Materials and methods for SLC6A1 gene therapy

Abstract

The present disclosure relates to methods for treating conditions associated with the requirement for electrogenic sodium and chloride-binding gamma aminobutyric acid transporter (GAT-1) protein due to a defective SLC6A1 gene, for example as seen in encephalography of childhood epilepsy. Specifically, the present disclosure provides gene therapy vectors for specifically treating loss of expression of GAT-1 protein and / or reduced levels of GAT-1 protein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 278,905, filed November 12, 2021, which is incorporated by reference in its entirety.

[0002] The present disclosure relates to methods for treating conditions associated with the requirement for electrogenic sodium and chloride-binding gamma aminobutyric acid transporter (GAT-1) protein due to a defective SLC6A1 gene, for example as seen in encephalography of childhood epilepsy. Specifically, the present disclosure provides gene therapy vectors for specifically treating loss of expression of GAT-1 protein and / or reduced levels of GAT-1 protein.

[0003] Incorporation by reference of sequence listing This application contains, as a separate part of the disclosure, a sequence listing in computer-readable format (Filename: 56330_Seqlisting.XML, 59,174 bytes, ASCII text file created on November 8, 2022), which is incorporated by reference in its entirety herein. [Background technology]

[0004] The SLC6A1 gene encodes the electrogenic sodium and chloride-binding gamma-aminobutyric acid (GABA) transporter GAT-1. The GAT-1 protein is localized in the plasma membrane of GABAergic neurons and astrocytes. There, it is involved in the reuptake of the inhibitory neurotransmitter GABA from synapses, i.e., its removal from the synaptic cleft.

[0005] Autosomal dominant mutations in the SLC6A1 gene cause a form of childhood epileptic encephalopathy, the main symptoms of which include various forms of seizures, impaired cognitive development, and ataxia. The majority of genetic variants arise de novo, and the functional impact of some mutations has been recently confirmed. A study of 460 epilepsy patients identified eight patients with disease-associated SLC6A1 variants, including five missense mutations, one nonsense, one splice site, and one in-frame deletion. Introduction of the identified SLC6A1 variants into the rat GAT-1 sequence resulted in a range of loss to up to a 27% reduction in transport activity of wild-type active GABA [Non-Patent Document 1].

[0006] Treatment for patients is currently limited to symptomatic treatment, primarily through the use of antiepileptic drugs. These drugs do not address the underlying genetic defect and therefore are unlikely to halt or slow the progression of the disease and may result in a loss of efficacy when administered over long periods of time.

[0007] Thus, there remains a need in the art for treatments of conditions, including childhood epileptic encephalopathies, in which the GABA transporter activity of the GAT-1 protein is required. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Mattison et al.,SLC6A1 variants identified in epilepsy patients reduce gamma-aminobutyric acid transport,Epilepsia,59(9):e135-e41(2018) Summary of the Invention

[0009] The present disclosure provides a gene therapy vector that expresses a functional GAT-1 protein. The gene therapy vector is useful for delivering a transgene encoding a GAT-1 protein to a subject who requires the GABA transporter activity of GAT-1 (i.e., the activity of removing GABA from the synaptic cleft).

[0010] The provided methods treat conditions associated with decreased GAT-1 protein levels, including, but not limited to, childhood epileptic encephalopathy. The present disclosure provides methods of treatment that involve delivering a gene therapy vector to the cerebrospinal fluid (CSF) of a subject via intraventricular, intracisternal, or lumbar intrathecal injection, or other injection methods that access the CSF, or via intravenous delivery, or via a combination of such routes.

[0011] The gene therapy vector is administered to a subject in need thereof using, for example, intrathecal delivery, and the subject is placed in the Trendelenburg position after administration of the gene therapy vector. Gene therapy vectors are useful for delivering transgenes to GABAergic neurons and / or astrocytes that have reduced levels of GAT-1 protein in a subject.

[0012] The gene therapy vector is, for example, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVTT, Anc80, AAV-7m8, Anc80L65, AAVRH10, AAVRH74, or AAV-B1, or any of their derivatives.The gene therapy vector is, for example, AAV2, AAV5, AAV6, AAV9, AAV8, or AAV10.The gene therapy vector is, for example, AAV9, AAV8, or AAV10.The gene therapy vector is, for example, AAV2, AAV5, or AAV6.

[0013] The transgene in the gene therapy vector contains a promoter that drives expression of the GAT-1 protein, which has GABA transporter activity, for example in neurons and astrocytes.

[0014] The gene therapy vector comprises, for example, SLC6A1 cDNA. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows an exemplary transgene flanked by AAV ITRs for expression of GAT-1 protein from SLC6A1 cDNA. [Diagram 2] FIG. 2 shows expression in HEK293 cells of mRNA derived from an rAAV containing the transgene of FIG. [Figure 3-1] 3A-E show expression in wild-type mice of mRNA derived from rAAV containing the transgene of FIG. 1. [Figure 3-2] Same as above. [Figure 3-3] Same as above. [Figure 4-1] 4A-B show the body weights of treated S295L mice. [Figure 4-2] Same as above. [Diagram 5] 5A-D show the results of behavioral testing of treated S295L mice: A: rotarod day 40, B: cage hanging day 40, D: cage hanging day 140, and E: cage hanging day 140. [Figure 6] FIG. 6 shows the results of the clasping test of treated S295L mice. [Figure 7] 7A-C show the results of additional mouse behavioral testing. [Figure 8] FIG. 8 shows the results of EEG studies of treated S295L mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Gene Therapy Vectors Adeno-associated virus (AAV) is an example of a gene therapy vector. It is a replication-defective parvovirus, whose single-stranded DNA genome is approximately 4.7 kb long, including two 145-nucleotide inverted terminal repeats (ITRs), and can be used to refer to the virus itself or its derivatives. The term includes all subtypes, and both naturally occurring and recombinant forms, unless otherwise specified. There are multiple serotypes of AAV. Each serotype of AAV is associated with a specific clade, whose members share serological and functional similarities. Thus, AAV can also be referred to by clade. For example, the AAV9 sequence is referred to as a "clade F" sequence (Gao et al., J. Virol., 78:6381-6388 (2004). The present disclosure contemplates the use of any sequence within a particular clade, e.g., clade F. The nucleotide sequences of the genomes of the 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 Srivastava et al. al., J. Virol., 45:555-564 (1983), the complete genome of AAV-3 is provided under GenBank accession number NC_1829, the complete genome of AAV-4 is provided under GenBank accession number NC_001829, the AAV-5 genome is provided under GenBank accession number AF085716, the complete genome of AAV-6 is provided under GenBank accession number NC_00 1862, at least portions of the AAV-7 and AAV-8 genomes are provided under GenBank accession numbers AX753246 and AX753249, respectively, and the AAV-9 genome is provided by Gao et al. al., J. Virol., 78:6381-6388 (2004), the AAV-10 genome is provided in Mol. Ther., 13(1):67-76 (2006), the AAV-11 genome is provided in Virology, 330(2):375-383 (2004), a portion of the AAV-12 genome is provided in Genbank accession number DQ813647, and a portion of the AAV-13 genome is provided in Genbank accession number EU285562.The sequence of the AAV rh.74 genome is provided in U.S. Patent No. 9,434,928, which is incorporated herein by reference. The sequence of the AAV-B1 genome is provided in Choudhury et al., Mol. Ther., 24(7):1247-1257 (2016). The sequence of Anc80 is provided in Zinn et al., Cell Reports 12:1056-1068, 2015 and Vandenberghe et al., PCT / US2014 / 060163, both of which are incorporated herein by reference in their entireties, and GenBank Accession Nos. KT235804-KT235812.

[0017] Cis acting sequences that direct viral DNA replication, encapsidation / packaging, and host cell chromosomal integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map positions) drive the expression of two AAV internal open reading frames encoding the rep and cap genes. The two rep promoters (p5 and p19), coupled with alternative splicing of a single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep78, rep68, rep52, and rep40) from the rep gene. The Rep proteins have 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 three capsid proteins, VP1, VP2, and VP3. Alternative splicing and non-consensus translation start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located in the AAV genome at map position 95. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158:97-129 (1992).

[0018] AAV has unique characteristics that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is non-cytopathic, 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 non-dividing cells and persist essentially for the life of those cells as transcriptionally active nuclear episomes (extrachromosomal elements). The native AAV proviral genome is infectious as cloned DNA in a plasmid making the construction of recombinant genomes feasible. Furthermore, because signals directing AAV replication, genome encapsidation, and integration are contained within the ITRs of the AAV genome, part or all of the internal ∼4.3 kb genome (encoding replication and structural capsid proteins, rep-cap) can be replaced with foreign DNA, such as a gene cassette containing a promoter, DNA of interest, and a polyadenylation signal. Rep and cap proteins can be provided in trans. Another important feature of AAV is that it is an extremely stable and robust virus. AAV easily survives the conditions used to inactivate adenovirus (56°C-65°C for several hours), making cryopreservation of AAV less important. AAV can even be lyophilized. Finally, cells infected with AAV do not exhibit resistance to superinfection.

[0019] The term "AAV" as used herein refers to a wild-type AAV virus or virus particle. The terms "AAV", "AAV virus", and "AAV virus particle" are used interchangeably herein. The term "rAAV" refers to a recombinant, infectious, encapsidated virus or virus particle. The terms "rAAV", "rAAV virus", and "rAAV virus particle" are used interchangeably herein.

[0020] The term "rAAV genome" refers to a polynucleotide sequence derived from a modified native AAV genome. Provided are rAAV genomes modified to remove the cap and rep genes of native AAV. The rAAV genome comprises at least one or both endogenous 5' and 3' inverted repeat repeats (ITRs). The rAAV genome can comprise an ITR from an AAV serotype different from the AAV serotype from which the AAV genome was derived. The rAAV genome can comprise three ITRs (e.g., as in scAAV).

[0021] Provided herein is a rAAV genome that contains a transgene flanked at the 5' and 3' ends by AAV ITRs. SEQ ID NO: 1 shows the polynucleotide sequence of SLC6A1 cDNA. SEQ ID NO: 2 shows the amino acid sequence of GAT-1 protein encoded by SEQ ID NO: 1.

[0022] The transgenes provided herein include, but are not limited to, transgenes comprising an SLC6A1 cDNA or polynucleotide encoding a GAT-1 protein having GABA transporter activity, wherein the polynucleotide is 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide of SEQ ID NO:1.

[0023] Transgenes provided herein include, but are not limited to, transgenes set forth in SEQ ID NOs: 3, 4, 5, 6, 7, and 8 (each of these SEQ ID NOs also including 5' and 3' AAV ITRs flanking the transgene), each of which comprises the SLC6A1 cDNA of SEQ ID NO: 1. Also provided herein are transgenes encoding GAT-1 proteins having GABA transporter activity, which transgenes are at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 3, 4, 5, 6, 7, or 8.

[0024] The transgenes provided herein can encode a GAT-1 protein having GABA transporter activity that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the GAT-1 protein of SEQ ID NO:2, for example. The transgenes provided herein include polynucleotides that encode a GAT-1 protein having GABA transporter activity and that hybridize under stringent conditions to a transgene comprising SEQ ID NO:1, or a transgene of SEQ ID NO:3, 4, 5, 6, 7, or 8, or its complement.

[0025] The term "stringent" is used to refer to conditions that are generally understood in the art as stringent. Hybridization stringency is determined primarily by temperature, ionic strength, and the concentration of denaturing agents such as formamide. Examples of stringent conditions for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, (Cold Spring Harbor, NY1989).

[0026] Exemplary promoters are the chicken beta actin promoter (CBA) (SEQ ID NO: 9), the truncated methyl-CpG binding protein 2 (MeCP2) promoter, also referred to as the P546 MeCP2 promoter (SEQ ID NO: 10) (e.g., for driving expression in neurons and astrocytes), the human synapsin (hSyn) promoter (SEQ ID NO: 12) (e.g., for driving expression in neurons), the human somatostatin (hSST) promoter (SEQ ID NO: 13) (e.g., for driving expression in inhibitory neurons), the small glial fibrillary acidic protein [gfaABC(1)D] promoter (SEQ ID NO: 11) (e.g., for driving expression in astrocytes), the glial fibrillary acidic protein (GFAP) promoter (SEQ ID NO: 14), the CMV promoter, and the Myo7A promoter. Additional promoters are contemplated herein, including, but not limited to, the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters (e.g., actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter, and creatine kinase promoter). Additionally, provided herein are CBA promoter, P546 MeCP2 promoter, hSyn promoter, hSST promoter, gfaABC(1)D promoter, and GFAP promoter that are at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the promoter nucleotide sequences of the corresponding SEQ ID NOs: 9 to 14, respectively, and have transcription-promoting activity.

[0027] Examples of transcriptional regulators are tissue-specific regulators, such as promoters that allow expression specifically in neurons or specifically in astrocytes. Examples include the neuron-specific enolase and astrocyte-specific glial fibrillary acidic protein promoters. Inducible promoters are also contemplated. Non-limiting examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters. The gene cassette may also include an intron sequence to facilitate processing of the transgene RNA transcript when expressed in mammalian cells. One example of such an intron is the SV40 intron.

[0028] "Packaging" refers to the series of intracellular events that result in the assembly and encapsidation of AAV particles. The term "production" refers to the process of production of rAAV (infectious, encapsidated rAAV particles) by the packaging cell.

[0029] AAV "rep" and "cap" genes refer to polynucleotide sequences that encode the replication and encapsidation proteins, respectively, of the adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

[0030] AAV "helper virus" refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A variety of such AAV helper viruses are known in the art, including adenoviruses, herpesviruses, baculoviruses, and poxviruses such as vaccinia. Adenoviruses can encompass several different subgroups, with adenovirus type 5 of subgroup C being the most commonly used. Numerous adenoviruses of human, non-human mammalian, and avian origin are known and available from depositories such as the ATCC. Herpes family viruses include, for example, herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), also available from depositories such as the ATCC.

[0031] "Helper virus functions" refers to functions encoded in the helper virus genome that enable replication and packaging of AAV (in conjunction with other requirements for replication and packaging as described herein). As described herein, "helper virus functions" can be provided in a number of ways, including by providing a helper virus or, for example, by providing polynucleotide sequences encoding the necessary functions to the producer cell in trans.

[0032] The rAAV genome provided herein lacks AAV rep and cap DNA. The AAV DNA (e.g., ITR) in the rAAV genome contemplated herein can be derived from any AAV serotype suitable for obtaining recombinant virus, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVTT, Anc80, AAV-7M8, Anc80L65, AAVRH10, AAVRH74, and AAV-B1, and derivatives thereof. As mentioned above, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. rAAV with capsid mutations are also contemplated. See, e.g., Marsic et al., Molecular Therapy, 22(11):1900-1909 (2014). Modified capsids herein are also contemplated, including capsids with various post-translational modifications, such as glycosylation and deamidation. Deamidation of asparagine or glutamine side chains to convert asparagine residues to aspartic acid or isoaspartic acid residues, and conversion of glutamine to glutamic acid or isoglutamic acid are contemplated in the rAAV capsids provided herein. See, e.g., Giles et al., Molecular Therapy, 26(12):2848-2862 (2018). Modified capsids herein are also contemplated to include targeting sequences that direct the rAAV to diseased tissues and organs in need of treatment.

[0033] The DNA plasmids provided herein contain the rAAV genome described herein. The DNA plasmids can be introduced into cells permissive for infection with AAV helper viruses (e.g., adenovirus, E1-deleted adenovirus, or herpesvirus) to assemble the rAAV genome into infectious viral particles using AAV9 capsid proteins. Techniques for producing rAAV in which the rAAV genome to be packaged, the rep and cap genes, and the helper virus functions are provided to the cell are standard in the art. The production of rAAV particles requires the presence of the following components in a single cell (referred to herein as a packaging cell): the rAAV genome, the AAV rep and cap genes separated from (i.e., not present in) the rAAV genome, and the helper virus functions. The AAV rep and cap genes may be from any AAV serotype from which a recombinant virus may be derived, or may be from an AAV serotype different from the rAAV genome ITRs. The production of pseudotyped rAAV is disclosed, for example, in WO01 / 83692, the entire disclosure of which is incorporated herein by reference. AAV capsid proteins can be modified to enhance the delivery of recombinant rAAV. Modifications to capsid proteins are generally known in the art. See, for example, US2005 / 0053922 and US2009 / 0202490, the entire disclosures of which are incorporated herein by reference.

[0034] The method for generating packaging cells is to create a cell line that stably expresses all the components necessary for rAAV production. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, the AAV rep and cap genes separated from the rAAV genome, and a selectable marker such as a neomycin resistance gene may be integrated into the genome of the cell. The rAAV genome may be introduced into a bacterial plasmid by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of a synthetic linker containing a restriction endonuclease cleavage site (Laughlin et al., 1983, Gene, 23:65-73), or direct blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line can then be infected with a helper virus, such as adenovirus. The advantage of this method is that the cells are selectable and are suitable for large-scale production of rAAV. Other non-limiting examples of suitable methods use adenovirus, herpesvirus or baculovirus, rather than plasmids, to introduce the rAAV genome and / or rep and cap genes into the packaging cells.

[0035] The general principles of rAAV particle production are reviewed in, for example, Carter, Current Opinions in Biotechnology, 1533-1539 (1992), and Muzyczka, Curr. Topics in Microbial. and Immunol., 158:97-129 (1992). Various approaches have been proposed by Ratschin et al., Mol. Cell. Biol. 4:2072 (1984), Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984), Tratschin et al., Mol. Cell. Biol. al., J. Virol., 62:1963 (1988), and Lebkowski et al., Mol. Cell. Biol., 7:349 (1988), Samulski et al. al., J. Virol., 63: 3822-3828 (1989), U.S. Pat. No. 5,173,414, WO95 / 13365 and corresponding U.S. Pat. No. 5,658.776, WO95 / 13392, WO96 / 17947, PCT / US98 / 18600, WO97 / 09441 (PCT / US96 / 14423), WO97 / 08298 (PCT / US96 / 13872), WO97 / 21825 (PCT / US96 / 20777), WO97 / 06243 (PCT / FR96 / 01064), WO99 / 11764, Perrin et al., Vaccine 13: 1244-1250 (1995), Paul et al. al., Human Gene Therapy, 4:609-615 (1993), Clark et al. (Gene Therapy 3:1124-1132 (1996), U.S. Patent No. 5,786,211, U.S. Patent No. 5,871,982, and U.S. Patent No. 6,258,595. The above documents are incorporated herein by reference in their entireties, with particular emphasis being placed on the sections of the documents relevant to rAAV particle production.

[0036] Further provided herein are packaging cells that produce infectious rAAV particles. In one embodiment, the packaging cells can be stably transformed cancer cells, such as HeLa cells, 293 cells, and PerC.6 cells (synonymous 293 cells). In another embodiment, the packaging cells can be cells that are not transformed cancer cells, such as low-passage 293 cells (human fetal kidney cells transformed with adenovirus E1), MRC-5 cells (human fetal fibroblast cells), WI-38 cells (human fetal fibroblast cells), Vero cells (monkey kidney cells), and FRhL-2 cells (fetal rhesus lung cells).

[0037] Also provided herein are rAAVs (e.g., infectious, encapsidated rAAV particles) comprising the rAAV genome of the present disclosure. The genome of the rAAV lacks AAV rep and cap DNA, i.e., there is no AAV rep or cap DNA between the ITRs of the rAAV genome. The rAAV genome can be a self-complementary (sc) genome. An rAAV having a sc genome is referred to herein as a scAAV. The rAAV genome can be a single-stranded (ss) genome. An rAAV having a single-stranded genome is referred to herein as a ssAAV.

[0038] rAAV can be purified by methods standard in the art, such as column chromatography and / or cesium chloride gradients. Methods for purifying rAAV from helper viruses are known in the art and include, for example, those disclosed in Clark et al., Hum. Gene Ther., 10(6):1031-1039 (1999), Schenpp and Clark, Methods Mol. Med., 69:427-443 (2002), U.S. Patent No. 6,566,118, and WO 98 / 09657.

[0039] composition Also provided is a composition comprising rAAV. The composition comprises a rAAV encoding a polypeptide of interest, including, but not limited to, GAT-1 protein. The composition may comprise two or more rAAV encoding different polypeptides of interest.

[0040] The compositions provided herein comprise rAAV and one or more pharma- ceutically acceptable excipients. Acceptable excipients are non-toxic to recipients and preferably inert at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, e.g., phosphate-buffered saline (PBS), citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as Tween, copolymers such as poloxamer 188, Pluronic (e.g., Pluronic F68), or polyethylene glycol (PEG). The compositions provided herein can include a pharma- ceutically acceptable aqueous excipient containing a non-ionic, hypo-osmolar compound or contrast agent, such as iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, or ioxilan, and the aqueous excipient containing the non-ionic, hypo-osmolar compound can have one or more of the following properties: a vapor pressure osmolality of about 180 mgI / mL, about 322 mOsm / kg water, an osmolality of about 273 mOsm / L, an absolute viscosity of about 2.3 cp at 20° C. and about 1.5 cp at 37° C., and a specific gravity of about 1.164 at 37° C. Exemplary compositions include about 20-40% non-ionic, hypo-osmolar compound, or about 25-35% non-ionic, hypo-osmolar compound. An exemplary composition is 20 mM Tris (pH 8.0), 1 mM MgCl 2, 200 mM NaCl, 0.005% poloxamer 188, and about 25% to about 35% of a non-ionic, hypoosmolar compound. Another exemplary composition includes scAAV formulated in 1× PBS and 0.001% Pluronic F68.

[0041] For CSF delivery, including but not limited to intrathecal delivery, the viral vector can be mixed with a contrast agent (Omnipaque or the like). For example, the composition can include a non-ionic, low osmolarity contrast agent, including but not limited to iobitridol, iohexol, iomeprol, iopamidol, iopentol, iopromide, ioversol, ioxilan, or combinations thereof.

[0042] Dosages may be expressed in units of viral genomes (vg). Doses contemplated herein are approximately 1×10 7 vg, approx. 1×10 8 vg, approx. 1×10 9 vg, approx. 5×10 9 vg, approx. 6×10 9 vg, approx. 7×10 9 vg, approx. 8×10 9 vg, approx. 9×10 9 vg, approx. 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. 1×10 11 vg, approx. 1.1×10 11 vg, approx. 1.2×10 11 vg, approx. 1.3×10 11 vg, approx. 1.2×10 11 vg, approx. 1.3×10 11 vg, approx. 1.4×10 11 vg, approx. 1.5×10 11 vg, approx. 1.6×10 11 vg, approx. 1.7×10 11 vg, approx. 1.8×10 11 vg, approx. 1.9×10 11 vg, approx. 2×1011 vg, approx. 3×10 11 vg, approx. 4×10 11 vg, approx. 5×10 11 vg, approx. 1×10 12 vg, approx. 1×10 13 vg, approx. 1.1×10 13 vg, approx. 1.2×10 13 vg, approx. 1.3×10 13 vg, approx. 1.5×10 13 vg, approx. 2×10 13 vg, approx. 2.5×10 13 vg, approx. 3×10 13 vg, approx. 3.5×10 13 vg, approx. 4×10 13 vg, approx. 4.5×10 13 vg, approx. 5×10 13 vg, approx. 6×10 13 vg, approx. 1×10 14 vg, approx. 2×10 14 vg, approx. 3×10 14 vg, approx. 4×10 14 vg, approx. 5×10 14 vg, approx. 1×10 15 vg~approx. 1×10 16 vg, or more of the entire viral genome. Approximately 1 × 10 9 vg~approx. 1×10 10 vg, approx. 5×10 9 vg~approx. 5×10 10 vg, approx. 1×10 10 vg~approx. 1×10 11 vg, approx. 1×10 11 vg~approx. 1×10 15 vg, approx. 1×10 12 vg~approx. 1×10 15 vg, approx. 1×10 12 vg~approx. 1×10 14 vg, approx. 1×10 13 vg~approx. 6×10 14 vg, and approximately 6 × 10 13 vg~approx. 1.0×10 14 vg, 2.0×10 14 vg, 3.0×10 14 vg, 5.0×10 14 Also contemplated is a dose of 1.65×1011 vg.

[0043] For example, CSF doses range from approximately 1×10 13 vg / patient ~ approx. 1×10 15 vg / patient. For example, an intravenously delivered dose can range from 1×10 13 vg / kilogram (kg) body weight ~2×10 14 vg / kg range.

[0044] Treatment method The therapeutic method herein targets cells with reduced GABA transporter activity of GAT-1 protein. The therapeutic method herein can target cells with a "defective" SLC6A1 gene, i.e., a gene with at least one "defective" (i.e., mutated) allele that encodes a GAT-1 protein that lacks GABA transporter activity. As understood in the art, a diploid subject, such as a human subject, generally has two copies of each gene, referred to as alleles. Methods of transduction are provided that target such cells in a subject (e.g., a human subject). Methods are provided that transduce, for example, one or more of neurons, astrocytes, and / or central nervous system tissues in a subject (e.g., a human subject).

[0045] The use of the therapeutic methods described herein is indicated for example for reducing the GABA transporter activity of GAT-1 protein in epileptic encephalopathies, such as pediatric epileptic encephalopathies, by increasing the expression levels of GAT-1 mRNA and protein.

[0046] The terms "transducing" and "transduction" are used to refer to the administration / delivery of a rAAV of the present disclosure that encodes a GAT-1 protein having GABA transporter activity either in vivo or in vitro to a target cell, resulting in expression of functional GAT-1 protein by the target cell. Transduction of a cell with a rAAV of the present disclosure results in sustained expression of the polypeptide encoded by the rAAV.

[0047] The methods provided herein transduce target cells with one or more rAAVs as described herein. In some embodiments, the rAAV viral particles containing the transgene are administered or delivered to the CSF of a subject, for example, by intraventricular injection, intracisternal injection, or lumbar intrathecal injection, or by other injection methods that access the CSF, or via intravenous delivery, or via a combination of such routes. Intrathecal administration refers to delivery to the subarachnoid space of the brain or spinal cord. In particular, intrathecal administration to the brain can be performed by intraventricular injection. The brain regions that are intended to be delivered include, but are not limited to, the motor cortex, the visual cortex, the cerebellum, and the brain stem.

[0048] For intrathecal administration, the subject may be maintained in Trendelenburg position (head down) after injection of the rAAV (e.g., for about 5, about 10, about 15, or about 20 minutes). For example, the patient may be tilted about 1 degree to about 30 degrees, about 15 to about 30 degrees, about 30 to about 60 degrees, about 60 to about 90 degrees, or about 90 to about 180 degrees in the head down position.

[0049] The therapeutic methods provided herein include administering an effective dose or effective doses of a composition comprising a rAAV provided herein to a subject (e.g., an animal, including, but not limited to, a human patient) in need thereof. If the dose is administered prior to the onset of symptoms, the administration is prophylactic. If the dose is administered after the onset of symptoms, the administration is therapeutic. An effective dose is one that alleviates (eliminates or reduces) at least one symptom associated with a condition of reduced levels of GABA transporter activity of GAT-1, slows or prevents the progression of the condition, reduces the severity of the condition, results in remission (partial or complete) of the condition, and / or prolongs survival.

[0050] An effective dose for the treatment of epileptic encephalopathy is one that ameliorates (eliminates or reduces) seizures, impaired cognitive development, and ataxia. EXAMPLES

[0051] The following examples illustrate specific embodiments, but variations and modifications will occur to those skilled in the art. Accordingly, only such limitations as appear in the claims should be placed on the invention.

[0052] Example 1 Production of rAAV expressing GAT-1 For use in gene therapy re-expression approaches for SLC6A1 pathology, a transgene expressing human SLC6A1 cDNA (SEQ ID NO: 1) (Origene catalog number SC126769) was used to generate rAAV. At 4494 base pairs, SLC6A1 falls within the bounds of AAV.

[0053] AAV9 targets neurons primarily and astrocytes secondarily. Expression levels and cell type specific transduction are further tuned by using different promoters to drive transgene expression. The first construct uses the CAG promoter to drive SLC6A1 expression, resulting in strong ubiquitous expression. CAG was chosen over other ubiquitous promoters such as CBA because of its strong bias towards inhibitory neurons compared to excitatory neurons [Nathanson et al., Neuroscience, 161(2):441-450(2009)]. Next, a truncated version of the MECP2 promoter known as p546 is used to target neurons and astrocytes, but with low efficacy. The third construct restricts expression to neurons through the use of the synapsin promoter (Nathanson, Neuroscience, see above) and further restricts expression to inhibitory GABAergic neurons through the somatostatin promoter [Nagai et al., Biochem Biophys Res Commun., 518(4):619-624(2019); Nathanson et al., Front Neural Circuits., 3:19(2009)]. Finally, GFAP is used to target astrocytes [Lawlor et al., Mol Ther., 17(10):1692-1702(2009)]. After cloning, five constructs were sequenced, expression was confirmed in 293 cells, and small-scale AAV9 viral preparations were generated.

[0054] The transgenes depicted in Figure 1 use the aforementioned promoters to drive expression of GAT-1 protein from human SLC6A1 cDNA. The transgene of SEQ ID NO:3 contains the CBA promoter (ubiquitous promoter), the transgene of SEQ ID NO:4 contains the P546 MeCP2 promoter (ubiquitous promoter), the transgene of SEQ ID NO:6 contains the hSyn promoter (neuron-specific promoter), the transgene of SEQ ID NO:7 contains the hSST promoter (inhibitory neuron-specific promoter), the transgene of SEQ ID NO:5 contains the gfaABC(1)D promoter (astrocyte-specific promoter), and the transgene of SEQ ID NO:8 contains the GFAP promoter (astrocyte-specific promoter). The promoters contemplated for use in the methods herein are of a size that allows the transgene construct to fit well into the rAAV and are focused on driving transgene expression to increase GAT-1 protein expression in transduced cells by at least 10% and increase expression in neurons and astrocytes.

[0055] Transgenes were subcloned into AAV9 production plasmids, i.e., pscAAV.SLC6A1.CBA, pscAAV.SLC6A1.P546, pscAAV.SLC6A1.hSyn, pscAAV.SLC6A1.SST, pscAAV.SLC6A1.gfaABC(1)D, pssAAV.SLC6A1.GFAP, and scAAV and ssAAV were produced by transient triple transfection of 293 cells with a double-stranded AAV2-ITR-based production plasmid, a plasmid encoding the Rep2Cap9 sequence, and the adenoviral helper plasmid pHelper, as described in Foust et al., Nat Biotechnol., 27(1):59-65 (2009).

[0056] The resulting SLC6A1 rAAVs were: ssAAV.CBA.SLC6A1, ssAAV.P546.SLC6A1, ssAAV.hSyn.SLC6A1, ssAAV.hSST.SLC6A1, ssAAV.gfaABC(1)D.SLC6A1, ssAAA.GFAP.SLC6A1, scAAV.CBA.SLC6A1 (sometimes referred to herein as scAAV.CAG.SLC6A1), scAAV.P546.SLC6A1, scAAV.hSyn.SLC6A1 (sometimes referred to herein as scAAV.Syn.SLC6A1 (hereinafter sometimes referred to as scAAV.SST.SLC6A1), scAAV.gfaABC(1)D.SLC6A1, and scAAA.GFAP.SLC6A1.

[0057] Example 2 In vitro expression analysis The expression of SLC6A1 mRNA from the AAV production plasmid generated in Example 1 was evaluated in HEK293T cells. HEK293T cells were transfected with one of the AAV production plasmids expressing SLC6A1 cDNA or a control scAAV expressing green fluorescent protein (GFP). Cells were harvested 72 hours after transfection and the expression of SLC6A1 mRNA was analyzed by PCR and qPCR.

[0058] The expression levels of SLC6A1 mRNA are shown in FIG. 2 as fold changes relative to the expression levels of control GFP mRNA. Example 3 Expression in wild-type mice The expression of SLC6A1 mRNA from the scAAV and ssAAV generated in Example 1 was evaluated in wild-type mice. Each of the AAV9 constructs expressing SLC6A1 was injected into the wild-type mice at 2.95 × 10 10 ~1.5×10 11Four neonatal wild-type mice (n=4 for each rAAV9) were administered rAAV9 vg / mouse by intracerebroventricular injection. WT mice were sacrificed along with uninjected WT control mice at approximately 4 weeks of age (short-term) or approximately 4 months of age (long-term). Tissues were harvested for SLC6A1 mRNA expression, protein expression, and histology.

[0059] The expression levels of SLC6A1 mRNA in injected mice are shown as relative fold change compared to the mRNA expression levels in age-matched control non-injected mice in Figures 3A-E. There were no signs of toxicity either clinically or by blood tests.

[0060] Example 4 Administration of AAV gene therapy constructs in a mouse model of SLC6A1 While GAT-1 knockout mice are viable [Jensen et al., J Neurophysiol, 90(4):2690-2701 (2003) and Chiu et al., J Neurosci, 22(23):10251-10266 (2002)], heterozygous mice have no phenotype and therefore do not recapitulate the disease in human patients. Homozygous mice exhibit behavioral and cognitive deficits and seizures beginning on postnatal day 19 that can be quantified by EEG. Two additional mouse models have missense mutations A288V and S295L that are equivalent to known human point mutations. Unlike knockout mice, both A288V and S295L mice are phenotypically abnormal as heterozygotes, including the presence of seizures. The A288V and S295L mouse models of SLC6A1 are useful for evaluating the safety and efficacy of gene replacement strategies.

[0061] All viral vectors were evaluated using the S295L mouse model. The effects of scAAV.P546.SLC6A1, scAAV.hSyn.SLC6A1, scAAV.hSST.SLC6A1, scAAV.gfaABC(1)D.SLC6A1 (harboring the gfaABC(1)D.v2 transgene of SEQ ID NO:5) and ssAAA.GFAP.SLC6A1 were compared to control empty viral particles. Each of the rAAV9 vectors was administered at 3×10 10 Neonatal mice were administered the drug by intracerebroventricular injection of 1000 mg / vg / mouse.

[0062] Mice were subjected to weekly weight measurements and biweekly rotarod, cage hang, and various behavioral tests including clasping tests starting from days 21 to 28. Animals were humanely or at predetermined time points sacrificed for postmortem biochemical, molecular, and histological analyses, including transgene expression analysis.

[0063] The results are shown in Figures 4A to 4D (body weight), Figures 5A to 5C (rotarod and cage hang), and Figure 6 (clasping). Weight gain in males and females was compared between wild-type, heterozygous, and homozygous animals containing the S295L mutation in the SLC6A1 gene. Untreated homozygous mutant male mice showed reduced weight gain. Importantly, weight gain in homozygous mutant male mice was normalized when treated with AAV-p546-SLC6A1 (i.e., scAAV.P546.SLC6A1). No differences were seen in female homozygous mutant mice treated with any construct compared to wild-type animals, which was expected since there was also no difference between untreated and wild-type females. See Figure 4A-D.

[0064] In the rotarod assay performed, mice were placed on a rotating wheel that continued to rotate at an accelerating rate. The time it took for the mouse to fall off was measured. A highly significant difference was seen in the ability to stay on the rotarod between 40-day-old wild-type and homozygous mutant animals. In addition, a wire-hang test was performed in which mice were placed on a cage lid containing a metal wire, and then the lid was inverted. The time the animal could hang on the grid before falling off was measured. At day 150, a highly significant difference was seen in both males and females. At that time point, homozygous mutant mice showed a large reduction in their ability to hang on the cage. Different AAV-SLC6A1 constructs had different effects on improving the mutant mice's ability to hang for longer periods of time. The most effective construct was AAV-P546-SLC6A1 (i.e., scAAV.P546.SLC6A1), which reduced the latency to normal wild-type levels. See Figures 5A-C. Figures 7A-C show data from additional mice.

[0065] Untreated homozygous mutant mice display a clasping phenotype in which they are unable to properly spread their hind limbs when suspended by their tails, and as shown in Figure 6, are indistinguishable from wild-type mice (left image) and treated mutant mice (right image), both of which exhibited normal hind limb positioning in this assay.

[0066] Example 5 Quantification of epileptiform discharges in mice As seizure activity is a common disease phenotype in SLC6A1 subjects, these mice were also tested by electroencephalogram (EEG).

[0067] Mice were premedicated using 1 mg / kg injectable buprenorphine HCL and 5 mg / kg carprofen. The animals were then induced using isoflurane via an induction chamber. Anesthesia was maintained using 1-3% isoflurane via a nose cone. Once the animals had reached a surgical depth of anesthesia, the surgical site was aseptically prepared. An incision was made approximately 2.5 cm long extending cranially from the scapula to just caudal to the base of the eye. A DSI teleoperated implant was inserted subcutaneously. For EMG monitoring, biopotential leads were surgically placed in the trapezius muscle. Additionally, for EEG monitoring, holes were drilled into the skull and electrodes were placed using a stereotaxic device at the following coordinates: AP+1.0 / ML-1.5 (LH) and AP-2.0 / ML+2.0 (RH). The incision was closed using a simple continuous suture pattern.

[0068] After surgery, mice were given 1 mL of warm NaCl, orally administered 2 mg / kg capromorelin, and placed in an incubator for at least 12 hours. Mice were allowed to recover for at least 72 hours and maintained on oral carpophen through their drinking water. Data was then acquired by placing the mice directly on a radio telemetry receiver plate system for 24 hours. Data was then analyzed with DSI NeuroScore™ software.

[0069] The results are shown in Figure 8. Homozygous mutant mice exhibited a significant increase in spike trains / 24 hours compared to heterozygous control mice. Homozygous mutant mice treated with AAV9-P546-SLC6A1 (i.e., scAAV.P546.SLC6A1) exhibited a significant decrease in spike trains / 24 hours compared to untreated homozygous mutant mice.

[0070] Example 6 AAV gene therapy for human patients AAV gene therapy provides patients with a wild-type copy of SLC6A1 to address haploinsufficiency caused by mutations in one copy of the SLC6A1 gene.

[0071] While the present invention has been described with respect to specific embodiments, it is understood that variations and modifications thereof will occur to those skilled in the art, and therefore only such limitations as appear in the claims should be placed on the invention.

[0072] All documents referenced in this application are incorporated herein by reference in their entirety.

Claims

1. 1. A composition for delivering a transgene encoding a GAT-1 protein having GABA transporter activity to a subject in need of the GABA transporter activity of the GAT-1 protein, the composition comprising a gene therapy vector containing the transgene; the gene therapy vector is administered to the cerebrospinal fluid of the subject by intraventricular injection, intracisternal injection, lumbar intrathecal injection, or by intravenous delivery; The composition, wherein the transgene comprises a polynucleotide of SEQ ID NO: 1, or a polynucleotide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, encoding a GAT-1 protein having GABA transporter activity, or wherein the transgene comprises a polynucleotide of SEQ ID NO: 3, 4, 5, 6, 7, or 8, or a polynucleotide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 4, 5, 6, 7, or 8, encoding a GAT-1 protein having GABA transporter activity.

2. The composition of claim 1 , wherein the subject has at least one defective SLC6A1 allele.

3. 10. The composition of claim 1, wherein the transgene is delivered to neurons or astrocytes, or both, of the subject.

4. 10. The composition of claim 1, wherein said administering treats at least one of seizures, cognitive development disorders, and ataxia in said subject.

5. 10. The composition of claim 1, wherein said administering treats epileptic encephalopathy in said subject.

6. The composition of claim 5, wherein the epileptic encephalopathy is pediatric epileptic encephalopathy.

7. 2. The composition of claim 1, wherein the gene therapy vector is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVTT, Anc80, AAV-7m8, Anc80L65, AAVRH10, AAVRH74, or AAV-B1, or a derivative of any of them.

8. The composition of claim 1 , wherein the gene therapy vector is administered by intrathecal delivery.

9. 10. The composition of claim 1, wherein the gene therapy vector is administered by intrathecal delivery and the subject is placed in the Trendelenburg position after administration of the gene therapy vector.

10. The composition of claim 1 , wherein the gene therapy vector is administered by intracerebroventricular injection.

11. The composition of claim 1 , wherein the gene therapy vector is administered by intracisternal injection.

12. The composition of claim 1 , wherein the gene therapy vector is administered by lumbar intrathecal injection.

13. The composition of claim 1 , wherein the gene therapy vector is administered by intravenous delivery.

14. A transgene comprising a polynucleotide of SEQ ID NO: 1, or a polynucleotide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, encoding a GAT-1 protein having GABA transporter activity.

15. A transgene comprising a polynucleotide of SEQ ID NO: 3, 4, 5, 6, 7, or 8, or a polynucleotide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 4, 5, 6, 7, or 8, which encodes a GAT-1 protein having GABA transporter activity.

16. A recombinant adeno-associated virus (rAAV) having a genome comprising a transgene comprising a polynucleotide of SEQ ID NO: 1, or a polynucleotide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, encoding a GAT-1 protein having GABA transporter activity.

17. A recombinant adeno-associated virus (rAAV) having a genome comprising a transgene of SEQ ID NO: 3, 4, 5, 6, 7, or 8, or a polynucleotide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, 4, 5, 6, 7, or 8, encoding a GAT-1 protein having GABA transporter activity.

18. 18. The recombinant adeno-associated virus (rAAV) of claim 16 or 17, wherein the recombinant adeno-associated virus is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV11, AAV12, AAV13, AAVTT, Anc80, AAV-7m8, Anc80L65, AAVRH10, AAVRH74, or AAV-B1 serotype vector, or a derivative thereof.

19. 19. The recombinant adeno-associated virus (rAAV) of claim 18, which is AAV9.

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