Adeno-associated viral vectors for expressing GLUT1 and uses thereof

Abstract

Provided herein is a gene therapy for GLUT1 deficiency syndrome and related disorders using recombinant adeno-associated virus (rAAV) virion as a vector to express GLUT1 protein or its functional variant.The capsid can be AAV-BR1 capsid or its functional variant.Other promoters or capsids can be used.The rAAV virion can use an endothelial specific promoter, such as FLT-1 promoter.Further provided are methods of treatment, such as by administering rAAV virion into the brain and / or into a vein, as well as other compositions and methods. TIFF2025505662000042.tif203166

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 307,963, filed February 8, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] SEQUENCE LISTING STATEMENT The Sequence Listing XML associated with this application is provided in XML file format and is incorporated herein by reference. The XML file containing the Sequence Listing XML is named ROPA_027_01WO_ST26.xml. The XML file is 223m012 bytes, was created on Feb. 8, 2023, and has been submitted electronically via the USPTO Patent Center. [Background technology]

[0003] background Mutations in the SLC2A1 gene, which encodes glucose transporter 1 (GLUT1), are associated with a neurodevelopmental disorder termed GLUT1 deficiency syndrome (GLUT1 DS). GLUT1 DS is an autosomal dominant disorder and often manifests as a sporadic disease in which de novo mutations result in haploinsufficiency and syndromic heterozygosity.

[0004] GLUT1 is an insulin-independent glucose transporter. Patients with typical GLUT1 DS, also known as De Vivo disease, suffer from low brain glucose levels and display a phenotype characterized by early-onset seizures (median age 12 months), developmental delay, acquired microcephaly (slowed head growth), complex movement disorders (spasticity, ataxia, dystonia); paroxysmal eye-head movements; and CSF glycopenia, i.e., low glucose concentrations in the cerebrospinal fluid (CSF). The clinical course of the disease highlights the importance of early treatment. Alter et al. J. Child Neurol. 30(2):160-169 (2015). GLUT1 is involved in endothelial cell function, including transport of glucose across the blood-brain barrier (BBB), angiogenesis, and maintenance of the BBB. However, studies in haploinsufficient mouse models have provided conflicting evidence regarding the role of GLUT1 in maintaining the physical integrity of the BBB. Endothelial lineage-specific knockout of GLUT1 reduces endothelial energy availability and reduces proliferation without affecting migration, thereby delaying developmental angiogenesis (Veys et al., Circ. Res. 2020; 127:466-482), but the effect of restoring GLUT1 expression specifically in endothelial cells has not been tested.

[0005] Treatment strategies for this disease are outlined in Tang et al. Ann. Clin. Trans. Neurol. 2019; 6(9):1923-1932. The current standard of care is the ketogenic diet, which elevates the levels of ketone bodies, which substitute for glucose in the blood, making them available to the brain. Treatment with the triglyceride triheptanoin has been proposed as an alternative to the ketogenic diet. Gene therapy using adeno-associated virus (AAV) vectors has also been attempted. To target neuronal GLUT1 deficiency, AAV9 vectors encoding GLUT1 under the control of a neuron-specific promoter (e.g., synapsin) have been tested in postnatal juvenile mouse models. Other studies have utilized constitutive promoters (e.g., CMV promoter) or the promoter of the endogenous GLUT1 gene. Various small molecules have also been tested, including the carbonic anhydrase inhibitor acetazolamide, an anticonvulsant.

[0006] Haploinsufficiency of GLUT1 halts cerebral angiogenesis, resulting in a relatively small cerebral microvasculature, which may be related to the glucose dependence of endothelial tip cells, although Tang et al. observe that whether low GLUT1 in endothelial cells causes this pathology remains to be investigated. GLUT1 protein is expressed in additional brain cells, including oligodendrocytes, microglia, and ependymal cells.

[0007] There is an unmet need for a treatment for GLUT1 deficiency syndrome. The gene therapy provided herein addresses this need. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Alter et al. J. Child Neurol. 30(2):160-169 (2015) [Non-Patent Document 2] Veys et al., Circ. Res. 2020; 127:466-482 [Non-Patent Document 3] Tang et al. Ann. Clin. Trans. Neurol. 2019; 6(9):1923-1932 Summary of the Invention

[0009] overview The present invention relates generally to gene therapy for neurological diseases or disorders using adeno-associated virus (AAV)-based delivery of a polynucleotide encoding GLUT1 or a functional variant thereof.

[0010] GLUT1 Deficiency Syndrome (DS) is a neurodevelopmental disorder with clinical symptoms rooted in a lack of proper neuronal function, but the gene therapy of the present invention, without being bound by theory, may target endothelial cells, which are responsible for directing angiogenesis and vasculature development in the central nervous system (CNS). AAV can be delivered to the developing CNS vasculature via intravenous, direct delivery to the intraventricular system, or by a combination of both routes, followed by expression of GLUT1 protein in endothelial tip cells, promoting the growth and formation of blood vessels throughout the CNS during a critical period of angiogenesis and neurodevelopment. Furthermore, AAV can be delivered to the CNS vasculature, followed by expression of GLUT1 protein in mature endothelial cells, promoting increased glucose availability to neurons by increasing glucose transport across the blood-brain barrier.

[0011] In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a vector genome and a capsid, wherein the vector genome comprises an expression cassette flanked by 5' and 3' inverted terminal repeats (ITRs), the expression cassette comprises a polynucleotide sequence encoding GLUT1 or a functional variant thereof operably linked to a promoter, and the capsid is a BR1 capsid or a functional variant thereof.

[0012] In some embodiments, the capsid is a BR1 capsid.

[0013] In some embodiments, the capsid comprises a polypeptide sequence motif XDGXXWX (SEQ ID NO: 107), where each X is any amino acid.

[0014] In some embodiments, the capsid comprises the polypeptide sequence ADGVQWT (SEQ ID NO:108), DDGVSWK (SEQ ID NO:109), SDGLTWS (SEQ ID NO:110) or SDGLAWV (SEQ ID NO:111).

[0015] In some embodiments, the capsid comprises the polypeptide sequence NRGTEWD ​​or a functional variant thereof having one, two, three or more substitutions therein.

[0016] In some embodiments, the capsid comprises the polypeptide sequence NRGTEWD ​​(SEQ ID NO:112).

[0017] In some embodiments, the capsid comprises an insertion of the polypeptide sequence NRGTEWD ​​in the GH loop compared to the AAV2 VP1 reference sequence set forth in SEQ ID NO:76.

[0018] In some embodiments, the capsid comprises a VP3 polypeptide that shares at least 98%, at least 99%, or 100% identity with the AAV2 VP3 polypeptide sequence set forth in SEQ ID NO:106.

[0019] In some embodiments, the capsid comprises a VP2 polypeptide that shares at least 98%, at least 99%, or 100% identity with the AAV2 VP2 polypeptide sequence set forth in SEQ ID NO:105.

[0020] In some embodiments, the capsid comprises a VP1 polypeptide that shares at least 98%, at least 99%, or 100% identity with the AAV2 VP1 polypeptide sequence set forth in SEQ ID NO:104.

[0021] In some embodiments, the promoter is the FLT-1 promoter.

[0022] In some embodiments, the FLT-1 promoter is a human FLT-1 (hFLT-1) promoter.

[0023] In some embodiments, the hFLT-1 promoter shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:1.

[0024] In some embodiments, the expression cassette comprises a polyA signal, optionally a human growth hormone (hGH) polyA.

[0025] In some embodiments, the expression cassette comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), optionally a WPRE(x).

[0026] In some embodiments, the expression cassette comprises a 3' untranslated region (3' UTR) comprising a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:4.

[0027] In some embodiments, the polynucleotide sequence encoding GLUT1 is a SLC2A1 polynucleotide.

[0028] In some embodiments, the SLC2A1 polynucleotide is a human SLC2A1 polynucleotide.

[0029] In some embodiments, the polynucleotide sequence encoding GLUT1 shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:5.

[0030] In some embodiments, the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs), optionally AAV2 ITRs, optionally ITRs that share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:6 or SEQ ID NO:7.

[0031] In some embodiments, the expression cassette shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with any one of SEQ ID NOs:8-16, SEQ ID NO:97, SEQ ID NO:99 and SEQ ID NO:101.

[0032] In some embodiments, the rAAV virion is not an AAV2 virion.

[0033] In some embodiments, the disclosure provides a method of treating and / or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an rAAV virion.

[0034] In some embodiments, the disease or disorder is a neurological disorder.

[0035] In some embodiments, the disease or disorder is Glucose Transporter 1 Deficiency Syndrome (GLUT1 DS) or De Vivo Disease.

[0036] In some embodiments, the rAAV virions are administered by intracerebroventricular (ICV) injection.

[0037] In some embodiments, the rAAV virions are administered by intravenous (IV) injection.

[0038] In some embodiments, the rAAV virions are administered by ICV injection in combination with IV injection.

[0039] In some embodiments, administration results in expression of a polynucleotide sequence encoding GLUT1 in the brain, optionally at an increased level compared to a reference rAAV virion.

[0040] In some embodiments, administration results in increased expression of GLUT1 protein in the brain and / or increased glucose and / or lactate levels in the CSF, optionally at an increased level compared to a reference rAAV virion, where optionally the increase is an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or 100%.

[0041] In some embodiments, the reference rAAV virion is a variant of the AAV2 virion.

[0042] In some embodiments, the reference rAAV virion is an AAV-BR1 virion.

[0043] In some embodiments, the rAAV virions are 1×10 12 Vector genome (vg), 1 × 10 13 vg, 1×10 14 vg or 3×10 14 It is administered in a dose of vg.

[0044] In some embodiments, the method causes increased glucose uptake by brain microvascular endothelial cells compared to methods performed using the endogenous Glut1 promoter or a ubiquitous promoter.

[0045] In some embodiments, the disclosure provides a method of expressing GLUT1 in a cell, comprising contacting the cell with a rAAV virion.

[0046] In some embodiments, the cell is an endothelial cell.

[0047] In some embodiments, the endothelial cells are brain microvascular endothelial cells.

[0048] In some embodiments, the endothelial cells are in vivo endothelial cells.

[0049] In some embodiments, the cell is a neuron.

[0050] In some embodiments, the neuron is an in vivo neuron.

[0051] In some embodiments, the methods include in vivo administration of rAAV virions to a subject.

[0052] In some embodiments, the rAAV virion causes increased glucose uptake by the cell compared to a cell contacted with a rAAV virion containing an endogenous Glut1 promoter or a ubiquitous promoter.

[0053] In some embodiments, the disclosure provides a pharmaceutical composition comprising the rAAV virion.

[0054] In some embodiments, the disclosure provides a kit comprising the rAAV virions or pharmaceutical composition, and optionally instructions for use.

[0055] In further aspects, the disclosure provides polynucleotides (eg, vector genomes), pharmaceutical compositions, kits, and other compositions and methods.

[0056] Various other aspects and embodiments are disclosed in the following detailed description. The present invention is limited only by the scope of the appended claims. [Brief description of the drawings]

[0057] [Figure 1-1] FIG. 1 shows vector diagrams of various non-limiting examples of vector genomes. [Figure 1-2] See description of Figure 1-1. [Figure 2-1] Figure 2 shows a vector diagram of a non-limiting example of a vector genome. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 17. The uppercase portion is the expression cassette (SEQ ID NO: 8). [Figure 2-2] See description of Figure 2-1. [Figure 3-1] Figure 3 shows a vector diagram of a non-limiting example of a vector genome. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 19. The uppercase portion is the expression cassette (SEQ ID NO: 10). [Figure 3-2] See description of Figure 3-1. [Figure 4-1] Figure 4 shows a vector diagram of a non-limiting example of a vector genome. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 21. The uppercase portion is the expression cassette (SEQ ID NO: 12). [Figure 4-2] See description of Figure 4-1. [Figure 5-1]Figure 5 shows a vector diagram of a non-limiting example of a vector genome. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 96. The uppercase portion is the expression cassette (SEQ ID NO: 97). An alternative to the complete polynucleotide sequence of the vector genome is SEQ ID NO: 23. An alternative to the expression cassette is SEQ ID NO: 14. [Figure 5-2] See description of Figure 5-1. [Figure 6-1] Figure 6 shows a vector diagram of a non-limiting example of a vector genome. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 25. The uppercase portion is the expression cassette (SEQ ID NO: 16). [Figure 6-2] See description of Figure 6-1. [Figure 7-1] Figure 7 shows a vector diagram of a non-limiting example of a vector genome. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 98. The uppercase portion is the expression cassette (SEQ ID NO: 99). [Figure 7-2] See description of Figure 7-1. [Figure 8-1] Figure 8 shows a vector diagram of a non-limiting example of a vector genome. The complete polynucleotide sequence of the vector genome is SEQ ID NO: 100. The uppercase portion is the expression cassette (SEQ ID NO: 101). [Figure 8-2] See description of Figure 8-1. [Figure 9] AAV9-mediated expression of hGlut1 protein in CHO-Lec2 cells. CHO-Lec2 cells were transduced with AAV9 vectors expressing hGlut1 transgene protein driven by one of several endothelial-specific promoters (i.e., hFLT1, mTie1 or hGlut1) or by the ubiquitous CMV promoter. [SLC2A1 = GLUT1 gene]. [Figure 10]Figures 10A-10C: Expression of transgene protein (Glut1-GFP) after transfection into human brain microvascular endothelial cells (hCMEC / d3). Figure 10A: GFP fluorescence 72 hours after transfection with constructs containing one of several endothelial cell promoters driving expression of the Glut1-GFP transgene. Figure 10B: GFP fluorescence 72 hours after transfection with constructs containing one of two ubiquitous promoters (CMV or CAG), a control vector without Glut1 (CMV-GFP), or no transfection (no NFX). Images obtained using Operetta CLS™ (PerkinElmer®). Figure 10C: Schematic representation of an expression cassette containing the promoter of interest (hFLT1, mTie, hTie or hGlut1) and the GLUT1 (SLC2A1) gene (T2A-linked-GFP) and regulatory elements flanked by AAV2 inverted terminal repeats (ITRs). [Figure 11]11A-11C: 2-deoxy-D-glucose (glucose) uptake in hCMEC / d3 cells after human GLUT1 (SLC2A1) expression. Human brain microvascular endothelial cells (hCMEC / d3) were transfected with plasmids expressing either CAG-GFP (negative control) or the hGLUT1-t2A-eGFP transgene driven by one of several endothelial-specific promoters (i.e., hFLT1, mTie1 or hGlut1) or by the ubiquitous CMV promoter. Glucose uptake was measured using a luminescence-based kit (Promega®) with 0.5 mM 2-deoxy-D-glucose (2-DG) in the medium. Glucose uptake was normalized by total cell number (Total Cell) using phase contrast imaging [error bars represent SEM; n=6 replicates per condition]. FIG. 11A: Glucose (2-DG) uptake was measured 72 hours after transfection in the first experiment. Figure 11B: Glucose (2-DG) uptake was measured 72 hours after transfection in a second experiment. Figure 11C: Glucose (2-DG) uptake was measured 96 hours after transfection. [Figure 12]12A-12B: 2-deoxy-D-glucose (glucose) uptake in hCMEC / d3 cells after human GLUT1 (SLC2A1) expression. Human brain microvascular endothelial cells (hCMEC / d3) were transfected with a plasmid expressing the hGLUT1-t2A-eGFP transgene driven by one of several endothelial-specific promoters (i.e., hFLT1, mTie1 or hGlut1) or by the ubiquitous CMV promoter. Non-transfected hCMEC / d3 served as control (CON). Glucose uptake was measured using a luminescence-based kit (Promega®) with various concentrations of 2-deoxy-D-glucose in the medium (0 mM, 0.1 mM, 0.5 mM or 1.0 mM). Glucose uptake was normalized per cell through multiplexing with the RealTime-Glo MT Cell Viability Assay (Promega®), performed according to the manufacturer's recommendations. Figure 12A: shows glucose uptake in hCMEC / d3 cells after human Glut1 (SLC2A1) expression at 72 hours. Figure 12B: shows glucose uptake in hCMEC / d3 cells after human Glut1 (SLC2A1) expression at 96 hours. [Figure 13]2-Deoxy-D-glucose (glucose) uptake following AAV9-mediated hGLUT1 (SLC2A1) expression in hCMEC / d3 cells. Human brain microvascular endothelial cells (hCMEC / d3) were transduced with AAV9 vectors (3x105 vector genomes / cell) expressing either CAG-GFP (negative control) or an hGLUT1 transgene driven by one of several endothelial-specific promoters (i.e., hFLT1, mTie1 or hGlut1) or by the ubiquitous CMV promoter. Glucose (2-DG) uptake was measured 72 hours post-transduction using the luminescence-based Glucose Uptake-Glo kit (Promega®) and normalized per cell using the RealTime-Glo MT Cell Viability Assay (Promega®) [error bars represent SEM; n=4 replicates per condition]. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0058] Detailed Description of the Invention The present disclosure provides GLUT1 gene therapy vectors that deliver a polynucleotide encoding GLUT1 or a functional variant thereof with an AAV virion that has tropism for endothelial cells, such as AAV-BR1, along with methods of use and other compositions and methods. In some embodiments, the AAV vector genome includes an endothelial cell-specific promoter, such as the FLT-1 promoter.

[0059] The present disclosure further provides a method for treating a disease or disorder in a subject by administering the gene therapy vector of the present disclosure. In some embodiments, the disease or disorder is Glucose Transporter 1 Deficiency Syndrome (GLUT1 DS) or De Vivo Disease.

[0060] According to the present invention, polynucleotides encoding GLUT1 or functional variants thereof may be used in the generation of gene therapy vectors. The resulting vectors may be used in the treatment of diseases or disorders, such as Glucose Transporter 1 Deficiency Syndrome (GLUT1 DS), De Vivo Disease, etc.

[0061] definition The section headings are for organizational purposes only and are not to be construed as limiting the subject matter described to any particular aspect or embodiment.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, and suitable methods and materials are described below.All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will take precedence.In addition, the materials, methods and examples described herein are illustrative only and are not intended to be limiting.

[0063] All publications and patents mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.In case of conflict, this application, including any definitions herein, shall prevail.However, the mention of any references, articles, publications, patents, patent publications and patent applications cited in this specification is not intended to be, and should not be interpreted as, an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0064] In this description, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value in the recited range, and fractions thereof, where appropriate (e.g., tenths and hundredths of integers, etc.). The term "about", when immediately preceding a number or number, means that the number or number is within a range of plus or minus 10%. As used herein, the terms "a" and "an" should be understood to refer to "one or more" of the recited components, unless otherwise indicated. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. The term "and / or" should be understood to mean either one or both of the alternatives. As used herein, the terms "include" and "comprise" are used interchangeably.

[0065] The term "sequence identity" refers to the percentage of identity of a polypeptide or polynucleotide sequence of interest to a reference sequence, calculated as 100 times the number of perfect matches in an optimal alignment of the sequence of interest to the reference sequence, divided by the total length of the reference sequence (including gaps). Optimal alignment of sequences can be generated using the European Molecular Biology Open Software Suite (EMBOSS) Needle program, available at www.ebi.ac.uk, as described in Maderia et al. Nucleic Acids Res. 47(W1): W636-W641(2019). When comparing DNA and RNA sequences, thymine (T) and uracil (U) are counted as matches.

[0066] As used herein, "AAV vector" or "rAAV vector" refers to a recombinant vector that contains one or more polynucleotides of interest (or transgenes) flanked by AAV terminal repeats (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell transfected with a plasmid that encodes and expresses the rep and cap gene products. Alternatively, AAV vectors can be packaged into infectious particles using host cells that have been stably engineered to express the rep and cap genes.

[0067] As used herein, "AAV virion" or "AAV virus particle" or "AAV vector particle" refers to a virus particle that is composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector.As used herein, when a particle contains a heterologous polynucleotide (i.e., a polynucleotide other than the wild-type AAV genome, such as a transgene that is delivered to mammalian cells), it is usually referred to as "AAV vector particle" or simply "AAV vector".Therefore, the production of AAV vector particle necessarily includes the production of AAV vector, since the vector is contained within the AAV vector particle.

[0068] As used herein, "GH loop" refers to the loop formed between the G and H strands of the jelly-roll β-barrel of the AAV capsid protein VP1, as described in Xie et al. PNAS 99(16):10405-10410 (2002).

[0069] As used herein, "promoter" refers to a polynucleotide sequence capable of promoting the initiation of RNA transcription from a polynucleotide in a eukaryotic cell.

[0070] As used herein, "vector genome" refers to a polynucleotide sequence packaged by a vector (e.g., a rAAV virion), including flanking sequences (in AAV, inverted terminal repeats). The terms "expression cassette" and "polynucleotide cassette" refer to the portion of a vector genome between the flanking ITR sequences. "Expression cassette" means that the vector genome contains at least one gene encoding a gene product operably linked to an element (e.g., a promoter) that drives expression.

[0071] As used herein, the term "patient in need" or "subject in need" refers to a patient or subject at risk of or suffering from a disease, disorder, or condition that is suitable for treatment or improvement using the recombinant gene therapy vector or gene editing system disclosed herein. A patient or subject in need may be, for example, a patient or subject diagnosed with a disorder related to the central nervous system. The subject may have a mutation in the SLC2A1 gene, or a deletion of all or part of the SLC2A1 gene, or a deletion of a gene regulatory sequence, causing abnormal expression of GLUT1 protein. "Subject" and "patient" are used interchangeably herein. The subject treated by the methods described herein may be an adult or a child. The age of the subject may vary.

[0072] As used herein, the terms "variant" or "functional variant" refer interchangeably to a protein that has one or more amino acid substitutions, insertions or deletions compared to a parent protein that retains one or more desired activities of the parent protein.

[0073] As used herein, "gene disruption" refers to partial or complete loss of function or abnormal activity in gene.For example, subject may suffer from gene disruption of expression or function in SLC2A1 gene, which reduces the expression or causes loss or abnormality of function of GLUT1 protein in at least some cells (e.g., endothelial cells and / or neurons) of subject.

[0074] As used herein, "treat" refers to improve one or more symptoms of a disease or disorder. The term "prevent" refers to delay or halt the onset of one or more symptoms of a disease or disorder, or slow the progression of a neurological disease or disorder associated with SLC2A1, such as GLUT1 deficiency syndrome (GLUT1 DS).

[0075] GLUT1 protein or polynucleotide The present disclosure contemplates compositions and methods of use related to glucose transporter 1 (GLUT1) protein.Various mutations in SLC2A1 are known to be associated with GLUT1 DS.Both inherited mutations and de novo mutations have been observed.In some cases, heterozygous missense mutations are sufficient to cause disease.

[0076] The polypeptide sequence of GLUT1 is as follows: TIFF2025505662000002.tif51159

[0077] In some embodiments, the GLUT1 protein comprises a polypeptide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:26).

[0078] In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a vector genome, wherein the vector genome comprises a polynucleotide sequence encoding a GLUT1 protein or a functional variant thereof, operably linked to a promoter. In some embodiments, the disclosure provides a recombinant adeno-associated virus (rAAV) virion comprising a capsid and a genome, wherein the rAAV virion genome comprises a polynucleotide sequence encoding a GLUT1 protein, operably linked to a promoter. The polynucleotide encoding the GLUT1 protein is selected from the group consisting of: It may comprise a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to TIFF2025505662000003.tif129159.

[0079] In some embodiments, the polynucleotide sequence encoding the GLUT1 protein is a codon-optimized sequence. It may comprise a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to TIFF2025505662000004.tif129159.

[0080] Optionally, the polynucleotide sequence encoding the AAV virion genome may include a Kozak sequence, including but not limited to GCCACCATGG (SEQ ID NO: 28). The Kozak sequence may overlap with a polynucleotide sequence encoding a GLUT1 protein or a functional variant thereof. For example, the AAV virion genome may include the following: It may comprise a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to TIFF2025505662000005.tif129159 (underlined in Kozak).

[0081] In some embodiments, the Kozak sequence is as follows: TIFF2025505662000006.tif48128 or any one of these alternative Kozak sequences.

[0082] In some embodiments, the AAV virion genome does not contain a Kozak sequence. The polynucleotide sequence may be codon optimized.

[0083] AAV virion genome The AAV virion of the present disclosure comprises a vector genome. The vector genome may comprise an expression cassette (or a polynucleotide cassette for gene editing applications that do not require expression of a polynucleotide sequence). Any suitable inverted terminal repeat (ITR) may be used. The ITR may be from the same serotype as the capsid or a different serotype (e.g., AAV2 ITR may be used).

[0084] In some embodiments, the 5' ITR comprises the following: TIFF2025505662000007.tif21159.

[0085] In some embodiments, the 5' ITR comprises the following: TIFF2025505662000008.tif27159.

[0086] In some embodiments, the 5' ITR comprises the following: TIFF2025505662000009.tif27159.

[0087] In some embodiments, the 3' ITR comprises the following: TIFF2025505662000010.tif22159.

[0088] In some embodiments, the 3' ITR comprises the following: TIFF2025505662000011.tif27159.

[0089] In some embodiments, the AAV virion genome comprises, for example, the following: It contains one or more filler sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to TIFF2025505662000012.tif70159.

[0090] promoter In some embodiments, the polynucleotide sequence encoding a GLUT1 protein or a functional variant thereof is operably linked to a promoter.

[0091] The present disclosure contemplates the use of various promoters. Promoters useful in embodiments of the present disclosure include, but are not limited to, the cytomegalovirus (CMV) promoter, the phosphoglycerate kinase (PGK) promoter, or the promoter sequence consisting of the CMV enhancer and a portion of the chicken β-actin promoter and rabbit β-globin gene (CAG). In some cases, the promoter may be a synthetic promoter. An exemplary synthetic promoter is provided by Schlabach et al. PNAS USA. 107(6):2538-43 (2010). In some embodiments, the promoter is one of the following: TIFF2025505662000013.tif57159.

[0092] In some embodiments, the polynucleotide sequence encoding the GLUT1 protein or its functional variant is operably linked to an inducible promoter. The inducible promoter can be configured to transcribe or not transcribe the polynucleotide sequence in response to the addition or accumulation of an agent, or in response to the removal, degradation or dilution of the agent. The agent can be a drug. The agent can be tetracycline or one of its derivatives, such as, but not limited to, doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically regulated promoter, a physically regulated promoter (i.e., a promoter that responds to the presence or absence of light or to low or high temperatures). Inducible promoters include heavy metal ion inducible promoters (such as mouse mammary tumor virus (mMTV) promoter or various growth hormone promoters), and promoters from T7 phage that are active in the presence of T7 RNA polymerase. This list of inducible promoters is non-limiting.

[0093] In some cases, the promoter is a tissue-specific promoter, for example, a promoter that can drive expression in endothelial cells to a greater extent than in non-endothelial cells. In some embodiments, the tissue-specific promoter is an endothelial-specific promoter. In some embodiments, the tissue-specific promoter is selected from any of a variety of endothelial-specific promoters, including but not limited to FLT1 (vascular endothelial growth factor receptor 1), Tie1 (tyrosine kinase 1 with immunoglobulin-like and EGF-like domains), VE-cadherin (vascular endothelial cadherin), hSYN1 (human synapsin), INA (alpha-internexin), NES (nestin), TH (tyrosine hydroxylase), FOXA2 (forkhead box A2), CaMKII (calmodulin-dependent protein kinase II) and NSE (neuron-specific enolase). In some cases, the promoter is a ubiquitous promoter. "Ubiquitous promoter" refers to a promoter that is not tissue-specific under experimental or clinical conditions. In some cases, the ubiquitous promoter is any one of the CMV, CAG, UBC, PGK, EF1-α, GAPDH, SV40, HBV, chicken β-actin, and human β-actin promoters.

[0094] In some embodiments, the promoter sequence is selected from Table 3. In some embodiments, the promoter comprises a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs: 1-3 and 39-51.

[0095] [Table 3] TIFF2025505662000015.tif245166TIFF2025505662000016.tif245166TIFF202 5505662000017.tif245166TIFF2025505662000018.tif245166TIFF20255056620 00019.tif245166TIFF2025505662000020.tif245166TIFF2025505662000021.t if245166TIFF2025505662000022.tif245166TIFF2025505662000023.tif148166

[0096] In some embodiments, the AAV virion genome comprises a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 1. In some embodiments, the AAV virion genome comprises a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:2. In some embodiments, the AAV virion genome comprises a polynucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO:3.

[0097] Further illustrative examples of promoters are the SV40 late promoter from Simian Virus 40, the baculovirus polyhedron enhancer / promoter element, the herpes simplex virus thymidine kinase (HSV tk), the immediate early promoter from cytomegalovirus (CMV) and various retroviral promoters including the LTR element. A wide variety of other promoters are known in the art and are publicly available, and the sequences of many such promoters are available in sequence databases such as the GenBank database.

[0098] Other Regulatory Elements In some cases, the AAV virion of the present disclosure further comprises one or more regulatory elements selected from the group consisting of an enhancer, an intron, a polyA signal, a 2A peptide coding sequence, a WPRE (woodchuck hepatitis virus post-transcriptional regulatory element), and a HPRE (hepatitis B virus post-transcriptional regulatory element).

[0099] In some embodiments, the rAAV virion genome comprises a CMV enhancer.

[0100] In certain embodiments, the rAAV virion genome comprises one or more enhancers. In certain embodiments, the enhancer is a CMV enhancer sequence, a GAPDH enhancer sequence, a β-actin enhancer sequence, or an EF1-α enhancer sequence. The above sequences are known in the art. For example, the sequence of the CMV immediate early (IE) enhancer is as follows: The file is TIFF2025505662000024.tif52159.

[0101] In certain embodiments, the rAAV virion genome comprises one or more introns. In particular embodiments, the intron is a rabbit globin intron sequence, a chicken β-actin intron sequence, a synthetic intron sequence, or an EF1-α intron sequence.

[0102] In certain embodiments, the rAAV virion genome comprises a polyA sequence. In particular embodiments, the polyA sequence is a rabbit globin polyA sequence, a human growth hormone polyA sequence, a bovine growth hormone polyA sequence, a PGK polyA sequence, an SV40 polyA sequence, or a TK polyA sequence. In some embodiments, the polyA signal can be a bovine growth hormone polyadenylation signal (bGHpA).

[0103] In certain embodiments, the rAAV virion genome comprises one or more transcript stabilizing elements. In certain embodiments, the transcript stabilizing element is a WPRE sequence, a HPRE sequence, a scaffold attachment region, a 3' UTR or a 5' UTR. In certain embodiments, the rAAV virion genome comprises both a 5' UTR and a 3' UTR.

[0104] In some embodiments, the rAAV virion genome comprises a 5' untranslated region (UTR) selected from Table 4. In some embodiments, the rAAV virion genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 53-61.

[0105] [Table 4] TIFF2025505662000026.tif244166TIFF2025505662000027.tif242166TIFF2025505662000028.tif226166TIFF2025505662000029.tif110166

[0106] In some embodiments, the rAAV virion genome comprises a 3' untranslated region selected from Table 5. In some embodiments, the rAAV virion genome comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 62-70.

[0107] [Table 5] TIFF2025505662000031.tif244166TIFF2025505662000032.tif233166TIFF2025505662000033.tif242166TIFF2025505662000034.tif21166

[0108] In some embodiments, the rAAV virion genome comprises a polyadenylation (polyA) signal selected from Table 6. In some embodiments, the polyA signal comprises a polynucleotide sequence at least 75%, 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 71-75.

[0109] [Table 6] TIFF2025505662000036.tif110166

[0110] Exemplary rAAV virion genomes are depicted in Figures 2-8 and provided as SEQ ID NOs: 17-25. The capitalized portion of each sequence is the expression cassette (SEQ ID NOs: 8-16, SEQ ID NO: 97, SEQ ID NO: 99, and SEQ ID NO: 101). In some embodiments, the rAAV virion genome comprises a polynucleotide sequence that shares at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to any one of SEQ ID NOs:8-16, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO:101, optionally with or without lowercase ITR sequences, or any one of SEQ ID NOs:8-16, SEQ ID NO:97, SEQ ID NO:99, and SEQ ID NO: The coding sequences are underlined. Expression cassettes are written in capital letters.

[0111] Adeno-associated virus genome Adeno-associated virus (AAV) is a replication-deficient parvovirus whose single-stranded DNA genome is approximately 4.7 kb long and contains two approximately 145 nucleotide inverted terminal repeats (ITRs). There are several known variants of AAV, which are sometimes classified according to antigenic epitopes and are called serotypes. The nucleotide sequences of the genomes of AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077, the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and in Srivastava et al., J. Virol., 45: 555-564 (1983), the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829, the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829, the AAV-5 genome is provided in GenBank Accession No. AF085716, the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862, at least portions of the AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively, and the AAV-9 genome is provided in Gao et al., J. Virol., 78: The AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006), and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAVrh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the AAV ITR. Three AAV promoters (designated p5, p19 and p40 for their relative map positions) drive the expression of two AAV internal reading frames encoding the rep and cap genes.Two rep promoters (p5 and p19), coupled with differential splicing of a single AAV intron (at nucleotide positions 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins possess multiple enzymatic properties that are ultimately responsible for the replication of the viral genome. The cap gene is expressed from the p40 promoter and encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translation initiation sites are responsible for the production of the three associated capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0112] AAV possesses unique characteristics that make it attractive as a vehicle for delivering foreign DNA to cells, for example in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Furthermore, AAV can infect many mammalian cells, allowing the possibility of targeting many different tissues in vivo. Furthermore, AAV can transduce slowly dividing and nondividing cells and persist essentially for the life of these cells as transcriptionally active nuclear episomes (extrachromosomal elements). The AAV proviral genome is inserted as cloned DNA within a plasmid, making the construction of recombinant genomes feasible. Furthermore, signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, so that part or all of the internal ∼4.3 kb of the genome (encoding the replication and structural capsid protein, rep-cap) may be replaced with foreign DNA. To generate an AAV vector genome, the rep and cap proteins may be provided in trans. Another important feature of AAV is that it is a highly stable and potent virus. It easily survives the conditions used to inactivate adenovirus (56°C to 65°C for several hours), mitigating the importance of cryopreservation of AAV. AAV can also be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0113] Recombinant AAV virions useful in the practice of the present invention can be constructed using methodologies well known in the art of molecular biology. Typically, a transgene-carrying viral vector is constructed from a polynucleotide that codes for the transgene, appropriate regulatory elements, and elements required for the production of viral proteins, which mediate cell transduction. Such rAAV virions can be produced by techniques well known in the art, for example, by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of viral packaging cells include, but are not limited to, HeLa cells, SF9 cells (optionally with baculovirus helper vectors), 293 cells, etc. AAV vectors can be produced using a herpes virus-based system, as described in US2017 / 0218395A1. Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO95 / 14785, WO96 / 22378, U.S. Pat. No. 5,882,877, U.S. Pat. No. 6,013,516, U.S. Pat. No. 4,861,719, U.S. Pat. No. 5,278,056 and WO94 / 19478, the entire contents of each of which are incorporated herein by reference.

[0114] AAV vectors useful in the practice of the present invention can be packaged into AAV virions (virus particles) using a variety of systems, including adenovirus-based and helper-free systems. Standard methods in AAV biology include those described in Kwon and Schaffer. Pharm Res. 25(3):489-99 (2008); Wu et al. Mol. Ther. 14(3):316-27 (2006); Burger et al. Mol. Ther. 10(2):302-17 (2004); Grimm et al. Curr Gene Ther. 3(4):281-304 (2003); Deyle DR, Russell DW. Curr Opin Mol Ther. 11(4):442-447 (2009); McCarty et al. Gene Ther. 8(16):1248-54 (2001); and Duan et al. Mol Ther. 4(4):383-91 (2001). Helper-free systems include those described in U.S. Patent Nos. 6,004,797; 7,588,772; and 7,094,604;

[0115] The AAV DNA in the rAAV vector genome can be from any AAV variant or serotype from which recombinant virus can be derived, including, but not limited to, AAV variants or serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants are also contemplated, such as rAAV with capsid mutations. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0116] In some cases, the rAAV comprises a self-complementary genome. As defined herein, a rAAV comprising a "self-complementary" or "double-stranded" genome refers to a rAAV that has been engineered such that the coding region of the rAAV constitutes an intramolecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Therapy. 8 (16): 1248-54 (2001). The present disclosure contemplates the use of a rAAV comprising a self-complementary genome in some cases, because upon infection (such as transduction), rather than waiting for cell-mediated synthesis of the second strand of the rAAV vector genome, the two complementary halves of the scAAV associate to form one double-stranded DNA (dsDNA) unit, ready for immediate replication and transcription. It will be appreciated that instead of the full coding capacity found in rAAV (4.7-6 kb), rAAV containing self-complementary genomes may only retain about half that amount (approximately 2.4 kb).

[0117] In other cases, the rAAV vector genome is a single-stranded genome. As defined herein, a "single-stranded" genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAV has a single-stranded DNA genome. Some have indicated that the rAAV should be a scAAV to achieve efficient transduction of cells. However, the present disclosure contemplates that the rAAV vector genome may have a single-stranded genome rather than a self-complementary genome, with the understanding that other genetic modifications of the rAAV vector genome may also be beneficial to obtain optimal gene transcription in target cells. In some cases, the present disclosure relates to a single-stranded rAAV vector genome that can achieve efficient gene transfer to the anterior segment of the mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017).

[0118] In a preferred embodiment, the AAV is an AAV variant with high tropism for endothelial cells, such as AAV2 variant BR1. The production of pseudotyped rAAV is disclosed, for example, in WO 01 / 83692. Other types of rAAV variants, such as rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In some embodiments, the rAAV vector genome is of serotype AAV-BR1 and comprises a single-stranded genome. In some embodiments, the rAAV vector genome comprises the inverted terminal repeat (ITR) sequence of AAV2. In some embodiments, the rAAV vector genome comprises an AAV2 genome.

[0119] AAV genome may comprise wild-type AAV sequence, or may comprise one or more modifications to wild-type AAV sequence.In certain embodiments, AAV genome comprises one or more amino acid modifications, such as substitutions, deletions or insertions, in capsid protein, such as VP1, VP2 and / or VP3.In certain embodiments, the modifications provide reduced immunogenicity when AAV genome is provided to a subject.

[0120] The capsid protein of the rAAV may be modified so that the rAAV targets a specific target tissue of interest, such as endothelial cells and / or endothelial tip cells. In some embodiments, the rAAV is injected directly into the intraventricular lumen, intravenously, or by a combination of intraventricular and intravenous delivery to the subject.

[0121] In some embodiments, the rAAV virion is a variant of the AAV2 rAAV virion. The capsid can be an AAV2 capsid or a functional variant thereof. The polypeptide sequence of AAV2 VP1 is provided as SEQ ID NO: 76. In some embodiments, the AAV2 capsid is a reference AAV2 capsid, e.g., Shares at least 95% with TIFF2025505662000037.tif69159.

[0122] In some embodiments, the capsid protein is encoded by a polynucleotide provided on a plasmid in trans to a transfer plasmid. In some embodiments, the polynucleotide sequence of the AAV-BR1 capsid (cap) is SEQ ID NO: 103: shares at least 98%, at least 99% or 100% identity with TIFF2025505662000038.tif184159.

[0123] In some embodiments, the disclosure further provides a protein sequence of AAV-BR1 VP1 and functional variants thereof. The sequence of the AAV-BR1 VP1 polypeptide is provided as SEQ ID NO: 104. In some embodiments, the sequence of AAV VP1 is provided as SEQ ID NO: 104: shares at least 98%, at least 99% or 100% identity with TIFF2025505662000039.tif69159.

[0124] In some embodiments, the disclosure further provides protein sequences of AAV-BR1 VP2 and functional variants thereof. The sequence of the AAV-BR1 VP1 polypeptide is provided as SEQ ID NO: 105. In some embodiments, the sequence of AAV VP2 is provided as SEQ ID NO: 105: shares at least 98%, at least 99% or 100% identity with TIFF2025505662000040.tif63159.

[0125] In some embodiments, the disclosure further provides protein sequences of AAV-BR1 VP3 and functional variants thereof. The sequence of the AAV-BR1 VP1 polypeptide is provided as SEQ ID NO: 106. In some embodiments, the sequence of AAV VP3 is provided as SEQ ID NO: 106: shares at least 98%, at least 99% or 100% identity with TIFF2025505662000041.tif57159.

[0126] In some embodiments, the capsid comprises an insertion in the GH loop, approximately between amino acid residue number 588 and residue number 589 in the AAV2 VP1 reference sequence set forth in SEQ ID NO 76. In some embodiments, the insertion sequence is SEQ ID NO: 107: XDGXXWX (where each X is any amino acid) (SEQ ID NO: 107) Shares at least 98%, at least 99% or 100% identity with

[0127] In some embodiments, the capsid comprises an insertion in the GH loop between amino acid numbers 588 and 589 compared to the VP1 reference sequence set forth in SEQ ID NO: 76. In some embodiments, the insertion sequence is SEQ ID NO: 108: ADGVQWT (SEQ ID NO: 108) Shares at least 98%, at least 99% or 100% identity with

[0128] In some embodiments, the capsid comprises an insertion in the GH loop between amino acid numbers 588 and 589 compared to the VP1 reference sequence set forth in SEQ ID NO 76. In some embodiments, the inserted sequence is SEQ ID NO: 109: DDGVSWK (SEQ ID NO: 109) Shares at least 98%, at least 99% or 100% identity with

[0129] In some embodiments, the capsid comprises an insertion in the GH loop between amino acid numbers 588 and 589 compared to the VP1 reference sequence set forth in SEQ ID NO: 76. In some embodiments, the insertion sequence is SEQ ID NO: 110: SDGLTWS (SEQ ID NO: 110) Shares at least 98%, at least 99% or 100% identity with

[0130] In some embodiments, the capsid comprises an insertion in the GH loop between amino acid numbers 588 and 589 compared to the VP1 reference sequence set forth in SEQ ID NO: 76. In some embodiments, the insertion sequence is SEQ ID NO: 111: SDGLAWV (SEQ ID NO: 111) Shares at least 98%, at least 99% or 100% identity with

[0131] In some embodiments, the capsid comprises an insertion in the GH loop between amino acid numbers 588 and 589 compared to the VP1 reference sequence set forth in SEQ ID NO: 76. In some embodiments, the insertion sequence is SEQ ID NO: 112: NRGTEWD (SEQ ID NO: 112) Shares at least 98%, at least 99% or 100% identity with

[0132] Additional AAV capsids for use in the rAAV virions of the present disclosure include those disclosed in Patent Publication Nos. WO 2009 / 012176 A2 and WO 2015 / 168666 A2.

[0133] The use of AAV-BR1 may result in endothelial cell-specific expression of the Glut1 transgene and therefore enhance transgene efficacy.

[0134] In some embodiments, the rAAV vector genome is not the AAV2 genome.Without being bound by theory, the use of the AAV2 genome results in the transduction of neural cells in addition to or instead of endothelial cells.Without being bound by theory, the spread of the AAV2 genome in the CNS and related cerebral vasculature is restricted by its interaction with heparan sulfate proteoglycan (HSPG) receptor.

[0135] Pharmaceutical Compositions and Kits In one aspect, the disclosure provides a pharmaceutical composition comprising a rAAV virion of the disclosure and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0136] For purposes of administration, e.g., administration by injection, a variety of solutions can be utilized, including sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent can be first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or pharmacologically acceptable salts can be prepared, for example, at least 0.001% or 0.01%, in water suitably mixed with a surfactant, such as poloxamer 188. Dispersions of rAAV can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, all sterile aqueous media utilized are readily available by standard techniques well known to those skilled in the art.

[0137] Pharmaceutical forms suitable for injectable use include, but are not limited to, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0138] Sterile injectable solution can be prepared by incorporating the required amount of rAAV in a suitable solvent with various other components as listed above as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating sterilized active ingredient into a sterile vehicle that contains basic dispersion medium and other components as listed above.In the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying technique, which produces powder of active ingredient plus any additional desired components from its solution that has been previously sterilized and filtered.

[0139] In another aspect, the disclosure includes a kit comprising a rAAV virion of the disclosure and instructions for use.

[0140] How to use In one aspect, the present disclosure provides a method for increasing GLUT1 activity in a cell, comprising contacting the cell with the rAAV of the present disclosure.In another aspect, the present disclosure provides a method for increasing GLUT1 activity in a subject, comprising administering the rAAV of the present disclosure to the subject.In some embodiments, the cell and / or the subject is deficient in SLC2A1 messenger RNA or GLUT1 protein expression level and / or activity, and / or comprises loss-of-function mutation of SLC2A1.The cell can be an endothelial cell, for example, an endothelial edge cell or a cerebrovascular endothelial cell.

[0141] In some embodiments, the method restores normal function of endothelial tip cells. In some embodiments, the method restores GLUT1 transporter protein expression levels in cell culture and / or in vivo. In some embodiments, the method restores normal glucose transport and metabolism (e.g., glycolysis, lactate production) in cell culture and / or in vivo. In some embodiments, the method restores normal angiogenesis and / or development of microvasculature in the central nervous system (CNS).

[0142] In some embodiments, the method restores normal function of cerebrovascular endothelial cells. In some embodiments, the method restores GLUT1 transporter protein expression levels in cell culture and / or in vivo. In some embodiments, the method restores normal glucose transport and metabolism (e.g., glycolysis, lactate production) in cell culture and / or in vivo. In some embodiments, the method restores normal angiogenesis and / or development of microvasculature in the central nervous system (CNS).

[0143] Treatment In another aspect, the present disclosure provides a method for treating disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of the rAAV virion of the present disclosure.In some embodiments, the disease or disorder is a neurological disease or disorder.In some embodiments, the subject suffers from genetic disruption of SLC2A1 expression or function.In some embodiments, the disease or disorder is GLUT1 deficiency syndrome (GLUT1 DS).

[0144] AAV-mediated delivery of GLUT1 protein to the vasculature of the CNS may increase lifespan and prevent, attenuate, alleviate or attenuate neurodegeneration, early-onset seizures, developmental delay, acquired microcephaly (slowed head growth), complex movement disorders (spasticity, ataxia, dystonia), paroxysmal eye-head movements, and / or low lactose and / or glucose concentrations in the cerebrospinal fluid (CSF glycemia). In some embodiments, the methods provide treatment early in the disease course, for example in newborns, infants or young children.

[0145] In some embodiments, the disclosed methods result in an increase in wild-type GLUT1 protein expression in a subject (e.g., an increase of about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 70%, or about 70% to about 100%). In certain embodiments, the disclosed methods result in an increase in the ratio of wild-type GLUT1 protein to mutant GLUT1 protein in a subject (e.g., an increase of about 5% to about 25%, about 25% to about 50%, about 50% to about 100%, or about 100% to about 200%).

[0146] In some embodiments, the disclosed methods result in an increase in wild-type GLUT1 protein expression in endothelial cells (e.g., about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 70%, or about 70% to about 100% increase). In certain embodiments, the disclosed methods result in an increase in the ratio of wild-type GLUT1 protein to mutant GLUT1 protein in endothelial cells (e.g., about 5% to about 25%, about 25% to about 50%, about 50% to about 100%, or about 100% to about 200% increase).

[0147] The methods disclosed herein can provide efficient biodistribution in the vasculature of the brain and / or CNS. They can result in sustained expression in all or a significant portion of endothelial cells (e.g., endothelial tip cells). Notably, the methods disclosed herein can provide long-term expression of GLUT1 protein throughout the development and aging of subjects after administration of rAAV virions.

[0148] Combination therapy is also contemplated by the present invention. The combination of the method of the present invention with standard medical treatment (e.g., corticosteroids or topical decompression agents) is particularly contemplated, as is the combination with novel treatments. In some cases, subjects may be treated with a combination of steroids and / or immunosuppressants to suppress or reduce immune response to administration of rAAV as described herein.

[0149] For example, a therapeutically effective amount of rAAV vector genome for intracerebroventricular (ICV), intracisternal (ICM) injection, or intravenous (IV) injection is approximately 1×10 by brain weight. 12 vg / kg ~ approx. 5×10 12 vg / kg, or approximately 1 × 10 13 vg / kg ~ approx. 5×10 13 vg / kg, or approximately 1 × 10 14 vg / kg ~ approx. 5×10 14 vg / kg, or approximately 1 × 10 15 vg / kg ~ approx. 5×10 15 vg / kg of rAAV. The invention also includes compositions containing rAAV vector genomes in these ranges.

[0150] For example, in certain embodiments, a therapeutically effective amount of a rAAV vector genome is about 1×10 10 vg, approx. 2×10 10 vg, approx. 3×10 10 vg, approx. 4×10 10 vg, approx. 5×10 10 vg, approx. 6×10 10 vg, approx. 7×10 10 vg, approx. 8×10 10 vg, approx. 9×10 10 vg, approx. 1×10 12 vg, approx. 2×10 12 vg, approx. 3×10 12 vg, approx. 4×10 12 vg, approx. 4×10 13 vg and approximately 4 × 10 14 The invention also includes compositions containing these doses of the rAAV vector genome.

[0151] In some embodiments, for example when ICV injection is performed, a therapeutically effective amount of the rAAV vector genome is greater than 1×10 10 vg / hemisphere~2×10 14 vg / hemisphere, or approximately 1×10 10 vg / hemisphere, approx. 1×10 11 vg / hemisphere, approx. 1×10 12 vg / hemisphere, 1×10 13vg / hemisphere, or approximately 1×10 14 In some embodiments, for example when ICM injections are performed, the therapeutically effective amount of the rAAV vector genome is in the range of 2×10 10 vg total ~2×10 14 vg total, or approximately 2 × 10 10 Total vg, about 2×10 11 Total vg, about 2×10 12 Total vg, about 2×10 13 vg total, or approximately 2 × 10 14 Doses in the vg total range.

[0152] In some embodiments, the therapeutic composition is administered at about 1×10 per volume of the therapeutic composition injected. 9 , 1×10 10 or 1×10 11 In one embodiment, the therapeutic composition comprises approximately 1×10 rAAV vector genomes per mL. 11 , 1×10 12 , 1×10 13 or 1×10 14 In certain embodiments, the therapeutic composition comprises about 1 x 10 rAAV genomes per mL. 14 , 1×10 13 or 1×10 12 Contains an rAAV genome of less than 1000 ng / ml.

[0153] Evidence of functional improvement, clinical benefit or efficacy in patients may be assessed by: paroxysmal eye and head movements, surrogate markers of reduction in seizure frequency (generalized tonic-clonic and myoclonic seizures), analysis of lactose and / or glucose concentrations in cerebrospinal fluid (CSF), assessment of developmental delay, chorea, dystonia and microcephaly. Measurement of cognitive, motor, speech and language function using standard disease assessment scales such as Columbia Neurological Score, Composite Intelligence Assessment, Adaptive Behaviour Composite, Verbal and Non-verbal Cognitive Skills and Visual-Motor Integration, and the 6-Minute Walk Test. Cognitive and developmental assessments including the Peabody Developmental Motor Scales, 2nd Edition (PDMS-2) and Bayley Scales of Infant Development, 3rd Edition, as appropriate for the child's level of disability. Gross Motor Function Scale (GFMF-88), Pediatric Disability Scale (PEDI). These or similar scales, as well as patient-reported quality of life outcomes such as the Caregiver Global Impression of Change in Seizure Duration (CGICSD), Pediatric Quality of Life Inventory (PedsQL™) and Vineland Adaptive Behavior Scales-2nd edition, on a three-point scale (decreased, unchanged, or increased mean duration), may demonstrate improvement in disease components. Baseline and post-treatment brain magnetic resonance imaging may show improvement or normalization of brain volume for the patient's age compared with age-matched patient control data and historical data from patients with GLUT1 deficiency.

[0154] Clinical efficacy may be observed as an extension of life span, achievement of normal neurodevelopmental milestones, normalization of CSF glucose concentrations, reduction in frequency or magnitude of paroxysmal eye and head movements, reduction or absence of epileptic seizure activity (including myoclonic, clonic, generalized tonic-clonic and / or epileptic convulsions), improvement or absence of development of complex movement disorders such as spasticity, dystonia and / or ataxia, and improvement or normalization of Columbia Neurological Score and / or 6-minute walk test performance. Evidence of neuroprotective and / or neurorestorative effects may be evident in all of the metrics mentioned above and / or on magnetic resonance imaging (MRI) by characterizing overall brain size, absence of microcephaly and / or cortical atrophy and / or cerebellar atrophy.

[0155] In some embodiments, the method causes an increase in glucose uptake by the cell compared to a cell contacted with a vector comprising an endogenous Glut1 promoter or a ubiquitous promoter, or a cell of a subject administered with a vector comprising an endogenous Glut1 promoter or a ubiquitous promoter. In some cases, the increase is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40% or at least 50%. In some cases, the increase is at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold or at least 1.8-fold. The vector can be any vector disclosed herein. The cell can be an endothelial cell or a neuronal cell. For example, the method can increase glucose uptake by human brain microvascular endothelial cells, either in vitro or in vivo.

[0156] Administration of the Composition The administration of an effective dose of the composition can be by a route standard in the art, including, but not limited to, intravenous, intracerebral, intrathecal, intracisternal or intraventricular administration. In some cases, administration includes intravenous, intracerebral, intrathecal, intracisternal or intraventricular injection. Administration can be performed by intrathecal injection with or without Trendelenburg gradient. Intracisternal (ICM) delivery can be achieved by catheter insertion in the intrathecal (IT) space. Intraventricular injection can be achieved by magnetic resonance imaging (MRI) guided neurosurgical targeting.

[0157] In some embodiments, the present disclosure provides for systemic administration of an effective dose of the rAAV and compositions of the present invention.For example, systemic administration can be administration to the circulatory system so that the entire body is affected.Systemic administration includes intravenous administration by injection or infusion.

[0158] In some embodiments, administration of the rAAV of the present invention can be accomplished using any physical method that delivers the rAAV recombinant vector to the target tissue of an animal, including, but not limited to, injection into the central nervous system (CNS) or cerebrospinal fluid (CSF) and / or intravenous injection.

[0159] In some embodiments, the disclosed methods include intraventricular, intracisternal, intrathecal, or intraparenchymal delivery. Injection can be performed using specialized cannulas, catheters, syringes / needles with an infusion pump. Optionally, targeting of the injection site can be achieved with MRI-guided imaging. Administration can include delivery of an effective amount of rAAV virions, or pharmaceutical compositions comprising rAAV virions, to the CNS. These can be achieved, for example, by unilateral intraventricular injection, bilateral intraventricular injection, intracisternal injection with or without Trendelenburg tilting, intrathecal injection with or without Trendelenburg tilting. The disclosed compositions can also be administered intravenously.

[0160] In some embodiments, the methods of the disclosure include administration of rAAV by intravenous (IV) injection. In some embodiments, the methods of the disclosure include administration of rAAV by intracerebroventricular (ICV) injection. In some embodiments, the methods of the disclosure include administration of rAAV by IV injection in combination with ICV injection.

[0161] Direct delivery to the CNS may involve targeting of unilateral or bilateral intraventricular cavities, specific neuronal regions, or more general brain regions including neuronal targets. Selection of an individual patient's intraventricular cavities, brain regions, and / or neuronal targets and subsequent intraoperative delivery of AAV may be accomplished utilizing any number of software planning programs (e.g., Stealth System, Clearpoint Neuronavigation System, Brainlab, Neuroinspire, etc.) using a number of imaging techniques (MRI, CT, CT combined with MRI). Targeting and delivery to the intraventricular cavities or brain regions may involve the use of a standard stereotactic frame (Leksell, CRW) or a frameless approach with or without intraoperative MRI. The actual delivery of AAV may be by injection through a needle or cannula with or without a lumen (e.g., Smartflow cannula, MRI interventional cannula) lined with a material to prevent rAAV adsorption. The delivery device consists of a syringe and an automatic infusion pump or a microinfusion pump with preprogrammed infusion rates and volumes. The syringe / needle combination, or just the guide cannula for the injection needle, may be directly connected to the stereotactic frame. Injections may involve a constant flow rate or a variable flow rate with convection-enhanced delivery. EXAMPLES

[0162] Example 1: Preclinical bioactivity and efficacy The vector genomes disclosed in Figures 2-8 are used to produce recombinant AAV virions with AAV-BR1 capsids. These are evaluated in mouse models of disease as a result of GLUT1 deficiency disease. One model utilizes a floxed GLUT1 gene crossed with transgenic animals expressing Cre / lox from a constitutive or endothelial-specific promoter (e.g., Tie-2). The resulting mice are heterozygous null at the GLUT1 locus and display a developmental phenotype that mimics the human disease. The second GLUT1 DS mouse model is heterozygous haploinsufficient mice generated by targeted disruption of the promoter and exon 1 region of the mouse GLUT-1 gene (GLUT-1 + / - Additional animal models may include the GLUT1 DS model, in which the GLUT1 gene has a S324P point mutation.

[0163] Gene expression and dose response are evaluated in vitro (using endothelial and neuronal cell lines) and in vivo (using wild-type and GLUT1 DS model mice). Cultured cells (human embryonic kidney cells 293, HEK293; human umbilical vein endothelial cells, HUVEC; human brain-derived endothelial cells, bEND3; human brain microvascular endothelial cells, HBEC-5i; human brain microvascular endothelial cell line, hCMEC / D3 (blood-brain barrier model); human glial oligodendrocyte hybrid cells, MO3.13; human neuroblastoma, SH-SY5Y) transfected with SLC2A1 expression vectors are analyzed to determine transduction efficiency by quantitative real-time PCR analysis and GLUT1 levels by ELISA and / or Western blot. Proof of concept and efficacy of the AAV vector construct is demonstrated in vivo using GLUT1 DS mice by expression of the transgene (GLUT1 protein) in the CNS and CNS vasculature by immunolabeling, enhanced brain capillary density and / or increased blood vessel size in the CNS, increased brain glucose uptake using positron emission tomography (PET), increased CSF glucose or lactate levels and / or increased CSF / blood glucose ratio, increased CSF lactate levels, and improved motor performance using standard assays such as rotarod and / or vertical post assays compared to GLUT1 DS mutant mouse controls. Gene expression and efficacy in vivo using the GLUT1 DS mouse model is demonstrated following delivery of the AAV vector construct by intravenous injection or direct injection into the intraventricular lumen, utilizing either these routes of administration alone and / or in combination.

[0164] Example 2: In vitro evaluation of Glut1 expression using an endothelial promoter Gene expression was assessed in vitro using human brain microvascular endothelial cells (hCMEC / D3). Expression of Glut1 by hCMEC / D3 cells transfected with AAV9 vectors encoding SLC2A1 under the control of the hFLT1, mTIE1, hGlut1 or CMV promoters (as diagrammed in FIG. 10C) was assessed (FIG. 9). Expression from the endothelial promoters (hFLT1 and mTIE1) was comparable to that from the Glut1 promoter and much lower than that from the CMV promoter. Similar patterns of expression levels between these constructs were observed by immunofluorescence microscopy (FIGS. 10A and 10B).

[0165] Surprisingly, 2-deoxy-D-glucose (2-DG) uptake by human brain microvascular endothelial cells transfected or transduced with genes under the control of an endothelial promoter was greater than that of the control Glut1 promoter, with the hFLT-1 promoter showing the highest levels of 2-DG uptake (Figures 11A-11C, 12, and 13). This finding of greater 2-DG uptake with the hFLT-1 promoter construct was also observed across a range of 2-DG concentrations (Figure 12A; 0, 0.1, 0.5, and 1 mM) and various time points post-transfection (Figure 12B), and in some cases was found to be comparable or slightly greater than that observed with the CMV promoter (Figures 11A-11C; Figures 12A, 12B; Figure 13).

[0166] Figure 9: Expression of transgene protein (Glut1-GFP) after transfection into human brain microvascular endothelial cells (hCMEC / d3).

[0167] FIG. 10A: GFP fluorescence 72 hours after transfection with constructs containing one of several endothelial cell promoters driving expression of the Glut1-GFP transgene.

[0168] (B) GFP fluorescence 72 hours after transfection with constructs containing one of two ubiquitous promoters (CMV or CAG), a control vector without Glut1 (CMV-GFP), or no transfection (no NFX). Images obtained using Operetta CLS™ (PerkinElmer®).

[0169] FIG. 10C: Schematic representation of an expression cassette containing the promoter of interest (hFLT1, mTie, hTie or hGlut1) and GLUT1 (SLC2A1) gene (T2A-linked-GFP) and regulatory elements flanked by AAV2 inverted terminal repeats (ITRs).

[0170] 11A-11C: 2-deoxy-D-glucose (Glucose) uptake in hCMEC / d3 cells after human GLUT1 (SLC2A1) expression. Human brain microvascular endothelial cells (hCMEC / d3) were transfected with plasmids expressing either CAG-GFP (CON; negative control) or the hGLUT1-t2A-eGFP transgene driven by one of several endothelial-specific promoters (i.e., hFLT1, mTie, hTie, or hGlut1) or by the ubiquitous CMV or CAG promoter. Glucose uptake was measured using a luminescence-based kit (Promega®) with 0.5 mM 2-deoxyglucose (2-DG) in the medium. Glucose (2-DG) uptake was normalized by total cell number (Total Cell) using phase contrast imaging [error bars represent SEM; n=6 replicates per condition].

[0171] FIG. 11A: In the first experiment, glucose (2-DG) uptake was measured 72 hours after transfection.

[0172] FIG. 11B: In a second experiment, glucose (2-DG) uptake was measured 72 hours after transfection.

[0173] FIG. 11C: Glucose (2-DG) uptake was measured 96 hours after transfection.

[0174] FIG. 12A: Glucose (2-DG) uptake in hCMEC / D3 cells after human Glut1 (SLC2A1) expression at 72 hours.

[0175] FIG. 12B: Glucose (2-DG) uptake in hCMEC / D3 cells after human Glut1 (SLC2A1) expression at 96 hours.

[0176] Figure 13: 2-Deoxy-D-glucose (glucose) uptake following AAV9-mediated expression of hGLUT1 (SLC2A1) in hCMEC / D3 cells. Human brain microvascular endothelial cells (hCMEC / d3) were transfected with AAV9 vectors (3x10) expressing either CAG-GFP (negative control) or an hGLUT1 transgene driven by one of several endothelial-specific promoters (i.e., hFLT1, mTie1 or hGlut1) or by the ubiquitous CMV promoter. 5 Cells were transduced with 100 μg / mL of vector genome / cell. Glucose (2-DG) uptake was measured 72 h post-transduction using the luminescence-based Glucose Uptake-Glo kit (Promega®) and normalized per cell using the RealTime-Glo MT Cell Viability Assay (Promega®) [error bars represent SEM; n=4 replicates per condition].

[0177] Example 3: In vivo evaluation of AAV-BR1 capsid-mediated Glut1 expression using an endothelial promoter in a GLUT1-deficient animal model We conduct a series of experiments to evaluate the in vivo effects of AAV-BR1 capsid-mediated Glut1 transporter protein expression in a mouse model of GLUT1 deficiency syndrome (DS). This model utilizes mice that are heterozygous haploinsufficient due to targeted disruption of the promoter and exon 1 region of the mouse GLUT-1 gene (GLUT-1+ / - mice), which exhibit characteristic features of human GLUT DS, including seizure activity, CSF glycopenia, cerebellar myelopathy, and impaired motor function (Wang et al, Hum Mol Gen, 2006; Tang et al., Nat Comm, 2016). AAV-BR1 virions are evaluated at different doses and different routes of administration (intravenous or intracerebroventricular), with expression of the GLUT1 transgene driven by either a ubiquitous promoter (CMV) or one of several endothelial cell promoters (e.g., hFLT-1). We evaluate to what extent endothelial cell promoter-mediated GLUT1 transgene expression following delivery with AAV-BR1 virions can prevent or reduce functional and pathological impairments in this mouse model. Potential beneficial effects of AAV-BR1-mediated Glut1 protein expression when administered to heterozygous haploinsufficient mice will be revealed by comparison with untreated GLUT-1+ / - control mice, consisting of improved or normalized weight gain, behavioral performance on exercise tests (e.g., rotarod, vertical post assay), CSF glucose levels, brain weight, and the integrity and size of the cerebral microvasculature (e.g., cerebral capillary density, vessel size, number of vascular branch points).

Claims

1. A recombinant adeno-associated virus (rAAV) virion comprising a vector genome and a capsid, the vector genome comprises an expression cassette flanked by 5' and 3' inverted terminal repeats (ITRs); The expression cassette comprises a polynucleotide sequence encoding GLUT1 or a functional variant thereof operably linked to a promoter; and The capsid is a BR1 capsid or a functional variant thereof; The rAAV virion.

2. The rAAV virion of claim 1, wherein the capsid is a BR1 capsid.

3. The rAAV virion of claim 1, wherein the capsid comprises a polypeptide sequence motif XDGXXWX (SEQ ID NO: 107), where each X is any amino acid.

4. The rAAV virion of claim 1, wherein the capsid comprises the polypeptide sequence ADGVQWT (SEQ ID NO: 108), DDGVSWK (SEQ ID NO: 109), SDGLTWS (SEQ ID NO: 110) or SDGLAWV (SEQ ID NO: 111).

5. The rAAV virion of claim 1, wherein the capsid comprises the polypeptide sequence NRGTEWD ​​or a functional variant thereof having one, two, three or more substitutions therein.

6. The rAAV virion of claim 1, wherein the capsid comprises the polypeptide sequence NRGTEWD ​​(SEQ ID NO:112).

7. The rAAV virion of claim 1, wherein the capsid comprises an insertion of the polypeptide sequence NRGTEWD ​​in the GH loop compared to the AAV2 VP1 reference sequence set forth in SEQ ID NO 76.

8. The rAAV virion of claim 1, wherein the capsid comprises a VP3 polypeptide that shares at least 98%, at least 99%, or 100% identity with the AAV-BR1 VP3 polypeptide sequence set forth in SEQ ID NO:

106.

9. The rAAV virion of claim 1, wherein the capsid comprises a VP2 polypeptide that shares at least 98%, at least 99%, or 100% identity with the AAV-BR1 VP2 polypeptide sequence set forth in SEQ ID NO:

105.

10. The rAAV virion of claim 1, wherein the capsid comprises a VP1 polypeptide that shares at least 98%, at least 99%, or 100% identity with the AAV-BR1 VP1 polypeptide sequence set forth in SEQ ID NO:

104.

11. The rAAV virion of any one of claims 1 to 10, wherein the promoter is an FLT-1 promoter.

12. The rAAV virion of claim 11, wherein the FLT-1 promoter is a human FLT-1 (hFLT-1) promoter.

13. The rAAV virion of claim 12, wherein the hFLT-1 promoter shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:

1.

14. The rAAV virion of any one of claims 1 to 13, wherein the expression cassette comprises a polyA signal, optionally a human growth hormone (hGH) polyA.

15. The rAAV virion of any one of claims 1 to 14, wherein the expression cassette comprises a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE), optionally a WPRE(x).

16. The rAAV virion of any one of claims 1 to 15, wherein the expression cassette comprises a 3' untranslated region (3' UTR) comprising a sequence that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to SEQ ID NO:

4.

17. The rAAV virion of any one of claims 1 to 16, wherein the polynucleotide sequence encoding GLUT1 is a SLC2A1 polynucleotide.

18. The rAAV virion of claim 17, wherein the SLC2A1 polynucleotide is a human SLC2A1 polynucleotide.

19. The rAAV virion of any one of claims 16-18, wherein the polynucleotide sequence encoding GLUT1 shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO:

5.

20. The rAAV virion of any one of claims 1 to 19, wherein the expression cassette is flanked by 5' and 3' inverted terminal repeats (ITRs), optionally AAV2 ITRs, optionally ITRs that share at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO: 6 or SEQ ID NO:

7.

21. The rAAV virion of any one of claims 1-20, wherein the expression cassette shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with any one of SEQ ID NOs: 8-16, SEQ ID NO: 97, SEQ ID NO: 99 and SEQ ID NO:

101.

22. 22. The rAAV virion of claim 20 or claim 21, which is not an AAV2 virion.

23. A method for treating and / or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject a rAAV virion described in any one of claims 19 to 22.

24. 24. The method of claim 23, wherein the disease or disorder is a neurological disorder.

25. 25. The method of claim 23 or claim 24, wherein the disease or disorder is Glucose Transporter 1 Deficiency Syndrome (GLUT1 DS) or De Vivo Disease.

26. The method of any one of claims 23 to 25, wherein the rAAV virions are administered by intracerebroventricular (ICV) injection.

27. The method of any one of claims 23 to 25, wherein the rAAV virions are administered by intravenous (IV) injection.

28. The method of any one of claims 23 to 25, wherein the rAAV virions are administered by intracerebroventricular (ICV) injection in combination with intravenous (IV) injection.

29. The method of any one of claims 23 to 28, wherein the administration results in expression of a polynucleotide sequence encoding GLUT1 in the brain, optionally at an increased level compared to a reference rAAV virion.

30. The method of any one of claims 23 to 29, wherein the administration results in increased expression of GLUT1 protein in the brain and / or increased glucose and / or lactate levels in the CSF, optionally at an increased level compared to a reference rAAV virion, wherein optionally the increase is an increase of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or 100%.

31. The method of claim 30, wherein the reference rAAV virion is an AAV2 virion.

32. The method of claim 30, wherein the reference rAAV virion is an AAV9 virion.

33. 1 x 10 rAAV virions 12 Vector genome (vg), 1 × 10 13 vg, 1×10 14 vg or 3×10 14 The method of any one of claims 23 to 32, wherein the administration is at a dose of vg.

34. The method of any one of claims 23 to 33, which causes increased glucose uptake by brain microvascular endothelial cells compared to a method performed using an endogenous Glut1 promoter or a ubiquitous promoter.

35. A method for expressing GLUT1 in a cell, comprising contacting the cell with a rAAV virion of any one of claims 23 to 34.

36. 36. The method of claim 35, wherein the cell is an endothelial cell.

37. 37. The method of claim 36, wherein the endothelial cells are brain microvascular endothelial cells.

38. 38. The method of claim 36 or claim 37, wherein the endothelial cells are in vivo endothelial cells.

39. 36. The method of claim 35, wherein the cell is a neuron.

40. 40. The method of claim 39, wherein the neuron is an in vivo neuron.

41. The method of any one of claims 35 to 40, comprising in vivo administration of the rAAV virion to a subject.

42. The method of any one of claims 35 to 41, wherein the rAAV virion causes increased glucose uptake by the cell compared to a cell contacted with a rAAV virion containing an endogenous Glut1 promoter or a ubiquitous promoter.

43. A pharmaceutical composition comprising the rAAV virion of any one of claims 23 to 34.

44. A kit comprising the rAAV virion of any one of claims 23 to 34 or the pharmaceutical composition of claim 43, and optionally instructions for use.

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