPase activating protein 1 (SynGAPl), wherein the encoded SynGAPl comprises one or more non-naturally occurring glycosylation sites.

[0033] In certain embodiments, the encoded SynGAPl comprises from 1-20 non-naturally occurring glycosylation sites.

[0034] In certain embodiments, the encoded SynGAPl comprises from 4-10 non-naturally occurring glycosylation sites.

[0035] In certain embodiments, the encoded SynGAPl comprises from 6-10 non-naturally occurring glycosylation sites.

[0036] In certain embodiments, the encoded SynGAPl comprises from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) non-naturally occurring glycosylation sites.

[0037] In certain embodiments, the encoded SynGAPl comprises 8 non-naturally occurring glycosylation sites.

[0038] In certain embodiments, the polynucleotide further comprises a polynucleotide encoding a secretion signal.

[0039] In certain embodiments, the encoded secretion signal is selected from MYRMQLLSCIALSLALVTNS (SEQ ID NO:29), MKRMQLLSCIALSLALVTNS (SEQ ID NO:30), MRMQLLSCIALSLALVTNS (SEQ ID NO:31), MRRMQLLSCIALSLALVTNS (SEQ ID NO:32), MRRKKMQLLSCIALSLALVTN (SEQ ID NO:33), MYRMQLLLLIALSLALVTNS (SEQ ID NO:34), MYRMQLLLSCIALSLALVTNS (SEQ ID NO:35), MYRMQLLLSCIALLLALVTNS (SEQ ID NO:36), MYRMQLLLLIALSLALVTNS (SEQ ID NO: 37), MRMQLLLLIALSLALVTNS (SEQ ID NO: 38), MRRMQLLLLIALSLALVTNS (SEQ ID NO: 39) and MRRKKMQLLLLIALSLALVTNS (SEQ ID NO:40).

[0040] In certain embodiments, the polynucleotide further comprises a regulatory sequence that directs expression of the encoded SynGAPl.

[0041] In certain embodiments, the regulatory sequence comprises one or more of the following: a promoter, an intron, an enhancer, a polyadenylation signal, a terminator, a silencer, a TATA box, or a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE).

[0042] In certain embodiments, the polynucleotide comprises a microglia-specific promoter.

[0043] In certain embodiments, the promoter is an MNDU3 promoter, a Syn-1 (Synapsin 1) promoter, a CMV promoter, a CD68 promoter, a human or mouse PGK promoter, a MNDU promoter, or a long or short EFl alpha promoter.

[0044] In certain embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the nucleic acid sequence of SEQ ID NO:7, SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15.

[0045] In certain embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO:5.

[0046] In certain embodiments, the polynucleotide comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the nucleic acid sequence of SEQ ID NO:7.

[0047] In certain embodiments, the polynucleotide comprises a nucleic acid sequence encoding a SynGAPl protein having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO: 8, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

[0048] In certain embodiments, the polynucleotide comprises a nucleic acid sequence encoding a SynGAPl having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

[0049] In certain embodiments, the polynucleotide comprises a nucleic acid sequence encoding a SynGAPl having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO: 8.

[0050] Certain embodiments provide a vector comprising a polynucleotide described herein.

[0051] In certain embodiments, the vector is a viral vector or a non-viral vector.

[0052] In certain embodiments, the vector is for expression in a prokaryotic host cell system.

[0053] In certain embodiments, the vector is for expression in a eukaryotic host cell system.

[0054] In certain embodiments, the vector is a plasmid, or a viral vector such as baculovirus, a retroviral vector, an adenoviral vector, an AAV vector, or a lentiviral vector.

[0055] In one embodiment, the AAV refers to of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV12, AAV13, AAV PHP.B, or AAV rh74.

[0056] In certain embodiments, the vector is a retroviral vector, an adenoviral vector, an adeno- associated viral vector, or a Herpes viral vector.

[0057] In certain embodiments, the vector is a lentiviral vector.

[0058] In certain embodiments, the lentiviral vector is a self-inactivating lentiviral vector.

[0059] Certain embodiments provide a SynGAPl protein encoded by a polynucleotide described herein.

[0060] In certain embodiments, the SynGAPl protein comprises any combination of the following glycosylation sites, where the aa numbering is with reference to SEQ ID NO:6: aa 79-aa81; aa222-aa224; aa256-aa258; aa293-aa295; aa315-aa317; aa456-aa458; aa556-aa558; and / or aa689-aa691.

[0061] Certain embodiments provide a population of hematopoietic stem and progenitor cells (HSPCs) that have been transduced with a vector described herein.

[0062] Certain embodiments provide a population of hematopoietic stem and progenitor cells (HSPCs) that comprise a polynucleotide described herein.

[0063] Certain embodiments provide a population of hematopoietic stem and progenitor cells (HSPCs) that comprise a protein described herein.

[0064] In certain embodiments, the population of HSPCs comprises CD34+ HSPCs. Certain embodiments provide a method of treating a human subject with SYNGAP1- related intellectual disability, comprising administering to a human subject in need thereof an effective amount of a vector described herein.

[0065] Certain embodiments provide the use of a vector described herein in the treatment of a SYNGAP1 -related intellectual disability.

[0066] Certain embodiments provide a method of treating a human subject with SYNGAP1- related intellectual disability, comprising administering to a human subject in need thereof an effective amount of the population of HSPCs described herein.

[0067] Certain embodiments provide the use of the population of a population of HSPCs described herein in the treatment of a SYNGAP1 -related intellectual disability.

[0068] Polynucleotides are provided that comprise a nucleotide sequence that encodes a polypeptide described herein. In some embodiments, the polynucleotide is DNA. In some embodiments, the polynucleotide is cDNA. In some embodiments, the polynucleotide is RNA.

[0069] In some embodiments, the polynucleotide is included within a nucleic acid construct. In some embodiments, the construct is a replicable vector. In some embodiments, the vector is selected from a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non- episomal mammalian vector.

[0070] In some embodiments, the polynucleotide is operably linked to one or more regulatory nucleotide sequences in an expression construct. In one series of embodiments, the nucleic acid expression constructs are adapted for expression of the polypeptide in a system that permits isolation of the polypeptide in milligram or gram quantities. In some embodiments, the system is a mammalian cell expression system. In some embodiments, the system is a yeast cell expression system.

[0071] An enhancer is a regulatory element that increases the expression of a target sequence. A "promoter / enhancer" is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer / promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer / promoter is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer / promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (e.g., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer / promoter. In some embodiments, a mutated amino acid (aa) or nucleotide (nt) residue refers to the aa residue or nt residue is different from the residue at a corresponding position in a reference sequence. In some embodiments, a mutated protein, polypeptide, or polynucleotide comprises a mutated aa or nt residue. In some embodiments, the reference sequence is a naturally occurring and / or wildtype sequence.

[0072] In some embodiments, the polynucleotide further comprises a regulatory sequence which direct expression of the SynGAPl polypeptide, protein or the biological equivalent thereof. In some embodiments, wherein the regulatory sequence comprises one or more of the following: a promoter, an intron, an enhancer, a polyadenylation signal, a terminator, a silencer, a TATA box, or a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). In a further embodiment, a polynucleotide as disclosed herein further comprises a promoter operatively linked to the polynucleotide for expression of the polynucleotide. Nonlimiting examples of such include a promoter selected from a pol II promoter, e.g., an MNDU3 promoter, a Syn-1 (Synapsin 1) promoter, a CMV promoter, a PGK promoter, and an EFl alpha promoter. Sequences of these and other pol II promoters are known in the art. The polynucleotides can further comprise an enhancer element operatively linked to the polynucleotide encoding the mutated SynGAPl protein, to increase or enhance expression of the polynucleotide.

[0073] In a further aspect, the polynucleotide further comprises a polynucleotide encoding a signal peptide and / or a secretion signal located 5’ to the polynucleotide encoding the modified SynGAPl protein. Non-limiting examples of such signal peptide and / or secretion signal include a single chain fragment variable signal peptide, a twin-arginine transport protein signal peptide, an IL-4 secretion signal, an IL-2 secretion signal and an IL-10 secretion signal.

[0074] In some embodiments, the sequence further comprises one or more of the following: a polypurine tract sequence (PPT), a central PPT (cPPT), an R region, a U5, an encapsidation signal (Psi), a Rev-Responsive Element (RRE), a full-length U3 or a fragment thereof, a detectable or selection maker, a polynucleotide encoding a detectable or selection polypeptide, a regulatory sequence directing expression of the detectable or selection polypeptide, or a coding sequence for a cleavable peptide located between the coding sequence for the detectable or selection polypeptide and the sequence encoding the SynGAPl polypeptide or protein or a biological equivalent thereof. In some embodiments, the cleavable peptide is a self-cleaving peptide, optionally a 2A self-cleaving peptide. In some embodiments, the 2A self-cleaving peptide is selected from P2A, T2A, E2A, F2A and BmCPV2A. In some embodiments, the genetic information of the vector (which is also referred to herein as a vector genome or a viral genome) is RNA which comprises, or alternatively consists essentially of, or yet further consists of, on the 5’ and 3’ ends, the minimal LTR regions required for integration of the vector, and a polynucleotide as disclosed herein between the two LTR regions. In some embodiments, between the two LTR regions further comprises an encapsidation signal (a psi region) which is required for packaging of the vector RNA into the particle. In some embodiments, the psi region is followed by a Rev-Responsive Element (RRE) and a central polypurine tract sequence (cPPT) that enhance vector production by transporting the full-length vector transcript out of the nucleus for efficient packaging into the vector particle.

[0075] Further provided are polynucleotides which are equivalents, complements, reverse sequences, or reverse complements to the modified SynGAPl coding polynucleotides. In some embodiments, an equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 70% sequence identity , or alternatively at least 75% sequence identity, or alternatively at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 91 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 93 % sequence identity, or alternatively at least 94 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 96 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity, or alternatively at least 99 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide. Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide hybridizes under conditions of high stringency to any one of the reference polynucleotide, its complement, or its reverse complement.

[0076] Additionally or alternatively, the equivalent nucleic acid, polynucleotide or oligonucleotide must encode a functional SynGAPl protein, polypeptide or a biological equivalent thereof that optionally can be identified through one or more assays described herein. In some embodiments, an equivalent nucleic acid, polynucleotide, or oligonucleotide has at least 70% sequence identity , or alternatively at least 75% sequence identity, or alternatively at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least

[0077] 90 % sequence identity, or alternatively at least 91 % sequence identity, or alternatively at least

[0078] 92 % sequence identity, or alternatively at least 93 % sequence identity, or alternatively at least

[0079] 94 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least

[0080] 96 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least

[0081] 98 % sequence identity, or alternatively at least 99 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide. Additionally or alternatively, an equivalent nucleic acid, polynucleotide or oligonucleotide hybridizes under conditions of high stringency to any one of the reference polynucleotide, its complement, or its reverse complement.

[0082] In a further aspect, the polynucleotide further comprises a promoter operatively linked to the polynucleotide. Non-limiting examples of such include pol II promoters selected from the group of an MNDU3 promoter, a Syn-1 (Synapsin 1) promoter, a CMV promoter, a PGK promoter, and an EFl alpha promoter. The promoter can be operatively linked to the coding polynucleotides to drive expression in a suitable host system. In a further aspect, the polynucleotide further comprises a polynucleotide encoding a secretion signal located 5’ to the polynucleotide encoding the modified protein. Non-limiting examples of secretion signals include a single chain fragment variable secretion signal, a twin-arginine transport protein secretion signal, an IL-4 secretion signal, an IL-2 secretion signal and an IL- 10 secretion signal. The polynucleotides can further comprise a polynucleotide that is, or encodes, a detectable or purification marker.

[0083] Expression vehicles for production of a recombinant polypeptides include plasmids and other vectors. For instance, suitable vectors include plasmids of the following types: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells, such as E. coli. The pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg-derived vectors are examples of mammalian expression vectors suitable for transfection of eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived, and p205) can be used for transient expression of polypeptides in eukaryotic cells. In some embodiments, it may be desirable to express the recombinant polypeptide by the use of a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac- derived vectors. Additional expression systems include adenoviral, adeno-associated virus, and other viral expression systems.

[0084] Vectors may be transformed into any suitable host cell. In some embodiments, the host cells, e.g., bacteria or yeast cells, may be adapted for use as a surface expression library. In some cells, the vectors are expressed in host cells to express relatively large quantities of the polypeptide. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the cells are mammalian cells, such as Chinese Hamster Ovary (CHO) cell, baby hamster kidney (BHK) cell, NSO cell, YO cell, HEK293 cell, COS cell, Vero cell, or HeLa cell.

[0085] Cell penetrating peptides or cell penetrating domains (CPPs) or cell penetrating domains, as used herein, refer to short peptides that facilitate cellular uptake of various molecular cargos (from small chemical molecules to nanosize particles and large fragments of DNA), the use of which is contemplated. A “cargo”, such as a modified protein as disclosed herein, is associated with the peptides either through chemical linkage via covalent bonds or through non-covalent interactions. The function of the CPPs are to deliver the cargo into target cells, a process that commonly occurs through endocytosis with the cargo delivered to the endosomes of living mammalian cells. In some embodiments, the target cell is a neuron. CPPs typically have an amino acid composition containing either a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences that contain an alternating pattern of polar / charged amino acids and non-polar, hydrophobic amino acids. It was previously reported that the human immunodeficiency virus transactivator of transcription (HIV- TAT) protein can be delivered to cells using a CPP.

[0086] A CPP may also be chemically modified, such as prenylated near the C-terminus of the CPP. Prenylation is a post-translation modification resulting in the addition of a 15 (fameysyl) or 20 (geranylgeranyl) carbon isoprenoid chain on the peptide. A chemically modified CPP can be even shorter and still possess the cell penetrating property.

[0087] A host cell transfected with an expression vector(s) encoding the polypeptide as described herein can be cultured under appropriate conditions to allow expression of the polypeptides to occur. The polypeptides may be secreted and isolated from a mixture of cells and medium containing the polypeptides. Alternatively, the polypeptides may be retained in the cytoplasm or in a membrane fraction and the cells harvested, lysed, and the polypeptide isolated using a desired method.

[0088] In some embodiments, the polypeptide, protein or the biological equivalent thereof further comprises a signal peptide. In a further embodiment, the signal peptide is a secretion signal peptide (which is also referred to herein as a secretion signal). In some embodiments, the signal peptide or secretion signal is selected from an antibody heavy / light chain secretion signal, a twin-arginine transport protein secretion signal, an Interleukin-2 (IL2) secretion signal, an Interleukin-4 (IL4) secretion signal, Interleukin- 10 (IL10) secretion signal, an Interleukin-3 (IL3) secretion signal, an Interleukin-7 (IL7) secretion signal, an human IL2 secretion signal, a human OSM secretion signal, a VSV-G secretion signal, a Mouse Ig Kappa secretion signal, a Human IgG2 H secretion signal, a BM40 secretion signal, a Secrecon secretion signal, a Human IgKVIII secretion signal, a CD33 secretion signal, a tPA secretion signal, a Human Chymotrypsinogen secretion signal, a Human trypsinogen-2 secretion signal, a Gaussia luc secretion signal, a Albumin(HSA) secretion signal, an Influenza Haemagglutinin secretion signal, a Human insulin secretion signal, or a Silkworm Fibroin LC.

[0089] The polynucleotides and vectors can be contained in a host cell system for delivery or expression of the polynucleotides. The cell can be a prokaryotic or a eukaryotic cell. In one aspect, the host cell is a mammalian cell, e.g., a canine, feline, bovine, equine, murine, rat or human cell. The mammalian cell can be selected from, e.g., a stem cell, e.g., an induced pluripotent stem cell (iPSC), an embryonic stem cell, or an adult or somatic stem cell. In one aspect, the stem cell is a mesenchymal stem cell. In one aspect, the stem cell is a hematopoietic stem cell or a neuronal stem cell. In another aspect, the stem cell is a mesenchymal stem cell optionally identified by expressing the CD34+ marker.

[0090] Unmodified cells are sometimes referred to as “source cells” or “source stem cells”. The cells may be prokaryotic or eukaryotic, and include but are not limited to bacterial cells, yeast cells, plant cells, insect cells, animal cells, and mammalian cells, e.g., felines, canines, equines, murines, rats, simians, bovines, porcines and humans.

[0091] The term "stem cell" refers to a cell that is in an undifferentiated or partially differentiated state and has the capacity for self-renewal and / or to generate differentiated progeny. Self-renewal is defined as the capability of a stem cell to proliferate and give rise to more such stem cells, while maintaining its developmental potential (e.g., totipotent, pluripotent, multipotent, etc.). The term "somatic stem cell" is used herein to refer to any stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Natural somatic stem cells have been isolated from a wide variety of adult tissues including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Exemplary naturally occurring somatic stem cells include, but are not limited to, mesenchymal stem cells (MSCs) and neural or neuronal stem cells (NSCs). In some embodiments, the stem or progenitor cells can be embryonic stem cells. As used herein, "embryonic stem cells" refers to stem cells derived from tissue formed after fertilization but before the end of gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Most frequently, embryonic stem cells are pluripotent cells derived from the early embryo or blastocyst. Embryonic stem cells can be obtained directly from suitable tissue, including, but not limited to human tissue, or from established embryonic cell lines. “Embryonic-like stem cells” refer to cells that share one or more, but not all characteristics, of an embryonic stem cell.

[0092] “Differentiation” describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. “Directed differentiation” refers to the manipulation of stem cell culture conditions to induce differentiation into a particular cell type. “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell. As used herein, the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell. As used herein “a cell that differentiates into a mesodermal (or ectodermal or endodermal) lineage” defines a cell that becomes committed to a specific mesodermal, ectodermal or endodermal lineage, respectively. Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal.

[0093] As used herein, the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated”) position within the lineage of a cell. “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell. Induced pluripotent stem cells are examples of dedifferentiated cells.

[0094] As used herein, the "lineage" of a cell defines the heredity of the cell, i.e. its predecessors and progeny. The lineage of a cell places the cell within a hereditary scheme of development and differentiation.

[0095] A “multi -lineage stem cell” or “multipotent stem cell” refers to a stem cell that reproduces itself and at least two further differentiated progeny cells from distinct developmental lineages. The lineages can be from the same germ layer (e.g., mesoderm, ectoderm or endoderm), or from different germ layers. An example of two progeny cells with distinct developmental lineages from differentiation of a multilineage stem cell is a myogenic cell and an adipogenic cell (both are of mesodermal origin, yet give rise to different tissues). Another example is a neurogenic cell (of ectodermal origin) and adipogenic cell (of mesodermal origin).

[0096] A “precursor” or “progenitor cell” intends to mean cells that have a capacity to differentiate into a specific type of cell. A progenitor cell may be a stem cell. A progenitor cell may also be more specific than a stem cell. A progenitor cell may be unipotent or multipotent. Compared to adult stem cells, a progenitor cell may be in a later stage of cell differentiation. An example of progenitor cell includes, without limitation, a progenitor nerve cell.

[0097] As used herein, a “pluripotent cell” defines a less differentiated cell that can give rise to at least two distinct (genotypically and / or phenotypically) further differentiated progeny cells. In another aspect, a “pluripotent cell” includes an Induced Pluripotent Stem Cell (iPSC) which is an artificially derived stem cell from a non-pluripotent cell, typically an adult somatic cell, that has historically been produced by inducing expression of one or more stem cell specific genes. Such stem cell specific genes include, but are not limited to, the family of octamer transcription factors, i.e. Oct-3 / 4; the family of Sox genes, i.e., Soxl, Sox2, Sox3, Sox 15 and Sox 18; the family of Klf genes, i.e. Klfl, Klf2, Klf4 and Klf5; the family of Myc genes, i.e. c-myc and L- myc; the family of Nanog genes, i.e., OCT4, NANOG and REXI; or LIN28.

[0098] Provided herein is a cell comprising one or more of the following: a recombinant polynucleotide as disclosed herein, a vector as disclosed herein, a recombinant SynGAPl protein, polypeptide or the biological equivalent thereof as disclosed herein, thereby producing the polynucleotide, the vector, or the recombinant SynGAPl protein, polypeptide or biological equivalent thereof. In some embodiments, the cell is an isolated cell and / or an engineered cell. In some embodiments, the cell is a eukaryotic or a prokaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an in vitro and / or ex vivo cell. In some embodiments, the cell is an in vivo cell in a subject.

[0099] Also provided is a clonal population of a cell as disclosed herein.

[0100] Additionally provided is a method to express a SynGAPl protein, polypeptide or the biological equivalent thereof comprising growing a cell as disclosed herein under conditions that allow for the expression of the recombinant SynGAPl protein or polypeptide or a biological equivalent thereof.

[0101] Yet further provided is a cell or population of cells, comprising, or alternatively consisting essentially of, or yet further consisting of, one or more of: a polynucleotide as disclosed herein, a vector as disclosed herein, and a SynGAPl protein, polypeptide or a biological equivalent thereof as disclosed herein. In some embodiments, the vector is a viral particle. In some embodiment, the cell or population of cells further comprises a detectable marker.

[0102] In some embodiments, the cell is an isolated cell. In some embodiments, the cell is not a naturally occurring cell. In some embodiments, the cell is also referred to herein as a host cell. In some embodiments, the isolated host cell is a packaging cell line. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell. In further embodiments, the cell is a murine cell or a human cell.

[0103] Additionally or alternatively, the cell is a progenitor cell or a progeny thereof. In some embodiments, the cell is a stem cell, e.g., an embryonic stem cell, an induced pluripotent stem cell (iPSC), an adult stem cell, a mesenchymal stem cell, a neural stem cell, a hematopoietic stem cell (HSC), or a progeny of each thereof. In some embodiments, the vector and / or host cell can further comprise a detectable or purification label.

[0104] In some embodiments, the cell is a stem cell, such as a hematopoietic progenitor cell or a hematopoietic stem cell, e.g., a CD34+ cell. Alternatively, the stem cell is a neural stem cell or an iPSC.

[0105] In some embodiments, the cell is an immune cell, optionally selected from a B-cell, T- cell, Nature Killer (NK) cell, dendritic cell, a cell of the myeloid lineage, a neutrophil, a monocyte, a macrophage, and / or a microglia. In further embodiments, the immune cell is derived from a progenitor cell (such as a hematopoietic progenitor cell), a stem cell (e.g., an embryonic stem cell, an induced pluripotent stem cell (iPSC), an adult stem cell, a mesenchymal stem cell, a neural stem cell, a hematopoietic stem cell(HSC)), or a progeny of each thereof. In some embodiments, the T cell expresses CD4, i.e. is a CD4+ T cell. In some embodiments, the T cell expresses CD8, i.e. is a CD8+ T cell.

[0106] When used therapeutically, the cells can be allogeneic or autologous to the subject to be treated. The subjects can be mammalian, e.g., murine, canine, bovine, equine, ovine, feline or a human subject or patient.

[0107] In some embodiments, the cell expresses and / or secrets a recombinant SynGAPl protein or polypeptide or a biological equivalent thereof as disclosed herein.

[0108] Also provided is a population of cells as disclosed herein and / or a progeny thereof.

[0109] This disclosure further provides an isolated cell or an enriched population of cells, optionally, that are derived or differentiated from the stem cell described above. In some instances, the derived or differentiated cell or the enriched population of cells comprise, or consist essentially of, or yet further consist of an immune cell. In some instances, the immune cell is selected from a B-cell, T-cell, Nature Killer (NK) cell, dendritic cell, a cell of the myeloid lineage, and / or a neutrophil. In some embodiments, the T cell expresses CD4, i.e. is a CD4+ T cell. In some embodiments, the T cell expresses CD8, i.e. is a CD8+ T cell. In some instances, the isolated cell or an enriched population of immune cells comprise, or consist essentially of, or yet further consist of, a monocyte, a macrophage, and / or a microglia. In some cases, one or more types of the immune cells described herein are modified with a recombinant polynucleotide encoding an SynGAPl protein described herein to generate an SynGAPl expressing immune cell. In some cases, a B-cell, a T-cell, an NK cell, a dendritic cell, a neutrophil, or a cell of the myeloid lineage is modified (for example, is transduced or transfected) with a recombinant polynucleotide encoding a SynGAPl protein described herein to generate a modified cell expressing an SynGAPl protein, polypeptide or a biological equivalent thereof. In some cases, a macrophage is modified (for example, is transduced or transfected) with a recombinant polynucleotide encoding a modified SynGAPl protein described herein to generate an SynGAPl expressing macrophage in vivo and / or in vitro. In some cases, a CD34+ HSC is modified (for example, is transduced or transfected) with a recombinant polynucleotide encoding a modified SynGAPl protein described herein to generate an SynGAPl expressing HSC and / or macrophage in vivo and / or in vitro.

[0110] In some embodiments, the cell population expresses CD4, CD 14 and HLADR. In some embodiments, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92% , or at least about 93% , or at least about 94% , or at least about 95% , or at least about 96% , or at least about 97% , or at least about 98% , or at least about 99% of the cells in the population are CD4+, i.e. expressing CD4 optionally on the cell surface. Additionally or alternatively, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92% , or at least about 93% , or at least about 94% , or at least about 95% , or at least about 96% , or at least about 97% , or at least about 98% , or at least about 99% of the cell in the population are CD14+, i.e., expressing CD 14 optionally on the cell surface. Additionally or alternatively, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92% , or at least about 93% , or at least about 94% , or at least about 95% , or at least about 96% , or at least about 97% , or at least about 98% , or at least about 99% of the cells in the population are HL- ADR+, i.e., expressing HLA-DR optionally on the cell surface.

[0111] In some embodiments, the cell population comprises substantially macrophages, optionally derived from a stem cell such as a HSC. In some embodiments, the cell population comprises substantially a stem cell, such as HSCs, optionally deriving to macrophages.

[0112] In some embodiments, the cell population is substantially homogenous, for example, at least about 60%, or at least about 70%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 91%, or at least about 92% , or at least about 93% , or at least about 94% , or at least about 95% , or at least about 96% , or at least about 97% , or at least about 98% , or at least about 99% of the cells in the population are the same.

[0113] Guidance for preparing formulations for use in the present disclosure can be found in any number of handbooks for pharmaceutical preparation and formulation that are known to those of skill in the art. Dosages and desired drug concentration of pharmaceutical compositions described herein may vary depending on the particular use envisioned.

[0114] As described herein, certain embodiments of the invention provide autologous hematopoietic stem and progenitor cells transduced with a lentiviral vector expressing a modified form of SynGAPl, which can be useful in treating patients diagnosed with SynGAPl - related intellectual disability. The autologous hematopoietic stem and progenitor cells transduced with the SYNGAP1 lentiviral vector will develop into microglia that reside in the brains of infused patients. These microglia will then express and deliver therapeutic levels of SynGAPl protein to affected neurons.

[0115] Improvements in clinical phenotypes in patients transplanted with the SYNGAP1 vector transduced cells include decreased hyperactivity, improved motor functions, and decreased severity and frequency of seizures. Other efficacy endpoints can include: change in ORCA (Observer-Reported Communication Ability), change in VABS-III (Vineland Adaptive Behavior Scales, Third Edition), BSID-III (Bayley Scales of Infant Development, Third Edition), and WPPSI-IV (Wechsler Preschool and Primary Scale of Intelligence, Fourth Edition), the measurement of Intelligence Quotient (IQ) values above 55 at 24, 30 and 36 months after treatment, change in Caregiver QOL measures and Caregiver Global Impression of Change (CGIC), and an engraftment of the transduced cells above 5% in bone marrow-derived clonogenic progenitor cells at 12 months after the transplant.

[0116] In certain embodiments, treatment will include an autologous bone marrow transplant for infusion of the SYNGAP1 lentiviral vector transduced cells. Certain dosing regimens and routes of delivery will be utilized, including intravenous infusion to patients in Plasmalyte A, with equal or above to 3.0 xlO6SYNGAP1 vector transduced CD34+ cells / kg body weight after standard conditioning therapy. In some embodiments, the cell or cell population is administered at a dose of about 1 x 104to about 1 x 1015cells / kg body weight of the subject.

[0117] In some embodiments, the dose is at least about 1 x 104CD34+ cells / kg, or at least about 2 x4CD34+ cells / kg, or at least about 3 x 104CD34+ cells / kg, or at least about 4 x 104CD34+ cells / kg, or at least about 5 x 104CD34+ cells / kg, or at least about 6 x 104CD34+ cells / kg, or at least about 7 x 104CD34+ cells / kg, or at least about 8 x 104CD34+ cells / kg, or at least about 9 x 104CD34+ cells / kg, or at least about 1 x 105CD34+ cells / kg, or at least about 2 x 105CD34+ cells / kg, or at least about 3 x 105CD34+ cells / kg, or at least about 4 x 105CD34+ cells / kg, or at least about 5 x 105CD34+ cells / kg, or at least about 6 x 105CD34+ cells / kg, or at least about 7 x 105CD34+ cells / kg, or at least about 8 x 105CD34+ cells / kg, or at least about 9 x 105CD34+ cells / kg, or at least about 1 x 106CD34+ cells / kg, or at least about 2 x 106CD34+ cells / kg, or at least about 3 x 106CD34+ cells / kg, or at least about 4 x 106CD34+ cells / kg, or at least about 5 x 106CD34+ cells / kg, or at least about 6 x 106CD34+ cells / kg, or at least about 7 x 106CD34+ cells / kg, or at least about 8 x 106CD34+ cells / kg, or at least about 9 x 106CD34+ cells / kg, or at least about 1 x 107CD34+ cells / kg, or at least about 2 x 107CD34+ cells / kg, or at least about 3 x 107CD34+ cells / kg, or at least about 4 x 107CD34+ cells / kg, or at least about 5 x 107CD34+ cells / kg, or at least about 6 x 107CD34+ cells / kg, or at least about 7 x 107CD34+ cells / kg, or at least about 8 x 107CD34+ cells / kg, or at least about 9 x 107CD34+ cells / kg, or at least about 1 x 108CD34+ cells / kg, or at least about 2 x 108CD34+ cells / kg, or at least about 3 x 108CD34+ cells / kg, or at least about 4 x 108CD34+ cells / kg, or at least about 5 x 108CD34+ cells / kg, or at least about 6 x 108CD34+ cells / kg, or at least about 7 x 108CD34+ cells / kg, or at least about 8 x 108CD34+ cells / kg, or at least about 9 x 108CD34+ cells / kg, or at least about 1 x 109CD34+ cells / kg body weight of the subject.

[0118] SRID patients have normal bone marrow and would be able to accept an infusion of autologous gene modified CD34+ cells as a treatment strategy. Based on preliminary data, normal engraftment of the SynGAPl vector transduced cells has been observed in immunodeficient (IL2rg- / y) and SYNGAP1 deficient (+ / -) mice (BGS mice), which provides evidence that in patients, these cells will be tolerated. By genetically modifying a patient’s own HSPC with a lentiviral vector expressing functional SynGAPl, engraftment of the modified and transplanted HSPC would produce immune cell progeny that would secrete and deliver functional SynGAPl to affected neurons. There are several advantages of this stem cell gene therapy approach for SRID: a) This therapy would use autologous HSPC, which are safer than allogeneic transplants, which cause complicated immune reactions possibly leading to graft versus host disease. A lower intensity conditioning regimen may also be able to be used, b) Due to expression of SynGAPl encoded in the lentiviral vector, genetically engineered HSPC- derived cells are capable of secreting functional SynGAPl in large quantities to affected cells, c) The genetically modified immune cells circulate systemically and infiltrate the CNS as enzyme donors thus addressing both CNS and potential peripheral tissue pathology, d) Constitutive longterm expression of SynGAPl may translate to a one-time and life-long treatment unlike other treatment strategies which may require multiple injections / treatments for the life of the patient (i.e., ASOs). e) Neurons are not transduced directly, minimizing the potential for vector- mediated neurotoxicity.

[0119] For SRID, due to the lack of normal levels of SynGAPl, neuronal synapses do not function properly and develop too early, which presents with a wide array of clinical phenotypes and symptoms. These include intellectual disabilities, decreased motor skills, epilepsy, speech abnormalities, and sleep and behavioral disorders.1'5As such, treatments can be effective by, e.g., reducing or eliminating one or more symptoms or preventing or delaying the onset of one or more symptoms of SRID.

[0120] The therapy would be available to all those afflicted by SRID as the bone marrow is not affected in this disorder. Adequate numbers of CD34+ HSPC should be able to be collected from all participants. The therapy would include autologous CD34+ HSPC, so all patients requiring the therapy would be able to receive the therapy as their own HSPC would be used for development of their individual treatment. Adequate numbers of CD34+ HSPC can be collected from all participants.

[0121] In certain embodiments, the treatment could be performed at any time after birth, e.g., as an infant (e.g., from birth to about one year of age), a juvenile (e.g., from about one year of age to about 18 years of age), or an adult (e.g., older than about 18 years of age). In certain embodiments, treatment may be performed with an autologous bone marrow transplant or with an autologous peripheral blood infusion of vector transduced cells. Treatments using certain vectors, e.g., using an AAV vector, may be via intravenous or intracranial injection.

[0122] In certain embodiments, the modified SYNGAP1 sequence may be inserted into cells, e.g., as a treatment, using CRISPR technology.

[0123] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a polypeptide” may include two or more such molecules, and the like.

[0124] As used herein, the terms “about” and “approximately,” when used to modify an amount specified in a numeric value or range, indicate that the numeric value as well as reasonable deviations from the value known to the skilled person in the art, for example ± 20%, ± 10%, or ± 5%, are within the intended meaning of the recited value.

[0125] The term “subject,” “individual,” and “patient,” as used interchangeably herein, refer to a mammal, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one embodiment, the patient is a human. In one embodiment, the patient is a human male. In one embodiment, the patient is a human female. In some embodiments, the human is a patient in need of treatment for SRID. In some embodiments, the patient has one or more signs or symptoms of SRID. In another embodiment of the present disclosure, the human is a fetus, an infant, a pre-pubescent subject, an adolescent, a pediatric patient, or an adult. In one aspect, the subject is pre-symptomatic mammal or human. In another aspect, the subject has minimal clinical symptoms of the disease. The subject can be a male or a female, adult, an infant or a pediatric subject. The treatment is relevant for all age groups.

[0126] The term “isolated” as used herein with respect to nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively that are present in the natural source of the macromolecule. The term “isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term “isolated” is also used herein to refer to polypeptides, proteins and / or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term “isolated” means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require “isolation” to distinguish it from its naturally occurring counterpart.

[0127] In some embodiments, the term “engineered” or “recombinant” refers to having at least one modification not normally found in a naturally occurring protein, polypeptide, polynucleotide, strain, wild-type strain or the parental host strain of the referenced species. In some embodiments, the term “engineered” or “recombinant” refers to being synthetized by human intervention.

[0128] An “enriched population” of cells in certain embodiments is a substantially homogenous population of cells having certain defined characteristics. The cells are greater than 70 %, or alternatively greater than 75 %, or alternatively greater than 80 %, or alternatively greater than 85 %, or alternatively greater than 90 %, or alternatively greater than 95 %, or alternatively greater than 98% identical in the defined characteristics. The term “pharmaceutically acceptable excipient” refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically compatible for use in humans or animals, such as but not limited to a buffer, carrier, or preservative.

[0129] The term “administer” refers to a method of delivering agents compounds, or compositions (e.g., pharmaceutical composition) to the desired site of biological action. These methods include, but are not limited to, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, or intraperitoneal delivery.

[0130] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to clinical intervention to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.

[0131] The phrase “effective amount” in certain embodiments means an amount of a compound described herein that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0132] A “therapeutically effective amount” of a substance / molecule disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the substance / molecule, to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the substance / molecule are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.

[0133] An “effective amount” is in certain embodiments is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages.

[0134] Certain means of administration include intracerebroventricular (ICV), intraci sternal injection or infusion, subcutaneous injection, or implant administration. “Naturally occurring,” “native” or “wild type” is used to describe an object that can be found in nature as distinct from being artificially produced. For example, a nucleotide sequence present in an organism (including a virus), which can be isolated from a source in nature and which has not been intentionally modified in the laboratory, is naturally occurring. Furthermore, “wild-type” refers to the normal gene, or organism found in nature without any known mutation.

[0135] As used herein, the term “mutant” with respect to a mutant polypeptide or mutant polynucleotide is used interchangeably with “variant.” A variant with respect to a given wildtype reference sequence can include naturally occurring allelic variants. Accordingly, a “non- naturally” occurring polypeptide or polynucleotide refers to a variant or mutant polypeptide or polynucleotide that is not present in a cell in nature and that is produced, e.g., by genetic modification, e.g., using genetic engineering technology or mutagenesis techniques, of a native polynucleotide or polypeptide. A “variant” includes any domain comprising at least one amino acid mutation with respect to wild-type. Mutations may include substitutions, insertions, and deletions.

[0136] The presence of SynGAPl splice variants and isoforms is also contemplated herein (see, e.g., Gou et al., J Neurochem. 2020 Sep;154(6):618-634. doi: 10.1111 / jnc.14988. Epub 2020 Mar 10. PMID: 32068252; PMCID: PMC7754318).

[0137] In some embodiments, a starting or reference SynGAPl protein or polypeptide, such as a wild type as disclosed herein, an isoform thereof, a natural variant thereof, or a non-natural variant thereof, may be mutated to have at least one non-naturally occurring glycosylation site and one or more optional additional mutated residues that do not constitute a glycosylation site, thus resulting in an engineered and / or recombinant SynGAPl protein, polypeptide or a biological equivalent thereof. In further embodiments, the starting or reference SynGAPl protein or polypeptide may have an amino acid residue at any position mutated optionally to N with the proviso that the second amino acid residue on its C terminus side is a T or an S. Additionally or alternatively, the starting or reference SynGAPl protein or polypeptide may have an amino acid residue at any position mutated optionally to S to T with the proviso that the second amino acid residue on its N terminus side is an N. In some embodiments, any part of the starting or reference SynGAPl protein or polypeptide having a sequence of X1X2X3 may be engineered to be NXT / S (i.e., NXT and / or NXS), thus resulting in a recombinant SynGAPl protein, polypeptide or a biological equivalent thereof as disclosed herein, wherein XI, X2, X3 or X can be any amino acid residue. Exemplary glycosylation sites are provided herein. In some embodiments, the glycosylation sites are at amino acid positions of the polypeptide, protein or the equivalent thereof corresponding to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of those described in SEQ ID NO: 6, or a position shifting any one of the identified position herein to the C terminus or the N terminus on the polypeptide, protein or the equivalent by about 1 amino acid, about 2 amino acids, about 3 amino acids, about 4 amino acids, about 5 amino acids, about 6 amino acids, about 7 amino acids, about 8 amino acids, about 9 amino acids, or about 10 amino acids. For example, the glycosylation site(s), the sites with reference to SEQ ID NO:6, may be at any one of the following, or a combination thereof: aa 79-aa81; aa222-aa224; aa256-aa258; aa293-aa295; aa315-aa317; aa456-aa458; aa556-aa558; and / or aa689-aa691.

[0138] It is also contemplated that other glycosylations site(s) in the nucleotide sequence encoding SynGAPl may also or alternatively be designed.

[0139] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.

[0140] Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxy glutamate and O- phosphoserine. “Amino acid analogs” refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. “Amino acid mimetics” refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

[0141] Naturally occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Stereoisomers of a naturally-occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D- Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D-His), D-isoleucine (D- Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D-methionine (D-Met), D- asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D-serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D-Tyr), and combinations thereof.

[0142] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0143] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids.

[0144] The term “protein” as used herein refers to either a polypeptide or a dimer (z.e, two) or multimer (z.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions.

[0145] The term “protein”, “peptide” and “polypeptide” are used interchangeably and in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term “amino acid” refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.

[0146] The term equivalent and biological equivalent are used interchangeably, for example when referring to a protein or polypeptide as a reference. In some embodiments, an equivalent protein or polypeptide is one having at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the reference protein or polypeptide. In some embodiments, an equivalent protein or polypeptide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to a polypeptide or protein as disclosed herein. In some embodiments, an equivalent protein or polypeptide has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to polypeptide or protein encoded by an equivalent polynucleotide as noted herein. In addition or alternatively, the equivalent of a polynucleotide would encode a protein or polypeptide of the same or similar function as the reference or parent polynucleotide.

[0147] In some embodiments, the equivalent is a functional protein that optionally can be identified through one or more assays. In another aspect, an equivalent has at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%sequence identity to the reference protein or polypeptide. Some embodiments are with the proviso that one or more amino acids identified herein as mutated to a possible glycosylation site are mutated from a polypeptide or protein in the disclosed sequences. Some embodiments are with the proviso that one or more amino acids identified herein as mutated to a possible glycosylation site are not mutated from a polypeptide or protein in the disclosed sequences.

[0148] In some embodiments, the equivalent protein or polypeptide performs functions similar to a wildtype and / or at a similar level compared to a wildtype. For example, a biological equivalent of a protein or polypeptide may have similar functions compared to a wild type protein and / or any one or more of the functions of the biological equivalent of the protein or polypeptide at a similar level (such as having similar activity) compared to a wildtype protein or polypeptide. In further embodiments, for example, the equivalent’s function is at a level of at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 100%, at least about 1.5 folds, at least about 2 folds, at least about 3 folds, at least about 5 folds, at least about 10 folds of those wild type ones.

[0149] The term “conservative substitution,” “conservative mutation,” or “conservatively modified variant” refers to an alteration that results in the substitution of an amino acid with another amino acid that can be categorized as having a similar feature. Examples of categories of conservative amino acid groups defined in this manner can include: a “charged / polar group” including Glu (Glutamic acid or E), Asp (Aspartic acid or D), Asn (Asparagine or N), Gin (Glutamine or Q), Lys (Lysine or K), Arg (Arginine or R), and His (Histidine or H); an “aromatic group” including Phe (Phenylalanine or F), Tyr (Tyrosine or Y), Trp (Tryptophan or W), and (Histidine or H); and an “aliphatic group” including Gly (Glycine or G), Ala (Alanine or A), Vai (Valine or V), Leu (Leucine or L), He (Isoleucine or I), Met (Methionine or M), Ser (Serine or S), Thr (Threonine or T), and Cys (Cysteine or C). Within each group, subgroups can also be identified. For example, the group of charged or polar amino acids can be sub-divided into sub-groups including: a “positively-charged sub-group” comprising Lys, Arg and His; a “negatively-charged sub-group” comprising Glu and Asp; and a “polar sub-group” comprising Asn and Gin. In another example, the aromatic or cyclic group can be sub-divided into subgroups including: a “nitrogen ring sub-group” comprising Pro, His and Trp; and a “phenyl subgroup” comprising Phe and Tyr. In another further example, the aliphatic group can be subdivided into sub-groups, e.g., an “aliphatic non-polar sub-group” comprising Vai, Leu, Gly, and Ala; and an “aliphatic slightly-polar sub-group” comprising Met, Ser, Thr, and Cys. Examples of categories of conservative mutations include amino acid substitutions of amino acids within the sub-groups above, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free -OH can be maintained; and Gin for Asn or vice versa, such that a free -NH2 can be maintained. In some embodiments, hydrophobic amino acids are substituted for naturally occurring hydrophobic amino acid, e.g., in the active site, to preserve hydrophobicity.

[0150] In certain embodiments, codon optimization can be utilized to further engineer the nucleotide sequences of the invention.

[0151] The terms “identical” or percent “identity,” in the context of two or more polypeptide or polynucleotides sequence, refer to two or more sequences or subsequences that are the same or have a specified percentage of residues, e.g., at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or greater, that are identical over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection. In some embodiments, a sequence that has a specified percent identity relative to a reference sequence differs from the reference sequence by one or more conservative substitutions.

[0152] For sequence comparison of polypeptides, typically one amino acid sequence acts as a reference sequence, to which a candidate sequence is compared. Alignment can be performed using various methods available to one of skill in the art, e.g., visual alignment or using publicly available software using known algorithms to achieve maximal alignment. Such programs include the BLAST programs, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.) or Megalign (DNASTAR). The parameters employed for an alignment to achieve maximal alignment can be determined by one of skill in the art. For sequence comparison of polypeptide sequences for purposes of this application, the BLASTP algorithm standard protein BLAST for aligning two proteins sequence with the default parameters is used.

[0153] The terms “corresponding to,” “determined with reference to,” or “numbered with reference to” when used in the context of the identification of a given amino acid residue in a polypeptide sequence, refers to the position of the residue of a specified reference sequence when the given amino acid sequence is maximally aligned and compared to the reference sequence. The polypeptide that is aligned to the reference sequence need not be the same length as the reference sequence.

[0154] The term “polynucleotide” and “nucleic acid” interchangeably refer to chains of nucleotides of any length and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules having mixtures of single- and double-stranded DNA and RNA.

[0155] The term “glycosylation site” refers to amino acid sequences having the motif “NXT / S”. In some embodiments, a glycosylation site comprises, or alternatively consists essentially of, or yet further consists of the consensus sequence of NXT / S (z.e., NXT and / or NXS) where X is any amino acid residue. In some embodiments, a glycosylation site comprises, or alternatively consists essentially of, or yet further consists of the consensus sequence of NXT / S (ie., NXT and / or NXS) where X is any amino acid residue except proline (P). In some embodiments, the glycosylation site has a consensus sequence of NXT or NXS, wherein X is any amino acid residue, optionally except proline (P). In some embodiments, the glycosylation is N-linked. Potential glycosylation sites can be identified by evaluating the wild-type sequence to locate areas where changes to the wildtype sequence can be minimized, e.g., to avoid negatively affecting the function of the resulting protein, e.g., to only make a mutation that changes one amino acid to create the glycosylation site.

[0156] The following Example is intended to be non-limiting. Further, certain aspects of the discoveries described here are included in Anderson et al., J Gene Med. 2024 Jul;26(7):e3717. doi: 10.1002 / jgm.3717. PMID: 38967915, which is hereby incorporated by reference for all that it contains.

[0157] EXAMPLE 1

[0158] As there are currently no effective treatments or cures for SRID, novel therapies are needed to help affected patients. Stem cell gene therapy approaches, especially those involving the hematopoietic system, offer the potential to supply therapeutic levels of SynGAPl through a process called cross-correction. If the HSPC and their cell progeny can be gene modified to deliver therapeutic levels of SynGAPl to affected neurons, disease treatment might be achieved.

[0159] To develop a stem cell gene therapy for SRID, a lentiviral vector expressing a modified form of mouse SynGAPl in human CD34+ HSPC was developed. The functionality, initial safety, and in vivo efficacy of this newly developed lentiviral vector was evaluated. As detailed herein, there are advantages to this approach including: a) the hematopoietic system is unaffected by SYNGAP1 and therefore, an autologous transplant setting can be utilized thus avoiding any allogeneic associated toxicities, b) as lentiviral vectors permanently integrate into the DNA of target cells, there is a potential for a one-time treatment for patients if enough gene modified cells engraft and therapeutic levels of SynGAPl is expressed and delivered to affected neurons, and c) this strategy does not rely on the correction of each individual neuron, as the engrafted gene modified microglia can continuously express and deliver functional SynGAPl to neurons throughout the brain. Since the SynGAPl protein is naturally found intracellularly in the synaptic junctions, the task of expressing and delivering SynGAPl to the brain to be delivered to neurons, and for it to function in a normal manner, was challenging. As described herein, modifications to the SYNGAP1 gene were made to allow for expression by the gene modified cells and uptake by neurons, while retaining normal SynGAPl function. These modifications included (1) adding a secretion signal so that the SynGAPl protein is expressed and secreted by the gene modified cells and (2) adding N-glycosylation sites to the SynGAPl protein sequence to allow for uptake by neurons via the mannose-6-phospahate receptor. To initially evaluate this strategy, expression of SynGAPl was successfully detected in synl8 vector transduced cells, both B lymphocytes obtained from a SRID patient and also in normal human CD34+ HSPC derived macrophages as demonstrated by QPCR and Western blots. Normal differentiation and expansion of the synl8 vector transduced cells was also observed when cultured in a CFU assay. To further evaluate this therapeutic strategy and determine whether in vivo efficacy could be observed, an immunodeficient (IL2rg- / y) version of the heterozygous SYNGAP1+ / - mouse model (named BGS) was developed, which allowed for transplant and engraftment human CD34+ HSPC.see 26-30The knockout of the IL2 receptor gamma gene, as observed in human patients with X-linked severe combined immunodeficiency, causes an immunodeficiency in the mice by halting the activation of T and B cells thus creating a limited number of mature lymphocytes.32'34It also causes defects in natural killer cell activity allowing for the transplanted human CD34+ HSPC to robustly engraft in the bone marrow, lymphoid organs, and in the brain.26'29This strategy of introducing an immunodeficiency into disease-specific mouse models has been successful in prior work and allows use of the exact cell type that would be used in a clinical trial setting.17'18The ILr2g- / y genotype was used in making the BGS mice.18Both male and female heterozygous SYNGAP1+ / - mice were used when generating the BGS line since they display similar phenotypes, while homozygous SYNGAP1- / - mice die within two weeks of birth and do not allow for adequate efficacy studies. Phenotypic differences are well defined between wild type SYNGAP1+ / + and heterozygous SYNGAP1+ / - mice.30Heterozygous SYNGAP1+ / - mice exhibit hyperactivity displaying increased movements and higher total distance traveled values. Heterozygous SYNGAP1+ / - mice exhibit an impaired grasping ability causing a decreased latency to fall from the accelerating rod. After introducing the IL2rg- / y genotype into the SYNGAP1+ / - mice, effects of the added immunodeficiency on the phenotypes observed in the original immunocompetent heterozygous SYNGAP1+ / - mice were not observed. Nontransplanted BGS mice demonstrated their typical hyperactivity in the open field assay displaying increased total distance, increased horizontal activity, and increased vertical episodes compared to their wild type littermates. They also displayed a lack of grasping ability in the rotorod assay compared to their wild type littermates. However, upon transplantation of the SynGAPl expressing vector transduced cells, a significant decrease and thus improvement in the open field assay measurements including total distance, horizontal activity, and vertical episodes was observed, as compared to the nontransplanted and empty vector transduced cell engrafted mice. This was also observed when evaluating grasping ability in a rotorod assay as a significant increase in latency time to fall was observed in the mice transplanted with the synl8 vector transduced cells compared to the nontransplanted and empty vector control mice. Even though improvement in these motor skills was observed, a complete reversion to wild type levels was not observed. Since these mice were transplanted as eight- week-old adults, it is possible that a level of neurodegeneration or neuronal dysfunction was already present and irreversible. It is also possible that a certain therapeutic threshold of SynGAPl expression needs to be reached in observe a complete reversion to wild type phenotypes.

[0160] In an effort to determine whether the levels of expression of SynGAPl correlated with performance in the open field and rotorod assays, the synl8 mice were separated based on average VCN per cell, either >2.0 VCN (synl8 high) with a range of 2.33-3.60 (average of 2.98) or <2.0 VCN (synl8 low) with a range of 0.42 to 1.23 (average of 0.88). Analysis of the open field and rotorod data was then repeated and compared to the control groups. It was observed that the synl8 high group performed closer to wild type levels unlike the synl8 low group. This suggested that VCN and an inferred increase in expression of SynGAPl by the gene modified cells have a direct correlation with phenotypic improvement. This suggests a possible therapeutic threshold that needs to be reached with SynGAPl expression and delivery to be therapeutically effective in helping patients.

[0161] As a first step in explaining the results, immunohistochemistry was performed on the brain tissues of the mice that were transplanted with synl8 vector transduced cells. A significant increase in SynGAPl positive cells was detected in the synl8 brain tissues and can potentially explain why these mice performed better in the motor assays compared to the control mice. However, in comparison to what was observed in the open field and rotorod data when separating the synl8 group into high and low subgroups based on VCN, a correlation in positive cell counts within the IHC data was not observed. The synl8 high subgroup did not have a higher number of SynGAPl positive cells compared to the synl8 low subgroup. The positive cell intensity data was also evaluated, and similar levels were observed between the synl8 high and synl8 low subgroups. This observation could be due to the sensitivity of the assay not being strong enough to detect lower areas of concentration of SynGAPl that were expressed by the gene modified cells and secreted into the extracellular space. The results presented here also are only for positive cells and not the total concentration of SynGAPl in the brain tissues of these mice.

[0162] When initially evaluating the feasibility of this stem cell gene therapy approach, a strong MNDU3 promoter was used to express the modified SYNGAP1 gene. However, other promoters (e.g., strong promoters) may also be used in place of the MNDU3 promoter. For example, other promoters include: a Syn-1 (Synapsin 1) promoter, human PGK, mouse PGK, CMV, long EFl alpha, short EFl alpha, CD68, and microglia-specific promoters.

[0163] MATERIALS AND METHODS

[0164] SYNGAP1 Lentiviral vector

[0165] A third-generation self-inactivating lentiviral vector backbone, CCLc-X, was used to generate the SYNGAP1 expressing vector (synl8). A modified mouse SYNGAP1 gene was manually synthesized, cloned under the control of an MNDU3 promoter, and inserted into the lentiviral vector backbone (Figure 1(A)). The sequence of this vector was then verified. A control empty vector (EV), only containing the CCLc-X backbone was also used in the experiments to control for vector transduction.

[0166] Lentiviral vectors were generated in human embryonic kidney (HEK)-293 cells by transfection with a 1 : 1 :5:5 ratio of the envelope vesicular stomatitis virus glycoprotein (VSVG) plasmid, a plasmid expressing the Rev gene, a packaging plasmid containing a capsid gene and a reverse transcriptase gene, and one of the transfer plasmids, either the control EV or the synl8 plasmids. Forty-eight hours post-transfection, supernatants were concentrated by ultrafiltration. Titers for total vector transducing units were calculated by transduction of HEK-293 cells. Total DNA was extracted from the transduced cells, at least 48 hours post-transduction, using a Promega Wizard Genomic DNA Purification kit (Promega, Madison, WI, USA). Quantitative PCR (QPCR) was then performed using primers and a probe specific for the vector backbone: forward primer: 5’-ACCTGAAAGCGAAAGGGAAAC-3’ (SEQ ID NO: 17) reverse primer: 5’- CGCACCCATCTCTCTCCTTCT-3’ (SEQ ID NO: 18) probe: 5’- AGCTCTCTCGACGCAGGACTCGGC-3’ (SEQ ID NO: 19). The QPCRs were performed on a StepOne Real Time PCR System (ThermoFisher, Carlsbad, CA, USA).

[0167] SYNGAPl-affected B lymphocytes and human CD34+ HSPC

[0168] SYNGAP1 -affected B cells were purchased from a commercial source (Cori ell, Camden, NJ, USA). The cells were cultured in complete RPMI media (ThermoFisher, Carlsbad, CA, USA) containing 15% fetal bovine serum and 5% L-glutamine. Cells were left either nontransduced, transduced with the EV vector, or transduced with the synl8 vector at a multiplicity of infection (MOI) of 5 with the addition of 8 mg / ml protamine sulfate for a minimum of 3 hours at 37 degrees Celsius. Human CD34+ HSPC were purchased from a commercial source (Stem Cell Technologies, Vancouver, BC, Canada). These CD34+ cells were cultured for 24 hours in XVIVO-IO media (Lonza, Anaheim, CA, USA) containing 100 ng / ml Flt-3 ligand, thrombopoietin (TPO), and stem cell factor (SCF) (R&D Systems, Minneapolis, MN). After this culture period, the cells were left either nontransduced, transduced with the EV vector, or transduced with the synl8 vector at an MOI of 5 with the addition of 8 mg / ml protamine sulfate and a 1 : 100 dilution of LentiBoost (Sirion Biotech, Cambridge, MA, USA) for a minimum of 3 hours at 37 degrees Celsius.

[0169] Colony-forming unit (CFU) assays

[0170] The human CD34+ cells, either NT, EV control vector transduced, or synl8 vector transduced (500 total cells) were cultured for 14 days in cytokine enriched MethoCult medium (Stemcell Technologies, Vancouver, BC, Canada). After this culture period, total granulocyte / macrophage (GM), granulocyte / erythrocyte / megakaryocyte / macrophage (GEMM), and burst forming unit-erythroid colonies (BFU-E), were counted by microscopy. Total cell counts post-culture were also evaluated. Experiments were performed in triplicate.

[0171] Derivation of mature macrophages and phenotypic analyses

[0172] After the CFU assays were completed, the cells were further cultured and differentiated into mature macrophages, in vitro, by plating the cells in DMEM supplemented with 10% FBS, 10 ng / ml of granulocyte / macrophage colony stimulating factor (GM-CSF), and 10 ng / ml macrophage colony stimulating factor (M-CSF) (R&D Systems, Minneapolis, MN, USA) for 4 days. Macrophages were further analyzed by flow cytometry for surface expression of the macrophage cell surface markers, CD 14, CD4, and HLA-DR. Macrophages were stained with a phycoerythrin (PE)-conjugated CD14 (catalog# 555398), a PE-conjugated CD4 (catalog# 555347), or a PE-conjugated HLADR antibody (catalog# 555561) (BD Biosciences, San Jose, CA, USA). Flow cytometry was performed on a Becton Dickinson Canto I flow cytometer.

[0173] Quantitative PCR

[0174] To detect SYNGAP1 mRNA expression in the vector transduced cells, quantitative PCR (QPCR) was performed. Total RNA was extracted from the cells using RNA STAT-60 (amsbio, Cambridge, MA, USA) according to the manufacturer’s protocol. cDNA synthesis was then performed using a Taqman Reverse Transcription Reagents kit (ThermoFisher, Carlsbad, CA, USA). QPCR was then performed using a mouse SYNGAP1 -specific primer and probe set: forward primer: 5'-CATCGAGCGAGAAGAGTACAAG-3' (SEQ ID NO:20), reverse primer: 5'-CCTCTCCTTCAGCGAATGTATC-3 (SEQ ID N0:21)', probe: 5'- TGGACAGGGTGAAGGAGTATGAGGA-31(SEQ ID NO:22). Experiments were performed in triplicate.

[0175] Western blot

[0176] For Western blots, 25 pg protein was separated on a 4-20% Mini-PROTEAN TGX gel (BioRad, Hercules, CA, USA) with Tris-Glycine SDS Running Buffer (Invitrogen, Carlsbad, CA, USA). Protein was subsequently transferred onto a nitrocellulose membrane. The membrane was rinsed with deionized water and incubated in blocking solution (5% bovine serum albumin in TBST; 50 mm Tris, 150 mm NaCl, 0.1% Tween- 20) for 30 min at room temperature. Membranes were incubated with primary rabbit SynGAP polyclonal antibody (catalog# PA1- 046) (ThermoFisher, Carlsbad, CA, USA) at 1 : 1000 dilution in blocking solution at 4 degrees Celsius overnight. After three 10-min washes with TBST the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (catalog# NEF812001EA) (PerkinElmer, Shelton, CT, USA) (1 : 1000 dilution in blocking solution) for 45 min at room temperature. After three more washes in TBST, proteins were visualized with Prometheus Prosignal Pico ECL Reagent (Genesee Scientific, El Cajon, CA, USA) using the ChemiDoc XRS Imaging System (BioRad, Hercules, CA, USA). GAPDH was used as an internal loading control (primary antibody: mouse anti-human GAPDH (catalog# ab8245) (abeam, Boston, MA, USA), secondary antibody: goat anti-mouse IgGl HRP (catalog# ab97240) (abeam, Boston, MA, USA)).

[0177] Generation of the immunodeficient SYNGAP1 mouse model

[0178] To generate an immunodeficient version of the SYNGAP 1 -affected mouse model, the B6-SYNGAP1+ / - heterozygous mice (JAX stock# 008890) was crossed with the B6-IL2rg- / y homozygous mice (JAX stock# 003175).26-30Since male and female heterozygous SYNGAP1+ / - mice behave the same and their breeding is unaffected by the loss of wild type SynGAP 1 expression, both sexes were used to create the immunodeficient mouse model (B6-IL2rg- / ySYNGAPU- / -) (BGS). Genotyping was performed by normal PCR with the following primer sets and according to the protocols listed by the Jackson Laboratory for the respective genes: SYNGAP1 common 5’-AGGGAACATAAGTCTTGGCTCTGTC-3’ (SEQ ID NO:23), SYNGAP1 wild type 5’-ACCTCAAATCCACACTCCTCTCCAG-3’ (SEQ ID NO:24), SYNGAP1 mutant 5’-ATGCTCCAGACTGCCTTGGGAAAAG-3’ (SEQ ID NO:25), il2rg common 5’-GTGGGTAGCCAGCTCTTCAG-3’ (SEQ ID NO:26), IL2rg wild type reverse 5’- CCTGGAGCTGGACAACAAAT-3’ (SEQ ID NO:27), IL2rg mutant reverse 5’- GCCAGAGGCCACTTGTGTAG-3’ (SEQ ID NO:28).

[0179] Transplantation and generation of the humanized BGS mice

[0180] Eight-week old BGS (IL2rg- / ySYNGAPl+ / -) mice (N=9 per group containing a mix of males and females) were conditioned with busulfan (20 mg / kg body weight) on day -2 and -1 pre-transplant by intraperitoneal injection. On day 0, post-busulfan conditioning, mice were transplanted intravenously with 500,000 total human CD34+ HSPC transduced with either the EV or the synl8 vector. Nontransplanted wild type (WT) (IL2rg- / ySYNGAPl+ / +) and nontransplanted heterozygous SYNGAP1 (NT) mice (IL2rg- / ySYNGAPl+ / -) were used as controls. The WT and NT mice that were not transplanted did not receive any busulfan conditioning. Sixteen weeks post-transplant to allow for the transplanted cells to fully engraft, the mice were subjected to the open field and rotorod assays. At the end of these experiments, the mice were euthanized and the peripheral blood was analyzed for lentiviral vector copy number by QPCR as described above to determine the levels of engraftment of the gene modified cells. Brain tissues were also collected and analyzed for SynGAPl expression by immunohistochemistry. All mouse handling and experiments were performed following the UC Davis IACUC policies. A power analysis was performed to determine that an estimated sample size required was eight animals per group. Both male and female heterozygous SYNGAP1 mice display similar SYNGAP1 -related phenotypes and therefore, were combined into each group during analysis. There was no set method of randomization, however, when choosing a mouse for transplant in the EV or synl8 group, a random mouse with the correct genotype required was used. Also, NT and WT littermates were always used as controls.

[0181] Motor skill evaluation

[0182] Open field assay: To evaluate whether the SYNGAP1 vector transduced human CD34+ HSPC improved motor phenotypes, mice were subjected to a 15 -minute open field assay to evaluate total distance moved, total horizontal activity, and total vertical episodes. Mice were first allowed to acclimate to the room for one hour. Mice were then placed in a 45 * 45 cm photocell-equipped Accuscan which automatically recorded and analyzed parameters. The Student’ s t-test was used for statistical analyses to compare the synl8 mice to either the NT, EV, or WT groups.

[0183] Rotarod assay: Motor coordination, balance, grasping ability, and learning were evaluated on an accelerating rotarod apparatus. Mice were first allowed to acclimate to the room for one hour. The mice were then placed on a rotating cylinder that slowly accelerated from 5 to 40 revolutions per minute over a 5-minute testing period (Rotamex-5, Columbus Instruments, Columbus, OH, USA). Testing was performed on three consecutive days. Performance was scored as latency time to fall off of the rotating rod with a maximum time of 5 minutes. If a mouse fell off within 3 seconds of placement, a second trial was allowed. The Student’s t test was used for statistical analyses to compare the synl8 mice to either the NT, EV, or WT groups using the same rationale as above to test the hypothesis. Statistical variance was similar between groups compared. Personnel performing the open field and rotorod experiments were blinded by only having access to the mouse identification numbers.

[0184] Immunohistochemistry

[0185] Upon completion of the open field and rotorod assays, mice were perfused and the brain tissues were collected and fixed in 4% paraformaldehyde. Tissues were further trimmed and embedded as paraffin blocks. These tissue blocks were then sagittally sectioned at 5 pm directly onto SuperFrost slides. Staining of the slides was performed using a Leica Bond automated immunostainer with a rabbit anti-mouse SynGAP polyclonal antibody (catalog# PAI -046) (ThermoFisher, Carlsbad, CA, USA) at a 1 :400 dilution in Citrate Epitope Retrieval buffer 1 at pH 6.0. A secondary anti-rabbit Poly-HRP-IgG antibody (catalog# RE7280-CE) (Leica Biosystems, Deer Park, IL, USA) was then added followed by the addition of DAB (3,3'- Diaminobenzidine) and hematoxylin as a counterstain. Whole slide images were generated using a Pannoramic SCAN (3D Histech). Whole sections were then enumerated for total positive cells and total positive cell intensity using imageDx software. The Student’s t test was used for statistical analyses to directly compare the synl8 mice to either the NT, EV, or WT groups.

[0186] RESULTS

[0187] SYNGAP1 vector functionality and detection of expression in transduced cells

[0188] To evaluate whether the SYNGAP 1 vector was capable of expressing SynGAP 1 in transduced cells, QPCR and Western blots were performed to detect SynGAPl mRNA and protein, respectively. Vector expression was initially in patient-derived SYNGAP 1 -affected B lymphocytes containing a nonsense mutation in exon 17. As displayed in Figure 1(B), overexpression (>27-fold) of SYNGAP1 transcripts was detected in the vector transduced B lymphocytes (synl8) compared to the nontransduced (NT) and empty vector (EV) transduced cells. The data is presented as fold-increase with the NT cell value normalized to 1.0. To further evaluate expression of SynGAPl in the vector transduced cells, Western blots were performed. As displayed in Figure 1(C), successful expression of SynGAPl (-135 kDa band) was demonstrated in the synl8 vector transduced cells compared to the NT and EV cells. GAPDH was used as an internal loading control.

[0189] For the HSPC gene therapy approach to be effective, the cells need to be able to divide and differentiate into the myeloid lineage to effectively express functional SynGAPl to nearby neurons. Therefore, to initially evaluate the safety of the SYNGAP1 expressing vector, human CD34+ HSPC were transduced with the synl8 vector and subjected to a colony forming unit (CFU) assay. Total numbers of granulocyte / macrophage (GM), granulocyte / erythrocyte / megakaryocyte / macrophage (GEMM), and burst forming unit-erythroid colonies (BFU-E) were counted and compared to control cells. The total number of cells obtained after 14 days of culture in cytokine-enriched methylcellulose media were also calculated to determine fold expansion of the cells. As displayed in Figure 2(A) and 2(B), similar levels of all colonies (GM, GEMM, and BFU-E) and a similar level of total cell expansion were observed in the synl8 (68- fold expansion) cultures as compared to the NT (73-fold expansion, p=0.22) and EV (69-fold expansion, p=0.83) cultures. After the CFU assay was completed, these cells were further differentiated into mature macrophages, in vitro, and evaluated for SynGAPl expression by Western blots. As displayed in Figure 2(C), expression of SynGAPl was observed in the synl8 vector transduced cells as compared to the NT and EV cells which did not show any expression. Detection of SynGAPl in the NT and EV lanes was not expected as it is not normally expressed in macrophages. GAPDH was used as an internal loading control.

[0190] To further evaluate normal differentiation of the synl8 vector transduced CD34+ cell derived macrophages, flow cytometry was performed to compare levels of the cell surface phenotypic markers CD 14, CD4, and HLADR were present. Similar levels of these markers (CD14+ 82.4-93.0%, CD4+ 79.0-88.4%, and HLADR+ 63.8-73.1%) were observed in the NT, EV, and synl8 macrophage cultures.

[0191] Together, these results highlight the functionality of the SYNGAP1 expressing lentiviral vector and the ability of the vector transduced human CD34+ HSPC to divide and differentiate into myeloid cells in a manner similar to control cells. Improved motor function in syn!8 vector transduced cell engrafted BGS mice

[0192] As a first step in evaluating the efficacy of a stem cell gene therapy approach in rescuing SYNGAP1 -related phenotypes, SYNGAP1 vector transduced human CD34+ HSPC were transplanted into immunodeficient (IL2rg- / y) mice heterozygous for the SYNGAP1+ / - mutation (BGS). Eight-week-old BGS mice were left either nontransplanted (N=9) or transplanted with either the EV (N=9) or SYNGAP1 (synl8) (N=9) expressing vector transduced human CD34+ HSPC. Wild type nontransplanted B6-IL2rg- / ySYNGAPl+ / + mice were used as controls (N=9). Sixteen weeks post-transplant, mice were subjected to an openfield assay to determine if improvement of the SYNGAP1 -related phenotype of hyperactivity and increased total activity had occurred. As displayed in Figure 3(A), a significant decrease in total distance traveled was observed in the synl8 vector transduced cell engrafted mice compared to the NT (p=0.036) and the EV (p=0.021) groups. As displayed in Figure 3(B), a significant decrease in horizontal activity was observed in the synl8 vector transduced cell engrafted mice compared to the NT (p=0.049) and the EV (p=0.022) groups. As displayed in Figure 3(C), a significant decrease in vertical episodes was also observed in the synl8 vector transduced cell engrafted mice compared to the NT (p=0.029) and the EV (p=0.037) groups. As compared to the WT mice, total distance traveled and horizontal activity was not completely rescued to WT levels in the synl8 mice as these values were still significantly different (p=0.0038 and p=0.0013, respectively). However, when comparing WT and synl8 mice in total vertical episodes, there was no significant difference (p=0.34) between the two groups.

[0193] When analyzing the data, a subgroup of synl8 mice performed closer to WT mice than other synl8 mice, and this subgroup had a higher lentiviral vector copy number (VCN) upon collection and analysis of the peripheral blood for post-euthanization. Therefore, whether there was a correlation between VCN and performance in the open field assay was evaluated. With a higher average VCN per cell, there is a greater chance of obtaining a higher level of expression of SynGAPl and potentially greater efficacy. Subsequently, mice were separated by peripheral blood VCN with those having either >2.0 VCN (N=4) with a range of 2.33-3.60 (average of 2.98) or <2.0 VCN (N=5) per cell with a range of 0.42 to 1.23 (average of 0.88). As displayed in Figure 3(D)-3(F), an increased level of improvement of SYNGAP1 -related phenotypes was observed in the four synl8 mice with the highest VCN (average VCN of 2.98) as compared to the five synl8 mice with the lowest VCN (average VCN of 0.88). Significance in phenotypic improvement of the individual phenotypes also changed when the synl8 mice were separated based on average VCN. An increased level of significance was observed in total distance traveled when comparing synl8 high mice to NT (p=0.0084) and EV (p=0.0032) groups. However, when comparing synl8 low mice to NT (p=0.29) and EV (p=0.22) groups, no significant difference was observed. When comparing the synl8 high mice to the WT group, full rescue to WT levels was not achieved as there was still a significant difference (p=0.021) between groups, however, the synl8 high group performed better than when all synl8 mice were analyzed together (p=0.0038). This was found to also be true when evaluating the synl8 high and synl8 low mice for total horizontal activity and total vertical episodes. Significance increased between the synl8 high subgroup compared to the NT and EV groups for horizontal activity (p=0.0077 and p=0.0065) and vertical episodes (p<0.001 and p=0.018), respectively. For the synl8 low group, no significant difference was observed between the NT and EV groups when looking at horizontal activity (p=0.45 and p=0.26) and vertical episodes (p=0.22 and p=0.21), respectively. These results highlighted the potential of greater phenotypic improvement when a higher level of the SYNGAP1 expressing vector is present in the gene modified cells.

[0194] To further evaluate motor skills upon transplantation of SYNGAP1 vector transduced human CD34+ HSPC, a rotorod assay was performed to measure grasping ability by remaining on a rotating rod for as long as possible while its speed accelerates over time. This assay can also measure learning as it is performed on three consecutive days and any improvement in latency to fall can be attributed to a mouse’s learning how to stay on the rod over time. As displayed in Figure 4(A), a significant increase in latency time to fall was observed on day 3 of the experiment in the mice transplanted with the SynGAPl vector transduced cells (synl8) as compared to the NT (p= 0.036) and EV (p=0.049) groups. Reversion to WT activity was not observed as the synl8 mice were still significantly different (p=0.021) than WT mice. As the latency time to fall also increased from days 2 to 3 in the synl8 group, this suggests that a level of learning was established compared to the NT and EV groups which demonstrated no increase in latency time to fall from days 2 to 3. As with the open field assay, whether the level of peripheral blood VCN per cell had any correlation with performance in the rotorod assay was assessed. As displayed in Figure 4(B), upon separating the synl8 group into high and low VCN subgroups (N=4 and N=5, respectively), a significant increase in latency time to fall was observed with the synl8 high mice compared to the NT and EV groups on day 1 (p=0.013 and p=0.020), day 2 (p=0.0057 and p=0.0012), and day 3 (p=0.011 and p=0.013) and not just on day 3 when evaluating the entire synl8 group together. When comparing the synl8 high group to WT mice, phenotypic improvement to WT levels was observed since no significant difference in latency time to fall was observed on day 1 (p=0.80), day 2 (p=0.16), or day 3 (p=0.44). There was still a significant difference on day 3 between the syn low group compared to the NT (p=0.021) and EV (p=0.039) groups. Again, learning in the synl8 high and low groups can be inferred as the latency time to fall increased from day 2 to day 3.

[0195] At the conclusion of these studies, the mice were bled via the tail vein and VCN per cell was calculated by QPCR. For the EV mice, VCN ranged from 0.31 -2.1 and for the synl 8 mice, VCN ranged from 0.42-3.6. These VCN values were used to separate the synl 8 group into high and low subgroups. Together, these results demonstrated that a significant level of improvement in these phenotypes could be accomplished in mice transplanted with the SYNGAP1 vector transduced cells.

[0196] Detection of SynGAPl expression in the brains of synl8 mice

[0197] To determine whether an increased level of SynGAPl was present in the mice transplanted with the SYNGAP1 vector transduced cells, immunohistochemistry was performed on brain tissue obtained from the mice post-evaluation of the open field and rotorod assays. As displayed in Figure 5(A), a significant increase in SynGAPl was detected in the brains of synl8 mice (N=9) compared to NT (N=6) (p=0.019) and EV (N=6) (p=0.013) groups. The BGS mice are heterozygous for SynGAPl expression so some level of detection in the NT and EV groups in the brain tissues was anticipated. As compared to WT mice (N=5), no significant difference (p=0.19) was observed between these two groups. Unlike what was observed in the motor assays, however, the syn 18 high subgroup did not show an increased level of expression compared to the synl 8 low subgroup. Representative images of a region of the frontal lobe from the labeled brain tissues from each group are presented in Figure 5(B). These results confirm that transplantation and engraftment of the SYNGAP1 vector transduced cells resulted in higher levels of SynGAPl expression in the brains of BGS mice.

[0198] The following sequences illustrate particular nucleotide and amino acid sequences.

[0199] Wild type human SynGAPl sequence (SEQ ID NO: 1). atgagcaggt ctcgagcctc catccatcgg gggagcatcc ccgcgatgtc ctatgccccc ttcagagatg tacggggacc ctctatgcac cgaacccaat acgttcattc cccgtatgat cgtcctggtt ggaaccctcg gttctgcatc atctcgggga accagctgct catgctggat gaggatgaga tacaccccct actgatccgg gaccggagga gcgagtccag tcgcaacaaa ctgctgagac gcacagtctc cgtgccggtg gaggggcggc cccacggcga gcatgaatac cacttgggtc gctcgaggag gaagagtgtc ccagggggga agcagtacag catggagggt gcccctgctg cgcccttccg gccctcgcaa ggcttcctga gccgacggct aaaaagctcc atcaaacgaa cgaagtcaca acccaaactt gaccggacca gcagctttcg ccagatcctg cctcgcttcc gaagtgctga ccatgaccgg gcccggctga tgcaaagctt taaggagtca cactctcatg agtccttgct gagtcctagc agtgcagctg aggcattgga gctcaacttg gatgaagatt ccattatcaa gccagtgcac agctccatcc tgggccagga gttctgtttt gaggtaacaa cttcatcagg aacaaaatgc tttgcctgtc ggtctgcggc cgaaagagac aaatggattg agaatctgca gcgggcagta aagcccaaca aggacaacag ccgccgggta gacaatgtgc taaagctgtg gatcatagag gcccgggagc tgccccccaa gaagcggtac tactgtgagc tctgcctgga tgacatgctg tatgcacgca ccacctccaa gccccgctct gcctctgggg acaccgtctt ctggggcgag cacttcgagt ttaacaacct gccggctgtc cgtgccctgc ggctgcatct gtaccgtgac tcagacaaaa agcgcaagaa ggacaaggca ggctatgtcg gcctggtgac tgtgccagtg gccaccctgg ctgggcgcca cttcacagag cagtggtacc ctgtaaccct gccaacaggc agtgggggat ctgggggcat gggttcggga gggggagggg gctcgggggg tggctcaggg ggcaagggca aaggaggttg cccggctgtg cggctgaaag cacgttacca gacaatgagc atcttgccca tggagctata taaagagttt gcagagtatg tcaccaacca ttatcggatg ctgtgtgcag tcttggagcc cgccctgaat gtcaaaggca aggaggaggt tgccagtgca ctagttcaca tcctgcagag tacaggcaag gccaaggact tcctttcaga catggccatg tctgaggtag accggttcat ggaacgggag cacctcatat tccgcgagaa cacgcttgcc actaaagcca tagaagagta tatgagactg attggtcaga aatacctcaa ggatgccatt ggagaattca tccgtgctct gtatgaatct gaggaaaact gcgaggtaga ccctatcaag tgcacagcat ccagtttggc agagcaccag gccaacctgc gaatgtgctg tgagttggcc ctgtgcaagg tggtcaactc ccactgcgtg ttcccgaggg agctgaagga ggtgtttgct tcgtggcggc tgcgctgcgc agagcgaggc cgggaggaca tcgcagacag gcttatcagc gcctcactct tcctgcgctt cctctgccca gcgattatgt cgcccagtct ctttgggctt atgcaggagt acccagatga gcagacctca cgaaccctca ccctcattgc caaggtcatc cagaacctgg ccaacttttc caagtttacc tcaaaggagg actttctggg cttcatgaat gagtttctgg agctggaatg gggttccatg cagcagtttt tgtatgagat ctccaatctg gacacgctaa ccaacagcag tagctttgag ggttacatcg acttgggccg agagctctcc acactgcatg ccctactctg ggaggtgctg ccccagctca gcaaggaagc cctcctgaag ctgggtccac tgccccggct cctcaacgac atcagcacag ctctgaggaa ccccaacatc caaaggcagc caagccgcca gagtgagcgg ccccggcctc agcctgtggt actgcggggg ccatcggctg agatgcaggg ctacatgatg cgggacctca acagctccat cgaccttcag tccttcatgg ctcgaggcct caacagctct atggacatgg ctcgcctccc ctccccaacc aaggaaaagc cacccccacc accgcctggt ggtggtaaag acctgttcta tgtaagccgt ccacccctgg cccgttcctc accagcatac tgcacgagca gctcggacat cacagagcca gagcagaaga tgctgagtgt caacaagagt gtgtccatgc tggacttaca gggtgatggg cctggtggcc gcctcaacag cagcagtgtt tcgaacctgg cggccgtagg ggacctgctg cactcaagcc aggcctcgct gacagcagcc ttggggctac ggcctgcgcc tgccggacgc ctctcccagg ggagtggctc atccatcacg gcggctggca tgcgcctcag ccagatgggt gtcaccacag acggtgtccc tgcccagcaa ctgcgaatcc ccctctcctt ccagaaccct ctcttccaca tggctgctga tgggccaggt cccccaggcg gccatggagg gggcggtggc catggcccac cttcctccca tcaccaccac caccaccatc accaccaccg aggtggagag ccccctgggg acacctttgc cccattccat ggctatagca agagtgagga cctctcttcc ggggtcccca agccccctgc tgcctccatc cttcatagcc acagctacag tgatgagttt ggaccctctg gcactgactt cacccgtcgg cagctttcac tccaggacaa cctgcagcac atgctgtccc ctccccagat caccattggt ccccagaggc cagccccctc agggcctgga ggtgggagcg gtgggggcag cggtgggggt ggcgggggcc agccgcctcc attgcagagg ggcaagtctc agcagttgac agtcagcgca gcccagaaac cccggccatc cagcgggaat ctattgcagt ccccagagcc aagttatggc cccgcccgtc cacggcaaca gagcctcagc aaggagggca gcattggggg cagcgggggc agcggtggcg gagggggtgg ggggctgaag ccctccatca ccaagcagca ttctcagaca ccatccacat tgaaccccac aatgccagcc tctgagcgga cagtggcctg ggtctccaac atgcctcacc tgtcggctga catcgagagt gcccacatcg agcgggaaga gtacaagctc aaggagtact caaaatcgat ggatgagagc cggctggata gggtgaagga gtacgaggag gagattcact cactgaaaga gcggctgcac atgtccaacc ggaagctgga agagtatgag cggaggctgc tgtcccagga agaacaaacc agcaaaatcc tgatgcagta tcaggcccga ctggagcaga gtgagaagag gctaaggcag cagcaggcag agaaggattc ccagatcaag agcatcattg gcaggctgat gctggtggag gaggagctgc gccgggacca ccccgccatg gctgagccgc tgccagaacc caagaagagg ctgctcgacg ctcaggagag gcagcttccc cccttgggtc caacaaaccc gcgtgtgacg ctggccccac cgtggaatgg cctggccccc ccagccccac cacccccacc ccggctgcag attacggaga acggcgagtt ccgaaacacc gcagaccac tag

[0200] Wild type human SynGAPl amino acid sequence (SEQ ID NO:2).

[0201] MSRSRASIHRGSI PAMSYAPFRDVRGPSMHRTQYVHSPYDRPGWNPRFCI ISGNQLLMLDEDEIHPLLIRDRRSESS

[0202] RNKLLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEGAPAAPFRPSQGFLSRRLKSSIKRTKSQPKLDRTS

[0203] SFRQILPRFRSADHDRARLMQSFKESHSHESLLSPSSAAEALELNLDEDSI IKPVHSSILGQEFCFEVTTSSGTKCF

[0204] ACRSAAERDKWIENLQRAVKPNKDNSRRVDNVLKLWI IEARELPPKKRYYCELCLDDMLYARTTSKPRSASGDTVFW

[0205] GEHFEFNNLPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPVTLPTGSGGSGGMGSGGGGGS

[0206] GGGSGGKGKGGCPAVRLKARYQTMSILPMELYKEFAEYVTNHYRMLCAVLEPALNVKGKEEVASALVHILQSTGKAK

[0207] DFLSDMAMSEVDRFMEREHLI FRENTLATKAIEEYMRLIGQKYLKDAIGEFIRALYESEENCEVDPIKCTASSLAEH

[0208] QANLRMCCELALCKVVNSHCVFPRELKEVFASWRLRCAERGREDIADRLISASLFLRFLCPAIMSPSLFGLMQEYPD

[0209] EQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNEFLELEWGSMQQFLYEISNLDTLTNSSSFEGYIDLGRELSTLH

[0210] ALLWEVLPQLSKEALLKLGPLPRLLNDISTALRNPNIQRQPSRQSERPRPQPVVLRGPSAEMQGYMMRDLNSSIDLQ

[0211] SFMARGLNSSMDMARLPSPTKEKPPPPPPGGGKDLFYVSRPPLARSSPAYCTSSSDITEPEQKMLSVNKSVSMLDLQ

[0212] GDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSSITAAGMRLSQMGVTTDGVPAQQLRI P

[0213] LSFQNPLFHMAADGPGPPGGHGGGGGHGPPSSHHHHHHHHHHRGGEPPGDTFAPFHGYSKSEDLSSGVPKPPAASIL

[0214] HSHSYSDEFGPSGTDFTRRQLSLQDNLQHMLSPPQITIGPQRPAPSGPGGGSGGGSGGGGGGQPPPLQRGKSQQLTV SAAQKPRPSSGNLLQS PE PSYGPARPRQQSLSKEGS I GGSGGSGGGGGGGLKPS ITKQHSQTPSTLNPTMPASERTV

[0215] AWVSNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEE IHSLKERLHMSNRKLEEYERRLLSQEEQTSK

[0216] I LMQYQARLEQSEKRLRQQQAEKDSQIKS I I GRLMLVEEELRRDHPAMAE PLPE PKKRLLDAQERQLPPLGPTNPRV

[0217] TLAPPWNGLAPPAPPPPPRLQITENGE FRNTADH

[0218] Human ss-wt-syn: Wild type human SynGAPl sequence with secretion signal (underlined) (SEQ ID NO:3). atgtacaggatgcaa ctcctgtctt gcattgcact aagtcttgca cttgtcacaa acagt atgagcaggt ctcgagcctc catccatcgg gggagcatcc ccgcgatgtc ctatgccccc ttcagagatg tacggggacc ctctatgcac cgaacccaat acgttcattc cccgtatgat cgtcctggtt ggaaccctcg gttctgcatc atctcgggga accagctgct catgctggat gaggatgaga tacaccccct actgatccgg gaccggagga gcgagtccag tcgcaacaaa ctgctgagac gcacagtctc cgtgccggtg gaggggcggc cccacggcga gcatgaatac cacttgggtc gctcgaggag gaagagtgtc ccagggggga agcagtacag catggagggt gcccctgctg cgcccttccg gccctcgcaa ggcttcctga gccgacggct aaaaagctcc atcaaacgaa cgaagtcaca acccaaactt gaccggacca gcagctttcg ccagatcctg cctcgcttcc gaagtgctga ccatgaccgg gcccggctga tgcaaagctt taaggagtca cactctcatg agtccttgct gagtcctagc agtgcagctg aggcattgga gctcaacttg gatgaagatt ccattatcaa gccagtgcac agctccatcc tgggccagga gttctgtttt gaggtaacaa cttcatcagg aacaaaatgc tttgcctgtc ggtctgcggc cgaaagagac aaatggattg agaatctgca gcgggcagta aagcccaaca aggacaacag ccgccgggta gacaatgtgc taaagctgtg gatcatagag gcccgggagc tgccccccaa gaagcggtac tactgtgagc tctgcctgga tgacatgctg tatgcacgca ccacctccaa gccccgctct gcctctgggg acaccgtctt ctggggcgag cacttcgagt ttaacaacct gccggctgtc cgtgccctgc ggctgcatct gtaccgtgac tcagacaaaa agcgcaagaa ggacaaggca ggctatgtcg gcctggtgac tgtgccagtg gccaccctgg ctgggcgcca cttcacagag cagtggtacc ctgtaaccct gccaacaggc agtgggggat ctgggggcat gggttcggga gggggagggg gctcgggggg tggctcaggg ggcaagggca aaggaggttg cccggctgtg cggctgaaag cacgttacca gacaatgagc atcttgccca tggagctata taaagagttt gcagagtatg tcaccaacca ttatcggatg ctgtgtgcag tcttggagcc cgccctgaat gtcaaaggca aggaggaggt tgccagtgca ctagttcaca tcctgcagag tacaggcaag gccaaggact tcctttcaga catggccatg tctgaggtag accggttcat ggaacgggag cacctcatat tccgcgagaa cacgcttgcc actaaagcca tagaagagta tatgagactg attggtcaga aatacctcaa ggatgccatt ggagaattca tccgtgctct gtatgaatct gaggaaaact gcgaggtaga ccctatcaag tgcacagcat ccagtttggc agagcaccag gccaacctgc gaatgtgctg tgagttggcc ctgtgcaagg tggtcaactc ccactgcgtg ttcccgaggg agctgaagga ggtgtttgct tcgtggcggc tgcgctgcgc agagcgaggc cgggaggaca tcgcagacag gcttatcagc gcctcactct tcctgcgctt cctctgccca gcgattatgt cgcccagtct ctttgggctt atgcaggagt acccagatga gcagacctca cgaaccctca ccctcattgc caaggtcatc cagaacctgg ccaacttttc caagtttacc tcaaaggagg actttctggg cttcatgaat gagtttctgg agctggaatg gggttccatg cagcagtttt tgtatgagat ctccaatctg gacacgctaa ccaacagcag tagctttgag ggttacatcg acttgggccg agagctctcc acactgcatg ccctactctg ggaggtgctg ccccagctca gcaaggaagc cctcctgaag ctgggtccac tgccccggct cctcaacgac atcagcacag ctctgaggaa ccccaacatc caaaggcagc caagccgcca gagtgagcgg ccccggcctc agcctgtggt actgcggggg ccatcggctg agatgcaggg ctacatgatg cgggacctca acagctccat cgaccttcag tccttcatgg ctcgaggcct caacagctct atggacatgg ctcgcctccc ctccccaacc aaggaaaagc cacccccacc accgcctggt ggtggtaaag acctgttcta tgtaagccgt ccacccctgg cccgttcctc accagcatac tgcacgagca gctcggacat cacagagcca gagcagaaga tgctgagtgt caacaagagt gtgtccatgc tggacttaca gggtgatggg cctggtggcc gcctcaacag cagcagtgtt tcgaacctgg cggccgtagg ggacctgctg cactcaagcc aggcctcgct gacagcagcc ttggggctac ggcctgcgcc tgccggacgc ctctcccagg ggagtggctc atccatcacg gcggctggca tgcgcctcag ccagatgggt gtcaccacag acggtgtccc tgcccagcaa ctgcgaatcc ccctctcctt ccagaaccct ctcttccaca tggctgctga tgggccaggt cccccaggcg gccatggagg gggcggtggc catggcccac cttcctccca tcaccaccac caccaccatc accaccaccg aggtggagag ccccctgggg acacctttgc cccattccat ggctatagca agagtgagga cctctcttcc ggggtcccca agccccctgc tgcctccatc cttcatagcc acagctacag tgatgagttt ggaccctctg gcactgactt cacccgtcgg cagctttcac tccaggacaa cctgcagcac atgctgtccc ctccccagat caccattggt ccccagaggc cagccccctc agggcctgga ggtgggagcg gtgggggcag cggtgggggt ggcgggggcc agccgcctcc attgcagagg ggcaagtctc agcagttgac agtcagcgca gcccagaaac cccggccatc cagcgggaat ctattgcagt ccccagagcc aagttatggc cccgcccgtc cacggcaaca gagcctcagc aaggagggca gcattggggg cagcgggggc agcggtggcg gagggggtgg ggggctgaag ccctccatca ccaagcagca ttctcagaca ccatccacat tgaaccccac aatgccagcc tctgagcgga cagtggcctg ggtctccaac atgcctcacc tgtcggctga catcgagagt gcccacatcg agcgggaaga gtacaagctc aaggagtact caaaatcgat ggatgagagc cggctggata gggtgaagga gtacgaggag gagattcact cactgaaaga gcggctgcac atgtccaacc ggaagctgga agagtatgag cggaggctgc tgtcccagga agaacaaacc agcaaaatcc tgatgcagta tcaggcccga ctggagcaga gtgagaagag gctaaggcag cagcaggcag agaaggattc ccagatcaag agcatcattg gcaggctgat gctggtggag gaggagctgc gccgggacca ccccgccatg gctgagccgc tgccagaacc caagaagagg ctgctcgacg ctcaggagag gcagcttccc cccttgggtc caacaaaccc gcgtgtgacg ctggccccac cgtggaatgg cctggccccc ccagccccac cacccccacc ccggctgcag attacggaga acggcgagtt ccgaaacacc gcagaccac tag

[0219] Human ss-wt-syn: Wild type human SynGAPl amino acid sequence with secretion signal (underlined) (SEQ ID NO:4). MYRMQLLSCIALSLALVTNSMSRSRAS IHRGS I PAMS YAPFRDVRGPSMHRTQYVHS PYDRPGWNPRFCI I SGNQLL

[0220] MLDEDE IHPLLIRDRRSESSRNKLLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEGAPAAPFRPSQGFLS

[0221] RRLKSS IKRTKSQPKLDRTSS FRQI LPRFRSADHDRARLMQS FKESHSHESLLS PSSAAEALELNLDEDS I IKPVHS

[0222] S I LGQE FCFEVTTSSGTKCFACRSAAERDKWIENLQRAVKPNKDNSRRVDNVLKLWI IEARELPPKKRYYCELCLDD

[0223] MLYARTTSKPRSASGDTVFWGEHFE FNNLPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPV

[0224] TLPTGSGGSGGMGSGGGGGSGGGSGGKGKGGCPAVRLKARYQTMS I LPMELYKE FAEYVTNHYRMLCAVLE PALNVK

[0225] GKEEVASALVHI LQSTGKAKDFLSDMAMSEVDRFMEREHLI FRENTLATKAIEEYMRLI GQKYLKDAI GE FIRALYE

[0226] SEENCEVDPIKCTASSLAEHQANLRMCCELALCKVVNSHCVFPRELKEVFASWRLRCAERGREDIADRLI SASLFLR

[0227] FLCPAIMS PSLFGLMQEYPDEQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNE FLELEWGSMQQFLYE I SNLDTL

[0228] TNSSS FEGYIDLGRELSTLHALLWEVLPQLSKEALLKLGPLPRLLNDI STALRNPNIQRQPSRQSERPRPQPVVLRG

[0229] PSAEMQGYMMRDLNSS IDLQS FMARGLNSSMDMARLPS PTKEKPPPPPPGGGKDLFYVSRPPLARSS PAYCTSSSDI

[0230] TE PEQKMLSVNKSVSMLDLQGDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSS ITAAGM

[0231] RLSQMGVTTDGVPAQQLRI PLS FQNPLFHMAADGPGPPGGHGGGGGHGPPSSHHHHHHHHHHRGGE PPGDTFAPFHG

[0232] YSKSEDLSSGVPKPPAAS I LHSHSYSDE FGPSGTDFTRRQLSLQDNLQHMLS PPQITI GPQRPAPSGPGGGSGGGSG

[0233] GGGGGQPPPLQRGKSQQLTVSAAQKPRPSSGNLLQS PE PSYGPARPRQQSLSKEGS I GGSGGSGGGGGGGLKPS ITK

[0234] QHSQTPSTLNPTMPASERTVAWVSNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEE IHSLKERLHMS

[0235] NRKLEEYERRLLSQEEQTSKI LMQYQARLEQSEKRLRQQQAEKDSQIKS I I GRLMLVEEELRRDHPAMAE PLPE PKK

[0236] RLLDAQERQLPPLGPTNPRVTLAPPWNGLAPPAPPPPPRLQITENGE FRNTADH

[0237] Human synl8: Human SynGAPl sequence with 8x N-glycan sites (bolded) (SEQ ID NO: 5) atgagcaggt ctcgagcctc catccatcgg gggagcatcc ccgcgatgtc ctatgccccc ttcagagatg tacggggacc ctctatgcac cgaacccaat acgttcattc cccgtatgat cgtcctggtt ggaaccctcg gttctgcatc atctcgggga accagctgct catgctggat gaggatgaga tacaccccct actgatccgg gaccggagga gcgagtccag tcgc aac aaa ACT ctg agac gcacagtctc cgtgccggtg gaggggcggc cccacggcga gcatgaatac cacttgggtc gctcgaggag gaagagtgtc ccagggggga agcagtacag catggagggt gcccctgctg cgcccttccg gccctcgcaa ggcttcctga gccgacggct aaaaagctcc atcaaacgaa cgaagtcaca acccaaactt gaccggacca gcagctttcg ccagatcctg cctcgcttcc gaagtgctga ccatgaccgg gcccggctga tgcaaagctt taaggagtca cactctcatg agtccttgct gagtcctagc agtgcagctg aggcattgga gctcaacttg gatgaagatt ccattatcaa gccagtgcac agctccatcc tgggccagga gttctgtttt gag AAT aca act tcatcagg aacaaaatgc tttgcctgtc ggtctgcggc cgaaagagac aaatggattg agaatctgca gcgggcagta aagcccaaca aggac aac age ACT cgggtagacaatgtgctaaagctgtg gatcatagag gcccgggagc tgccccccaa gaagcggtactactgtgagctctgcctggatgacatgctgtatgca AAC a ccacctccaa gccccgctct gcctctgggg acaccgtctt ctggggcgag cacttcgagt tt aac aac ACG ccggctgtccgtgccctgcggctgcatctgtaccgtgactcagacaaaa agcgcaagaa ggacaaggca ggctatgtcg gcctggtgac tgtgccagtg gccaccctgg ctgggcgcca cttcacagag cagtggtacc ctgtaaccct gccaacaggc agtgggggat ctgggggcat gggttcggga gggggagggg gctcgggggg tggctcaggg ggcaagggca aaggaggttg cccggctgtg cggctgaaag cacgttacca gacaatgagc atcttgccca tggagctata taaagagttt gcagagtatg tcaccaacca ttatcggatg ctgtgtgcag tcttggagcc cgccctgaat gtcaaaggca aggaggaggt tgccagtgca ctagttcaca tcctg AAT agt aca ggcaaggccaaggacttcctttcagacatggccatg tctgaggtag accggttcat ggaacgggag cacctcatat tccgcgagaa cacgcttgcc actaaagcca tagaagagta tatgagactg attggtcaga aatacctcaa ggatgccatt ggagaattca tccgtgctct gtatgaatct gaggaaaact gcgaggtaga ccctatcaag tgcacagcat ccagtttggc agagcaccag gccaacctgc gaatgtgctg tgagttggcc ctgtgcaagg tggtc aac tcc ACC tgcgtgttcccgagggagctgaaggaggtgtttgcttcgtggcggc tgcgctgcgc agagcgaggc cgggaggaca tcgcagacag gcttatcagc gcctcactct tcctgcgctt cctctgccca gcgattatgt cgcccagtct ctttgggctt atgcaggagt acccagatga gcagacctca cgaaccctca ccctcattgc caaggtcatc cagaacctgg ccaacttttc caagtttacc tcaaaggagg actttctggg cttcatgaat gagtttctgg agctggaatg gggttccatg cagcagtttt tgtatgagat ctccaatctg gacacgctaa ccaacagcag tagctttgag ggttacatcg acttgggc cga gag AAC tcc acactgcatg ccctactctg ggaggtgctg ccccagctca gcaaggaagc cctcctgaag ctgggtccac tgccccggct cctcaacgac atcagcacag ctctgaggaa ccccaacatc caaaggcagc caagccgcca gagtgagcgg ccccggcctc agcctgtggt actgcggggg ccatcggctg agatgcaggg ctacatgatg cgggacctca acagctccat cgaccttcag tccttcatgg ctcgaggcct caacagctct atggacatgg ctcgcctccc ctccccaacc aaggaaaagc cacccccacc accgcctggt ggtggtaaag acctgttcta tgtaagccgt ccacccctgg cccgttcctc accagcatac tgcacgagca gctcggacat cacagagcca gagcagaaga tgctgagtgt caacaagagt gtgtccatgc tggacttaca gggtgatggg cctggtggcc gcctcaacag cagcagtgtt tcgaacctgg cggccgtagg ggacctgctg cactcaagcc aggcctcgct gacagcagcc ttggggctac ggcctgcgcc tgccggacgc ctctcccagg ggagtggctc atccatcacg gcggctggca tgcgcctcag ccagatgggt gtcaccacag acggtgtccc tgcccagcaa ctgcgaatcc ccctctcctt ccagaaccct ctcttccaca tggctgctga tgggccaggt cccccaggcg gccatggagg gggcggtggc catggcccac cttcctccca tcaccaccac caccaccatc accaccaccg aggtggagag ccccctgggg acacctttgc cccattccat ggctatagca agagtgagga cctctcttcc ggggtcccca agccccctgc tgcctccatc cttcatagcc acagctacag tgatgagttt ggaccctctg gcactgactt cacccgtcgg cagctttcac tccaggacaa cctgcagcac atgctgtccc ctccccagat caccattggt ccccagaggc cagccccctc agggcctgga ggtgggagcg gtgggggcag cggtgggggt ggcgggggcc agccgcctcc attgcagagg ggcaagtctc agcagttgac agtcagcgca gcccagaaac cccggccatc cagcgggaat ctattgcagt ccccagagcc aagttatggc cccgcccgtc cacggcaaca gagcctcagc aaggagggca gcattggggg cagcgggggc agcggtggcg gagggggtgg ggggctgaag ccctccatca ccaagcagca ttctcagaca ccatccacat tgaaccccac aatgccagcc tctgagcgga cagtggcctg ggtctccaac atgcctcacc tgtcggctga catcgagagt gcccacatcg agcgggaaga gtacaagctc aaggagtact caaaatcgat ggatgagagc cggctggata gggtgaagga gtacgaggag gagattcact cactgaaaga gcggctgcac atgtccaacc ggaagctgga agagtatgag cggaggctgc tgtcccagga agaacaaacc agcaaaatcc tgatgcagta tcaggcccga ctggagcaga gtgagaagag gctaaggcag cagcaggcag agaaggattc ccagatcaag agcatcattg gcaggctgat gctggtggag gaggagctgc gccgggacca ccccgccatg gctgagccgc tgccagaacc caagaagagg ctgctcgacg ctcaggagag gcagcttccc cccttgggtc caacaaaccc gcgtgtgacg ctggccccac cgtggaatgg cctggccccc ccagccccac cacccccacc ccggctgcag attacggaga acggcgagtt ccgaaacacc gcagaccac tag

[0238] Human synl8: Human SynGAPl sequence 8x N-glycan sites (bolded and underlined) (SEQ ID

[0239] NO: 6)

[0240] MSRSRASIHRGSIPAMSYAPFRDVRGPSMHRTQYVHSPYDRPGWNPRFCIISGNQLLMLDEDEIHPLLIRDRRSESS

[0241] RNKTLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEGAPAAPFRPSQGFLSRRLKSSIKRTKSQPKLDRTS

[0242] SFRQILPRFRSADHDRARLMQSFKESHSHESLLSPSSAAEALELNLDEDSIIKPVHSSILGQEFCFENTTSSGTKCF

[0243] ACRSAAERDKWIENLQRAVKPNKDNSTRVDNVLKLWIIEARELPPKKRYYCELCLDDMLYANTTSKPRSASGDTVFW

[0244] GEHFEFNNTPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPVTLPTGSGGSGGMGSGGGGGS

[0245] GGGSGGKGKGGCPAVRLKARYQTMSILPMELYKEFAEYVTNHYRMLCAVLEPALNVKGKEEVASALVHILNSTGKAK

[0246] DFLSDMAMSEVDRFMEREHLIFRENTLATKAIEEYMRLIGQKYLKDAIGEFIRALYESEENCEVDPIKCTASSLAEH

[0247] QANLRMCCELALCKVVNSTCVFPRELKEVFASWRLRCAERGREDIADRLISASLFLRFLCPAIMSPSLFGLMQEYPD

[0248] EQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNEFLELEWGSMQQFLYEISNLDTLTNSSSFEGYIDLGRENSTLH

[0249] ALLWEVLPQLSKEALLKLGPLPRLLNDISTALRNPNIQRQPSRQSERPRPQPVVLRGPSAEMQGYMMRDLNSSIDLQ

[0250] SFMARGLNSSMDMARLPSPTKEKPPPPPPGGGKDLFYVSRPPLARSSPAYCTSSSDITEPEQKMLSVNKSVSMLDLQ

[0251] GDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSSITAAGMRLSQMGVTTDGVPAQQLRIP

[0252] LSFQNPLFHMAADGPGPPGGHGGGGGHGPPSSHHHHHHHHHHRGGEPPGDTFAPFHGYSKSEDLSSGVPKPPAASIL

[0253] HSHSYSDEFGPSGTDFTRRQLSLQDNLQHMLSPPQITIGPQRPAPSGPGGGSGGGSGGGGGGQPPPLQRGKSQQLTV

[0254] SAAQKPRPSSGNLLQSPEPSYGPARPRQQSLSKEGSIGGSGGSGGGGGGGLKPSITKQHSQTPSTLNPTMPASERTV

[0255] AWVSNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEEIHSLKERLHMSNRKLEEYERRLLSQEEQTSK

[0256] ILMQYQARLEQSEKRLRQQQAEKDSQIKSIIGRLMLVEEELRRDHPAMAEPLPEPKKRLLDAQERQLPPLGPTNPRV

[0257] TLAPPWNGLAPPAPPPPPRLQITENGEFRNTADH

[0258] Human synl8: Human SynGAPl sequence with secretion signal (underlined) and 8xN-glycan sites (bolded) (SEQ ID NO: 7) atgtacaggatgcaa ctcctgtctt gcattgcact aagtcttgca cttgtcacaa acagt atgagcaggt ctcgagcctc catccatcgg gggagcatcc ccgcgatgtc ctatgccccc ttcagagatg tacggggacc ctctatgcac cgaacccaat acgttcattc cccgtatgat cgtcctggtt ggaaccctcg gttctgcatc atctcgggga accagctgct catgctggat gaggatgaga tacaccccct actgatccgg gaccggagga gcgagtccag tcgc aac aaa ACT ctg agac gcacagtctc cgtgccggtg gaggggcggc cccacggcga gcatgaatac cacttgggtc gctcgaggag gaagagtgtc ccagggggga agcagtacag catggagggt gcccctgctg cgcccttccg gccctcgcaa ggcttcctga gccgacggct aaaaagctcc atcaaacgaa cgaagtcaca acccaaactt gaccggacca gcagctttcg ccagatcctg cctcgcttcc gaagtgctga ccatgaccgg gcccggctga tgcaaagctt taaggagtca cactctcatg agtccttgct gagtcctagc agtgcagctg aggcattgga gctcaacttg gatgaagatt ccattatcaa gccagtgcac agctccatcc tgggccagga gttctgtttt gag AAT aca act tcatcagg aacaaaatgc tttgcctgtc ggtctgcggc cgaaagagac aaatggattg agaatctgca gcgggcagta aagcccaaca aggac aac age ACT cgggtagacaatgtgctaaagctgtg gatcatagag gcccgggagc tgccccccaa gaagcggtactactgtgagctctgcctggatgacatgctgtatgca AAC a ccacctccaa gccccgctct gcctctgggg acaccgtctt ctggggcgag cacttcgagt tt aac aac ACG ccggctgtccgtgccctgcggctgcatctgtaccgtgactcagacaaaa agcgcaagaa ggacaaggca ggctatgtcg gcctggtgac tgtgccagtg gccaccctgg ctgggcgcca cttcacagag cagtggtacc ctgtaaccct gccaacaggc agtgggggat ctgggggcat gggttcggga gggggagggg gctcgggggg tggctcaggg ggcaagggca aaggaggttg cccggctgtg eggetgaaag cacgttacca gacaatgagc atcttgccca tggagetata taaagagttt gcagagtatg tcaccaacca ttatcggatg ctgtgtgcag tettggagee cgccctgaat gteaaaggea aggaggaggt tgccagtgca ctagttcaca teetg AAT agt aca ggcaaggccaaggacttcctttcagacatggccatg tetgaggtag accggttcat ggaacgggag cacctcatat teegegagaa cacgcttgcc actaaagcca tagaagagta tatgagactg attggteaga aatacctcaa ggatgccatt ggagaattca tccgtgctct gtatgaatet gaggaaaact gegaggtaga ccctatcaag tgcacagcat ccagtttggc agagcaccag gccaacctgc gaatgtgctg tgagttggcc etgtgeaagg tggte aac tee ACC tgcgtgttcccgagggagctgaaggaggtgtttgcttcgtggcggc tgcgctgcgc agagegagge cgggaggaca tcgcagacag gcttatcagc gcctcactct tcctgcgctt cctctgccca gegattatgt cgcccagtct ctttgggctt atgeaggagt acccagatga gcagacctca cgaaccctca ccctcattgc caaggtcatc cagaacctgg ccaacttttc caagtttacc tcaaaggagg actttctggg cttcatgaat gagtttctgg agctggaatg gggttccatg cagcagtttt tgtatgagat ctccaatctg gacacgctaa ccaacagcag tagetttgag ggttacatcg acttgggc ega gag AAC tee acactgcatg ccctactctg ggaggtgctg ccccagctca gcaaggaagc cctcctgaag ctgggtccac tgccccggct cctcaacgac atcagcacag ctctgaggaa ccccaacatc caaaggcagc caagccgcca gagtgagegg ccccggcctc agcctgtggt actgcggggg ccatcggctg agatgeaggg ctacatgatg cgggacctca acagctccat cgaccttcag tccttcatgg ctcgaggcct caacagctct atggacatgg ctcgcctccc ctccccaacc aaggaaaagc cacccccacc accgcctggt ggtggtaaag acctgttcta tgtaageegt ccacccctgg cccgttcctc accagcatac tgeaegagea gctcggacat cacagagcca gagcagaaga tgctgagtgt caacaagagt gtgtccatgc tggacttaca gggtgatggg cctggtggcc gcctcaacag cagcagtgtt tcgaacctgg cggccgtagg ggacctgctg cactcaagcc aggcctcgct gacagcagcc ttggggctac ggcctgcgcc tgccggacgc ctctcccagg ggagtggctc atccatcacg gcggctggca tgcgcctcag ccagatgggt gtcaccacag acggtgtccc tgcccagcaa ctgcgaatcc ccctctcctt ccagaaccct ctcttccaca tggctgctga tgggccaggt cccccaggcg gccatggagg gggcggtggc catggcccac cttcctccca tcaccaccac caccaccatc accaccaccg aggtggagag ccccctgggg acacctttgc cccattccat ggctatagca agagtgagga cctctcttcc ggggtcccca agccccctgc tgcctccatc cttcatagcc acagctacag tgatgagttt ggaccctctg gcactgactt cacccgtcgg cagctttcac tccaggacaa cctgcagcac atgctgtccc ctccccagat caccattggt ccccagaggc cagccccctc agggcctgga ggtgggagcg gtgggggcag cggtgggggt ggcgggggcc agccgcctcc attgcagagg ggcaagtctc agcagttgac agtcagcgca gcccagaaac cccggccatc cagcgggaat ctattgcagt ccccagagcc aagttatggc cccgcccgtc cacggcaaca gagcctcagc aaggagggca gcattggggg cagcgggggc agcggtggcg gagggggtgg ggggctgaag ccctccatca ccaagcagca ttctcagaca ccatccacat tgaaccccac aatgccagcc tctgagcgga cagtggcctg ggtctccaac atgcctcacc tgtcggctga catcgagagt gcccacatcg agcgggaaga gtacaagctc aaggagtact caaaatcgat ggatgagagc cggctggata gggtgaagga gtacgaggag gagattcact cactgaaaga gcggctgcac atgtccaacc ggaagctgga agagtatgag cggaggctgc tgtcccagga agaacaaacc agcaaaatcc tgatgcagta tcaggcccga ctggagcaga gtgagaagag gctaaggcag cagcaggcag agaaggattc ccagatcaag agcatcattg gcaggctgat gctggtggag gaggagctgc gccgggacca ccccgccatg gctgagccgc tgccagaacc caagaagagg ctgctcgacg ctcaggagag gcagcttccc cccttgggtc caacaaaccc gcgtgtgacg ctggccccac cgtggaatgg cctggccccc ccagccccac cacccccacc ccggctgcag attacggaga acggcgagtt ccgaaacacc gcagaccac tag

[0259] Human synl8: Human SynGAPl sequence with secretion signal (underlined) and 8x N-glycan sites (bolded and underlined) (SEQ ID NO: 8)

[0260] MYRMQLLSCIALSLALVTNSMSRSRAS IHRGS I PAMS YAPFRDVRGPSMHRTQYVHS PYDRPGWNPRFCI I SGNQLL

[0261] MLDEDE IHPLLIRDRRSESSRNKTLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEGAPAAPFRPSQGFLS

[0262] RRLKSS IKRTKSQPKLDRTSS FRQI LPRFRSADHDRARLMQS FKESHSHESLLS PSSAAEALELNLDEDS I IKPVHS

[0263] S I LGQE FCFENTTSSGTKCFACRSAAERDKWIENLQRAVKPNKDNSTRVDNVLKLWI IEARELPPKKRYYCELCLDD

[0264] MLYANTTSKPRSASGDTVFWGEHFE FNNTPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPV

[0265] TLPTGSGGSGGMGSGGGGGSGGGSGGKGKGGCPAVRLKARYQTMS I LPMELYKE FAEYVTNHYRMLCAVLE PALNVK

[0266] GKEEVASALVHI LNSTGKAKDFLSDMAMSEVDRFMEREHLI FRENTLATKAIEEYMRLI GQKYLKDAI GE FIRALYE

[0267] SEENCEVDPIKCTASSLAEHQANLRMCCELALCKVVNSTCVFPRELKEVFASWRLRCAERGREDIADRLI SASLFLR

[0268] FLCPAIMS PSLFGLMQEYPDEQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNE FLELEWGSMQQFLYE I SNLDTL

[0269] TNSSS FEGYIDLGRENSTLHALLWEVLPQLSKEALLKLGPLPRLLNDI STALRNPNIQRQPSRQSERPRPQPVVLRG PSAEMQGYMMRDLNSSIDLQSFMARGLNSSMDMARLPSPTKEKPPPPPPGGGKDLFYVSRPPLARSSPAYCTSSSDI

[0270] TEPEQKMLSVNKSVSMLDLQGDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSSITAAGM

[0271] RLSQMGVTTDGVPAQQLRI PLSFQNPLFHMAADGPGPPGGHGGGGGHGPPSSHHHHHHHHHHRGGEPPGDTFAPFHG

[0272] YSKSEDLSSGVPKPPAASILHSHSYSDEFGPSGTDFTRRQLSLQDNLQHMLSPPQITIGPQRPAPSGPGGGSGGGSG

[0273] GGGGGQPPPLQRGKSQQLTVSAAQKPRPSSGNLLQSPEPSYGPARPRQQSLSKEGSIGGSGGSGGGGGGGLKPSITK

[0274] QHSQTPSTLNPTMPASERTVAWVSNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEEIHSLKERLHMS

[0275] NRKLEEYERRLLSQEEQTSKILMQYQARLEQSEKRLRQQQAEKDSQIKSI IGRLMLVEEELRRDHPAMAEPLPEPKK

[0276] RLLDAQERQLPPLGPTNPRVTLAPPWNGLAPPAPPPPPRLQITENGEFRNTADH

[0277] Wild type mouse SynGAPl sequence (SEQ ID NO: 9) atgagcag gtctcgagcc tccatccatc gggggagcat

[0278] 241 ccccgcgatg tcctatgccc ccttcagaga tgtacgggga ccccctatgc accgaaccca

[0279] 301 atacgttcat tccccgtacg atcgtcctgg ctggaaccct cggttctgca tcatctcggg

[0280] 361 gaaccagctg ctcatgctgg atgaggatga gatacacccc cttctgatcc gcgaccggag

[0281] 421 gagcgagtcc agccgaaaca aactgctgag acgcaccgtc tctgtgccag tggaggggcg

[0282] 481 gccccacggc gagcatgaat accacttggg tcgctcgagg aggaagagtg tcccaggggg

[0283] 541 gaaacagtac agcatggagg ccgcccccgc tgcgcccttc cggccctcgc aaggcttcct

[0284] 601 gagccggagg ctaaaaagct ctatcaaacg tacaaagtca caacccaaac ttgaccggac

[0285] 661 cagcagcttt cgacagatcc tgcctcgctt ccgaagtgct gaccatgacc gggcccggct

[0286] 721 gatgcagagc ttcaaggagt cacattccca cgagtccctg ctgagtccca gtagtgctgc

[0287] 781 tgaggccttg gagctcaacc tggatgaaga ctccattatc aagccagtac acagctccat

[0288] 841 cctgggtcag gagttctgct ttgaggtaac aacatcatct ggaacaaaat gctttgcctg

[0289] 901 tcggtctgca gctgaaaggg acaaatggat tgagaatctg cagagggctg taaaacccaa

[0290] 961 caaggacaac agccgccgag tagataacgt gctgaagcta tggatcatag aggctcgaga

[0291] 1021 gctgcccccc aagaagagat attactgtga gctgtgcctg gacgacatgc tgtatgcacg

[0292] 1081 aaccacctcc aagccccgct cggcttcagg agacaccgtc ttttggggcg agcactttga

[0293] 1141 gtttaacaac ctgcctgccg tccgggccct tcggctgcat ctgtaccgtg actcagacaa

[0294] 1201 aaagcggaag aaggacaagg ctggctacgt tggcctggtg actgttccag tggccaccct

[0295] 1261 agctgggcgc cacttcacag agcagtggta ccccgtgacc ttgccgacag gcagtggggg

[0296] 1321 ctctgggggc atgggctcgg ggggaggagg agggtcaggg ggtggctcag ggggcaaagg

[0297] 1381 gaaaggaggt tgtcctgctg tgagactgaa agcccgttac cagacgatga gcatcctgcc

[0298] 1441 catggagctg tataaggagt ttgcagagta tgtgaccaac cattaccgga tgctatgtgc

[0299] 1501 agtgctggag cctgccctga atgtcaaagg caaggaagag gtggccagtg cactggttca

[0300] 1561 catcctgcag agcacaggca aggccaagga cttcctttca gacatggcca tgtcagaggt

[0301] 1621 agaccgattc atggagcggg aacacctcat attccgagag aacacgctcg ccactaaagc

[0302] 1681 catagaagag tatatgagac tgattggcca gaaatacctc aaggatgcca ttggggagtt

[0303] 1741 cattcgtgct ctgtatgaat ctgaggagaa ctgtgaagta gaccccatca agtgcacagc

[0304] 1801 gtccagtctg gcagagcacc aggccaacct gcggatgtgc tgtgagttgg ccctgtgcaa

[0305] 1861 ggtggtcaac tcccattgcg tgttcccgag ggagctgaag gaggtgtttg catcttggcg

[0306] 1921 gctgcgctgc gcagagcggg gccgagagga cattgctgac aggctgatca gcgcctcgct 1981 cttcctgcgc ttcctctgcc cggccattat gtcgcccagt ctatttgggc ttatgcagga

[0307] 2041 gtacccagat gagcagacct cacgaaccct caccctcatc gccaaggtca tccagaacct

[0308] 2101 ggccaacttt tccaagttta cctcaaagga ggacttcctg ggcttcatga atgagtttct

[0309] 2161 ggagctggag tggggctcta tgcagcagtt cttgtatgag atatccaacc tggacacctt

[0310] 2221 gaccaacagc agcagttttg agggctatat agacttgggc cgcgagctct ccacacttca

[0311] 2281 cgccctgctc tgggaggtgc tgccccagct cagcaaggaa gccctcctga agctgggccc

[0312] 2341 actgccccgg ctcctcaatg acatcagcac agccctgagg aaccctaaca tccaaaggca

[0313] 2401 gccaagccgc cagagtgaac ggactcggtc tcagcccatg gtgctgcgag ggccatcagc

[0314] 2461 cgagatgcag ggctacatga tgcgggacct caatagctcc atcgaccttc aatccttcat

[0315] 2521 ggctcgaggc ctcaacagct ctatggacat ggctcgcctc ccctccccaa ccaaggaaaa

[0316] 2581 accaccacca ccaccacccg gtgggggtaa agaccttttc tatgtgagcc ggccaccact

[0317] 2641 ggcccggtcc tccccagcat actgcacgag cagctcggac atcacagagc cagagcagaa

[0318] 2701 gatgctgagt gtcaacaaga gtgtgtccat gctggacctt cagggcgacg ggcctggagg

[0319] 2761 tcgccttaac agtagcagtg tttccaacct ggcagctgtt ggggacttgt tgcattccag

[0320] 2821 ccaggcctcg ctgacagcag ctttggggtt gcggcctgca cctgccgggc gcctctccca

[0321] 2881 ggggagtggc tcttccatca cagcagctgg catgcgcctc agccagatgg gggtcactac

[0322] 2941 agatggtgtc cccgcccagc aactgcgcat ccccctttcc ttccagaacc ctctcttcca

[0323] 3001 tatggctgct gatgggccag ggcccccagc aggccatgga gggagcagtg gtcatggtcc

[0324] 3061 accttcctcc catcaccacc accaccacca tcaccaccac cgagggggag aacccccagg

[0325] 3121 ggacactttt gccccattcc atggctatag caagagcgag gacctctctt caggggtccc

[0326] 3181 taagcccccc gccgcctcca tccttcacag ccacagctac agcgacgagt ttggaccctc

[0327] 3241 tggcactgat tttacccgcc ggcagctctc gcttcaggac agtctacagc acatgctctc

[0328] 3301 ccctccccag attaccatcg gtccccagag gccagctccc tcagggccgg gagggggcag

[0329] 3361 cggcgggggc agcggtgggg gccagccacc ccccttgcag aggggcaagt ctcaacagtt

[0330] 3421 gacagtgagt gccgcccaga aacctcggcc gtctagcggg aacctgttgc agtccccgga

[0331] 3481 gccaagctac ggacctgccc gtcctcggca gcagagcctc agcaaagagg gcagcattgg

[0332] 3541 gggcagcggg ggcagcgggg gcggaggggg tggggggctc aagccctcca tcaccaagca

[0333] 3601 gcattcccag actccatcca cgcttaaccc cacaatgcca gcctcggagc ggaccgtagc

[0334] 3661 ctgggtctcc aacatgcctc acctgtcggc tgacatcgag agtgcacaca tcgagcgaga

[0335] 3721 agagtacaag ctcaaggagt actccaagtc catggacgaa agccggctgg acagggtgaa

[0336] 3781 ggagtatgag gaggagatac attcgctgaa ggagaggcta cacatgtcca accggaagct

[0337] 3841 ggaagagtat gagcggaggt tgctgtccca ggaagaacag accagcaaga tcctgatgca

[0338] 3901 gtaccaagcc cgcctggagc agagtgagaa gcgcttgaga cagcagcagg tggagaagga

[0339] 3961 ctcccagatc aagagcatca ttggcaggct gatgctggtg gaggaggagc tgcgtcggga

[0340] 4021 ccaccccgcc atggctgagc cgctgcctga gcccaagaag aggctgctcg acgctcagga

[0341] 4081 gaggcagctt ccccccttgg gtccaacaaa cccgcgtgtg acgctggccc caccttggaa

[0342] 4141 cggcctggcc cccccagccc cacccccccc accccggctg cagatcacag agaacggcga

[0343] 4201 gttccggaac accgcagacc ac tag Wild type mouse SynGAPl amino acid sequence (SEQ ID NO: 10)

[0344] MSRSRASIHRGSI PAMSYAPFRDVRGPPMHRTQYVHSPYDRPGWNPRFCI ISGNQLLMLDEDEIHPLLIRDRRSESS

[0345] RNKLLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEAAPAAPFRPSQGFLSRRLKSSIKRTKSQPKLDRTS

[0346] SFRQILPRFRSADHDRARLMQSFKESHSHESLLSPSSAAEALELNLDEDSI IKPVHSSILGQEFCFEVTTSSGTKCF

[0347] ACRSAAERDKWIENLQRAVKPNKDNSRRVDNVLKLWI IEARELPPKKRYYCELCLDDMLYARTTSKPRSASGDTVFW

[0348] GEHFEFNNLPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPVTLPTGSGGSGGMGSGGGGGS

[0349] GGGSGGKGKGGCPAVRLKARYQTMSILPMELYKEFAEYVTNHYRMLCAVLEPALNVKGKEEVASALVHILQSTGKAK

[0350] DFLSDMAMSEVDRFMEREHLI FRENTLATKAIEEYMRLIGQKYLKDAIGEFIRALYESEENCEVDPIKCTASSLAEH

[0351] QANLRMCCELALCKVVNSHCVFPRELKEVFASWRLRCAERGREDIADRLISASLFLRFLCPAIMSPSLFGLMQEYPD

[0352] EQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNEFLELEWGSMQQFLYEISNLDTLTNSSSFEGYIDLGRELSTLH

[0353] ALLWEVLPQLSKEALLKLGPLPRLLNDISTALRNPNIQRQPSRQSERTRSQPMVLRGPSAEMQGYMMRDLNSSIDLQ

[0354] SFMARGLNSSMDMARLPSPTKEKPPPPPPGGGKDLFYVSRPPLARSSPAYCTSSSDITEPEQKMLSVNKSVSMLDLQ

[0355] GDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSSITAAGMRLSQMGVTTDGVPAQQLRI P

[0356] LSFQNPLFHMAADGPGPPAGHGGSSGHGPPSSHHHHHHHHHHRGGEPPGDTFAPFHGYSKSEDLSSGVPKPPAASIL

[0357] HSHSYSDEFGPSGTDFTRRQLSLQDSLQHMLSPPQITIGPQRPAPSGPGGGSGGGSGGGQPPPLQRGKSQQLTVSAA

[0358] QKPRPSSGNLLQSPEPSYGPARPRQQSLSKEGSIGGSGGSGGGGGGGLKPSITKQHSQTPSTLNPTMPASERTVAWV

[0359] SNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEEIHSLKERLHMSNRKLEEYERRLLSQEEQTSKILM

[0360] QYQARLEQSEKRLRQQQVEKDSQIKSI IGRLMLVEEELRRDHPAMAEPLPEPKKRLLDAQERQLPPLGPTNPRVTLA

[0361] PPWNGLAPPAPPPPPRLQITENGEFRNTADH

[0362] Mouse ss-wt-syn: Wild type mouse SynGAPl sequence with secretion signal (underlined) (SEQ ID NO: 11) atgtacaggatgcaa ctcctgtctt gcattgcact aagtcttgca cttgtcacaa acagt atgagcag gtctcgagcc tccatccatc gggggagcat

[0363] 241 ccccgcgatg tcctatgccc ccttcagaga tgtacgggga ccccctatgc accgaaccca 301 atacgttcat tccccgtacg atcgtcctgg ctggaaccct cggttctgca tcatctcggg 361 gaaccagctg ctcatgctgg atgaggatga gatacacccc cttctgatcc gcgaccggag 421 gagcgagtcc agccgaaaca aactgctgag acgcaccgtc tctgtgccag tggaggggcg 481 gccccacggc gagcatgaat accacttggg tcgctcgagg aggaagagtg tcccaggggg 541 gaaacagtac agcatggagg ccgcccccgc tgcgcccttc cggccctcgc aaggcttcct 601 gagccggagg ctaaaaagct ctatcaaacg tacaaagtca caacccaaac ttgaccggac 661 cagcagcttt cgacagatcc tgcctcgctt ccgaagtgct gaccatgacc gggcccggct

[0364] 721 gatgcagagc ttcaaggagt cacattccca cgagtccctg ctgagtccca gtagtgctgc 781 tgaggccttg gagctcaacc tggatgaaga ctccattatc aagccagtac acagctccat 841 cctgggtcag gagttctgct ttgaggtaac aacatcatct ggaacaaaat gctttgcctg 901 tcggtctgca gctgaaaggg acaaatggat tgagaatctg cagagggctg taaaacccaa 961 caaggacaac agccgccgag tagataacgt gctgaagcta tggatcatag aggctcgaga 1021 gctgcccccc aagaagagat attactgtga gctgtgcctg gacgacatgc tgtatgcacg 1081 aaccacctcc aagccccgct cggcttcagg agacaccgtc ttttggggcg agcactttga 1141 gtttaacaac ctgcctgccg tccgggccct tcggctgcat ctgtaccgtg actcagacaa 1201 aaagcggaag aaggacaagg ctggctacgt tggcctggtg actgttccag tggccaccct

[0365] 1261 agctgggcgc cacttcacag agcagtggta ccccgtgacc ttgccgacag gcagtggggg

[0366] 1321 ctctgggggc atgggctcgg ggggaggagg agggtcaggg ggtggctcag ggggcaaagg

[0367] 1381 gaaaggaggt tgtcctgctg tgagactgaa agcccgttac cagacgatga gcatcctgcc

[0368] 1441 catggagctg tataaggagt ttgcagagta tgtgaccaac cattaccgga tgctatgtgc

[0369] 1501 agtgctggag cctgccctga atgtcaaagg caaggaagag gtggccagtg cactggttca

[0370] 1561 catcctgcag agcacaggca aggccaagga cttcctttca gacatggcca tgtcagaggt

[0371] 1621 agaccgattc atggagcggg aacacctcat attccgagag aacacgctcg ccactaaagc

[0372] 1681 catagaagag tatatgagac tgattggcca gaaatacctc aaggatgcca ttggggagtt

[0373] 1741 cattcgtgct ctgtatgaat ctgaggagaa ctgtgaagta gaccccatca agtgcacagc

[0374] 1801 gtccagtctg gcagagcacc aggccaacct gcggatgtgc tgtgagttgg ccctgtgcaa

[0375] 1861 ggtggtcaac tcccattgcg tgttcccgag ggagctgaag gaggtgtttg catcttggcg

[0376] 1921 gctgcgctgc gcagagcggg gccgagagga cattgctgac aggctgatca gcgcctcgct

[0377] 1981 cttcctgcgc ttcctctgcc cggccattat gtcgcccagt ctatttgggc ttatgcagga

[0378] 2041 gtacccagat gagcagacct cacgaaccct caccctcatc gccaaggtca tccagaacct

[0379] 2101 ggccaacttt tccaagttta cctcaaagga ggacttcctg ggcttcatga atgagtttct

[0380] 2161 ggagctggag tggggctcta tgcagcagtt cttgtatgag atatccaacc tggacacctt

[0381] 2221 gaccaacagc agcagttttg agggctatat agacttgggc cgcgagctct ccacacttca

[0382] 2281 cgccctgctc tgggaggtgc tgccccagct cagcaaggaa gccctcctga agctgggccc

[0383] 2341 actgccccgg ctcctcaatg acatcagcac agccctgagg aaccctaaca tccaaaggca

[0384] 2401 gccaagccgc cagagtgaac ggactcggtc tcagcccatg gtgctgcgag ggccatcagc

[0385] 2461 cgagatgcag ggctacatga tgcgggacct caatagctcc atcgaccttc aatccttcat

[0386] 2521 ggctcgaggc ctcaacagct ctatggacat ggctcgcctc ccctccccaa ccaaggaaaa

[0387] 2581 accaccacca ccaccacccg gtgggggtaa agaccttttc tatgtgagcc ggccaccact

[0388] 2641 ggcccggtcc tccccagcat actgcacgag cagctcggac atcacagagc cagagcagaa

[0389] 2701 gatgctgagt gtcaacaaga gtgtgtccat gctggacctt cagggcgacg ggcctggagg

[0390] 2761 tcgccttaac agtagcagtg tttccaacct ggcagctgtt ggggacttgt tgcattccag

[0391] 2821 ccaggcctcg ctgacagcag ctttggggtt gcggcctgca cctgccgggc gcctctccca

[0392] 2881 ggggagtggc tcttccatca cagcagctgg catgcgcctc agccagatgg gggtcactac

[0393] 2941 agatggtgtc cccgcccagc aactgcgcat ccccctttcc ttccagaacc ctctcttcca

[0394] 3001 tatggctgct gatgggccag ggcccccagc aggccatgga gggagcagtg gtcatggtcc

[0395] 3061 accttcctcc catcaccacc accaccacca tcaccaccac cgagggggag aacccccagg

[0396] 3121 ggacactttt gccccattcc atggctatag caagagcgag gacctctctt caggggtccc

[0397] 3181 taagcccccc gccgcctcca tccttcacag ccacagctac agcgacgagt ttggaccctc

[0398] 3241 tggcactgat tttacccgcc ggcagctctc gcttcaggac agtctacagc acatgctctc

[0399] 3301 ccctccccag attaccatcg gtccccagag gccagctccc tcagggccgg gagggggcag

[0400] 3361 cggcgggggc agcggtgggg gccagccacc ccccttgcag aggggcaagt ctcaacagtt

[0401] 3421 gacagtgagt gccgcccaga aacctcggcc gtctagcggg aacctgttgc agtccccgga

[0402] 3481 gccaagctac ggacctgccc gtcctcggca gcagagcctc agcaaagagg gcagcattgg

[0403] 3541 gggcagcggg ggcagcgggg gcggaggggg tggggggctc aagccctcca tcaccaagca 3601 gcattcccag actccatcca cgcttaaccc cacaatgcca gcctcggagc ggaccgtagc 3661 ctgggtctcc aacatgcctc acctgtcggc tgacatcgag agtgcacaca tcgagcgaga 3721 agagtacaag ctcaaggagt actccaagtc catggacgaa agccggctgg acagggtgaa 3781 ggagtatgag gaggagatac attcgctgaa ggagaggcta cacatgtcca accggaagct 3841 ggaagagtat gagcggaggt tgctgtccca ggaagaacag accagcaaga tcctgatgca

[0404] 3901 gtaccaagcc cgcctggagc agagtgagaa gcgcttgaga cagcagcagg tggagaagga 3961 ctcccagatc aagagcatca ttggcaggct gatgctggtg gaggaggagc tgcgtcggga 4021 ccaccccgcc atggctgagc cgctgcctga gcccaagaag aggctgctcg acgctcagga 4081 gaggcagctt ccccccttgg gtccaacaaa cccgcgtgtg acgctggccc caccttggaa 4141 cggcctggcc cccccagccc cacccccccc accccggctg cagatcacag agaacggcga

[0405] 4201 gttccggaac accgcagacc ac tag

[0406] Mouse ss-wt-syn: Wild type mouse SynGAPl amino acid sequence with secretion signal (underlined) (SEQ ID NO: 12)

[0407] MYRMQLLSCIALSLALVTNSMSRSRAS IHRGS I PAMS YAPFRDVRGPPMHRTQYVHS PYDRPGWNPRFCI I SGNQLL

[0408] MLDEDE IHPLLIRDRRSESSRNKLLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEAAPAAPFRPSQGFLS

[0409] RRLKSS IKRTKSQPKLDRTSS FRQI LPRFRSADHDRARLMQS FKESHSHESLLS PSSAAEALELNLDEDS I IKPVHS

[0410] S I LGQE FCFEVTTSSGTKCFACRSAAERDKWIENLQRAVKPNKDNSRRVDNVLKLWI IEARELPPKKRYYCELCLDD

[0411] MLYARTTSKPRSASGDTVFWGEHFE FNNLPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPV

[0412] TLPTGSGGSGGMGSGGGGGSGGGSGGKGKGGCPAVRLKARYQTMS I LPMELYKE FAEYVTNHYRMLCAVLE PALNVK

[0413] GKEEVASALVHI LQSTGKAKDFLSDMAMSEVDRFMEREHLI FRENTLATKAIEEYMRLI GQKYLKDAI GE FIRALYE

[0414] SEENCEVDPIKCTASSLAEHQANLRMCCELALCKVVNSHCVFPRELKEVFASWRLRCAERGREDIADRLI SASLFLR

[0415] FLCPAIMS PSLFGLMQEYPDEQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNE FLELEWGSMQQFLYE I SNLDTL

[0416] TNSSS FEGYIDLGRELSTLHALLWEVLPQLSKEALLKLGPLPRLLNDI STALRNPNIQRQPSRQSERTRSQPMVLRG

[0417] PSAEMQGYMMRDLNSS IDLQS FMARGLNSSMDMARLPS PTKEKPPPPPPGGGKDLFYVSRPPLARSS PAYCTSSSDI

[0418] TE PEQKMLSVNKSVSMLDLQGDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSS ITAAGM

[0419] RLSQMGVTTDGVPAQQLRI PLS FQNPLFHMAADGPGPPAGHGGSSGHGPPSSHHHHHHHHHHRGGE PPGDTFAPFHG

[0420] YSKSEDLSSGVPKPPAAS I LHSHSYSDE FGPSGTDFTRRQLSLQDSLQHMLS PPQITI GPQRPAPSGPGGGSGGGSG

[0421] GGQPPPLQRGKSQQLTVSAAQKPRPSSGNLLQS PE PSYGPARPRQQSLSKEGS I GGSGGSGGGGGGGLKPS ITKQHS

[0422] QTPSTLNPTMPASERTVAWVSNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEE IHSLKERLHMSNRK

[0423] LEEYERRLLSQEEQTSKI LMQYQARLEQSEKRLRQQQVEKDSQIKS I I GRLMLVEEELRRDHPAMAE PLPE PKKRLL

[0424] DAQERQLPPLGPTNPRVTLAPPWNGLAPPAPPPPPRLQITENGE FRNTADH

[0425] Mouse synl8: Mouse SynGAPl sequence 8x N-glycan sites (bolded) (SEQ ID NO: 13) atgagcag gtctcgagcc tccatccatc gggggagcat ccccgcgatg tcctatgccc ccttcagaga tgtacgggga ccccctatgc accgaaccca atacgttcat tccccgtacg atcgtcctgg ctggaaccct cggttctgca tcatctcggg gaaccagctg ctcatgctgg atgaggatga gatacacccc cttctgatcc gcgaccggag gagcgagtcc agccgaaaca aa ACT ctgag acgcaccgtc tctgtgccag tggaggggcg gccccacggc gagcatgaat accacttggg tcgctcgagg aggaagagtg tcccaggggg gaaacagtac agcatggagg ccgcccccgc tgcgcccttc cggccctcgc aaggcttcct gagccggagg ctaaaaagct ctatcaaacg tacaaagtca caacccaaac ttgaccggac cagcagcttt cgacagatcc tgcctcgctt ccgaagtgct gaccatgacc gggcccggct gatgcagagc ttcaaggagt cacattccca cgagtccctg ctgagtccca gtagtgctgc tgaggccttg gagctcaacc tggatgaaga ctccattatc aagccagtac acagctccat cctgggtcag gagttctgct tt gag AAT ac aacatcatct ggaacaaaat gctttgcctg tcggtctgca gctgaaaggg acaaatggat tgagaatctg cagagggctg taaaacccaa caaggacaac age ACT ega gta gataaegt getgaageta tggatcatag aggetegaga gctgcccccc aagaagagat attactgtga gctgtgcctg gacgacatgc tg tat gca AAC acc acc tee aagccccgct cggcttcagg agacaccgtc ttttggggcg agcactttga gttt aac aac ACG cctgccg tccgggccct teggetgeat ctgtaccgtg actcagacaa aaageggaag aaggacaagg etggetaegt tggcctggtg actgttccag tggccaccct agctgggcgc cacttcacag agcagtggta ccccgtgacc ttgccgacag gcagtggggg ctctgggggc atgggctcgg ggggaggagg agggtcaggg ggtggctcag ggggcaaagg gaaaggaggt tgtcctgctg tgagactgaa agcccgttac cagacgatga gcatcctgcc catggagctg tataaggagt ttgcagagta tgtgaccaac cattaccgga tgctatgtgc agtgctggag cctgccctga atgteaaagg caaggaagag gtggccagtg cactggttca cate etg AAT age aca ggca aggccaagga cttcctttca gacatggcca tgtcagaggt agaccgattc atggagcggg aacacctcat attccgagag aacacgctcg ccactaaagc catagaagag tatatgagac tgattggcca gaaatacctc aaggatgcca ttggggagtt cattcgtgct etgtatgaat etgaggagaa ctgtgaagta gaccccatca agtgcacagc gtccagtctg gcagagcacc aggccaacct gcggatgtgc tgtgagttgg ccctgtgcaa ggtggtcaac tee ACC tgc gtg ttcccgag ggagetgaag gaggtgtttg catcttggcg gctgcgctgc gcagagcggg geegagagga cattgctgac aggetgatea gcgcctcgct cttcctgcgc ttcctctgcc cggccattat gtcgcccagt ctatttgggc ttatgeagga gtacccagat gagcagacct cacgaaccct caccctcatc gccaaggtca tccagaacct ggccaacttt tccaagttta cctcaaagga ggacttcctg ggcttcatga atgagtttct ggagctggag tggggctcta tgcagcagtt ettgtatgag atatccaacc tggacacctt gaccaacagc agcagttttg agggetatat agacttgggc egegag AAC tee aca ett ca cgccctgctc tgggaggtgc tgccccagct cagcaaggaa gccctcctga agctgggccc actgccccgg ctcctcaatg acatcagcac agccctgagg aaccctaaca tccaaaggca gccaagccgc cagagtgaac ggactcggtc tcagcccatg gtgetgegag ggccatcagc egagatgeag ggctacatga tgcgggacct caatagctcc atcgaccttc aatccttcat ggetegagge ctcaacagct ctatggacat ggctcgcctc ccctccccaa ccaaggaaaa accaccacca ccaccacccg gtgggggtaa agaccttttc tatgtgagee ggccaccact ggcccggtcc tccccagcat aetgeaegag cagctcggac atcacagagc cagagcagaa gatgetgagt gtcaacaaga gtgtgtccat gctggacctt cagggcgacg ggcctggagg tcgccttaac agtagcagtg tttccaacct ggcagctgtt ggggacttgt tgcattccag ccaggcctcg ctgacagcag ctttggggtt gcggcctgca cctgccgggc gcctctccca ggggagtggc tcttccatca cagcagctgg catgcgcctc agccagatgg gggtcactac agatggtgtc cccgcccagc aactgcgcat ccccctttcc ttccagaacc ctctcttcca tatggctgct gatgggccag ggcccccagc aggccatgga gggagcagtg gtcatggtcc accttcctcc catcaccacc accaccacca tcaccaccac cgagggggag aacccccagg ggacactttt gccccattcc atggctatag caagagcgag gacctctctt caggggtccc taagcccccc gccgcctcca tccttcacag ccacagctac agcgacgagt ttggaccctc tggcactgat tttacccgcc ggcagctctc gcttcaggac agtctacagc acatgctctc ccctccccag attaccatcg gtccccagag gccagctccc tcagggccgg gagggggcag cggcgggggc agcggtgggg gccagccacc ccccttgcag aggggcaagt ctcaacagtt gacagtgagt gccgcccaga aacctcggcc gtctagcggg aacctgttgc agtccccgga gccaagctac ggacctgccc gtcctcggca gcagagcctc agcaaagagg gcagcattgg gggcagcggg ggcagcgggg gcggaggggg tggggggctc aagccctcca tcaccaagca gcattcccag actccatcca cgcttaaccc cacaatgcca gcctcggagc ggaccgtagc ctgggtctcc aacatgcctc acctgtcggc tgacatcgag agtgcacaca tcgagcgaga agagtacaag ctcaaggagt actccaagtc catggacgaa agccggctgg acagggtgaa ggagtatgag gaggagatac attcgctgaa ggagaggcta cacatgtcca accggaagct ggaagagtat gagcggaggt tgctgtccca ggaagaacag accagcaaga tcctgatgca gtaccaagcc cgcctggagc agagtgagaa gcgcttgaga cagcagcagg tggagaagga ctcccagatc aagagcatca ttggcaggct gatgctggtg gaggaggagc tgcgtcggga ccaccccgcc atggctgagc cgctgcctga gcccaagaag aggctgctcg acgctcagga gaggcagctt ccccccttgg gtccaacaaa cccgcgtgtg acgctggccc caccttggaa cggcctggcc cccccagccc cacccccccc accccggctg cagatcacag agaacggcga gttccggaac accgcagacc ac tag

[0426] Mouse synl8: Mouse SynGAPl amino acid sequence 8x N-glycan sites (bolded and underlined) (SEQ ID NO: 14).

[0427] MSRSRAS IHRGS I PAMSYAPFRDVRGPPMHRTQYVHS PYDRPGWNPRFCI I SGNQLLMLDEDE IHPLLIRDRRSESS

[0428] RNKTLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEAAPAAPFRPSQGFLSRRLKSS IKRTKSQPKLDRTS

[0429] S FRQI LPRFRSADHDRARLMQS FKESHSHESLLS PSSAAEALELNLDEDS I IKPVHSS I LGQE FCFENTTSSGTKCF

[0430] ACRSAAERDKWIENLQRAVKPNKDNSTRVDNVLKLWI IEARELPPKKRYYCELCLDDMLYANTTSKPRSASGDTVFW

[0431] GEHFE FNNTPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPVTLPTGSGGSGGMGSGGGGGS

[0432] GGGSGGKGKGGCPAVRLKARYQTMS I LPMELYKE FAEYVTNHYRMLCAVLE PALNVKGKEEVASALVHI LNSTGKAK

[0433] DFLSDMAMSEVDRFMEREHLI FRENTLATKAIEEYMRLI GQKYLKDAI GE FIRALYESEENCEVDPIKCTASSLAEH

[0434] QANLRMCCELALCKVVNSTCVFPRELKEVFASWRLRCAERGREDIADRLI SASLFLRFLCPAIMS PSLFGLMQEYPD

[0435] EQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNE FLELEWGSMQQFLYE I SNLDTLTNSSS FEGYIDLGRENSTLH

[0436] ALLWEVLPQLSKEALLKLGPLPRLLNDI STALRNPNIQRQPSRQSERTRSQPMVLRGPSAEMQGYMMRDLNSS IDLQ

[0437] S FMARGLNSSMDMARLPS PTKEKPPPPPPGGGKDLFYVSRPPLARSS PAYCTSSSDITE PEQKMLSVNKSVSMLDLQ

[0438] GDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSS ITAAGMRLSQMGVTTDGVPAQQLRI P LSFQNPLFHMAADGPGPPAGHGGSSGHGPPSSHHHHHHHHHHRGGEPPGDTFAPFHGYSKSEDLSSGVPKPPAASIL

[0439] HSHSYSDEFGPSGTDFTRRQLSLQDSLQHMLSPPQITIGPQRPAPSGPGGGSGGGSGGGQPPPLQRGKSQQLTVSAA

[0440] QKPRPSSGNLLQSPEPSYGPARPRQQSLSKEGSIGGSGGSGGGGGGGLKPSITKQHSQTPSTLNPTMPASERTVAWV

[0441] SNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEEIHSLKERLHMSNRKLEEYERRLLSQEEQTSKILM

[0442] QYQARLEQSEKRLRQQQVEKDSQIKSI IGRLMLVEEELRRDHPAMAEPLPEPKKRLLDAQERQLPPLGPTNPRVTLA

[0443] PPWNGLAPPAPPPPPRLQITENGEFRNTADH

[0444] Mouse synl8: Mouse SynGAPl sequence with secretion signal (underlined) and 8x N-glycan sites (bolded) (SEQ ID NO: 15). atgtacaggatgcaa ctcctgtctt gcattgcact aagtcttgca cttgtcacaa acagt atgagcag gtctcgagcc tccatccatc gggggagcat ccccgcgatg tcctatgccc ccttcagaga tgtacgggga ccccctatgc accgaaccca atacgttcat tccccgtacg atcgtcctgg ctggaaccct cggttctgca tcatctcggg gaaccagctg ctcatgctgg atgaggatga gatacacccc cttctgatcc gcgaccggag gagcgagtcc agccgaaaca aa ACT ctgag acgcaccgtc tctgtgccag tggaggggcg gccccacggc gagcatgaat accacttggg tcgctcgagg aggaagagtg tcccaggggg gaaacagtac agcatggagg ccgcccccgc tgcgcccttc cggccctcgc aaggcttcct gagccggagg ctaaaaagct ctatcaaacg tacaaagtca caacccaaac ttgaccggac cagcagcttt cgacagatcc tgcctcgctt ccgaagtgct gaccatgacc gggcccggct gatgcagagc ttcaaggagt cacattccca cgagtccctg ctgagtccca gtagtgctgc tgaggccttg gagctcaacc tggatgaaga ctccattatc aagccagtac acagctccat cctgggtcag gagttctgct tt gag AAT ac aacatcatct ggaacaaaat gctttgcctg tcggtctgca gctgaaaggg acaaatggat tgagaatctg cagagggctg taaaacccaa caaggacaac age ACT ega gta gataaegt getgaageta tggatcatag aggetegaga gctgcccccc aagaagagat attactgtga gctgtgcctg gacgacatgc tg tat gca AAC acc acc tee aagccccgct cggcttcagg agacaccgtc ttttggggcg agcactttga gttt aac aac ACG cctgccg tccgggccct teggetgeat ctgtaccgtg actcagacaa aaageggaag aaggacaagg etggetaegt tggcctggtg actgttccag tggccaccct agctgggcgc cacttcacag agcagtggta ccccgtgacc ttgccgacag gcagtggggg ctctgggggc atgggctcgg ggggaggagg agggtcaggg ggtggctcag ggggcaaagg gaaaggaggt tgtcctgctg tgagactgaa agcccgttac cagacgatga gcatcctgcc catggagctg tataaggagt ttgcagagta tgtgaccaac cattaccgga tgctatgtgc agtgctggag cctgccctga atgteaaagg caaggaagag gtggccagtg cactggttca cate etg AAT age aca ggca aggccaagga cttcctttca gacatggcca tgtcagaggt agaccgattc atggagcggg aacacctcat attccgagag aacacgctcg ccactaaagc catagaagag tatatgagac tgattggcca gaaatacctc aaggatgcca ttggggagtt cattcgtgct etgtatgaat etgaggagaa ctgtgaagta gaccccatca agtgcacagc gtccagtctg gcagagcacc aggccaacct gcggatgtgc tgtgagttgg ccctgtgcaa ggtggtcaac tee ACC tgc gtg ttcccgag ggagetgaag gaggtgtttg catcttggcg gctgcgctgc gcagagcggg gccgagagga cattgctgac aggctgatca gcgcctcgct cttcctgcgc ttcctctgcc cggccattat gtcgcccagt ctatttgggc ttatgcagga gtacccagat gagcagacct cacgaaccct caccctcatc gccaaggtca tccagaacct ggccaacttt tccaagttta cctcaaagga ggacttcctg ggcttcatga atgagtttct ggagctggag tggggctcta tgcagcagtt cttgtatgag atatccaacc tggacacctt gaccaacagc agcagttttg agggctatat agacttgggc cgcgag AAC tcc aca ctt ca cgccctgctc tgggaggtgc tgccccagct cagcaaggaa gccctcctga agctgggccc actgccccgg ctcctcaatg acatcagcac agccctgagg aaccctaaca tccaaaggca gccaagccgc cagagtgaac ggactcggtc tcagcccatg gtgctgcgag ggccatcagc cgagatgcag ggctacatga tgcgggacct caatagctcc atcgaccttc aatccttcat ggctcgaggc ctcaacagct ctatggacat ggctcgcctc ccctccccaa ccaaggaaaa accaccacca ccaccacccg gtgggggtaa agaccttttc tatgtgagcc ggccaccact ggcccggtcc tccccagcat actgcacgag cagctcggac atcacagagc cagagcagaa gatgctgagt gtcaacaaga gtgtgtccat gctggacctt cagggcgacg ggcctggagg tcgccttaac agtagcagtg tttccaacct ggcagctgtt ggggacttgt tgcattccag ccaggcctcg ctgacagcag ctttggggtt gcggcctgca cctgccgggc gcctctccca ggggagtggc tcttccatca cagcagctgg catgcgcctc agccagatgg gggtcactac agatggtgtc cccgcccagc aactgcgcat ccccctttcc ttccagaacc ctctcttcca tatggctgct gatgggccag ggcccccagc aggccatgga gggagcagtg gtcatggtcc accttcctcc catcaccacc accaccacca tcaccaccac cgagggggag aacccccagg ggacactttt gccccattcc atggctatag caagagcgag gacctctctt caggggtccc taagcccccc gccgcctcca tccttcacag ccacagctac agcgacgagt ttggaccctc tggcactgat tttacccgcc ggcagctctc gcttcaggac agtctacagc acatgctctc ccctccccag attaccatcg gtccccagag gccagctccc tcagggccgg gagggggcag cggcgggggc agcggtgggg gccagccacc ccccttgcag aggggcaagt ctcaacagtt gacagtgagt gccgcccaga aacctcggcc gtctagcggg aacctgttgc agtccccgga gccaagctac ggacctgccc gtcctcggca gcagagcctc agcaaagagg gcagcattgg gggcagcggg ggcagcgggg gcggaggggg tggggggctc aagccctcca tcaccaagca gcattcccag actccatcca cgcttaaccc cacaatgcca gcctcggagc ggaccgtagc ctgggtctcc aacatgcctc acctgtcggc tgacatcgag agtgcacaca tcgagcgaga agagtacaag ctcaaggagt actccaagtc catggacgaa agccggctgg acagggtgaa ggagtatgag gaggagatac attcgctgaa ggagaggcta cacatgtcca accggaagct ggaagagtat gagcggaggt tgctgtccca ggaagaacag accagcaaga tcctgatgca gtaccaagcc cgcctggagc agagtgagaa gcgcttgaga cagcagcagg tggagaagga ctcccagatc aagagcatca ttggcaggct gatgctggtg gaggaggagc tgcgtcggga ccaccccgcc atggctgagc cgctgcctga gcccaagaag aggctgctcg acgctcagga gaggcagctt ccccccttgg gtccaacaaa cccgcgtgtg acgctggccc caccttggaa cggcctggcc cccccagccc cacccccccc accccggctg cagatcacag agaacggcga gttccggaac accgcagacc ac tag Mouse synl8: Mouse SynGAPl amino acid sequence with secretion signal (underlined) and 8x N-glycan sites (bolded and underlined) (SEQ ID NO: 16).

[0445] MYRMQLLSCIALSLALVTNSMSRSRAS IHRGS I PAMS YAPFRDVRGPPMHRTQYVHS PYDRPGWNPRFCI I SGNQLL MLDEDEIHPLLIRDRRSESSRNKTLRRTVSVPVEGRPHGEHEYHLGRSRRKSVPGGKQYSMEAAPAAPFRPSQGFLS RRLKSSIKRTKSQPKLDRTSSFRQILPRFRSADHDRARLMQSFKESHSHESLLSPSSAAEALELNLDEDSIIKPVHS SILGQEFCFENTTSSGTKCFACRSAAERDKWIENLQRAVKPNKDNSTRVDNVLKLWIIEARELPPKKRYYCELCLDD MLYANTTSKPRSASGDTVFWGEHFEFNNTPAVRALRLHLYRDSDKKRKKDKAGYVGLVTVPVATLAGRHFTEQWYPV TLPTGSGGSGGMGSGGGGGSGGGSGGKGKGGCPAVRLKARYQTMSILPMELYKEFAEYVTNHYRMLCAVLEPALNVK GKEEVASALVHILNSTGKAKDFLSDMAMSEVDRFMEREHLIFRENTLATKAIEEYMRLIGQKYLKDAIGEFIRALYE SEENCEVDPIKCTASSLAEHQANLRMCCELALCKVVNSTCVFPRELKEVFASWRLRCAERGREDIADRLISASLFLR FLCPAIMSPSLFGLMQEYPDEQTSRTLTLIAKVIQNLANFSKFTSKEDFLGFMNEFLELEWGSMQQFLYEISNLDTL TNSSSFEGYIDLGRENSTLHALLWEVLPQLSKEALLKLGPLPRLLNDISTALRNPNIQRQPSRQSERTRSQPMVLRG PSAEMQGYMMRDLNSSIDLQSFMARGLNSSMDMARLPSPTKEKPPPPPPGGGKDLFYVSRPPLARSSPAYCTSSSDI TEPEQKMLSVNKSVSMLDLQGDGPGGRLNSSSVSNLAAVGDLLHSSQASLTAALGLRPAPAGRLSQGSGSSITAAGM RLSQMGVTTDGVPAQQLRIPLSFQNPLFHMAADGPGPPAGHGGSSGHGPPSSHHHHHHHHHHRGGEPPGDTFAPFHG YSKSEDLSSGVPKPPAASILHSHSYSDEFGPSGTDFTRRQLSLQDSLQHMLSPPQITIGPQRPAPSGPGGGSGGGSG GGQPPPLQRGKSQQLTVSAAQKPRPSSGNLLQSPEPSYGPARPRQQSLSKEGSIGGSGGSGGGGGGGLKPSITKQHS QTPSTLNPTMPASERTVAWVSNMPHLSADIESAHIEREEYKLKEYSKSMDESRLDRVKEYEEEIHSLKERLHMSNRK LEEYERRLLSQEEQTSKILMQYQARLEQSEKRLRQQQVEKDSQIKSIIGRLMLVEEELRRDHPAMAEPLPEPKKRLL DAQERQLPPLGPTNPRVTLAPPWNGLAPPAPPPPPRLQITENGEFRNTADH

[0446] REFERENCES

[0447] 1) Kilinc, M, Creson, T, Rojas, C, Aceti, M, Ellegood, J, Vaissiere, T, et al. Species-conserved SYNGAP1 phenotypes associated with neurodevelopmental disorders. Mol Cell Neurosci. 2018;91:140-150.

[0448] 2) Holder, JL, Hamdan, FF, Michaud, JL, Adam, MP, Mirzaa, GM, Pagon, RA, et al. SYNGAP1- Related Intellectual Disability. 2019; In: Adam MP, et al. editors. GeneReviews® Seattle (WA): University of Washington, Seattle.

[0449] 3) Gamache, TR, Araki, Y, Huganir, RL. Twenty Years of SynGAP Research: From Synapses to Cognition. J Neurosci.2020;40:1596-1605.

[0450] 4) Jeyabalan, N, Clement, JP. SYNGAP1: Mind the Gap. Front Cell Neurosci.2016; 10:32.

[0451] 5) Kim, JH, Lee, H-K, Takamiya, K, Huganir, RL. The role of synaptic GTPase-activating protein in neuronal development and synaptic plasticity. J Neurosci.2003;23 : 1119-1124.

[0452] 6) Kim, JH, Lee, H-K, Takamiya, K, Huganir, RL. The role of synaptic GTPase-activating protein in neuronal development and synaptic plasticity. J Neurosci.2003;23: 1119-1124. 7) Rumbaugh, G, Paige Adams, J, Kim, JH, Huganir, RL. SynGAP regulates synaptic strength and mitogen-activated protein kinases in cultured neurons. PNAS USA. 2006;103:4344-4351.

[0453] 8) Chen, HJ, Rojas-Soto, M, Oguni, A, Kennedy, MB. A synaptic Ras-GTPase activating protein (p!35 SynGAP) inhibited by CaM kinase II. Neuron. 1998;20:895-904.

[0454] 9) Gamache, TR, Araki, Y, Huganir, RL. Twenty Years of SynGAP Research: From Synapses to Cognition. J Neurosci. 2020;40: 1596-1605.

[0455] 10) Capotondo, A, Milazzo, R, Politi, LS, Quattrini, A, Palini, A, Plati, T, et al. Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation. Proc Natl Acad Sci USA. 2012;109: 15018-15023.

[0456] 11) Sergijenko, A, Langford- Smith, A, Liao, AY, Pickford, CE, McDermott, J, Nowinski, G, et al. Myeloid / Microglial driven autologous hematopoietic stem cell gene therapy corrects a neuronopathic lysosomal disease. Mol Ther. 2013;21 : 1938-1949.

[0457] 12) Andoh, M, Koyama, R. Microglia regulate synaptic development and plasticity. Dev Neurobiol. 2021;81 :568-590.

[0458] 13) Lim, SH, Park, E, You, B, Jung, Y, Park, AR, Park, SG, et al. Neuronal synapse formation induced by microglia and interleukin 10. PLoS ONE. 2013;8:e81218.

[0459] 14) Parkhurst, CN, Yang, G, Ninan, I, Savas, JN, Yates, JR, Lafaille, JJ, et al. Microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor. Cell. 2013; 155: 1596-1609.

[0460] 15) Miyamoto, A, Wake, H, Ishikawa, AW, Eto, K, Shibata, K, Murakoshi, H, et al. Microglia contact induces synapse formation in developing somatosensory cortex. Nature Communications. 2016;7: 12540.

[0461] 16) Cavazzana, M, Bushman, FD, Miccio, A, Andre- Schmutz, I, Six, E. Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges. Nat Rev Drug Discov. 2019;18:447-462.

[0462] 17) Beegle, J, Hendrix, K, Maciel, H, Nolta, JA, Anderson, JS. Improvement of motor and behavioral activity in Sandhoff mice transplanted with human CD34+ cells transduced with a HexA / HexB expressing lentiviral vector. J Gene Med. 2020;22:e3205.

[0463] 18) Adhikari, A, Copping, NA, Beegle, J, Cameron, DL, Deng, P, O’Geen, H, et al. Functional rescue in an Angelman syndrome model following treatment with lentivector transduced hematopoietic stem cells. Hum Mol Genet. 2021;30: 1067-1083.

[0464] 19) Biffi, A, Montini, E, Lorioli, L, Cesani, M, Fumagalli, F, Plati, T, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science. 2013;341 : 1233158.

[0465] 20) Tucci, F, Scaramuzza, S, Aiuti, A, Mortellaro, A. Update on Clinical Ex Vivo Hematopoietic Stem Cell Gene Therapy for Inherited Monogenic Diseases. Mol Ther. 2021;29:489-504.

[0466] 21) Biffi, A, Aubourg, P, Cartier, N. Gene therapy for leukodystrophies. Hum Mol Genet. 2011;20:R42-53.

[0467] 22) Eichler, F, Duncan, C, Musolino, PL, Orchard, PJ, Oliveira, SD, Thrasher, AJ, et al. Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. N Engl J Med. 2017;377: 1630-1638.

[0468] 23) Cartier, N, Hacien-Bey-Abina, S, Bartholomae, CC, Veres, G, Schmidt, M, Kutschera, I, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science. 2009;326:818-823.

[0469] 24) Fraldi, A, Serafini, M, Sorrentino, NC, Gentner, B, Aiuti, A, Bernardo, ME. Gene therapy for mucopolyssacharidoses: in vivo and ex vivo approaches. Ital J Pediatr. 2018;44: 130.

[0470] 25) Penati, R, Fumagelli, F, Calbi, V, Bernardo, ME, Aiuti, A. Gene therapy for lysosomal storage disorders: recent advances for metachromatic leukodystrophy and mucopolysaccaridosis I. J Inherit Metab Dis. 2017; 40:543-554.

[0471] 26) Cao, X, Shores, EW, Hu-Li, J, Anver, MR, Kelsall, BL, Russell, SM, et al. Defective lymphoid development in mice lacking expression of the common cytokine receptor gamma chain. Immunity. 1995;2:223-238.

[0472] 27) Schorle, H, Holtschke, T, Hunig, T, Schimpl, A, Horak, I. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature. 1991;352:621-624.

[0473] 28) Shultz, LD, Lyons, BL, Burzenski, LM, Gott, B, Chen, X, Chaleff, S, et al. Human lymphoid and myeloid cell development in NOD / LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174:6477-6489.

[0474] 29) Pearson, T, Shultx, LD, Miller, D, King, M, Laning, J, Fodor, W, et al. Non-obese diabeticrecombination activating gene-1 (NOD-Ragl null) interleukin (IL)-2 receptor common gamma chain (IL2r gamma null) null mice: a radioresistant model for human lymphohaematopoietic engraftment. Clin Exp Immunol. 2008;154:270-284.

[0475] 30) Nakajima, R, Takao, K, Hattori, S, Shoji, H, Komiyama, NH, Grant, SGN, et al. Comprehensive behavioral analysis of heterozygous Syngapl knockout mice. Neuropsychopharmacol Rep. 2019;39:223-237.

[0476] 31) Creson, TK, Rojas, C, Hwaun, E, Vaissiere, T, Kilinc, M, Jimenez-Gomez, A, et al. Re- expression of SynGAP protein in adulthood improves translatable measures of brain function and behavior. 2019; eLife. 8:e46752.

[0477] 32) Fischer, A. Severe combined immunodeficiencies. Immunodefic Rev. 1992;3:83-100.

[0478] 33) Cavazzana, M, Six, E, Lagresle-Peyrou, C, Andre- Schmutz, I, Hacein-Bey-Abina, S. Gene Therapy for X-Linked Severe Combined Immunodeficiency: Where Do We Stand? Hum Gene Ther. 2016;2: 108-116.

[0479] 34) Shaw, KL, Kohn, DB. A tale of two SCIDs. Sci Transl Med. 201 l;97:97ps36.

[0480] 35) Barclay, SL, Yang, Y, Zhang, S, Fong, R, Barraza, A, Nolta, JA, et al. Safety and Efficacy of a tCD25 Preselective Combination Anti-HIV Lentiviral Vector in Human Hematopoietic Stem and Progenitor Cells. Stem Cells. 2015;33:870-879.

[0481] 36) Larkin, HD. Autologous Gene Therapy Approved for Childhood CALD. JAMA. 2022;17: 1679-1680.

[0482] All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The present disclosure has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

1. CLAIMSWhat is claimed is:

1. A synaptic Ras GTPase activating protein 1 (SynGAPl) protein having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16, wherein the SynGAPl protein comprises one or more non-naturally occurring glycosylation sites.

2. The SynGAPl protein of claim 1 having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO: 8, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

3. The SynGAPl protein of claim 2 having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO: 8.

4. A polynucleotide comprising a nucleic acid sequence that encodes a synaptic Ras GTPase activating protein 1 (SynGAPl), wherein the encoded SynGAPl comprises one or more non-naturally occurring glycosylation sites.

5. The polynucleotide of claim 4, wherein the encoded SynGAPl comprises from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) non-naturally occurring glycosylation sites.

6. The polynucleotide of claim 5, wherein the encoded SynGAPl comprises 8 non- naturally occurring glycosylation sites.

7. The polynucleotide of any one of claims 4-6, wherein the polynucleotide further comprises a polynucleotide encoding a secretion signal.

8. The polynucleotide of claim 7, wherein the secretion signal is selected from MYRMQLLSCIALSLALVTNS (SEQ ID NO:29), MKRMQLLSCIALSLALVTNS (SEQ ID NO:30), MRMQLLSCIALSLALVTNS (SEQ ID NO:31), MRRMQLLSCIALSLALVTNS (SEQ ID NO:32), MRRKKMQLLSCIALSLALVTN (SEQ ID NO:33),MYRMQLLLLIALSLALVTNS (SEQ ID NO:34), MYRMQLLLSCIALSLALVTNS (SEQ ID NO:35), MYRMQLLLSCIALLLALVTNS (SEQ ID NO:36), MYRMQLLLLIALSLALVTNS (SEQ ID NO: 37), MRMQLLLLIALSLALVTNS (SEQ ID NO: 38), MRRMQLLLLIALSLALVTNS (SEQ ID NO: 39) and MRRKKMQLLLLIALSLALVTNS (SEQ ID NO:40).

9. The polynucleotide of any one of claims 4-8, wherein the polynucleotide further comprises a regulatory sequence that directs expression of the encoded SynGAPl.

10. The polynucleotide of claim 9, wherein the regulatory sequence comprises one or more of the following: a promoter, an intron, an enhancer, a polyadenylation signal, a terminator, a silencer, a TATA box, or a Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE).

11. The polynucleotide of claim 10, wherein the polynucleotide comprises a microgliaspecific promoter.

12. The polynucleotide of claim 7, wherein the promoter is an MNDU3 promoter, a Syn-1 (Synapsin 1) promoter, a CMV promoter, a CD68 promoter, a human or mouse PGK promoter, a MNDU promoter, or a long or short EFl alpha promoter.

13. The polynucleotide of any one of claims 4-12, wherein the polynucleotide comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the nucleic acid sequence of SEQ ID NO:7, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NOV, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO:15.

14. The polynucleotide of claim 13, wherein the polynucleotide comprises a nucleic acid sequence having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the nucleic acid sequence of SEQ ID NO: 7, SEQ ID NO: 1, SEQ ID NO:3 or SEQ ID NO:5.

15. The polynucleotide of claim 14, wherein the polynucleotide comprises a nucleic acidsequence having at least 90% identity (e.g, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the nucleic acid sequence of SEQ ID NO:7.

16. The polynucleotide of any one of claims 4-15, wherein the polynucleotide comprises a nucleic acid sequence encoding a SynGAPl protein having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO:8, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 or SEQ ID NO: 16.

17. The polynucleotide of claim 16, wherein the polynucleotide comprises a nucleic acid sequence encoding a SynGAPl having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO: 8, SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.

18. The polynucleotide of claim 17, wherein the polynucleotide comprises a nucleic acid sequence encoding a SynGAPl having at least 90% identity (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) with the amino acid sequence of SEQ ID NO: 8.

19. A vector comprising the polynucleotide of any one of claims 4 to 18.

20. The vector of claim 19, wherein the vector is a viral vector or a non-viral vector.

21. The vector of claim 19, wherein the vector is a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a Herpes viral vector.

22. The vector of claim 19, wherein the vector is a lentiviral vector.

23. The vector of claim 22, wherein the lentiviral vector is a self-inactivating lentiviral vector.

24. A SynGAPl protein encoded by the polynucleotide of any one of claims 4-18.

25. The SynGAPl protein of claim 24, which comprises any combination of the following glycosylation sites, where the aa numbering is with reference to SEQ ID NO:6: aa 79-aa81; aa222-aa224; aa256-aa258; aa293-aa295; aa315-aa317; aa456-aa458; aa556-aa558; and / or aa689-aa691.

26. A population of hematopoietic stem and progenitor cells (HSPCs) that have been transduced with the vector of any one of claims 19-23.

27. A population of hematopoietic stem and progenitor cells (HSPCs) that comprise the polynucleotide of any one of claims 4-18.

28. A population of hematopoietic stem and progenitor cells (HSPCs) that comprise the protein of any one of claims 1-3, 24 or 25.

29. The population of HSPCs of any one of claims 25-28, which comprises CD34+ HSPCs.

30. A method of treating a human subject with SYNGAP1 -related intellectual disability, comprising administering to a human subject in need thereof an effective amount of the vector of any one of claims 19-23.

31. The use of a vector of any one of claims 19-23 in the treatment of a SYNGAP1 -related intellectual disability.

32. A method of treating a human subject with SYNGAP1 -related intellectual disability, comprising administering to a human subject in need thereof an effective amount of the population of HSPCs of any one of claims 26-29.

33. The use of the population of a population of HSPCs of any one of claims 26-29 in the treatment of a SYNGAP1 -related intellectual disability.

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