SLC2A1 LNCRNA AS A BIOLOGIC AND RELATED TREATMENTS AND

METHODS

CROSS REFERENCE TO RELATED APPLICATION

The present application claims priority to U.S. patent application serial No.

62/571,667 , filed October 12, 2017, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number NINDS NS057482 awarded by the National Institutes of Health. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates the discovery of a novel natural antisense transcript or long non-coding RNA to the human SLC2A1 (Glutl) gene and its use to modulate Glutl expression and serve as a therapeutic for treating or preventing Glut 1 deficiency syndrome, or other Glutl related conditions including certain cancers, diabetes, Alzheimer's disease, and retinitis pigmentosa. BACKGROUND OF THE INVENTION

The human cerebrum, despite its modest size (2% of body mass) has a voracious appetite for glucose, devouring 25% of the total amount metabolized by the body (Belanger et al. 2011). Curbing the supply of this nutrient to the brain has grave consequences for proper cerebral development and function. This is best exemplified by the rare but debilitating infantile-onset brain energy failure syndrome, Glucose Transporter- 1 deficiency syndrome (Glutl DS) (DeGiorgis and Veggiotti 2013). Caused by a heterozygous loss of the SLC2A1 (Glutl) gene and thus a corresponding reduction in the Glutl protein, Glutl DS is characterized by intractable infantile-onset seizures, neurodevelopmental delay and a complex movement disorder that combines elements of ataxia, dystonia and spasticity. An expanded phenotype that includes non-epileptic paroxysmal exercise-induced dyskinesia and hemolytic anemia (Brockmann 2012), in conjunction with emerging evidence that Glutl mutations underlie a significant proportion of the idiopathic generalized epilepsies (Arsoy et al. 2012), suggest that the disease is more prevalent than initially thought and could affect 6000 to 12,000 patients in the United States alone. Thus, it is a significant neurological disorder with correspondingly pronounced health as well as societal costs attached to it.

Although it has been roughly two decades since Glutl DS was found to result from mutations in the Glutl gene, little progress has been made in developing an optimal treatment for the disease. The most widely prescribed therapy for patients afflicted with Glutl DS involves a high-fat, ketogenic diet (DeGiorgis and Veggiotti 2013). Such a diet supplies the brain with an alternate, albeit imperfect energy source - ketone bodies (Klepper and Leiendecker 2013). The ketone bodies provide an alternate source of acetyl CoA that can be fed into the Krebs cycle. However, they fail to replenish glycolytic intermediates deriving from glucose. Moreover, entry of the ketone bodies into the parenchyma eventually declines as MCT1 (monocarboxylate transporter- 1), the relevant conduit used to deliver these molecules across the blood-brain barrier (BBB), is downregulated during early postnatal development (Pellerin et al. 1998). It is therefore little surprise that the ketogenic diet while effective in managing seizures in patients fails to avert cognitive dysfunction or the movement defects typically associated with Glutl DS (Pascual and Ronen 2015).

Considering the rather poor therapeutic outcome associated with the ketogenic diet, new methods for modulating and restoring Glutl in many conditions would be highly desirable. SUMMARY OF THE INVENTION

The present invention is based at least in part on the discovery of a natural antisense transcript (NAT) or long non-coding RNA (IncRNA) to the human SLC2A1 (Glutl) gene. This NAT or IncRNA regulates expression of the SLC2A1 (Glutl) gene.

In certain embodiments, the present invention provides a nucleic acid comprising SLC2A1-NAT or Glutl -NAT comprising the nucleotide sequence SEQ ID NO: 1.

In certain embodiments, the present invention relates to a nucleic acid comprising SLC2A1 -NAT or Glutl -NAT comprising the nucleotide sequence SEQ ID NO: 2.

The SLC2A1-NAT nucleic acids of the invention include those comprising the sequences SEQ ID NOs: 1 and 2 and variants and fragments thereof. The nucleic acids can be RNA or DNA. The DNA can be cDNA.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a nucleic acid comprising SLC2A1-NAT or Glutl-NAT described herein. In certain embodiments, the composition can further comprise one or more of pharmaceutically acceptable excipient, ligand, conjugate, vector, lipid, liposome, carrier, adjuvant or diluent.

In yet additional embodiments, the nucleic acid is provided in a recombinant adeno- associated vector (rAAV). In additional embodiments, the rAAV vector further comprises a chicken Beta-actin promoter and wherein the rAAV is capable of crossing the blood-brain barrier (BBB). In yet additional embodiments, the AAV is AAV8 or AAV9.

In certain embodiments, the present invention relates to kits comprising the nucleic acids and compositions described herein.

In further embodiments, the present invention relates to a method of restoring Glutl transport in the blood brain barrier (BBB) of a subject, comprising administering to the subject a therapeutically effective amount of a SLC2A1-NAT nucleic acid described herein or a pharmaceutical composition comprising a SLC2A1-NAT nucleic acid described herein.

In yet further embodiments, the invention provides for treating Glutl deficiency syndrome in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SLC2A1-NAT nucleic acid described herein or a pharmaceutical composition comprising a SLC2A1-NAT nucleic acid described herein.

In still further embodiments, the present invention provides for a method of alleviating in a subject, at least one of the symptoms associated with Glutl deficiency syndrome selected from the group consisting of hypoglycorrhachia, acquired microcephaly, ataxic and dystonic motor dysfunction, comprising administering to the subject a therapeutically effective amount of a SLC2A1-NAT nucleic acid described herein or a pharmaceutical composition comprising a SLC2A1-NAT nucleic acid described herein.

In a further embodiment, the present invention provides for a method of treating Glutl related condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a SLC2A1-NAT nucleic acid described herein or a pharmaceutical composition comprising a SLC2A1-NAT nucleic acid described herein.

The conditions include but are not limited to cancer, diabetes, Alzheimer's disease, and retinitis pigmentosa. BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted in drawings certain embodiments of the invention. However, the invention is not limited to the precise arrangements and instrumentalities of the embodiments depicted in the drawings.

Figure 1 are graphs and images showing Glutl repletion averts Glutl DS in model mice. Figure 1A shows brain Glutl transcript levels, assessed by Q-PCR, are increased in AAV9-Glutl -treated mice relative to concentrations in vehicle-treated cohorts. Figure IB shows Western blot analysis demonstrating a corresponding increase in Glutl protein. Figure 1C shows that restoring Glutl to mutants also mitigates hypoglycorrhachia as determined by a relative increase in CSF/blood glucose levels in mice treated with AAV9-Glutl. Figure ID are results of a PET imaging study depicting a normalization of signal in the brain of a mutant animal restored for the Glutl protein. Figure IE shows EEG analysis of mutant animals treated with vehicle or AAV9-Glutl as compared to wild- type mice. This analysis shows Glutl repletion also results in amelioration of seizure activity (denoted by arrows) in mutant animals. Note: *, *** P < 0.05, P < 0.001 respectively, one-way ANOVA, n > 5 of each genotype for results in Figures 1A and 1C. Data=Mean SEM.

Figure 2 are schematics, graphs and blots showing the novel natural antisense transcript regulates Glutl expression. Figure 2A shows the genomic organization of chromosome 1 locus harboring the Glutl gene and the IncRNA. Depicted are the two IncRNA alternatively spliced isoforms (Glutl-NATl and Glutl-NAT2). Figure 2B shows the expression of Glut- NAT 1 in various human tissue including brain tissue. Figure 2C shows the expression of Glutl-NAT2 in various human tissue including brain tissue. Figure 2D shows the expression of NAT1, and the Glutl gene in various tissues. Figure 2E is a graph showing the knockdown of the IncRNA using siRNAs common to the two isoforms results in concordant decrease of Glutl transcript. Figure 2F is a Western blot showing the knockdown of the IncRNA using siRNAs common to the two isoforms results in concordant decrease of Glutl protein. Note: The two Glutl bands in the blots correspond to differentially glycosylated proteins. The loading control is the ubiquitously expressed SMN protein. Figure 2G is a graph showing the overexpression of the IncRNA (Glutl-NAT2) results in an increase in Glutl transcript. Figure 2H is a Western blot showing the overexpression of the IncRNA (Glutl-NAT2) results in an increase in increase in Glutl protein. Note: The two Glutl bands in the blots correspond to differentially glycosylated proteins. The loading control is the ubiquitously expressed SMN protein. Figure 21 is a graph of the quantification of Glutl transcript (relative mRNA expression of Glutl versus β-actin) following domain- specific over-expression of the IncRNA (Glutl-NAT2) showing that exons 1-3 are as efficient as the full-length (FL) transcript at enhancing Glutl expression. Also note: *, ** P < 0.05 and 0.01, one-way ANOVA, n >3 samples.

Figure 3 is a graph showing the results of luciferase activity in HEK293 cells transfected with the indicated constructs, pGL4.1 promoter alone, pGL4.1 promoter + Glutl promoter, and pGL4.1 promoter + Glutl promoter + IncRNA.

Figure 4 shows a schematic and gel illustrating DsiRNA's design strategy targeting SLC2A1 LncRNA.

Figure 5 are gels and graphs illustrating SLC2A1 IncRNA expression in human fibroblast cells knocked down by DsiRNA. Figure 5A are representative blots and Figure 5B is a graph quantifying the amount of SLC2A1 RNA expression relative to a housekeeping gene (beta-actin).

Figure 6 are gels and graphs showing that human Glutl expression was decreased after SLC2A1 IncRNA was knocked down by DsiRNA. Figure 6A are representative blots and Figure 6B is a graph quantifying the human Glutl expression relative to a housekeeping gene (beta-actin).

Figure 7 is a schematic and gel of the hGlutl locus. Figure 7A shows the genomic structure of the BAC RP11-12501 depicting the Clal fragment harboring hGlutl and the NAT. Figure 7B is a gel of restriction digests of the BAC and the isolated ~79kb Clal fragment containing hGlutl and the NAT.

Figure 8 is a flow diagram of the breeding protocol to obtain transgenic mice to test the effects of the Glutl -NAT on Glutl expression in the intact organism.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms of words such as

"a," "an," and "the," include their corresponding plural references unless the context clearly dictates otherwise. The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

"Activation," "stimulation," and "treatment," as it applies to cells or to receptors, may have the same meaning, e.g., activation, stimulation, or treatment of a cell or receptor with a ligand, unless indicated otherwise by the context or explicitly. "Ligand" encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogues, muteins, and binding compounds derived from antibodies. "Ligand" also encompasses small molecules, e.g. , peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cell activation as regulated by internal mechanisms as well as by external or environmental factors. "Response," e.g., of a cell, tissue, organ, or organism, encompasses a change in biochemical or physiological behavior, e.g., concentration, density, adhesion, or migration within a biological compartment, rate of gene expression, or state of differentiation, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming.

"Activity" of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity; to the ability to stimulate gene expression or cell signaling, differentiation, or maturation; to antigenic activity, to the modulation of activities of other molecules, and the like. "Activity" of a molecule may also refer to activity in modulating or maintaining cell-to-cell interactions, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. "Activity" can also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological activity]/[mg protein], concentration in a biological compartment, or the like. "Activity" may refer to modulation of components of the innate or the adaptive immune systems.

"Administration" and "treatment," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human, including a human patient.

"Treat" or "treating" means to administer a therapeutic agent, such as a composition containing any of the nucleic acid or rAAV constructs or compositions of the present invention, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease or being at elevated at risk of acquiring a disease, for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom (also referred to as the "therapeutically effective amount") may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject.

The term "in need thereof would be a subject known or suspected of having or being at risk of a disease or disorder characterized by a Glutl deficiency.

"Isolated nucleic acid molecule" means a DNA or RNA of genomic, mRNA, cDNA, or synthetic origin or some combination thereof which is not associated with all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature or is linked to a polynucleotide to which it is not linked in nature. For purposes of this disclosure, it should be understood that "a nucleic acid molecule comprising" a particular nucleotide sequence does not encompass intact chromosomes. Isolated nucleic acid molecules "comprising" specified nucleic acid sequences may include, in addition to the specified sequences, coding sequences for up to ten or even up to twenty or more other proteins or portions or fragments thereof or may include operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences, and/or may include vector sequences. The term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, or virion, which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.

The phrase "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.

The phrases "operatively positioned," "operatively linked," "under control," or "under transcriptional control" means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene.

The term "expression vector or construct" means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or inhibitory RNA (e.g., shRNA, miRNA) from a transcribed gene.

A "host cell" refers to any cell that harbors, or is capable of harboring, a substance of interest. Often a host cell is a mammalian cell. A host cell may be used as a recipient of an AAV helper construct, an AAV minigene plasmid, an accessory function vector, or other transfer DNA associated with the production of recombinant AAVs or other expression vectors. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell" as used herein may refer to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

With respect to transfected host cells, the term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art.

With respect to cells, the term "isolated" refers to a cell that has been isolated from its natural environment (e.g., from a tissue or subject). The term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. As used herein, the terms "recombinant cell" refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

General Methods

Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2 ^nd Edition, 2001 3 ^rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 ^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.l- 4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

Abbreviations

IncRNA: Long non-coding RNA

NAT: natural antisense transcript SLC2A1-NAT, Glut-NAT, 5LC2A-lncRNA: long-noncoding RNA or natural antisense transcript that regulates the expression of SLC2A1 or Glutl gene. These terms are used interchangeable throughout the application to denote this novel nucleic acid (SEQ ID NO: 1) of the present invention.

Glutl : Glucose transporter 1, also known as solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1), is a uniporter protein that in humans is encoded by the SLC2A1 gene. Glutl facilitates the transport of glucose across the plasma membranes of mammalian cells. Glutl was the first glucose transporter to be characterized. Glutl 1 is highly conserved with the human Glutl protein (hGlutl) (Accession No: NP_006507.2) and mouse Glutl protein (mGlutl) (Accession No: NP_035530.2) sharing 98 % homology. Glutl exhibits 40% homology with other Gluts.

SLC2A1: Gene encoding the human glucose transporter 1 (hGlutl). Human SLC2A1

(Accession No: NG_008232.1 ; gene ID - 6513).

Slc2al: Gene encoding mouse Glutl (mGlutl) (Accession No: Genomic #:

NC_000070.6; gene ID - 20525).

GLUT1 DS: Glutl deficiency syndrome

AAV: adeno- associated virus

rAAV recombinant adeno-associated virus or viral vector

BBB : blood brain barrier

The Natural Antisense Transcript (NAT) to the Human SLC2A1 (Glutl) Gene Regulates Expression of the Gene

Mutations in the SLC2A1 (also referred to as Glutl) gene result in Glutl deficiency syndrome (Glutl DS), a rare but devastating neurodevelopmental disorder (DeVivo et al. 1991). The wild-type Glutl protein is widely expressed. However, its predominant cellular site of action appears to be the endothelial cells of the brain micro-vessels where it functions in the facilitated transport of glucose across the blood-brain barrier. Reduced levels or loss of the protein results in a complex phenotype whose signature features include hypoglycorrhachia, developmental delay and acquired microcephaly. Patients also exhibit a motor phenotype that is both ataxic as well as dystonic. Mice haploinsufficient for the slc2al gene exhibit many of the features of the human disease. A homozygous knockout of the murine slclal gene is embryonic lethal. Haplo-insufficient animal models exhibit many aspects of the human disease.

Considering the rather poor therapeutic outcome associated with the ketogenic diet, novel treatment options for the disease have been investigated. The inventors have previously demonstrated that augmenting Glutl levels using an AAV9-Glutl construct is remarkably effective in averting disease onset in Glutl DS model mice (Tang et al. 2017). See also Example 1, Figure 1. Restoring Glutl in a timely fashion therefore appears to constitute a safe, effective and reliable therapy for Glutl DS. Such repletion can be conveniently effected using viral vectors. Equally feasible is to enhance expression from the functional allele in diseases of haploinsufficiency such as Glutl DS. This latter approach has traditionally involved the use of small molecules that relieve transcriptional or translational repression. However, strategies employing small molecules often suffer the prospect of unwanted off- target effects. A more targeted approach involves non-coding RNAs, particularly IncRNAs (long non-coding RNAs), which have recently emerged as important regulators of gene activity (Marques and Ponting 2009).

The present invention is based at least in part on the discovery of a natural antisense transcript (NAT) to the human SLC2A1 (Glutl) gene. Unlike the ketogenic diet, the invention is based on specifically targeting the Glutl gene and therefore relies on specifically altering Glutl levels. The NAT is a potent regulator of Glutl; inhibiting NAT expression results in a fall in Glutl levels, while increasing NAT levels raises Glutl levels. This suggests that levels of the transcript can be modulated as a means of controlling Glutl concentrations.

In the process of investigating the Glutl promoter region, a novel IncRNA associated with the gene was found. 3' RACE analysis demonstrated that it is alternatively spliced, the two resulting transcripts differing from one another in the number of exons each harbors. The transcripts each overlap part of exon 1 of the Glutl (SLC2A1) gene but are expressed antisense to the protein coding gene. Accordingly, these were termed the IncRNA, SLC2A1- NAT (Natural Antisense Transcript) and its two isoforms SLC2A1-NAT1 and SLC2A1-NAT2 (Figure 2A). SLC2A1-NAT1 (NAT!) and SLC2A1-NAT2 (NAT2) share exons 1 and 2, but whereas exon 3 in NAT1 is large (approximately 2.5kb), its counterpart in NAT2 is much smaller (215bp) and followed by a fourth exon of similar size. Thus, the cDNAs of the two transcripts are approximately 3.1kb (NAT1) and approximately l. lkb (NAT2), and the corresponding genomic regions approximately 14kb and 23kb respectively. Importantly, bioinformatics analysis indicated that neither NAT has any protein coding potential. This, combined with established criteria pertaining to ncRNA length best defines them as bona fide IncRNAs (ncRNAs > 200bp in size).

Additional data pertinent to the IncRNA is as follows (see Example 2). First, bioinformatics analysis indicated that the NATs identified are restricted to primates and absent in rodents. This is consistent with the notion that IncRNAs are less evolutionarily conserved than are their protein coding mRNA counterparts (Belanger et al. 2011). Second, each NAT is widely expressed in humans. However, brain tissue and, particularly, cerebral cortex, expressed especially high levels (greater than 25 times that detected in fibroblasts) of the two (Figures 2B and C). Finally, relative to Glutl levels, the IncRNA as assessed by NATl, is more modestly expressed (Figure 2D). This data conformed to investigations of other IncRNAs and their expression relative to those of mRNAs (DeGiorgis and Veggiotti 2013)

Natural antisense transcripts have emerged as important regulators of their overlapping cognate protein coding genes (Brockmann 2009; Arsoy et al. 2012; Klepper and Leiendecker 2013). Accordingly, the effects of modulating the expression of SLC2A1-NAT on Glutl levels was examined. First siRNAs to suppress the two NATs in fibroblasts were used and it was found that Glutl transcripts as well as protein fell correspondingly (Figures 2E and 2F). This was confirmed using three different siRNAs in multiple fibroblast lines as well as in primary brain endothelial cells.

Next, SLC2A1-NAT2 was over-expressed in fibroblasts. In this instance, it was found that Glutl transcripts and protein were increased in a significant manner (Figures 2G and 2H). Finally, it was inquired if specific domains (exons) of NAT2 are responsible for mediating the increase in Glutl expression. It was found that exons 1-3 are as efficient as the full-length (exons 1-4) IncRNA in driving Glutl expression, whereas exon 1 or exons 1-2 fail to have any appreciable effect on Glutl levels (Figure 21). One interpretation of this finding is that the combination of exons 2 and 3 is critical in regulating/increasing Glutl expression.

Further evidence is shown herein that the natural antisense transcript regulates Glutl expression. The antisense transcript can enhance Glutl expression (Example 3; Figure 3). Accordingly, the knockdown of the antisense transcript decreased Glutl expression (Example 4; Figure 6). SLC2A1-NAT Nucleic Acids

These results illustrated that a novel long non-coding RNA (IncRNA) or natural antisense transcript (NAT) plays an important role in regulating the expression of the SLC2A1 or Glutl gene and its associated pathways.

Thus, the present invention provides for this natural antisense transcript denoted

SLC2A1-NAT nucleic acid. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is cDNA.

In some embodiments, the SLC2A1 -NAT is SLC2A1-NAT2.

SLC2A1-NAT comprises the nucleotide sequence SEQ ID NO: 1.

The present invention also provides for a nucleic acid comprising exons 1-3 of

SLC2A1-NAT comprising the sequence SEQ ID NO: 2. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is cDNA. As shown herein, the exons 1-3 are sufficient to regulate expression of the SLC2A1 gene.

For reference, exon 1 comprises nucleotides 1-529 of SEQ ID NOs: 1 and 2; exon 2 comprises nucleotides 530-675 of SEQ ID NOs: 1 and 2; and exon 3 comprises nucleotides

676-890 of SEQ ID NOs: 1 and 2.

The present invention also includes variants and fragments of the SLC2A1-NAT nucleic acids, including variants of SEQ ID NOs: 1 and 2, having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, and at least

99% identity to any of SEQ ID NOs: 1 and 2.

The present invention also provides for recombinant constructs comprising the

SLC2A1-NAT RNA or the DNA encoding the SLC2A1-NAT, and a vector that can be expressed in a transformed host cell. The present invention also includes the host cells transformed with the recombinant construct comprising the SLC2A1-NAT RNA or the DNA encoding the SLC2A1-NAT RNA, and a vector.

Expression vectors may be useful, comprising various nucleic acids, wherein the nucleic acid is operably linked to control sequences that are recognized by a host cell when the host cell is transfected with the vector.

Also provided are the virions comprising recombinant AAV and SLC2A1-NAT nucleic acid described herein, which serve to increase expression of Glut-1, and which may, in certain embodiments be under the direction of chicken- -actin promoter and a CMV enhancer. These sequences may be packaged with the AAV9 capsid to form the virions that are therapeutic to Glut-1 deficiency in the present invention. As shown in Example 1, rAAV-Glutl constructs were delivered to the appropriate tissue in mutant mice and restored Glutl expression and function. Thus it is expected that such constructs of rAAV and the SLC2A1-NAT nucleic acid described herein would be delivered to the appropriate tissue and restore Glut 1 expression and function.

Use of Glut-NAT as a Treatment for Glutl Deficiency Syndrome

As shown herein, SLC2A1-NAT increases the expression of Glutl. Thus the invention also provides for treating Glutl deficiency syndrome in a subject in need thereof, by administering a SLC2A1 -NAT nucleic acid as described herein.

The invention also provides for treating Glutl deficiency syndrome in a subject in need thereof, by administering a therapeutically effective amount of a pharmaceutical composition comprising a SLC2A1-NAT nucleic acid as described herein.

The present invention also provides for a method of alleviating in a subject, at least one of the symptoms associated with Glutl deficiency syndrome selected from the group consisting of hypoglycorrhachia, acquired microcephaly, ataxic and dystonic motor dysfunction, by administering a SLC2A1-NAT nucleic acid described herein.

The present invention also provides for a method of alleviating in a subject, at least one of the symptoms associated with Glutl deficiency syndrome selected from the group consisting of hypoglycorrhachia, acquired microcephaly, ataxic and dystonic motor dysfunction, by administering a therapeutically effective amount of a pharmaceutical composition comprising a SLC2A1-NAT nucleic acid described herein.

The present invention further provides for a method of restoring Glutl transport in the blood brain barrier (BBB) of a subject, by administering SLC2A1-NAT nucleic acid as described herein.

The present invention also provides for a method of restoring Glutl transport in the blood brain barrier (BBB) of a subject, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a SLC2A1-NAT nucleic acid described herein.

The SLC2A1-NAT nucleic acids for use in the methods of the invention include those comprising the sequences SEQ ID NOs: 1 and 2 and variants and fragments thereof. The nucleic acids can be RNA or DNA. The DNA can be cDNA.

The pharmaceutical compositions for use in the methods of the invention can further comprise one or more pharmaceutically acceptable excipient, ligand, a conjugate, a vector, a lipid, a nanoparticle, a liposome, a carrier, an adjuvant or a diluent. In yet additional embodiments, the nucleic acid is provided in a recombinant adeno- associated vector (rAAV). In additional embodiments, the rAAV vector further comprises a chicken Beta-actin promoter and wherein the rAAV is capable of crossing the blood-brain barrier (BBB). In yet additional embodiments, the AAV is AAV8 or AAV9.

Recombinant AAV Vectors

"Recombinant AAV (rAAV) vectors" described herein may be composed of, at a minimum, a nucleic acid comprising SLC2A1-NAT (SEQ ID NO: 1), variants and fragments thereof, and optionally useful regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).

Alternatively, the rAAV vectors described herein may be composed of, at a minimum, a nucleic acid comprising exons 1-3 of SLC2A1-NAT (SEQ ID NO: 2), variants and fragments thereof, and optionally useful regulatory sequences, and 5' and 3' AAV inverted terminal repeats (ITRs).

In certain embodiments, this recombinant AAV vector is packaged into a capsid protein and delivered to a selected target cell, and the nucleic acid comprising SLC2A1-NAT, and variants and fragments thereof, serves to increase expression of Glutl and provide therapeutic benefits to a Glutl deficient patient.

The AAV sequences of the vector may comprise the cis-acting 5' and 3' inverted terminal repeat sequences (See, e.g., B. J. Carter, in "Handbook of Parvoviruses", ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are typically about 145 bp in length. Preferably, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. (See, e.g., texts such as Sambrook et al, "Molecular Cloning. A Laboratory Manual", 2d ed., Cold Spring harbor Laboratory, New York (1989); and K. Fisher et al, J. Virol, 70:520 532 (1996)). An example of such a molecule is a "cis-acting" plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5' and 3' AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including presently identified mammalian AAV types.

In addition to the elements identified above for recombinant AAV vectors, the vector may also include conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and/or expression in a cell transfected with the plasmid vector or infected with the virus produced by the invention. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters which are native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.

As used herein, a nucleic acid sequence and regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. Similarly two or more coding regions are operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame. In some embodiments, operably linked coding sequences yield a fusion protein. In some embodiments, operably linked coding sequences yield a functional RNA (e.g., shRNA, miRNA).

For nucleic acids encoding proteins, a polyadenylation sequence generally is inserted following the transgene sequences and before the 3' AAV ITR sequence. An rAAV construct useful in the present invention may also contain an intron, desirably located between the promoter/enhancer sequence and the transgene. One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence. Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence is used to produce more than one polypeptide from a single gene transcript. An IRES sequence would be used to produce a protein that contain more than one polypeptide chains. Selection of these and other common vector elements are conventional and many such sequences are available (see, e.g., Sambrook et al, and references cited therein at, for example, pages 3.18 3.26 and 16.17 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989). In some circumstances, a Foot and Mouth Disease Virus 2A sequence may be included in a polyprotein; this is a small peptide (approximately 18 amino acids in length) that has been shown to mediate the cleavage of polyproteins. The cleavage activity of the 2A sequence has previously been demonstrated in artificial systems including plasmids and gene therapy vectors (AAV and retroviruses) (Ryan et al, EMBO, 1994; 4: 928-933; Mattion et al , J Virology, November 1996; p. 8124-8127; Furler et al, Gene Therapy, 2001 ; 8: 864- 873; and Halpin et al, The Plant Journal, 1999; 4: 453-459; de Felipe et al, Gene Therapy, 1999; 6: 198-208; de Felipe et al, Human Gene Therapy, 2000; 11 : 1921-1931.; and Klump et al, Gene Therapy, 2001 ; 8: 811-817).

The precise nature of the regulatory sequences needed for gene expression in host cells may vary between species, tissues or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer elements, and the like. Especially, such 5' non-transcribed regulatory sequences will include a promoter region that includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors may optionally include 5' leader or signal sequences.

Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41 :521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the 13-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clontech and Ariad. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al, Proc. Natl. Acad. Sci. USA, 93:3346- 3351 (1996)), the tetracycline-repressible system (Gossen et al. (1992) Proc. Natl. Acad. Sci. USA, 89:5547-5551), the tetracycline-inducible system (Gossen et al. (1995) Science, 268: 1766-1769, see also Harvey et al. (1998) Curr. Opin. Chem. Biol, 2:512-518), the RU486-inducible system (Wang et al. (1997) Nat. Biotech., 15:239-243 and Wang et al. (1997) Gene Ther., 4:432-441) and the rapamycin-inducible system (Magari et al. (1997) /. Clin. Invest., 100:2865-2872). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

In another embodiment, the native promoter, or fragment thereof, for the transgene will be used. The native promoter may be preferred when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene must be regulated temporally or developmental^, or in a tissue- specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

In some embodiments, the regulatory sequences impart tissue- specific gene expression capabilities. In some cases, the tissue-specific regulatory sequences bind tissue- specific transcription factors that induce transcription in a tissue specific manner. Such tissue- specific regulatory sequences {e.g., promoters, enhancers) are well known in the art. Exemplary tissue-specific regulatory sequences include but are not limited to the following tissue specific promoters: neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al. (1993) Cell. Mol. Neurobiol., 13:503-15), neurofilament light-chain gene promoter (Piccioli et al. (1991) Proc. Natl. Acad. Sci. USA, 88:5611-5), and the neuron- specific vgf gene promoter (Piccioli et al. (1995) Neuron, 15:373-84). In some embodiments, the tissue-specific promoter is a promoter of a gene selected from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), and ionized calcium-binding adapter molecule 1 (Iba-1). In some embodiments, the promoter is a chicken Beta-actin promoter.

Methods for obtaining recombinant AAVs having a desired capsid protein have been described (See, for example, US 2003/0138772, the contents of which are incorporated herein by reference in their entirety). A number of different AAV capsid proteins have been described, for example, those disclosed in Gao et al. (2004) /. Virol, 78(12):6381-6388; Gao et al. (2004) Proc Natl Acad Sci USA, 100(10):6081-6086. For the desired packaging of the presently described constructs and methods, the AAV9 vector and capsid is preferred. However, it is noted that other suitable AAVs such as rAAVrh.8 and rAAVrh.lO, or other similar vectors may be adapted for use in the present invention. Typically the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.

The components to be cultured in the host cell to package a rAAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. Most suitably, such a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters.

The recombinant AAV vector, rep sequences, cap sequences, and helper functions for producing the rAAV may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. See, e.g., Fisher et al. (1993) /. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the triple transfection method (e.g., as described in detail in U.S. Patent No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the "AAV helper function" sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (i.e. , AAV virions containing functional rep and cap genes). Non-limiting examples of vectors suitable for use with the present invention include pHLP19, described in U.S. Patent No. 6,001,650 and pRep6cap6 vector, described in U.S. Patent No. 6,156,303. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.

Pharmaceutical Compositions and Administration

Compositions and Methods for Administration of the SLC2A1-NAT Nucleic

Acids

Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to deliver the nucleic acid comprising SLC2A1 -NAT nucleic acid described herein.

The formation and use of liposomes is generally known to those of skill in the art.

Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Patent No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587).

Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. In addition, liposomes are free of the DNA length constraints that are typical of viral-based delivery systems. Liposomes have been used effectively to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals. In addition, several successful clinical trials examining the effectiveness of liposome-mediated drug delivery have been completed.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 μπι. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500A, containing an aqueous solution in the core.

Alternatively, nanocapsule or nanoparticle formulations may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. Nanoparticles can be used to transport drugs through the BBB when administered intravenously as well as the factors that influence its transportation.

NPs are colloidal carriers that can have a natural or synthetic origin and can vary from 1 to 1000 nm in size. Synthetic NPs may be prepared from polymeric materials such as poly(ethylenimine) (PEI), poly(alkylcyanoacrylates), poly(amidoamine) dendrimers (PAMAM), poly(e-caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), polyesters (poly(lactic acid) (PLA), or from inorganic materials such as gold, silicon dioxide (silica), among others. These carriers can transport drugs by adsorbing, entrapping or bounding covalently to them. Natural NPs are produced from natural polymers, such as polysaccharides (chitosan and alginate), amino acids (poly (lysine), poly(aspartic acid) (PASA)), or proteins (gelatin and albumin). Natural NPs have the advantage of providing biological signals to interact with specific receptors/transporters expressed by endothelial cells.

A number of ligands have been conjugated to NPs to facilitate BBB penetration. Such molecules can be grouped into four different types: (i) ligands that mediate the adsorption of proteins from the bloodstream that interact directly with BBB receptors or transporters; (ii) ligands that have direct interaction per se with BBB receptors or transporters; (iii) ligands that increase charge and hydrophobicity; and (iv) ligands that improve blood circulation time (e.g. PEG).

Other methods for assisting the NPs to cross the blood-brain barrier would include but are not limited to receptor mediated transport, transporter mediated transport, absorptive mediated transport, and cell penetrating transport. See generally Gao 2016.

Mammalian virus vectors that can be used to deliver the RNA include oncoretroviral vectors, adenovirus vectors, Herpes simplex virus vectors, and lentiviruses.

In particular, HSV vectors are tropic for the central nervous system (CNS) and can establish lifelong latent infections in neurons and thus, are a preferred vector for use in this invention.

Recombinant AAV Compositions and Methods of Administration

The rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. The rAAV, preferably suspended in a physiologically compatible carrier (e.g., in a composition), may be administered to a subject, e.g., a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate. In certain embodiments, compositions may comprise a rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes).

Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the rAAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

Optionally, the compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

The dose of rAAV virions required to achieve a desired effect or "therapeutic effect," e.g., the units of dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: the route of rAAV administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine a rAAV virion dose range to treat a subject having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. An effective amount of the rAAV is generally in the range of from about 10 μΐ to about 100 ml of solution containing from about 10 ⁹ to 10 ¹⁶ genome copies per subject. Other volumes of solution may be used. The volume used will typically depend, among other things, on the size of the subject, the dose of the rAAV, and the route of administration. For example, for intrathecal or intracerebral administration a volume in range of 1 μΐ to 10 μΐ or 10 μΐ to 100 μΐ may be used. For intravenous administration a volume in range of 10 μΐ to 100 μΐ, 100 μΐ to 1 ml, 1 ml to 10 ml, or more may be used. In some cases, a dosage between about 10 ¹⁰ to 10 ¹² rAAV genome copies per subject is appropriate. In certain embodiments, 10 ¹² rAAV genome copies per subject is effective to target CNS tissues. In some embodiments the rAAV is administered at a dose of 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , or 10 ¹⁵ genome copies per subject. In some embodiments the rAAV is administered at a dose of 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , or 10 ¹⁴ genome copies per kg. In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., about 10 ¹³ GC/ml or more). Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pll adjustment, salt concentration adjustment, etc. (See, e.g., Wright et al. (2005) Molecular Therapy 12:171- 178.)

Formulation of pharmaceutically-acceptable excipients and carrier solutions is well- known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Typically, these formulations may contain at least about 0.1% of the active ingredient or more, although the percentage of the active ingredient(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active ingredient in each therapeutically-useful composition may be prepared is such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is 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 a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity may 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 dispersion and by the use of surfactants. The 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 in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

Sterile injectable solutions are prepared by incorporating the active rAAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

The rAAV compositions disclosed herein may also be formulated in a neutral or salt form. Pharmaceutically-acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the rAAV compositions of the present invention into suitable host cells. In particular, the rAAV vector delivered components may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the rAAV compositions to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Patent No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Patent. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations, transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Patent No. 5,697,899).

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the compositions of the present invention, including the SLC2A1-NAT nucleic acid described herein in combination with one or more of a ligand, a conjugate, a vector, a lipid, a nanoparticle, a liposome, an adjuvant or a diluent may be further admixed with a pharmaceutically acceptable carrier or excipient. See, e.g. , Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

Formulations of therapeutic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Oilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

Toxicity and therapeutic efficacy of the therapeutic compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDso (the dose lethal to 50% of the population) and the EDso (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LDso/ EDso). In particular aspects, therapeutic compositions exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial.

In particular embodiments, the composition or therapeutic can be administered by an invasive route such as by injection. In further embodiments of the invention, the composition, therapeutic, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra- articularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery. Administration by noninvasive routes (e.g. , orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention.

In order to overcome any issue of the pharmacological agents crossing the blood/brain barrier, intrathecal administration is a further preferred form of administration. Intrathecal administration involves injection of the drug into the spinal canal, more specifically the subarachnoid space such that it reaches the cerebrospinal fluid. This method is commonly used for spinal anesthesia, chemotherapy, and pain medication. Intrathecal administration can be performed by lumbar puncture (bolus injection) or by a port-catheter system (bolus or infusion). The catheter is most commonly inserted between the laminae of the lumbar vertebrae and the tip is threaded up the thecal space to the desired level (generally L3-L4). Intrathecal formulations most commonly use water, and saline as excipients but EDTA and lipids have been used as well.

Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.

The pharmaceutical compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851 ; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of directly into the desired target site, often in a depot or sustained release formulation. Furthermore, one may administer the composition in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, the brain. The liposomes will be targeted to and taken up selectively by the desired tissue.

The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic composition, the level of symptoms, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic composition to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic composition and the severity of the condition being treated.

Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g., the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent.

As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of a nucleic acid SLC2A1-NAT based composition of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.

Kits

The present invention also provides kits comprising the components of the combinations of the invention in kit form. A kit of the present invention includes one or more components including, but not limited to, any SLC2A1-NAT nucleic acid described herein, in association with one or more additional components including, but not limited to a pharmaceutically acceptable ligand, a conjugate, a vector, a lipid, a nanoparticle, a liposome, an adjuvant, a diluent, carrier or excipient.

In one embodiment, a kit includes a SLC2A1-NAT nucleic acid, a SLC2A1-NAT nucleic acid based compound/composition of the invention or a pharmaceutical composition thereof in one container {e.g., in a sterile glass or plastic vial).

If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices as discussed above.

The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer/distributor information and patent information.

EXAMPLES

The present invention may be better understood by reference to the following non- limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention. Example 1- Glutl repletion averts Glutl DS in a mouse model of the disease

It has been demonstrated that restoring Glutl to Glutl DS model mice by means of an AAV9 vector harboring the Glutl gene was remarkably effective in thwarting disease (Tang et al. 2017). Briefly, the study demonstrated that systemic delivery of an AAV9-Glutl construct to PND3 mutant mice raised Glutl levels, improves motor performance, mitigates the hypoglycorrhachia (low cerebrospinal fluid - CSF - glucose) characteristically observed in the disease, enhanced the ability of the brain to absorb glucose, prevented the micrencephaly (reduced brain size) typical of Glutl DS and ameliorated the seizures that the mice suffer as a consequence of Glutl haploinsufficiency (Figure 1).

The study also revealed that low Glutl levels result in a profound diminution of the brain microvasculature. This last defect was also corrected upon Glutl repletion. Augmenting Glutl levels at two weeks, a time point representing the early symptomatic phase of the disease remained effective, albeit not to the extent observed when treatment was initiated at PND3 (PND3=post-natal day 3). In contrast, restoring Glutl during adulthood when the mutant mice were fully symptomatic failed to provide benefit. Nevertheless, these results showed that Glutl repletion when effected in a timely manner could translate into an effective and reliable means of treating Glutl DS. Restoring Glutl levels from the intact Glutl allele in Glutl DS patients, by targeting a regulatory non-coding RNA is expected to have a similar effect. Example 2- The expression of the Glutl gene is regulated by a novel natural antisense transcript

In the process of testing the AAV9 constructs in human fibroblasts and while simultaneously investigating the cause of a Glutl DS-like patient with no detectable mutations in the Glutl coding sequence, a natural antisense transcript partially overlapping exon 1 of Glutl and extending into the promoter region of the gene was discovered. 3 'RACE experiments indicated that there are, in fact, two alternatively spliced transcripts that differ in the number of exons that each contains (Figure 2A). Bioinformatic analysis suggested that neither transcript is capable of coding for a protein. This, combined with the size of the transcripts (greater than 200bp) and their orientation (antisense) relative to Glutl define them as long non-coding RNAs (IncRNAs). In keeping with the tendency of IncRNAs to be less evolutionarily conserved than protein coding genes (Marques and Ponting 2009; Johnsson et al. 2014) bioinformatic and follow-up analysis indicated that the transcripts are unique to primates and absent in mice. The larger, approximately 3.1kb transcript was denoted Glutl - NAT1 (or 5LC2A7-NAT1) and the smaller, approximately l. lkb one Glutl- NAT2 (or SLC2A1 -NAT2(.

Experiments to determine the expression profiles of the NATs demonstrated that the transcripts are widely expressed but especially abundant in the cerebral cortex (Figures 2B and 2C). Moreover, Glutl -NAT 1 was the predominant transcript, amounting to approximately 9 times as much Glutl-NAT2 in the cortex. Still, consistent with the lower abundance of IncRNAs relative to protein coding transcripts (Ravasi et al. 2006), it was found that Glutl -NAT 1 is not as highly expressed as Glutl (Figure 2D).

Long non-coding RNAs in close proximity to genes have, in many instances, been found to regulate the expression of the genes (Orom et al. 2010; Kim et al. 2010; Broskdorrf et al. 1992). Accordingly, it was inquired if the Glutl- NATs similarly functioned to regulate Glutl. First, the effect of inhibiting the NATs was determined by using siRNAs common to both NATs. It was found that lowering NAT expression had a corresponding effect on Glutl transcripts and protein (Figure 2E and 2F) - in fibroblasts as well as brain endothelial cells.

This study was extended to the effect of over-expressing Glutl -NAT2. Consistent with concordant regulation, it was found that raising Glutl -NAT2 correspondingly increased Glutl (Figures 2G and 2H).

Finally, it was investigated whether the truncated versions of Glutl-NAT2 recapitulated the effect of the full-length (FL) transcript in the over-expression studies. It was found that exons 1-3 of Glutl -NAT2 were just as effective as the FL IncRNA in increasing Glutl, while exon 1 or exons 1-2 failed to yield any effect (Figure 21). These results revealed a novel, alternatively spliced Glutl natural antisense transcript that concordantly regulates Glutl in cultured cells. Example 3- The Effects of SLCAl-IncRNA on Glut Activity

A promoter fragment containing 6kb of the Glutl upstream sequence was cloned into a luciferase-containing vector (pGL4.1). In parallel, exons 1-3 of the antisense transcript was closed into an expression vector. The 6kb Glutl promoter plasmid was then transfected into HEK-293 cells alone or with the plasmid containing the antisense transcript. The results showed, first that the 6kb Glutl promoter fragment was capable of driving strong lucif erase expression and, second, that such expression was enhanced approximately 40% in the presence of the antisense transcript (Figure 3). These results provided strong evidence, in vitro, that the antisense transcript was indeed able to regulate (enhance) expression from the promoter of the Glutl gene and showed that the transcript is one novel means of augmenting Glutl gene activity for the purpose of treating conditions such as Glutl deficiency syndrome that result from low Glutl levels. Additionally, the results showed that the exons 1-3 of Glutl-NAT was sufficient enough to regulate Glutl gene expression. Example 4- Human Glut-1 Expression was Decreased by a Knock-down of SLC2A1 LnRNA Dicer substrate interfering RNA (DsiRNA) was designed from the SLC2A1 LncRNA using the strategy shown in Figure 4. Three DsiRNAs were generated: DS1- DsiRNAl targeting exon 3; DS2- DsiRNA2 targeting exon 3; and DS6- DsiRNA6 targeting exon 2 of the SLC2A1 LncRNA.

The resulting DsiRNAs (10 nm) were transfected into human fibroblast cells and

RNA extracted 72 hours post-transfection. qPCR was performed for the expression of SLC2A1 LncRNA and human Glut-1 on the cells transfected with each DsiRNA.

As shown in Figure 5, the expression of the SLC2A1 LncRNA was knocked down by all three of the DsiRNAs.

More interestingly, is that all of the DsiRNAs greatly decreased the expression of human Glut-1 which again provided strong in vitro evidence that SLC2A1 LncRNA was indeed able to regulate (enhance) expression of the Glutl gene. See Figure 6. Additionally, the results showed that the exons 2 and 3 of Glutl-NAT are responsible for regulation of Glutl gene expression.

Example 5- Engineering a "humanized" Glutl mouse

The Glutl NAT is restricted to primates, posing a challenge to its study in rodents. The absence of a murine SMN2 gene posed a similar challenge when investigators attempted to model spinal muscular atrophy in mice, one that the inventors overcame by expressing the human SMN2 gene locus in the mice (Monani et al. 2017). The same strategy is used to generate the humanized Glutl mouse.

A bacterial artificial chromosome (BAC) clone, RP11-12501, which harbors the entire Glutl genomic sequence, the Glutl NAT and their flanking sequences has been identified. Additionally, it has been determined that an approximately 79kb Clal fragment contains this entire region (Figure 7A), offering a convenient means of extracting the human Glutl (hGlutl) locus out of the BAC for transfer into mice. Figure 7B depicts the isolated 79kb fragment that contains the human hGlutl locus. Accordingly, the fragment is injected into mouse oocytes to obtain mice transgenic for the hGlutl gene and its associated NAT to generate the humanized Glutl model mice. The transgene copy number and identification of the insertion site of the transgenes is assessed in the mouse as well as the expression profile of the hGlutl gene and its associated NAT. PCR and Q-PCR on a range of tissues including the brain, neuropil, and cerebral microvasculature fraction (a site of robust Glutl expression) are used to determine transcriptional activity of hGlutl and the NAT. Additionally, an hGlutl specific antibody is used to determine levels of expression of the hGlutl protein in the various mouse tissues.

Example 6- Generating hGlutl ^tg/tg ;mGlutl ^K0/K0 model mice

After the transgenic lines expressing a single copy of the approximately 79kb human fragment (hGlutl ^tg /-) are identified, studies are performed to determine if the NAT does regulate hGlutl expression. Model mice transgenic for the hGlutl locus are generated using the Glutl DS model mice previously described (Tang et al. 2017 and Wang et al. 2006), mutants heterozygous for murine Glutl (mGlutl ^+/K0 ), and the breeding strategy shown in Figure 8. Identification of viable mGlutl ^K0/K0 mice homozygous or hemizygous for hGlutl in the litters immediately imply that the human transgene is functional; prior studies have demonstrated that the mGlutl ^K0/K0 mutant exhibits an embryonic lethal phenotype resulting in the complete absence of viable animals with this genotype (Wang et al. 2006). These mice are assessed to determine the presence of two copies of the hGlutl gene in the hGlutl ^tg/tg ;mGlutl ^K0/K0 mice which renders them completely normal and then the mice are used in a detailed set of experiments to examine the effect of the NAT on hGlutl expression in the whole organism. The experience in assessing the Glutl DS phenotype in model mice (Tang et al. 2017) is used in ensuring that the hGlutl ^tg/tg ;mGlutl ^K0/K0 transgenics are no different from WT animals. Example 7- NAT knockdown experiments

Experiments to investigate the potential in vivo effects of the NAT on hGlutl expression are initiated by knocking down the expression of the antisense transcript by either using antisense oligonucleotides (ASOs) or by delivering a shRNA against the transcript by means of an AAV9 vector. These reagents are systemically administered to PND3 mice, with scrambled shRNA or ASOs as negative controls.

One to two weeks later, the expression of the NAT and hGlutl is determined and the knockdown of the NAT has a corresponding effect on hGlutl expression and triggers a Glutl DS disease phenotype. Briefly, the experiments are carried out on 6-8 week old mice and involve an assessment of the following parameters.

• Measurements of CSF glucose - Low CSF glucose levels are a reliable and widely- accepted biomarker of Glutl DS (DeGiorgis and Veggiotti 2013). Consequently, it is determined that CSF glucose concentrations decline in the hGlutl ^tg/tg ;mGlutl ^K0/K0 mice following knockdown of the NAT and hGlutl expression.

• Assessing brain size - It has been demonstrated that Glutl DS model mice have significantly smaller brains (micrencephaly) than WT control littermates. Consequently, mice in which the NAT has been knockdown have this phenotypic characteristic.

• Neurobehavioral outcome - Glutl haploinsufficiency in mice compromises motor performance (Tang et al. 2017 and Wang et al. 2006). Assays to measure this will constitute a final set of experiments to assess the physiological consequences of NAT knockdown. It has been previously found deficient performance on rotarod and gait analysis tests to reliably reflect Glutl DS in the mouse model. Both analyses show the knockdown mice have this neurobehavioral outcome.

Example 8- NAT over-expression studies

In order to extend the studies examining the effects of the Glutl -NAT on hGlutl in the whole organism, the expression of the natural antisense transcript is induced. Mice homozygous for the hGlutl transgene but completely ablated for mGlutl (hGlutl ^tg/tg ;mGlutl ^K0/K0 ) are adequate for these experiments. However, a more appealing and informative model is one lacking mGlutl but hemizygous for hGlutl (hGlutl ^tg/~ ;mGlutl ^K0/K0 ). Such animals, like mGlutl ^+/K0 mice, are expected to exhibit the signature features of Glutl DS as they are null for mGlutl and haploinsufficient for hGlutl. Triggering an increase in expression from the single hGlutl allele are, in these mice, not only detected at the RNA and protein level but is also expected to have a functional effect by ameliorating, or at least mitigating the severity, of the Glutl DS phenotype. For these studies, by means of an AAV9 construct, either the full-length Glutl-NAT2 RNA or an eGFP construct to PND3 mutants are delivered. Mice treated with either the Glutl-NAT2 or eGFP construct are assessed by subjecting them to the full battery of molecular and phenotypic assays described in the previous section.

The behavioral studies are carried out on sample sizes of 10 or more animals based on our prior experience (Tang et al. 2017). Studies involving an analysis of Glutl and NAT expression employ at least 4 animals. Mice of both genders are be utilized to avoid gender bias. However, results are reported separately for the two. Appropriate controls are gender and age-matched littermates. Finally, to avoid unintentional bias, whenever possible, the investigator is blind to mouse genotype.

These studies confirm that Glut NAT is a novel means of regulating Glutl expression.

REFERENCES

Arsov et al. (2012) Glucose transporter 1 deficiency in the idiopathic generalized epilepsies. Ann Neurol 72, 807-815.

Belanger et al. (2011) Brain energy metabolism: focus on astrocyte-neuron metabolic cooperation. Cell Metab.14- 24-3S

Brockdorff et al. (1992) The product of the mouse Xist gene is a 15 kb inactive X-specific transcript containing no conserved ORF and located in the nucleus. Cell. 71:515-26.

Brockmann (2009) The expanding phenotype of Glutl -deficiency syndrome. Brain Dev 31, 545-552.

De Giorgis and Veggiotti (2013) GLUT1 deficiency syndrome 2013: current state of the art. Seizure. 22:803-11

De Vivo et al. (1991). Defective glucose transport across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmental delay. N. Engl. J. Med., 325, 703- 709.

Gao (2016) Progress and Perspectives on Targeting Nanoparticles for Brain Drug Delivery. Acta Pharmaceutica Sinica B 6(4);268-286.

Johnsson et al. (2014) Evolutionary conservation of long non-coding RNAs; sequence, structure, function. Biochim Biophys Acta. 1840: 1063-71. Kim et al (2010) Widespread transcription at neuronal activity-regulated enhancers. Nature. 465: 182-7.

Klepper and Leiendecker (2013) Glutl deficiency syndrome and novel ketogenic diets. / Child Neurol. 28: 1045-8

Marques and Ponting (2009) Catalogues of mammalian long noncoding RNAs: modest conservation and incompleteness. Genome Biol. 10:R124.

Monani et al. (2000) The human centromeric survival motor neuron gene (SMN2) rescues embryonic lethality in Smn(-/-) mice and results in a mouse with spinal muscular atrophy. Hum Mol Genet. 9:333-9

Nagano et al. (2008) The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin. Science. 322:1717-20.

0rom et al. (2010) Long noncoding RNAs with enhancer-like function in human cells. Cell. 143:46-58.

Pascual and Ronen (2015) Glucose Transporter Type I Deficiency (G1D) at 25 (1990-2015): Presumptions, Facts, and the Lives of Persons With This Rare Disease. Pediatr Neurol. 53:379-93.

Pellerin et al. (1998) Expression of monocarboxylate transporter mRNAs in mouse brain: support for a distinct role of lactate as an energy substrate for the neonatal vs. adult brain. Proc Natl Acad Sci U S A. 95:3990-5

Ravasi et al. (2006) Experimental validation of the regulated expression of large numbers of non-coding RNAs from the mouse genome. Genome Res. 16:11-9

Tang et el. (2017) Brain microvasculature defects and Glutl deficiency syndrome averted by early repletion of the glucose transporter- 1 protein. Nat Commun. 8: 14152

Wang et al. (2006) A mouse model for Glut-1 haploinsufficiency. Hum Mol Genet. 15: 1169- 79