Human FRRS1l AAV9 viral vector

Abstract

Provided herein are compositions comprising viral vectors encoding the human FRRS1L gene and methods of using the compositions in treating FRRS1L diseases, for example, epileptic-dyskinetic encephalopathy.

Description

Atty. Docket No. UTSDP4429WO-1001354027TITLEHUMAN FRRS1L AAV9 VIRAL VECTORCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 687,222, filed August 26, 2024, and titled “HUMAN FRRS1 L AAV9 VIRAL VECTOR,” and U.S. Provisional Patent Application Serial No. 63 / 746,422, filed January 17, 2025, and titled “HUMAN FRRS1 LAAV9 VIRAL VECTOR,” which are incorporated by reference herein in their entireties.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in XML and is hereby incorporated by reference in its entirety. Said XML copy, created on August 23, 2025, is named “UTSD.P4429WO_SEQ_LIST.xml” and is about 7,000 bytes in size.BACKGROUND1. Field

[0003] The present disclosure relates to adeno associated vector expressing the human FRRS1 L gene.2. Discussion of Related Art

[0004] Epileptic-dyskinetic encephalopathy (EDE) is a rare genetic disorder that causes epilepsy and involuntary movements in infants. There is currently no treatment available for this disease. Gene therapy offers a unique therapeutic potential for treating such rare genetic disorders. EDE is caused by homozygous mutations in the FRRS1 L gene. The disease presents intellectual and developmental delay, epilepsy, chorea, and other progressive and debilitating motor impairments. FRRS1 L is a key determinant in the biogenesis of a-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the brain, essential to their maturation and recruitment into synaptic membranes. In the absence of FRRS1 L, AMPA receptors assembly line is compromised, causing impaired excitatory neurotransmission in the central nervous system (CNS) and a range of other neurological deficits. There is an unmet need for novel gene therapy treatment options for EDE and FRRS1 L related diseases.SUMMARY

[0005] In some aspects, the present disclosure encompasses a polynucleotide sequence comprising a nucleic acid sequence as set forth in SEQ ID NO: 1 , or a sequence at least about 60% identical thereto, and an operably linked promoter sequence. In some aspects, the nucleic acid sequence encodes a FRRS1 L protein as set forth in SEQ ID NO: 2 or a sequence1298854442Atty. Docket No. UTSDP4429WO-1001354027 at least about 80% identical thereto. In some aspects, the promoter sequence is a JeT promoter sequence. In some aspects, the JeT promoter comprises a sequence as set forth in SEQ ID NO: 3, or a sequence at least 90% identical thereto. In some aspects, the polynucleotide sequence further comprises one or more regulatory sequences operably linked to the nucleic acid sequence. In some aspects, the one or more regulatory sequences comprise, enhancers, polyadenylation signals, terminators, or any combination thereof. In some aspects, the polynucleotide sequence comprises a viral vector, or a plasmid. In some aspects, the viral vector comprises a lentiviral, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, herpes simplex viral vector, or a chimeric viral vector. In some aspects, the viral vector is an adeno-associated viral vector serotype 9. In some aspects, the viral vector is an adeno-associated viral vector serotype 9, comprising a sequence as set forth in SEQ ID NO: 1 , or a sequence at least 60% identical thereto.

[0006] Disclosed herein is a viral vector comprising a polynucleotide sequence, wherein the polynucleotide sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 1 or a nucleic acid sequence at least 80% identical thereto. In some aspects, the nucleic acid sequence encodes a FRRS1 L protein as set forth in SEQ ID NO: 2 or a sequence at least about 80% identical thereto. In some aspects, the viral vector is an adeno associated virus 9 (AAV9). In some aspects, the viral vector is neurotropic.

[0007] In some aspects, the present disclosure also encompasses a pharmaceutical composition comprising a disclosed polynucleotide sequence, or a disclosed viral vector, and at least one pharmaceutically acceptable excipient. In some aspects, the composition further comprises a nanoparticle, a liposome, a nanoconjugate, a nanocapsule, a micelle, an exosome, or a polymeric delivery vehicle. In some aspects, the pharmaceutical composition is formulated for intravenous, intracranial, intrathecal, subcutaneous, intramuscular, intranasal, cranial, transmucosal, trans-nasal, transcranial, or intracerebroventricular delivery. In some aspects, the pharmaceutical composition is formulated to cross the blood-brain barrier.

[0008] Also disclosed herein are methods of treating an FRRS1 L related disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the disclosed pharmaceutical composition. In some aspects, the FRRS1 L related disease or disorder is epileptic-dyskinetic encephalopathy. In some aspects, the subject is a human. In some aspects, the subject is less than 1 year of age. In some aspects, the administering of the pharmaceutical composition results in reduction in one or more of epileptic episodes, seizures, infantile spasms, and hyperkinetic movements by at least 25% or more.2298854442Atty. Docket No. UTSDP4429WO-1001354027BRIEF DESCRIPTION OF THE DRAWINGS

[0009] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. Aspects of the present disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein:

[0010] FIG. 1 provides a vector map of a self-complementary AAV9 vector encoding a human FRRS1 L gene operably linked to a Jet promoter sequence.

[0011] FIG. 2A provides a codon-optimized Human-FRRS1 L cDNA sequence (SEQ ID NO: 1) and its similarity to the mouse sequence (SEQ ID NO: 4).

[0012] FIG. 2B provides a sequence alignment of the wild-type human FRRS1 L protein (NCBI ID: NP_055149.3 with the translated codon optimized gene as provided in SEQ ID NO: 2. As shown in the figure, the codon-optimized protein product was 100% identical in amino acid sequences to the native Human FRRS1 L (NCBI sequence).

[0013] FIG. 3A provides a western blot analysis of FRRS1 L protein expression following HEK293 cell transfection with the FRRSI L-coding plasmid.

[0014] FIG. 3B provides a schematic showing intrathecal injection into FRRS1 L knock-out mouse.

[0015] FIG. 4A provides a schematic of experimental workflow for FRRS1 L gene-therapy pre- clinical study.

[0016] FIG. 4B-4C provide confirmation of transgene-specific expression following AAV9- FRRS1 L treatment in KO mice. FIG. 4B shows the relative expression of human FRRS1 L mRNA. FIG. 4C shows RT-qPCR analysis of endogenous mouse FRRS1 L in FRRS1 L-KO mice treated with either low (5x1O10vg) or high (3*1011vg) doses (AACt method). Fold changes were calculated using the 2-ddCt method and normalized using a mouse standard endogenous reference gene (RPL4) relative to wild-type genotype. Values shown as mean ± SEM (on a log10scale in FIG. 4B) with individual data points. Statistical analysis was performed using Kruskal-Wallis test with uncorrected Dunn’s post-hoc test. *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001.

[0017] FIG. 4D provides a western blot analysis of FRRS1 L protein expression in the spinal cord of FRRS1 L knock out mice, transfected with the FRRSI L-coding plasmid.

[0018] FIG. 4E provides a western blot analysis of FRRS1 L protein expression in the cerebellum of FRRS1 L knock out mice, transfected with the FRRSI L-coding plasmid.3298854442Atty. Docket No. UTSDP4429WO-1001354027

[0019] FIG. 4F provides a western blot analysis of FRRS1L protein expression in the kidney of FRRS1L knock out mice, transfected with the FRRSI L-coding plasmid.

[0020] FIG. 4G provides a western blot analysis of FRRS1L protein expression in the liver of FRRS1L knock out mice, transfected with the FRRSIL-coding plasmid.

[0021] FIG. 4H provides a western blot analysis of surface AMPA-R protein expression in the whole brain of FRRS1 L knock out mice, transfected with the FRRSIL-coding plasmid. Antibodies against GLUA1, GLUA4 and a pan-GLUA were used.

[0022] FIG. 4I provides a western blot analysis of surface AMPA-R protein expression in the cerebellum of FRRS1 L knock out mice, transfected with the FRRSIL-coding plasmid. Antibodies against GLUA1, GLUA4, pan-GLUA and GLUA2 / 3 were used.

[0023] FIG. 4J provides representative immunofluorescence images of the cerebellum using the FRRS1 L antibodies (red) and DAPI.

[0024] FIG. 4K provides representative immunofluorescence images of the hippocampus (CA1, CA3) using the FRRS1 L antibody (red), GLUA4 antibody (green) and DAPI.

[0025] FIG. 4L provides representative immunofluorescence images of the hippocampus - CA1 showing subsequent neuronal changes after injection using the FRRS1L antibody (red), GLUA4 antibody (green) and DAPI.

[0026] FIG. 4M provides representative immunofluorescence images of the cerebellum showing colocalization of AMPA-R (green) and PSD-95 (red). DAPI staining is shown in blue.

[0027] FIG. 5A provides the scoring template for behavioral tail suspension task (0- leg outward, 1- one leg retracted 50% of the time, 2- both leg retracted > 50% of the time, 3- entirely retracted all the time).

[0028] FIG. 5B shows the tail suspension task for FRRS1L+ / +and FRRSIL

[0029] FIG. 5C provides examples of horizontal ladder behavioral tasks.

[0030] FIG. 5D provides a bar graph showing the impact of transfection of the FRRSIL-coding plasmid into FRRS1L KO mice, on time to finish horizontal ladder task.

[0031] FIG. 5E provides a bar graph showing the impact of transfection of the FRRSIL-coding plasmid into FRRS1L KO mice, on leg error during the horizontal ladder task.

[0032] FIG. 5F is a photograph of the rotarod task.

[0033] FIG. 5G provides a bar graph showing data to study the impact of transfection of the FRRSIL-coding plasmid into FRRS1 L KO mice, on latency to fall during the rotarod task.

[0034] FIG. 5H is a photograph of the grip strength test.4298854442Atty. Docket No. UTSDP4429WO-1001354027

[0035] FIG. 5I provides a bar graph showing data to study the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on forelimb and hindlimb grip strength during a grip strength test.

[0036] FIG. 5J provides a bar graph showing data to study the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on total distance travelled during an open field test.

[0037] FIG. 5K provides a bar graph showing data to study the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on the run velocity during an open field test.

[0038] FIG. 5L provides a bar graph showing data to study the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on time in zone during anxiety and cognition tasks.

[0039] FIG. 5M provides a bar graph showing data to study the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on frequency to enter zone during anxiety and cognition tasks.

[0040] FIG. 5N is a schematic of the fear conditioning task for mice.

[0041] FIG. 50 provides a bar graph showing fear conditioning data to study the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on fear conditioning pretone and during tone.

[0042] FIG. 5P-5Q are photographs showing details of the digi-gate (gait analysis) task.

[0043] FIG. 5R shows the areas of the brain impacted during the digi-gate task.

[0044] FIG. 5S is a schematic of the digi-gate scoring methods.

[0045] FIG. 5T is a bar graph showing gait analysis data on the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on swing duration, brake in stance and propel in stance.

[0046] FIG. 5U is a bar graph showing gait analysis data on the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on paw-drag, ataxia, symmetry and stride.

[0047] FIG. 5V is a bar graph showing gait analysis data on the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on stride / stride (cm).

[0048] FIG. 5W is a bar graph showing gait analysis data on the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on sway width (paws only) and sway widthvariability (paws only).5298854442Atty. Docket No. UTSDP4429WO-1001354027

[0049] FIG. 5X is a bar graph showing gait analysis data on the impact of transfection of the FRRSI L-coding plasmid into FRRS1 L KO mice, on stride, stance and hind limb average shared stance.

[0050] FIG. 6A shows that AAV9 / H-FRRS1 L treatment helps normalizing irregular EEG- Pattern with a trait towards the ones in WT-mice.

[0051] FIG. 6B shows that AAV9 / H-FRRS1 L treatment has the potential to rescue brain atrophy. Consecutive transverse brain scans were processed using 3D-slicer. Data shown as Mean+SEM, with data-points for each animal. Data shown as Mean+SEM, with data-points for each animal. *P<0.05, **P<0.01 , ***P<0.001 , ****P<0.0001

[0052] FIG. 6C shows body weight follow-ups for the treated and un-treated mice.

[0053] The drawing figures do not limit the present disclosure to the specific aspects disclosed and described herein. The drawings are not necessarily to scale; emphasis instead being placed on clearly illustrating principles of certain aspects of the present disclosure.DETAILED DESCRIPTION

[0054] The following detailed description references the accompanying drawings that illustrate various aspects of the present disclosure. The drawings and description are intended to describe aspects and aspects of the present disclosure in sufficient detail to enable those skilled in the art to practice the present disclosure. Other components can be utilized, and changes can be made without departing from the scope of the present disclosure. The following description is, therefore, not to be taken in a limiting sense.

[0055] FRRS1 L disease is a rare inherited monogenic disorder with symptoms including epilepsy and seizures, progressive abnormal movements, developmental delay, diffuse hypotonia, cortical and cerebellar volume loss, and gradual loss of responsiveness to the environment, eventually leaving these patients in a debilitated state. There is currently no cure available for this disease and gene-replacement may be a suitable potential therapeutic option. FRRS1 L is a key determinant in the biogenesis of a-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors in the brain, essential to their maturation and recruitment into synaptic membranes. In the absence of FRRS1 L, AMPA receptors assembly line is compromised, causing impaired excitatory neurotransmission in the central nervous system (CNS) and a range of other neurological deficits, resulting in the disorder or diseased state. There is currently no cure for FRRS1 L related diseases / disorders.

[0056] In some aspects, the current disclosure overcomes limitations in the field of gene therapy by providing methods and compositions that may be used to specifically induce expression of FFRS1 L in the brain. The compositions and methods provided herein may be6298854442Atty. Docket No. UTSDP4429WO-1001354027 used, for example, in cell cultures, in the generation of genetically modified animals for research, or as a therapeutic to drive expression of FRRS1 L in a mammalian subject, such as a human or non-human animal. In an aspect, provided herein is an AAV9 viral vector encoding the FRRS1 L gene under a JeT promoter.

[0057] Disclosed herein is a polynucleotide sequence, wherein the polynucleotide sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 1 , or a nucleic acid sequence at least about 60% identical thereto. Described herein is a nucleic acid sequence encoding a FRRS1 L protein as provided in SEQ ID NO: 2, or a protein sequence at least about 80% identical thereto. In some aspects, the FRRS1 L protein is expressed from a JeT promoter sequence. In some aspects, the polynucleotide sequence encoding the FRRS1 L protein comprises a viral vector. In some aspects, the viral vector is an adenoviral vector, In some aspects, the adenoviral vector is a AAV9 vector. In some aspects, disclosed herein is a self- complementary adeno-associated-9 (AAV9) vector comprising a codon-optimized plasmid with a unique sequence, that drives the expression of human FRRS1 L gene using a JeT promoter. Also disclosed herein is a method of treating epileptic-dyskinetic encephalopathy and FRRS1 L disease by administering said viral vector.I. Terminology

[0058] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. For example, the use of a singular term, such as, “a” is not intended as limiting of the number of items. Also, the use of relational terms such as, but not limited to, “top,” “bottom,” “left,” “right,” “upper,” “lower,” “down,” “up,” and “side,” are used in the description for clarity in specific reference to the figures and are not intended to limit the scope of the present disclosure or the appended claims.

[0059] Any term of degree such as, but not limited to, “substantially” as used in the description and the appended claims, should be understood to include an exact, or a similar, but not exact configuration. For example, “a substantially planar surface” means having an exact planar surface or a similar, but not exact planar surface. Similarly, the terms “about” or “approximately,” as used in the description and the appended claims, should be understood to include the recited values or a value that is three times greater or one third of the recited values. For example, about 3 mm includes all values from 1 mm to 9 mm, and approximately 50 degrees includes all values from 16.6 degrees to 150 degrees. For example, they can refer to less than or equal to ± 5%, such as less than or equal to ± 2%, such as less than or equal to ± 1 %, such as less than or equal to ± 0.5%, such as less than or equal to ± 0.2%, such as less than or equal to ± 0.1%, such as less than or equal to ± 0.05%.7298854442Atty. Docket No. UTSDP4429WO-1001354027

[0060] The terms “comprising,” “including,” and “having” are used interchangeably in this disclosure. The terms “comprising,” “including,” and “having” mean to include, but not necessarily be limited to the things so described.

[0061] The terms “or” and “and / or,” as used herein, are to be interpreted as inclusive or meaning any one or any combination. Therefore, “A, B, or C” or “A, B, and / or C” mean any of the following: “A,” “B,” or “C”; “A and B”; “A and C”; “B and C”; “A, B, and C.” An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.

[0062] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs. The following references provide one of skill with a general definition of many of the terms used in this disclosure: Singleton et al., Dictionary of Microbiology and Molecular Biology (3rd ed. 2006); The Cambridge Dictionary of Science and Technology (Walker ed., 1990); The Glossary of Genetics, 5th Ed., R. Rieger et al. (2008), The Harper Collins Dictionary of Biology (1991), all of which are incorporated by reference herein. As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0063] The phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements. Wherever the terms “comprising” or “including” are used, it should be understood the disclosure also expressly contemplates and encompasses additional aspects “consisting of” the disclosed elements, in which additional elements other than the listed elements are not included.

[0064] The term “about” or “approximately,” as used herein, can mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” can mean an acceptable error range for the particular value, such as 10% of the value modified by the term “about.” As used herein, the term “about,” can mean relative to the recited value, e.g., amount, dose, temperature, time, percentage, etc., ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1 %.8298854442Atty. Docket No. UTSDP4429WO-1001354027

[0065] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.

[0066] The term “nucleic acid” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues See, e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991), the disclosure of which is incorporated in its entirety herein.

[0067] A “polynucleotide” described herein may comprise one or more nucleic acids each encoding a polypeptide, operably linked to a promoter (i.e., in a functional relationship with) and one or more regulatory sequences. Such a polynucleotide may alternatively be referred to herein as a “nucleic acid construct” or “construct”.9298854442Atty. Docket No. UTSDP4429WO-1001354027

[0068] As used herein, the term “operably linked” refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter, etc., operates to initiate, assist, affect, cause, and / or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain tissue(s), developmental stage(s), and / or condition(s).

[0069] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0070] Within the context of the application a protein is represented by an amino acid sequence and correspondingly a nucleic acid molecule or a polynucleotide represented by a nucleic acid sequence. Identity and similarity between sequences: throughout this application, each time one refers to a specific amino acid sequence SEQ ID NO (take SEQ ID NO: Y as example), one may replace it by a polypeptide represented by an amino acid sequence comprising a sequence that has at least 60% sequence identity or similarity with amino acid sequence SEQ ID NO: Y. Another preferred level of sequence identity or similarity is 65%. Another preferred level of sequence identity or similarity is 70%. Another preferred level of sequence identity or similarity is 75%. Another preferred level of sequence identity or similarity is 80%. Another preferred level of sequence identity or similarity is 85%. Another preferred level of sequence identity or similarity is 90%. Another preferred level of sequence identity or similarity is 95%. Another preferred level of sequence identity or similarity is 98%. Another preferred level of sequence identity or similarity is 99%.

[0071] Each amino acid sequence described herein by virtue of its identity or similarity percentage with a given amino acid sequence respectively has in a further preferred aspect an identity or a similarity of at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71 %, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%,10298854442Atty. Docket No. UTSDP4429WO-1001354027 at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with the given nucleotide or amino acid sequence, respectively. The terms “homology,” “sequence identity,” and the like are used interchangeably herein. Sequence identity is described herein as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In a preferred aspect, sequence identity is calculated based on the full length of two given SEQ ID NO’s or on a part thereof. Part thereof preferably means at least 50%, 60%, 70%, 80%, 90%, or 100% of both SEQ ID NO’s. In the art, “identity” also refers to the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. The degree of sequence identity between two sequences can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, such as global or local alignment algorithms. Non-limiting examples include BLASTp, BLASTn, Clustal W, MAFFT, Clustal Omega, AlignMe, Praline, GAP, BESTFIT, or another suitable method or algorithm. A Needleman and Wunsch global alignment algorithm can be used to align two sequences over their entire length or part thereof (part thereof may mean at least 50%, 60%, 70%, 80%, or 90% of the length of the sequence), maximizing the number of matches and minimizes the number of gaps. Default settings can be used and preferred program is Needle for pairwise alignment (in some aspects, EMBOSS Needle 6.6.0.0, gap open penalty 10, gap extent penalty: 0.5, end gap penalty: false, end gap open penalty: 10, end gap extent penalty: 0.5 is used) and MAFFT for multiple sequence alignment (in some aspects, MAFFT v7, default value is: BLOSUM62 [bl62] , Gap Open: 1.53, Gap extension: 0.123, Order: aligned, Tree rebuilding number: 2, Guide tree output: ON [true], Max iterate: 2, Perform FFTS: none is used).

[0072] “Similarity” between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. Similar algorithms used for determination of sequence identity may be used for determination of sequence similarity. Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called conservative amino acid substitutions. As used herein, “conservative” amino acid substitutions refer to the interchangeability of residues having similar side chains.

[0073] For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is11298854442Atty. Docket No. UTSDP4429WO-1001354027 asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gin or His; Asp to Glu; Cys to Ser or Ala; Gin to Asn; Glu to Asp; Gly to Pro; His to Asn or Gin; lie to Leu or Vai; Leu to lie or Vai; Lys to Arg; Gin or Glu; Met to Leu or lie; Phe to Met, Leu or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Vai to lie or Leu.

[0074] The term “gene therapy” refers to the transfer of genetic material (e.g., DNA or RNA) of interest into a cell to treat or prevent a genetic or acquired disease or condition. The genetic material of interest encodes a product (e.g., a protein polypeptide, peptide or functional RNA) whose production in vivo is desired. For example, the genetic material of interest can encode an enzyme, hormone, receptor, or polypeptide of therapeutic value.

[0075] The term “recombinant” as used herein to describe a nucleic acid molecule, means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.

[0076] As used herein, “regulatory elements” refer to any sequence elements that regulate, positively or negatively, the expression of an operably linked sequence. “Regulatory elements” include, without being limiting, a promoter, an enhancer, a leader, a transcription start site (TSS), a linker, 5’ and 3’ untranslated regions (UTRs), an intron, a polyadenylation signal, and a termination region or sequence, etc., that are suitable, necessary, or preferred for regulating or allowing expression of the gene or transcribable DNA sequence in a cell. Such additional regulatory element(s) can be optional and used to enhance or optimize expression of the gene or transcribable DNA sequence. A regulatory sequence can, for example, be inducible, noninducible, constitutive, cell-cycle regulated, metabolically regulated, and the like. A regulatory sequence may be a promoter. As used herein, the term “promoter” refers to a DNA sequence that contains an RNA polymerase binding site, a transcription start site, and / or a TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter12298854442Atty. Docket No. UTSDP4429WO-1001354027 sequence or other promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter of the present application can thus include variants of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. An “individual” or “subject,” as used interchangeably herein, is a mammal. In certain aspects, the individual or subject is a human.

[0077] The term “recombinant viral vector” or “viral vector” refers to an agent obtained from a naturally-occurring virus through genetic engineering techniques capable of transferring genetic material (e.g., DNA or RNA) of interest to a cell, which results in production of the product encoded by that said genetic material (e.g., a protein polypeptide, peptide or functional RNA) in the target cell.

[0078] The term “recombinant plasmid” or “plasmid” refers to a small, circular, doublestranded, self-replicating DNA molecule obtained through genetic engineering techniques capable of transferring genetic material of interest to a cell, which results in production of the product encoded by that said genetic material (e.g., a protein polypeptide, peptide or functional RNA) in the target cell. Furthermore, the term “recombinant plasmid” or “plasmid” also refers to a small, circular, double-stranded, self-replicating DNA molecule obtained through genetic engineering techniques used during the manufacturing of viral vectors as carriers of the recombinant vector genome.

[0079] “Adeno-associated virus” or AAV is a replication-deficient parvovirus, the singlestranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing et al., J Gen Virol, 75: 3385-3392 (1994). Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1 , VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).13298854442Atty. Docket No. UTSDP4429WO-1001354027

[0080] The term “administration” and variants thereof (e.g., “administering” a composition) in reference to a composition of the disclosure means introducing the composition or a prodrug of the composition into the system of the subject in need of treatment. When a composition of the disclosure or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), “administration” and its variants are each understood to include concurrent and sequential introduction of the composition or prodrug thereof and other agents. The present disclosure includes within its scope prodrugs of the compositions of this disclosure. In general, such prodrugs will be functional derivatives of the compositions of this disclosure which are readily convertible in vivo into the required composition. Thus, in the methods of treatment of the present disclosure, the term “administering” shall encompass the treatment of the various conditions described with the composition specifically disclosed or with a composition which may not be specifically disclosed, but which converts to the specified composition in vivo after administration to the patient.

[0081] The term “therapeutically effective amount” as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, or human that is being sought by a researcher, veterinarian, medical doctor or other clinician.

[0082] As used herein, the term “treating” refers to the application or administration of a composition including one or more active agents to a subject, who is in need of the treatment, for example, having a target disease or disorder, a symptom of the disease / disorder, or a predisposition toward the disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptom of the disease, or the predisposition toward the disease or disorder. Alleviating a target disease / disorder includes delaying the development or progression of the disease or reducing disease severity. Alleviating the disease does not necessarily require curative results. As used therein, “delaying” the development of a target disease or disorder means to defer, hinder, slow, retard, stabilize, and / or postpone progression of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individuals being treated. A method that “delays” or alleviates the development of a disease, or delays the onset of the disease, is a method that reduces probability of developing one or more symptoms of the disease in a given time frame and / or reduces extent of the symptoms in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a number of subjects sufficient to give a statistically significant result.

[0083] “Development” or “progression” of a disease means initial manifestations and / or ensuing progression of the disease. Development of the disease can be detectable and assessed using standard clinical techniques as well known in the art. However, development14298854442Atty. Docket No. UTSDP4429WO-1001354027 also refers to progression that may be undetectable. For purpose of this disclosure, development or progression refers to the biological course of the symptoms. “Development” includes occurrence, recurrence, and onset. As used herein “onset” or “occurrence” of a target disease or disorder includes initial onset and / or recurrence.

[0084] The term “adeno-associated virus” or “AAV” as used herein refers to a member of the class of viruses associated with this name and belonging to the genus Dependoparvovirus, family Parvoviridae. Adeno-associated virus is a single-stranded DNA virus that grows in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1 , pp. 169- 228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized after the publication dates of the reviews because it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. Multiple serotypes of this virus are known to be suitable for gene delivery; all known serotypes can infect cells from various tissue types. At least 11 sequentially numbered AAV serotypes are known in the art. Non-limiting exemplary serotypes useful in the methods disclosed herein include any of the 11 serotypes, e.g., AAV2, AAV8, AAV9, or variant serotypes, e.g., AAV-DJ and AAV PHP.B. The AAV particle comprises, consists essentially of, or consists of three major viral proteins: VPI, VP2 and VP3. In some aspects, the AAV refers to the serotype AAV1 , AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh74, or AAVrh.10.

[0085] Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to all serotypes (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh74, and AAVrh.10). Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, self-complementary AAV (scAAV) and AAV hybrids containing the genome of one serotype and the capsid of another serotype (e.g., AAV2 / 5, AAV-DJ and AAV-DJ8).15298854442Atty. Docket No. UTSDP4429WO-1001354027Exemplary adeno-associated viruses and recombinant adeno-associated viruses include, but are not limited to, rAAV-LK03, AAV-KP-1 (described in detail in Kerun et al. JCI Insight, 2019; 4(22):e131610) and AAV-NP59 (described in detail in Paulk et al. Molecular Therapy, 2018; 26(1): 289-303).

[0086] AAV Structure and Function

[0087] AAV is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length, including two 145-nucleotide inverted terminal repeat (ITRs). There are multiple serotypes of AAV. 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., J. Virol., 45: 555-564 (1983); the complete genome of AAV- 3 is provided in GenBank Accession No. NCJ829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_001862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Then, 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Patent 9,434,928, incorporated herein by reference in its entirety. U.S. Patent No. 9,434,928 also provides the sequences of the capsid proteins and a self-complementary genome. In one aspect, an AVV genome is a self-complementary genome. Cis-acting sequences directing viral DNA replication (rep), encapsidation / packaging, and host cell chromosome integration are contained within AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p19), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.

[0088] The cap gene is expressed from the p40 promoter and encodes the three capsid proteins, VPI, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. More specifically, after the single mRNA from which each of the VP1 , VP2 and VP3 proteins are translated is transcribed, it can be spliced in two different manners: either a longer or shorter intron can be excised, resulting in the formation of two pools of mRNAs: a 2.3 kb- and a 2.6 kb-long mRNA pool. The longer intron is often preferred and thus the 2.3-kb-long mRNA can be called the16298854442Atty. Docket No. UTSDP4429WO-1001354027 major splice variant. This form lacks the first AUG codon, from which the synthesis of VP1 protein starts, resulting in a reduced overall level of VP1 protein synthesis. The first AUG codon that remains in the major splice variant is the initiation codon for the VP3 protein. However, upstream of that codon in the same open reading frame lies an ACG sequence (encoding threonine) which is surrounded by an optimal Kozak (translation initiation) context. This contributes to a low level of synthesis of the VP2 protein, which is actually the VP3 protein with additional N terminal residues, as is VP1 , as described in Becerra SP et al., (December 1985). “Direct mapping of adeno-associated virus capsid proteins B and C: a possible ACG initiation codon”. Proceedings of the National Academy of Sciences of the United States of America. 82 (23): 7919-23, Cassinotti P et al., (November 1988). “Organization of the adeno- associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein 1”. Virology. 167 (1): 176-84, Muralidhar S et al., (January 1994). “Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity”. Journal of Virology. 68 (1): 170-6, and Trempe JP, Carter BJ (September 1988). “Alternate mRNA splicing is required for synthesis of adeno- associated virus VP1 capsid protein”. Journal of Virology. 62 (9): 3356-63, each of which is herein incorporated by reference. A single consensus polyA site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992).

[0089] Each VP1 protein contains a VP1 portion, a VP2 portion and a VP3 portion. The VP1 portion is the N-terminal portion of the VP1 protein that is unique to the VP1 protein. The VP2 portion is the amino acid sequence present within the VP1 protein that is also found in the N- terminal portion of the VP2 protein. The VP3 portion and the VP3 protein have the same sequence. The VP3 portion is the C-terminal portion of the VP1 protein that is shared with the VP1 and VP2 proteins.

[0090] The VP3 protein can be further divided into discrete variable surface regions l-IX (VR- l-IX). Each of the variable surface regions (VRs) can comprise or contain specific amino acid sequences that either alone or in combination with the specific amino acid sequences of each of the other VRs can confer unique infection phenotypes (e.g., decreased antigenicity, improved transduction and / or tissue-specific tropism relative to other AAV serotypes) to a particular serotype as described in DiMatta et al., “Structural Insight into the Unique Properties of Adeno-Associated Virus Serotype 9” J. Virol., Vol. 86 (12): 6947-6958, June 2012, the contents of which are incorporated herein by reference.

[0091] AAV possesses unique features 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 noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV17298854442Atty. Docket No. UTSDP4429WO-1001354027 infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA to generate AAV vectors. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65°C for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0092] Multiple studies have demonstrated long-term (> 1.5 years) recombinant AAV- mediated protein expression in muscle. See, Clark et al., Hum Gene Ther, 8: 659-669 (1997); Kessler et al., Proc Nat. Acad Sc. USA, 93: 14082-14087 (1996); and Xiao et al., J Virol, 70: 8098-8108 (1996). See also, Chao et al., Mol Ther, 2:619-623 (2000) and Chao et al., Mol Ther, 4:217-222 (2001). Moreover, because muscle is highly vascularized, recombinant AAV transduction has resulted in the appearance of transgene products in the systemic circulation following intramuscular injection as described in Herzog et al., Proc Natl Acad Sci USA, 94: 5804-5809 (1997) and Murphy et al., Proc Natl Acad Sci USA, 94: 13921- 13926 (1997). Moreover, Lewis et al., J Virol, 76: 8769-8775 (2002) demonstrated that skeletal myofibers possess the necessary cellular factors for correct antibody glycosylation, folding, and secretion, indicating that muscle is capable of stable expression of secreted protein therapeutics. Recombinant AAV (rAAV) genomes of the invention comprise, consist essentially of, or consist of a nucleic acid molecule encoding a therapeutic protein (e.g., SURF1) and one or more AAV ITRs flanking the nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12, AAV13, AAVPHP.B, AAVrh74, and AAVrh.10. Production of pseudotyped rAAVis disclosed in, for example, WO2001083692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, e.g., Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art.

[0093] Further, as the present disclosure is susceptible to aspects of many different forms, it is intended that the present disclosure be considered as an example of the principles of the18298854442Atty. Docket No. UTSDP4429WO-1001354027 present disclosure and not intended to limit the present disclosure to the specific aspects shown and described. Any one of the features of the present disclosure may be used separately or in combination with any other feature. References to the terms “aspect,” “aspects,” and / or the like in the description mean that the feature and / or features being referred to are included in, at least, one aspect of the description. Separate references to the terms “aspect,” “aspects,” and / or the like in the description do not necessarily refer to the same aspect and are also not mutually exclusive unless so stated and / or except as will be readily apparent to those skilled in the art from the description. For example, a feature, structure, process, step, action, or the like described in one aspect may also be included in other aspects but is not necessarily included. Thus, the present disclosure may include a variety of combinations and / or integrations of the aspects described herein. Additionally, all aspects of the present disclosure, as described herein, are not essential for its practice. Likewise, other systems, methods, features, and advantages of the present disclosure will be, or become, apparent to one with skill in the art upon examination of the figures and the description. It is intended that all such additional systems, methods, features, and advantages be included within this description, be within the scope of the present disclosure, and be encompassed by the claims.II. Compositions

[0094] In some aspects, the current disclosure encompasses compositions for use in the study and treatment of FRRS1 L gene related diseases or disorders. Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1 L), are associated with neurological conditions including severe intellectual disability, movement disorders, hypotonia, epilepsy, choreoathetosis, cognitive deficits, and epileptic encephalopathies. These clinical symptoms are associated with neurodegeneration in the cortex and cerebellum. The compositions disclosed herein provide gene therapy based solutions to tackle these diseases and disorders.

[0095] In some aspects, the current disclosure relates to a polynucleotide encoding the FRRSIL protein, wherein the polynucleotide comprises a nucleic acid sequence as set for in SEQ ID NO: 1 , or a sequence at least about 60% identical thereto. In some aspects, the nucleic acid sequence is at least about 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identical to the nucleotide sequence as set forth in SEQ ID NO: 1.

[0096] In some aspects, the nucleic acid sequence encodes FRRSIL as set forth in SEQ ID NO: 2, or a sequence at least about 80% identical thereto. In particular, the encoded amino19298854442Atty. Docket No. UTSDP4429WO-1001354027 acid sequence according to the disclosure has 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identity with the amino acid sequence as set forth in SEQ ID NO: 2.

[0097] In some aspects, the current disclosure also encompasses a polynucleotide sequence comprising the disclosed nucleic acid sequence. In some aspects, the nucleic acid sequence is operably linked to a promoter sequence. Thus, in some aspects, the polynucleotide comprises a nucleic acid sequence operably linked to one of the non-limiting example promoters described herein.

[0098] In some aspects, a promoter is an inducible promoter, a constitutive promoter, a mammalian cell promoter, a viral promoter, a chimeric promoter, an engineered promoter, a tissue-specific promoter, or any other type of promoter known in the art. In some aspects, a promoter is a RNA polymerase II promoter, such as a mammalian RNA polymerase II promoter. In some aspects, a promoter is a RNA polymerase III promoter, including, but not limited to, a HI promoter, a human U6 promoter, a mouse U6 promoter, or a swine U6 promoter. A promoter will generally be one that is able to promote transcription in a mammalian cell. A variety of promoters are known in the art, which in some aspects, can be used herein. Nonlimiting examples of promoters that can be used herein in some aspects include: human EFla, human cytomegalovirus (CMV), human ubiquitin C (UBC), mouse phosphoglycerate kinase 1 , polyoma adenovirus, simian virus 40 (SV40), p-globin, p-actin, a- fetoprotein, y-globin, p-interferon, y-glutamyl transferase, mouse mammary tumor virus (MMTV), Rous sarcoma virus, rat insulin, glyceraldehyde-3-phosphate dehydrogenase, metallothionein II (MT II), amylase, cathepsin, Ml muscarinic receptor, retroviral LTR (e.g., human T-cell leukemia virus HTLV), AAV ITR, interleukin-2, collagenase, platelet-derived growth factor, adenovirus 5 E2, stromelysin, murine MX gene, glucose regulated proteins (GRP78 and GRP94), a-2-macroglobulin, vimentin, MHC class I gene H-2K b, HSP70, proliferin, tumor necrosis factor, thyroid stimulating hormone a gene, immunoglobulin light chain, T-cell receptor, HLA DQa and DQ, interleukin-2 receptor, MHC class II, MHC class II HLA-DRa, muscle creatine kinase, prealbumin (transthyretin), elastase I, albumin gene, c-fos, c-HA-ras, neural cell adhesion molecule (NCAM), H2B (TH2B) histone, rat growth hormone, human serum amyloid (SAA), troponin I (TN I), duchenne muscular dystrophy, human immunodeficiency virus, and Gibbon Ape Leukemia Virus (GAL V) promoters. In some aspects, a promoter is the CMV immediate early promoter. In some aspects, the promoter is a CAG promoter and / or a CAG / CBA promoter.

[0099] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked with a nucleic acid encoding a gene, causes RNA to be transcribed from the nucleic acid in a cell under most or all physiological conditions. Examples of constitutive promoters20298854442Atty. Docket No. UTSDP4429WO-1001354027 include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter, the cytomegalovirus (CMV) promoter (see, e.g., Boshart et al., Cell 41 :521-530, 1985, which is incorporated herein by reference for the purposes described herein), the SV 40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFI-alpha promoter (Invitrogen).

[0100] 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. Additional examples of inducible promoters are known in the art. Examples of inducible promoters regulated by exogenously supplied compounds include the zinc-inducible sheep metallothionein (MT) promoter, the dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system; the ecdysone insect promoter , the tetracycline-repressible system , the tetracycline-inducible system, the RU486-inducible system, and the rapamycin-inducible system.

[0101] The term "tissue-specific" promoter refers to a promoter that is active only in certain specific cell types and / or tissues (e.g., transcription of a specific gene occurs only within cells expressing transcription regulatory and / or control proteins that bind to the tissue-specific promoter). In some aspects, regulatory and / or control sequences impart tissue-specific gene expression capabilities. In some cases, tissue-specific regulatory and / or control sequences bind tissue-specific transcription factors that induce transcription in a tissue-specific manner. In some aspects, a tissue-specific promoter is a neuron-specific promoter. In some aspects, a tissue-specific promoter is hematopoietic lineage cell-specific promoter. In some aspects, a tissue-specific promoter is an immune cell-specific promoter.

[0102] In some aspects, the promoter is a JeT promoter, a UsP promoter (JeTI), a Rous sarcoma virus (RSV) LTR promoter, a cytomegalovirus (CMV) promoter, an SV40 promoter, a dihydrofolate reductase promoter, a beta-actin promoter, a phosphoglycerol kinase (PGK) promoter, a U6 promoter, an H1 promoter, a CAG promoter, a hybrid chicken beta-actin promoter, an MeCP2 promoter, an EF1 promoter, a ubiquitous chicken p-actin hybrid (CBh) promoter, a U1a promoter, a U1 b promoter, an MeCP2 promoter, an MeP418 promoter, an MeP426 promoter, a minimal MeCP2 promoter, a VMD2 promoter, an mRho promoter, EFla promoter, llbc promoter, human p-actin promoter, TRE promoter, Ac5 promoter, Polyhedrin promoter, CaMKIla promoter, Gall promoter, TEF1 promoter, GDS promoter, ADH1 promoter, Libi promoter, or a- 1 -antitrypsin (hAAT) promoter, or a variant thereof.21298854442Atty. Docket No. UTSDP4429WO-1001354027

[0103] In some aspects, the polynucleotide comprises a JeT promoter operably linked to the disclosed nucleic acid sequence. The JeT promoter (see US6555674B2, the disclosure of which is incorporated herein in its entirety) is a recombinant promoter with transcriptional activity comparable to a number of strong mammalian promoters. The promoter consists of five key elements: (1) a TATA box; (2) a transcription initiation site (Inr); (3) a CAT consensus sequence in conjunction with (4) a CArG element and, (5) four Sp1 transcription binding sites (GGGCGG) arranged in two tandems. A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. In some aspects, the JET promoter comprises a sequence as set forth in SEQ ID NO: 3, or a sequence at least about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% (or any range derivable therein) identical thereto.

[0104] In some aspects, the polynucleotide sequence may further comprise other transcriptional and translational regulatory sequences. In some aspects, the transcriptional regulatory element constitutes a binding site for a transcriptional activator or repressor. A transcriptional activator is a protein that activates expression of the transgene when bound to the transcriptional regulatory element. A transcriptional repressor is a protein that prevents expression of the transgene when bound to the transcriptional regulatory element. Non-limiting examples of regulatory elements include transcription initiation, termination, enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; tissue specific regulatory sequences and when desired, sequences that enhance secretion of the encoded product. In some aspects, the regulatory signal is an enhancer sequence. By “enhancer” is meant a nucleic acid sequence that, when positioned proximate to a promoter, confers increased transcription activity relative to the transcription activity resulting from the promoter in the absence of the enhancer domain. Non-limiting examples of enhancers include CMV enhancer, MIE enhancer, GADD45G, HACNS1. In some aspects, the regulatory sequence is a viral posttranscriptional regulatory element for example woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), hepatitis B virus posttranscriptional regulatory element (HBVPRE), RNA transport element (RTE), or any variant thereof. In some aspects, the regulatory sequence is a transcription termination sequence, for example a SV40 late poly(A) sequence, a rabbit betaglobin poly(A) sequence, a bovine growth hormone poly(A) sequence, or any variant thereof. In some aspects, depending on the regulatory sequence, the sequence may be located anywhere on the recombinant polynucleotide sequence, for example before, or after the promoter sequence, between the promoter and the heterologous22298854442Atty. Docket No. UTSDP4429WO-1001354027 gene or cDNA, at the end of the gene or cDNA or after the start of the gene sequences. In some aspects, the regulatory sequence is a F2A, E2A, P2A, T2A Picornavirus IRES, Apthovirus IRES, Hepatitis A IRES, Pestivirus IRES, Hepesvirus IRES. In some aspects, the recombinant polynucleotide may comprise an untranslated regions (UTRs). In mRNA, the 5’IITR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas the 3’IITR starts immediately following the stop codon and continues until the transcriptional termination signal. In some aspects, any suitable naturally occurring or synthetic UTR sequence can be incorporated into the polynucleotides disclosed herein. Other non-UTR sequences may also be used as regions or subregions within the polynucleotides. For example, introns or portions of introns sequences may be incorporated into regions of the polynucleotides of the invention. Incorporation of intronic sequences may increase protein production as well as polynucleotide levels. Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5’ UTR which may contain a strong Kozak translational initiation signal and / or a 3’ UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5’UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes.23298854442Atty. Docket No. UTSDP4429WO-1001354027

[0105] In some aspects, the compositions of the current disclosure encompass vectors comprising the polynucleotide disclosed herein. A vector may comprise a nucleic acid construct or an expression construct as provided herein. A vector may be selected from any genetic element known in the art which can facilitate transfer of nucleic acids between cells, such as, but not limited to, plasmids, transposons, cosmids, chromosomes, artificial chromosomes, viruses, virions, and the like. A vector may also be a chemical vector, such as a lipid complex or naked DNA. “Naked DNA” or “naked nucleic acid” refers to a nucleic acid molecule that is not contained in encapsulating means that facilitates delivery of a nucleic acid into the cytoplasm of a target host cell. Naked DNA may be circular or linear (linearized DNA sequence). Optionally, a naked nucleic acid can be associated with standard means used in the art for facilitating its delivery of the nucleic acid to the target host cell, for example to facilitate the transport of the nucleic acid through the cell membrane.

[0106] A vector can be any genetic element, behaving either as an autonomous unit of polynucleotide replication within a cell, (i.e., capable of replication under its own control) or being rendered capable of replication by insertion into a cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and / or expression of the attached segment. Vectors can comprise nucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to affect the expression of the attached segment. Such sequences differ depending on the host organism; they include promoter sequences to effect transcription, enhancer sequences to increase transcription,24298854442Atty. Docket No. UTSDP4429WO-1001354027 ribosomal binding site sequences and transcription and translation termination sequences. Alternatively, expression vectors can be capable of directly expressing nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences.

[0107] In some aspects, constructs provided herein can be of different sizes. In some aspects, a construct is a plasmid and can include a total length of up to about 1 kb, up to about 2 kb, up to about 3 kb, up to about 4 kb, up to about 5 kb, up to about 6 kb, up to about 7 kb, up to about 8 kb, up to about 9 kb, up to about 10 kb, up to about 11 kb, up to about 12 kb, up to about 13 kb, up to about 14 kb, or up to about 15 kb. In some aspects, a construct is a plasmid and can have a total length in a range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 10 kb, about 1 kb to about 11 kb, about 1 kb to about 12 kb, about 1 kb to about 13 kb, about 1 kb to about 14 kb, or about 1 kb to about 15 kb.

[0108] In some aspects, the vector is a viral vector. In some aspects, the suitable delivery system may be a viral vector. In some aspects, the viral vector is an RNA viral vector. In some aspects, the viral vector is a DNA viral vector. In general, viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The viral construct is a nucleotide sequence that comprises sequences necessary for the production of recombinant virus in a packaging cell. In one aspect, the viral construct additionally comprise genetic elements that allow for the desired expression of a gene of interest in the host cell. Generation of the viral construct may be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PGR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y.; Coffin et al. (997) Retroviruses. Cold Spring Harbor Laboratory Press, N.Y.; and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, 2000).

[0109] Exemplary viral vectors include, for example, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors. In some aspects, viral vectors that integrate transgenes are used (e.g., virus other than adenoviral vectors). Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids may be transduced in any desired format that provides sufficiently efficient delivery levels, including in virus particles. A viral gene delivery vehicle may optionally25298854442Atty. Docket No. UTSDP4429WO-1001354027 comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences may be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus. In some aspects, the gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof are well known in the art. Numerous retroviral gene delivery vehicles may be utilized in the present invention.

[0110] Other viral vector systems that may be used to deliver a polynucleotide of the invention have been derived from herpes virus, e.g., Herpes Simplex, vaccinia virus, and several RNA viruses. Exemplary viruses include an alphavirus, a poxvirus, an arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (see, for example, Friedmann (1989) Science 244:1275-1281 ; Ridgeway (1988) supra; Baichwal and Sugden (1986) supra; and Horwich et al. (1990) J. Virol. 64:642-650).

[0111] In some aspects, a construct is a viral construct and can have a total number of nucleotides of up to 10 kb. In some aspects, a viral construct can have a total number of nucleotides in the range of about 4.5 kb to 5 kb, or about 4.7 kb. In some aspects, a viral construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 1 kb to about 9 kb, about 1 kb to about 1 O kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 2 kb to about 9 kb, about 2 kb to about 10 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 3 kb to about 9 kb, about 3 kb to about 10 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 4 kb to about 9 kb, about 4 kb to about 10 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 5 kb to about 9 kb, about 5 kb to about 10 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, about 6 kb to about 9 kb, about 6 kb to about 10 kb, about 7 kb to about 8 kb, about 7 kb to about 9 kb, about 7 kb to about 10 kb, about 8 kb to about 9 kb, about 8 kb to about 10 kb, or about 9 kb to about 10 kb.

[0112] In some aspects, a construct is a lentivirus construct and can have a total number of nucleotides of up to 8 kb. In some examples, a lentivirus construct can have a total number of nucleotides of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to26298854442Atty. Docket No. UTSDP4429WO-1001354027 about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, or about 7 kb to about 8 kb.

[0113] In some aspects, a construct is an adeno-associated virus construct and can have a total number of nucleotides of up to 8 kb. In some aspects, an adenovirus construct can have a total number of nucleotides in the range of about 1 kb to about 2 kb, about 1 kb to about 3 kb, about 1 kb to about 4 kb, about 1 kb to about 5 kb, about 1 kb to about 6 kb, about 1 kb to about 7 kb, about 1 kb to about 8 kb, about 2 kb to about 3 kb, about 2 kb to about 4 kb, about 2 kb to about 5 kb, about 2 kb to about 6 kb, about 2 kb to about 7 kb, about 2 kb to about 8 kb, about 3 kb to about 4 kb, about 3 kb to about 5 kb, about 3 kb to about 6 kb, about 3 kb to about 7 kb, about 3 kb to about 8 kb, about 4 kb to about 5 kb, about 4 kb to about 6 kb, about 4 kb to about 7 kb, about 4 kb to about 8 kb, about 5 kb to about 6 kb, about 5 kb to about 7 kb, about 5 kb to about 8 kb, about 6 kb to about 7 kb, about 6 kb to about 8 kb, or about 7 kb to about 8 kb.

[0114] Any of the constructs described herein can further include a control sequence, e.g., a control sequence selected from the group of a transcription initiation sequence, a transcription termination sequence, a promoter sequence, an enhancer sequence, an RNA splicing sequence, a polyadenylation (poly(A)) sequence, a Kozak consensus sequence, and / or additional untranslated regions which may house pre- or post-transcriptional regulatory and / or control elements. In some aspects, a promoter can be a native promoter, a constitutive promoter, an inducible promoter, and / or a tissue-specific promoter. Non-limiting examples of control sequences are described herein.

[0115] In some aspects, lentiviral vectors may be used. Numerous lentiviruses suitable for use in the present disclosure are well known in the art. “Lentivirus” refers to a genus of retroviruses that are capable of infecting dividing and non-dividing cells. Lentiviruses may infect nondividing cells owing to the karyophilic properties of their preintegration complex, which allow for its active import through the nucleopore. Several examples of lentiviruses include HIV (human immunodeficiency virus; including HIV type 1 , and HIV type 2), the etiologic agent of the human acquired immunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis (visna) or pneumonia (maedi) in sheep, the caprine arthritisencephalitis virus, which causes immune deficiency, arthritis, and encephalopathy in goats; equine infectious anemia virus, which causes autoimmune hemolytic anemia, and encephalopathy in horses; feline immunodeficiency virus (FIV), which causes immune deficiency in cats; bovine immune deficiency virus (BIV), which causes lymphadenopathy, lymphocytosis, and possibly central nervous system infection in cattle; and simian27298854442Atty. Docket No. UTSDP4429WO-1001354027 immunodeficiency virus (SIV), which cause immune deficiency and encephalopathy in subhuman primates.

[0116] A lentiviral genome is generally organized into a 5' long terminal repeat (LTR), the gag gene, the pol gene, the env gene, the accessory genes (nef, vif, vpr, vpu) and a 3' LTR. The viral LTR is divided into three regions called U3, R and U5. The U3 region contains the enhancer and promoter elements. The U5 region contains the polyadenylation signals. The R (repeat) region separates the U3 and U5 regions and transcribed sequences of the R region appear at both the 5' and 3' ends of the viral RNA. The 5' and 3' LTR’s serve to promote transcription and polyadenylation of the virion RNAs. The LTR contains all other cis-acting sequences necessary for viral replication. Lentiviruses have additional genes including vif, vpr, tat, rev, vpu, nef and vpx. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient encapsidation of viral RNA into particles (the Psi site). If the sequences necessary for encapsidation (or packaging of retroviral RNA into infectious virions) are missing from the viral genome, the cis defect prevents encapsidation of genomic RNA. However, the resulting mutant remains capable of directing the synthesis of all virion proteins.

[0117] In some aspects, the vector is a AAV vector comprising the recombinant polynucleotide. In some aspects, the AAV vector is a self- complementary AAV vector. In some aspects, the AAV vector is composed of, at a minimum, a polynucleotide as disclosed herein, and 5’ and 3’ AAV inverted terminal repeats (ITRs).

[0118] AAV possesses unique features 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 noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA such as a gene cassette containing a promoter, a DNA of interest and a polyadenylation signal. The rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold28298854442Atty. Docket No. UTSDP4429WO-1001354027 preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection.

[0119] Multiple serotypes of AAV exist and offer varied tissue tropism. Known serotypes include, for example, AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. AAV9 is described in U.S. Pat. No. 7,198,951 and in Gao et al., J. Virol., 78: 6381- 6388 (2004). Examples of suitable serotypes include AAV1 , 2, 5, 8, 9, PHP.eB. In some aspects, the AAV vector may be of any of the available serotypes. In some exemplary aspects, the serotype of the AAV vector is suitable for targeting microglia and / or macrophages. In some aspects, the viral vector is a recombinant adeno-associated virus of serotype 9.

[0120] The viral virus vectors may be pseudotyped. A “pseudotyped” virus is a viral particle having an envelope protein that is from a virus other than the virus from which the RNA genome is derived. The envelope protein may be from a different virus. For example, an envelope protein is the vesicular stomatitis virus G (VSV G) protein or from measles virus. However, to eliminate the possibility of human infection, viruses may alternatively be pseudotyped with ecotropic envelope protein that limit infection to a specific species, such as mice or birds. For example, in one aspect, a mutant ecotropic envelope protein is used, such as the ecotropic envelope protein 4.17 (see, for example, Powell et al. (2000) Nat. Biotech. 18:1279-1282).

[0121] The viral virus vectors may also be self-inactivating. For example, a “self-inactivating 3' LTR” is a 3' long terminal repeat (LTR) that contains a mutation, substitution or deletion that prevents the LTR sequences from driving expression of a downstream gene. A copy of the U3 region from the 3' LTR acts as a template for the generation of both LTR’s in the integrated provirus. Thus, when the 3' LTR with an inactivating deletion or mutation integrates as the 5' LTR of the provirus, no transcription from the 5' LTR is possible. This eliminates competition between the viral enhancer / promoter and any internal enhancer / promoter. For example, a deletion in the U3 region of the 3' LTR of the vector DNA, i.e., the DNA used to produce the vector RNA may be made. Thus, during reverse transcription, this deletion is transferred to the 5' LTR of the proviral DNA. It is desirable to eliminate enough of the U3 sequence to greatly diminish or abolish altogether the transcriptional activity of the LTR, thereby greatly diminishing or abolishing the production of full-length vector RNA in transduced cells. However, it is generally desirable to retain those elements of the LTR that are involved in polyadenylation of the viral RNA, a function spread out over U3, R and U5. Accordingly, it is desirable to eliminate as many of the transcriptionally important motifs from the LTR as possible while sparing the polyadenylation determinants. The LTR may be rendered about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95% 96% 97%, 98%, to about 99% transcriptionally inactive.29298854442Atty. Docket No. UTSDP4429WO-1001354027

[0122] Self-inactivating 3' LTRs and other viral self-inactivating methods and reagents are well known in the art.

[0123] Other elements commonly found in viral vectors and generally operably linked to genes of interest in order to enhance the expression or utility of the viral vectors are well known and described further below.

[0124] a) Enhancers, Promoters, and Inducible Forms Thereof

[0125] A “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter, or other regulatory element or useful element of the vector, is in a correct functional location and / or orientation in relation to a nucleic acid sequence to regulate the sequence (e.g., control transcriptional initiation and / or expression of that sequence).

[0126] A promoter generally comprises a sequence that functions to position the start site for RNA synthesis. The best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. To bring a coding sequence “under the control of” a promoter, the 5' end of the transcription initiation site of the transcriptional reading frame is placed “downstream” of (i.e. , 3' of) the chosen promoter. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements may be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements may function either cooperatively or independently to activate transcription.

[0127] In addition, a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals. It is well known that the initiation codon must be “in-frame” with the reading frame of the desired coding sequence to ensure translation of the entire insert.30298854442Atty. Docket No. UTSDP4429WO-1001354027The exogenous translational control signals and initiation codons may be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements.

[0128] A promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence. Enhancers were originally detected as genetic elements that increased transcription from a promoter located at a distant position on the same molecule of DNA. This ability to act over a large distance had little precedent in classic studies of prokaryotic transcriptional regulation. Subsequent work showed that regions of DNA with enhancer activity are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Aside from this operational distinction, enhancers and promoters are very similar entities. Promoters and enhancers have the same general function of activating transcription in the cell. They are often overlapping and contiguous, often seeming to have a very similar modular organization. Taken together, these considerations suggest that enhancers and promoters are homologous entities and that the transcriptional activator proteins bound to these sequences may interact with the cellular transcriptional machinery in fundamentally the same way. For example the CMV enhancer may be used in combination with the chicken p-actin promoter. Again, one of skill in the art will be able to select the appropriate enhancer based on the desired expression pattern.

[0129] A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and / or exon. Such a promoter may be referred to as “endogenous.” Similarly, an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence. Alternatively, certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment. Such promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different31298854442Atty. Docket No. UTSDP4429WO-1001354027 transcriptional regulatory regions, and / or mutations that alter expression. For example, promoters that are most commonly used in recombinant DNA construction include the - lactamase (penicillinase), lactose and tryptophan (trp) promoter systems. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including PCR™, in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906). Furthermore, it is contemplated the control sequences that direct transcription and / or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, may be employed as well. Control sequences comprising promoters, enhancers and other locus or transcription controlling / modulating elements are also referred to as “transcriptional cassettes”.

[0130] Naturally, it will be important to employ a promoter and / or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression. Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. (1989) supra). The promoters employed may be constitutive, tissuespecific, cell-specific, developmental stage-specific, inducible, and / or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous for gene therapy or for applications such as the large-scale production of recombinant proteins and / or peptides. The promoter may be heterologous or endogenous. Use of a T3, T7 or SP6 cytoplasmic expression system is another possible aspect. Eukaryotic cells may support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct. To determine whether a particular promoter is useful, a selected promoter may be tested in the construct in vim in an HSC lineage cell and, if the promoter is capable of promoting expression of the transgene at a detectable signal-to-noise ratio, it will generally be useful in accordance with the present invention. A desirable signal-to-noise ratio is one between about 10 and about 200, a more desirable signal-to-noise ratio is one 40 and about 200, and an even more desirable signal-to-noise ratio is one between about 150 and about 200. One means of testing such a promoter, described in more detail herein below, is through the use of a signal generating transgene such as a reporter, like a fluorescent protein such as the green fluorescent protein (GFP).

[0131] Non-limiting examples of promoters that may be used include the promoter for ubiquitin. CMV, p-actin, and pgk. Alternatively, the promoter may be a tissue specific promoter. Several non-limiting examples of tissue specific promoters that may be used include lek, and thy 1. In addition, promoters may be selected to allow for inducible expression of the32298854442Atty. Docket No. UTSDP4429WO-1001354027 transgene. In some aspects, the promoter is a JeT promoter. The JeT promoter is a recombinant promoter with transcriptional activity comparable to a number of strong mammalian promoters. The promoter consists of five key elements: (1) a TATA box; (2) a transcription initiation site (Inr); (3) a CAT consensus sequence in conjunction with (4) a CArG element and finally, (5) four Sp1 transcription binding sites (GGGCGG) arranged in two tandems.

[0132] Further, viral vector promoters, such as the RNA Polymerase III (Pol III) promoter or other promoters used as part of the viral vector, may be inducible. Any suitable inducible promoter may be used with the methods of the present invention and such promoters are well known in the art. Transcription-regulatory elements conferring inducibility on the promoters may be placed within the promoter region, such as between the proximal sequence element (PSE) and the transcription start site, upstream or downstream from the TATA box. Such sequences may also be placed outside the promoter, such as downstream from the end of an interfering RNA sequence. In addition, a viral vector contain 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more of such inducibility conferring elements in order to more or less tightly regulate transcription in response to the inducing signal.

[0133] Useful inducible Pol III promoters include tetracycline responsive promoters, operator sequences (tetO) of the E. coli tetracycline resistance operon. Many inducible promoters may be used as a cis- regulatory element and these commonly, but not necessarily, use an element that serves a landing pad function of providing a place to which a tethering factor (a sequencespecific DNA binding protein) may bind to the DNA and bring a diversification factor, fused to the tethering factor, into sufficient proximity of the coding region so that diversification of the coding region is capable of reversible regulation. A tethering factor is one that binds to the cis- regulatory element in a sequence-specific manner. In the aspects in which LacO serves as a cis-regulatory element, the Lac repressor, Lacl, may serve as the tethering factor, and its binding to the cis-regulatory element, LacO, may be regulated by isopropyl-p-D-thio- galactoside (IPTG). In the absence of IPTG, Lad binds LacO and diversification is accelerated (or otherwise regulated) by the presence of the diversification factor. IPTG may be added in the event that a halt or reduction in activity of the diversification factor is desired. In aspects in which TetO serves as the cis-regulatory element, TetR may be a suitable tethering factor, and the activity of the diversification factor may be regulated by tetracycline or doxycycline. Other transcription-regulatory elements that allow or inducible expression are well known in the art and may be inserted into the promoter region for controlled expression of genes of interest. For example, LPTG-inducible systems based on LacO and Lacl repressors are well known in the art, as are inducible systems based on Ore, GalO, MTII (phorbol ester, TFA), MMTV (glucocorticoids), beta-interferon (poly(rl) or poly(rc)), adenovirus 5 E2 (E1A), collagenase33298854442Atty. Docket No. UTSDP4429WO-1001354027(phorbol ester, TFA), and the like. For RNA Polymerase I- or Pol Ll-based transcription units, well-established inducible systems such as tetracycline transactivator systems, reverse tetracycline transactivator systems, and ecdysone systems may be used.

[0134] Additional regulatory elements are also well known that may enhance expression of the gene of interest. One type of posttranscriptional regulatory sequence is an intron positioned within the expression cassette, which may serve to stimulate gene expression. Since introns placed in such a manner may expose the RNA transcript of the gene of interest to the normal cellular splicing and processing mechanisms, it may be desirable to locate introncontaining transgenes in an orientation opposite to that of the vector genomic transcript. Alternatively, a method of enhancing expression of a gene of interest is through the use of a posttranscriptional regulatory element which does not rely on splicing events, such as the posttranscriptional processing element of herpes simplex virus, the posttranscriptional regulatory element of the hepatitis B virus (HPRE) or that of the woodchuck hepatitis virus (WPRE), which contains an additional cis-acting element not found in the HPRE. The regulatory element is positioned within the vector so as to be included in the RNA transcript of the transgene, but outside of stop codon of the transgene translational unit. The use of such regulatory elements are particularly preferred in the context of modest promoters, but may be contraindicated in the case of very highly efficient promoters.

[0135] b) Other Vector Elements

[0136] Vectors of the present invention may include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which may be used in conjunction with standard recombinant technology to digest the vector. “Restriction enzyme digestion” refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector. “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.

[0137] Most transcribed eukaryotic RNA molecules will undergo RNA splicing to remove introns from the primary transcripts. Vectors containing genomic eukaryotic sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression.34298854442Atty. Docket No. UTSDP4429WO-1001354027

[0138] The vectors useful for the present invention will generally comprise at least one termination signal. A “termination signal” or “terminator” is comprised of the DNA sequences involved in specific termination of an RNA transcript by an RNA polymerase. Thus, in certain aspects a termination signal that ends the production of an RNA transcript is contemplated. A terminator may be necessary in vivo to achieve desirable message levels.

[0139] In eukaryotic systems, the terminator region may also comprise specific DNA sequences that permit site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently. Thus, in other aspects involving eukaryotes, it is preferred that that terminator comprises a signal for the cleavage of the RNA, and it is more preferred that the terminator signal promotes polyadenylation of the message. The terminator and / or polyadenylation site elements may serve to enhance message levels and to minimize read through from the cassette into other sequences.

[0140] Terminators contemplated for use in the invention include any known terminator of transcription described herein or known to one of ordinary skill in the art, including but not limited to, for example, the termination sequences of genes, such as for example the bovine growth hormone terminator or viral termination sequences, such as for example the SV40 terminator. For example, Pol III terminators preferably comprise of stretches of 4 or more thymidine (“T”) residues. In a preferred aspect, a cluster of 5 consecutive Ts is linked immediately downstream of the RNA coding region to serve as the terminator. In such a construct pol III transcription is terminated at the second or third T of the DNA template, and thus only 2 to 3 uridine (“II”) residues are added to the 3' end of the coding sequence. In certain aspects, the termination signal may be a lack of transcribable or translatable sequence, such as due to a sequence truncation.

[0141] In eukaryotic gene expression, a polyadenylation signal is generally added in order to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed. Some examples include the SV40 polyadenylation signal or the bovine growth hormone polyadenylation signal, convenient and known to function well in various target cells. Polyadenylation may increase the stability of the transcript or may facilitate cytoplasmic transport.

[0142] In order to propagate a vector of the invention in a host cell, it may contain one or more origins of replication sites (often termed “ori”), which is a specific nucleic acid sequence at35298854442Atty. Docket No. UTSDP4429WO-1001354027 which replication is initiated. Alternatively an autonomously replicating sequence (ARS) may be employed if the host cell is yeast.

[0143] c) Production of Virus

[0144] Any method known in the art may be used to produce infectious viral particles whose genome comprises a copy of the viral construct described above. Preferably, the viral construct is introduced into a packaging cell line. The packaging cell line provides the viral proteins that are required in trans for the packaging of the viral genomic RNA into viral particles. The packaging cell line may be any cell line that is capable of expressing retroviral proteins. Useful packaging cell lines include 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430). The packaging cell line may stably express the necessary viral proteins (see, for example, U.S. Pat. No. 6,218,181). Alternatively a packaging cell line may be transiently transfected with plasmids comprising nucleic acid that encodes the necessary viral proteins. In one aspect a packaging cell line that stably expresses the viral proteins required for packaging the genome is transfected with a plasmid comprising the viral construct described above. In another aspect a packaging cell line that does not stably express the necessary viral proteins is co-transfected with two or more plasmids (see, for example, Yee e al. (1994) Meth. Cell. Biol. 43A:99-112). In some aspects, the packaging cell line may not express envelope gene products. In this case, the packaging cell line will package the viral genome into particles that lack an envelope protein. As the envelope protein is responsible, in part, for the host range of the viral particles, the viruses may be pseudotyped as described above. In other aspects, RNA interference activity of the packaging cells may be suppressed in order to improve the production of recombinant virus. This includes, without limitation, the use of co-transfection or stable transfection of constructs expressing siRNA molecules to inhibit Dicer, an RNase III family member of ribonuclease which is essential for RNA interference (Hammond et al. (20011) Nat. Rev. Genet. 2:110-119). The recombinant virus is then preferably purified from the packaging cells, titered and diluted to the desired concentration according to standard protocols well known in the art.

[0145] d) Delivery of Virus

[0146] Target cells may be transduced in any way that allows the virus to contact the target cells in which delivery of a sequence containing a gene of interest is desired according to well- known methods in the art (see, for example U.S. Pat. No. 8,552,150). In some aspects, a suitable amount of virus is introduced into a subject directly (in vivo), for example though injection into the host’s body. In some preferred aspects, the viral particles are injected into a subject’s peripheral blood stream. In other preferred aspects, the viral particles are injected36298854442Atty. Docket No. UTSDP4429WO-1001354027 into a subject through intra-dermal injection, subcutaneous injection, intra-peritoneal cavity injection, or intra-venal injection. The virus may be delivered using a subdermal injection device, such as those disclosed in U.S. Pat. Nos. 7,241 ,275, 7,115,108, 7,108,679, 7,083,599, 7,083,592, 7,047,070, 6,971 ,999, 6,808,506, 6,780,171 , 6,776,776, 6,689,118, 6,670,349, 6,569,143, 6,494,865, 5,997,501 , 5,848,991 , 5,328,483, 5,279,552, 4,886,499. Other injection locations also are suitable, such as directly into organs comprising target cells. For example intra-lymph node injection, intra-spleen injection, or intra-bone marrow injection may be used to deliver virus to the lymph node, the spleen and the bone marrow, respectively. Transduced cell populations of interest may then be selected.

[0147] In other aspects of the present invention, a suitable amount of virus is introduced into target cells obtained from a subject (ex vivo), for example through incubation of the virus with target primary cells or target cells in culture. The target cells may be cells obtained from bone marrow, fetal liver, peripheral blood, amniotic fluid, cord blood, and the like. Methods to obtain cells from a subject are well known in the art as described above. The virus may be suspended in media and added to the wells of a culture plate, tube or other container. The media containing the virus may be added prior to the plating of the cells or after the cells have been plated. Preferably cells are incubated in an appropriate amount of media to provide viability and to allow for suitable concentrations of virus in the media such that infection of the host cell occurs.

[0148] In still other aspects, target cells are provided and contacted with the virus in vitro, such as in culture plates.

[0149] The cells may be incubated with the virus for a sufficient amount of time to allow the virus to infect the cells. Preferably the cells are incubated with virus for at least 1 hour, more preferably at least 5 hours and even more preferably at least 10 hours.

[0150] In ex vivo, in vitro, and in vivo delivery aspects, any concentration of virus that is sufficient to infect the desired target cells may be used, as may be readily determined by the skilled artisan. When the target cell is to be cultured, the concentration of the viral particles is at least 1 PFU / pl, or at least 10 PFU / pl, or 400 PFU / pl and even more preferably at least 1 xio4PFU / pl. In some aspects, the concentration of the viral particles is about 1 x 102, 2 x 102, 3 x102, 4 X 102, 5 X 102, 6 X 102, 7 X 102, 8 X 102, 9 X 102, 1 X 103, 2 X 103, 3 X 103, 4 X 103, 5 X103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 104, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x104, 8 x 104, 9 x 104, 1 x 105, 2 x 105, 3 x 105, 4 x 105, 5 x 105, 6 x 105, 7 x 105, 8 x 105, 9 x105, 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x 106, 7 x 106, 8 x 106, 9 x 106, 1 x 107, 2 x107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, 2 x 108, 3 x 10s, 4 x108, 5 x 10s, 6 x 10s, 7 x 10s, 8 x 10s, 9 x 10s, 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x37298854442Atty. Docket No. UTSDP4429WO-1001354027109, 7 x 109, 8 x 109, 9 x 1 o9, 1 x io10PFU / pl. The titer of the virus may be adjusted to allow for, on average, 1 , 2, 3, 4, 5, or more independent cellular transductions with independent viral constructs. In one aspect, the viral titer is adjusted to allow for 1 or fewer such cellular transduction events in order to prevent multiple integration events.

[0151] The methods of infecting cells disclosed above do not depend upon individual-specific characteristics of the cells. As a result, they are readily extended to all mammals. In some aspects the recombinant virus is delivered to a human or to human HSC cell lineages. In other aspects, the recombinant virus is delivered to a mouse or to mouse HSC cell lineages. In still other aspects, the recombinant virus is delivered to an animal other than a human or a mouse, or to cells from an animal other than a human or a mouse.

[0152] As discussed above, the recombinant virus may be pseudotyped to confer upon it a broad host range as well as target cell specificity. One of skill in the art would also be aware of appropriate internal promoters to achieve the desired expression of a polynucleotide or gene of interest in a particular animal species. Thus, one of skill in the art will be able to modify the method of infecting dendritic cells derived from any species.

[0153] The transduced cells may be analyzed, for example for integration, transcription, and / or expression of genes of interest, the number of copies of the gene integrated, and the location of the integration. Such analysis may be carried out at any time and may be carried out by any methods known in the art. Incubator animals in which a recombinant virus or virus- infected target cells are administered may be analyzed for location of infected cells, expression of the virus-delivered gene of interest, modulation of an immune response, and / or monitored for symptoms associated with a disease or disorder by any methods known in the art.

[0154] Thus, in some aspects, the current disclosure also encompasses a viral vector comprising a polynucleotide sequence comprising a nucleic acid sequence as set forth in SEQ ID NO: 1 , or a sequence at least about 60% identical thereto. In some aspects, the nucleic acid sequence is at least about 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100% identical to the nucleotide sequence as set forth in SEQ ID NO: 1 . In some aspects, the viral vector is a recombinant adeno associated viral vector (rAAV). In some aspects, the viral vector is a recombinant adeno associated viral vector of serotype 9 (rAAV9). Exemplary vector map of a suitable rAAV9 vector is provided in FIG. 1 (scAAV9-JeT-h-FRRS1 L).III. Cells

[0155] In some aspects the current disclosure also encompasses a cell comprising the polynucleotide construct disclosed herein, or a vector, for example a viral vector, as disclosed38298854442Atty. Docket No. UTSDP4429WO-1001354027 herein. In some aspects, the cells are from a cell line commonly used to maintain and grow viral vectors and / or maintain polynucleotide constructs. In another aspect, contemplated are the use of host cells into which a polynucleotide construct, vector, or nucleic acid has been introduced. A polynucleotide construct encoding the protein can be transfected into cells according to a variety of methods known in the art. Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors. Host cells which may be used to maintain and produce disclosed viral vectors and / or the polynucleotide constructs include HEK293, HEK293T, HeLa, Sf9, BHK-21 , A549, Vero, CHO, PER.C6. In some aspects, the cell may be a transduced with the viral vector or the polynucleotide construct in vivo (for therapeutic purposes), ex vivo (for example, into patient derived cells) or in vitro for testing, production and / or maintenance.

[0156] Viral vectors may be introduced into a desired cell by direct infection, in which viral particles are simply added to the culture medium containing the target cells or administered in vivo. The viral vector binds to specific receptors on the cell surface, leading to entry via endocytosis or membrane fusion, depending on the virus type. Lentivirus, adenovirus, and adeno-associated virus (AAV) all utilize this method. The efficiency of direct transduction may be optimized by changing the multiplicity of infection (MOI): the ratio of viral particles to target cells, which should be carefully adjusted to balance efficiency and toxicity, the incubation time, or inclusion of various chemicals such as polybrene, protamine sulfate, or hexadimethrine bromide which increase viral binding and internalization, particularly for retroviral and lentiviral vectors. In some aspects, the current disclosure also encompasses a mammalian cell comprising the polynucleotide construct disclosed herein, or a vector, for example a viral vector, as disclosed herein. In some aspects, the animal cell may be in vitro, ex vivo or in vivo. In some aspects, the mammalian cell is a neuronal cell. In some aspects, the mammalian cell is from a subject suffering from Epileptic-dyskinetic encephalopathy (EDE).

[0157] In addition to viral delivery employing the viral vectors mentioned above, several non- viral methods for the transfer of polynucleotide constructs into cultured mammalian cells also are contemplated by the present disclosure. These include calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes and lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection. Some of these techniques may be successfully adapted for in vivo or ex vivo use.39298854442Atty. Docket No. UTSDP4429WO-1001354027

[0158] Once the polynucleotide construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement), or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or “episomes” encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the polynucleotide construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of construct employed.

[0159] In yet another embodiment, the polynucleotide construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.

[0160] In still another embodiment for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them. Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force. The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

[0161] In some embodiments, the expression construct is delivered directly to the neurons of a subject. This may require parenteral delivery, localized delivery, and / or localized surgical exposure of the tissue or cells. Again, DNA encoding a particular gene may be delivered via this method and still be incorporated by the present disclosure.IV. Pharmaceutical compositions

[0162] In addition to the compositions (polynucleotides / vectors, e.g., AAV vectors, comprising a nucleic acid sequence encoding the FRRS1L protein), provided herein are pharmaceutical compositions which are suitable for administration to mammals, for example humans. Thus, In some aspects, the current disclosure encompasses pharmaceutical compositions comprising the disclosed nucleic acid, polynucleotides and / or viral vectors and further comprising at least one pharmaceutically acceptable excipient. In some aspects, the40298854442Atty. Docket No. UTSDP4429WO-1001354027 pharmaceutical composition is suitable for delivery to humans. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0163] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0164] Relative amounts of the active ingredient (rAAV9 encoding FRRS1 L), the at least one pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered.

[0165] AAV vectors, comprising the disclosed nucleic acid sequence encoding the FRRS1 L polypeptide of the present invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit the sustained or delayed release; or (4) alter the biodistribution (e.g., target the viral vector to specific tissues or cell types such as brain and motor neurons).

[0166] Formulations of the present invention can include, without limitation, saline, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with viral vectors (e.g., for transplantation into a subject), nanoparticle mimics and combinations thereof. Further, the viral vectors of the present invention may be formulated using self-assembled nucleic acid nanoparticles.

[0167] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be41298854442Atty. Docket No. UTSDP4429WO-1001354027 administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0168] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and / or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1 % and 99% (w / w) of the active ingredient. By way of example, the composition may comprise between 0.1 % and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w / w) active ingredient.

[0169] In some aspects, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some aspects, an excipient is approved for use for humans and for veterinary use. In some aspects, an excipient may be approved by United States Food and Drug Administration. In some aspects, an excipient may be of pharmaceutical grade. In some aspects, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0170] In certain aspects, compositions disclosed herein may compromise one or more pharmaceutically acceptable excipient(s), diluents, and / or carrier(s). As used herein, a pharmaceutically acceptable diluent, excipient, or carrier, refers to a material suitable for administration to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable diluents, carriers, and excipients can include, but are not limited to, physiological saline, Ringer’s solution, phosphate solution or buffer, buffered saline, and other carriers known in the art.

[0171] In some aspects, pharmaceutical compositions herein may include stabilizers, antioxidants, colorants, other medicinal or pharmaceutical agents, carriers, adjuvants, preserving agents, stabilizing agents, wetting agents, emulsifying agents, solution promoters, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. Herein, the term “excipient” refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition,42298854442Atty. Docket No. UTSDP4429WO-1001354027 which is incorporated herein by reference. In certain aspects, pharmaceutical compositions described herein may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries to facilitate processing of genetically modified endothelial progenitor cells into preparations which can be used pharmaceutically. In some aspects, any of the well-known techniques, carriers, and excipients may be used as suitable and / or as understood in the art.

[0172] In certain aspects, pharmaceutical compositions described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some aspects, polymers that may comprise pharmaceutical compositions described herein include: water- soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose; waterinsoluble polymers such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylate), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or a combination thereof. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of polymers as suspending agent(s) by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of polymers as suspending agent(s) by total weight of the composition.

[0173] In certain aspects, pharmaceutical compositions disclosed herein may comprise a viscous formulation. In some aspects, viscosity of composition herein may be increased by the addition of one or more gelling or thickening agents. In some aspects, compositions disclosed herein may comprise one or more gelling or thickening agents in an amount to provide a sufficiently viscous formulation to remain on treated tissue. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agent(s) by total weight of the composition. In some aspects, suitable thickening agents for use herein can be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, viscosity enhancing agents can be acacia (gum arabic), agar, aluminum magnesium silicate, sodium43298854442Atty. Docket No. UTSDP4429WO-1001354027 alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellaran, gelatin, Ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, maize starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthum gum, gum tragacanth, ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethyl-cellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda (dextrose, maltodextrin and sucralose), or any combination thereof.

[0174] In certain aspects, pharmaceutical compositions disclosed herein may comprise additional agents or additives selected from a group including surface-active agents, detergents, solvents, acidifying agents, alkalizing agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antimicrobial agents, antibiotic agents, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancing agents, and the like. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of one or more agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more agents by total weight of the composition. In some aspects, one or more of these agents may be added to improve the performance, efficacy, safety, shelflife and / or other property of the muscarinic antagonist composition of the present disclosure. In some aspects, additives may be biocompatible, without being harsh, abrasive, and / or allergenic.

[0175] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more acidifying agents. As used herein, “acidifying agents” refers to compounds used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acid, citric acid, fumaric acid and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic acid may be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at44298854442Atty. Docket No. UTSDP4429WO-1001354027 least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more acidifying agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more acidifying agents by total weight of the composition.

[0176] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more alkalizing agents. As used herein, “alkalizing agents” are compounds used to provide alkaline medium. Such compounds include, by way of example and without limitation, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine and others known to those of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic base can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more alkalizing agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more alkalizing agents by total weight of the composition.

[0177] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more antioxidants. As used herein, “antioxidants” are agents that inhibit oxidation and thus can be used to prevent the deterioration of preparations by the oxidative process. Such compounds include, by way of example and without limitation, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophophorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite and other materials known to one of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more antioxidants by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more antioxidants by total weight of the composition.

[0178] In certain aspects, pharmaceutical compositions disclosed herein may comprise a buffer system. As used herein, a “buffer system” is a composition comprised of one or more buffering agents wherein “buffering agents” are compounds used to resist change in pH upon45298854442Atty. Docket No. UTSDP4429WO-1001354027 dilution or addition of acid or alkali. Buffering agents include, by way of example and without limitation, potassium metaphosphate, potassium phosphate, monobasic sodium acetate and sodium citrate anhydrous and dihydrate and other materials known to one of ordinary skill in the art. In some aspects, any pharmaceutically acceptable organic or inorganic buffer can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more buffering agents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more buffering agents by total weight of the composition.

[0179] In some aspects, the amount of one or more buffering agents may depend on the desired pH level of a composition. In some aspects, pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In some aspects, pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0180] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more preservatives. As used herein, “preservatives” refers to agents or combination of agents that inhibits, reduces or eliminates bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and a combination thereof. In some aspects, any pharmaceutically acceptable preservative can be used. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more preservatives by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more preservatives by total weight of the composition.

[0181] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more surface-acting reagents or detergents. In some aspects, surface-acting reagents or detergents may be synthetic, natural, or semi-synthetic. In some aspects, compositions disclosed herein may comprise anionic detergents, cationic detergents, zwitterionic detergents, ampholytic detergents, amphoteric detergents, nonionic detergents having a steroid skeleton, or a combination thereof. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about46298854442Atty. Docket No. UTSDP4429WO-100135402745%, at least about 50% total amount of one or more surface-acting reagents or detergents by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more surface-acting reagents or detergents by total weight of the composition.

[0182] In certain aspects, pharmaceutical compositions disclosed herein may comprise one or more stabilizers. As used herein, a “stabilizer” refers to a compound used to stabilize an active agent against physical, chemical, or biochemical process that would otherwise reduce the therapeutic activity of the agent. Suitable stabilizers include, by way of example and without limitation, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophonate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycols, sodium caprylate, and sodium saccharin and others known to those of ordinary skill in the art. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more stabilizers by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more stabilizers by total weight of the composition.

[0183] In some aspects, pharmaceutical compositions disclosed herein may comprise one or more tonicity agents. As used herein, a “tonicity agents” refers to a compound that can be used to adjust the tonicity of the liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose and others known to those or ordinary skill in the art. Osmolarity in a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolarity may be measured using methods commonly known in the art. In some aspects, a vapor pressure depression method is used to calculate the osmolarity of the compositions disclosed herein. In some aspects, the amount of one or more tonicity agents comprising a pharmaceutical composition disclosed herein may result in a composition osmolarity of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L or about 250 mOsm / L to about 320 mOsm / L. In some aspects, a composition herein may have an osmolality ranging from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg or from about 280 mOsm / kg to about 320 mOsm / kg. In some aspects, a pharmaceutical composition described herein may have an47298854442Atty. Docket No. UTSDP4429WO-1001354027 osmolarity of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L. In some aspects, pharmaceutical compositions disclosed herein may comprise at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% total amount of one or more tonicity modifiers by total weight of the composition. In some aspects, pharmaceutical compositions disclosed herein may comprise about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of one or more tonicity modifiers by total weight of the composition.

[0184] Dosage formulations

[0185] In certain aspects, the present disclosure provides compositions comprising one or more inhibitors disclosed herein, formulated for one or more routes of administration. Suitable routes of administration may, for example, include intravenous, intracranial, intrathecal, subcutaneous, intranasal route, cranial, transmucosal, trans-nasal, transcranial, intracerebroventricular, intestinal, and / or parenteral delivery. In some aspects, compositions herein formulated can be formulated for parenteral delivery. In some aspects, compositions herein formulated can be formulated intramuscular, subcutaneous, intramedullary, intravenous, intraperitoneal, intracranial intrathecal, and / or intranasal injections.

[0186] In certain aspects, pharmaceutical compositions of the present disclosure may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0187] In certain aspects, pharmaceutical compositions for use in accordance with the present disclosure thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of a pharmaceutical composition herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, physiological salt buffer, or any combination thereof.

[0188] In certain aspects, pharmaceutical compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection herein may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some aspects, compositions herein may48298854442Atty. Docket No. UTSDP4429WO-1001354027 be suspensions, solutions or emulsions in oily or aqueous vehicles, and / or may contain formulator agents such as suspending, stabilizing and / or dispersing agents.

[0189] In some aspects, compositions herein may comprise the active ingredient in a powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0190] Pharmaceutical compositions suitable for use in context of the present disclosure may include compositions wherein the active ingredients can be contained in an amount effective to achieve the intended purpose. In some aspects, a therapeutically effective amount means an amount of active ingredients effective to prevent, slow, alleviate or ameliorate symptoms of a disorder or prolong the survival of the subject being treated.

[0191] The active ingredient may be administered at once or may be divided into a number of smaller doses to be administered at intervals of time. The precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. Concentrations and dosage values may also vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. The compositions of the disclosure may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In some aspects, a composition comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different rAAVs each having one or more different transgenes.

[0192] The rAAVs are administered in sufficient amounts to transfect the cells of a desired tissue and to provide sufficient levels of gene transfer and expression without undue adverse effects.

[0193] The dose of rAAV virions required to achieve a particular “therapeutic effect,” e.g., the units of dose in genome copies / per kilogram of body weight (GC / kg), will vary based on several factors including, but not limited to: the route of rAAV virion 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 patient having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art.49298854442Atty. Docket No. UTSDP4429WO-1001354027

[0194] An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some aspects, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of the rAAV is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109to 1016genome copies. In some cases, a dosage between about 1011to 1013rAAV genome copies is appropriate. In certain aspects, 1012or 1013rAAV genome copies is effective to target CNS tissue. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

[0195] In some aspects, a dose of rAAV is administered to a subject no more than once per calendar day (e.g., a 24-hour period). In some aspects, a dose of rAAV is administered to a subject no more than once per 2, 3, 4, 5, 6, or 7 calendar days. In some aspects, a dose of rAAV is administered to a subject no more than once per calendar week (e.g., 7 calendar days). In some aspects, a dose of rAAV is administered to a subject no more than bi-weekly (e.g., once in a two calendar week period). In some aspects, a dose of rAAV is administered to a subject no more than once per calendar month (e.g., once in 30 calendar days). In some aspects, a dose of rAAV is administered to a subject no more than once per six calendar months. In some aspects, a dose of rAAV is administered to a subject no more than once per calendar year (e.g., 365 days or 366 days in a leap year).

[0196] In some aspects, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present. Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc.

[0197] 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.

[0198] Typically, these formulations may contain at least about 0.1% of the active compound 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 compound 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 pharmacological50298854442Atty. Docket No. UTSDP4429WO-1001354027 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.V. Method of treatment

[0199] In some aspects, the current disclosure also encompasses a method of treating a FRRS1 L related disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the disclosed pharmaceutical compositions. In some aspects the FRRS1 L related disease or disorder is epileptic-dyskinetic encephalopathy (EDE). Gene therapy as provided using the methods disclosed herein offers a unique therapeutic potential for treating EDE related to Ferric Chelate Reductase 1 Like (FRRS1 L), a severe pediatric neurodevelopmental disorder caused by homozygous mutations in the FRRS1 L gene. The disease presents with intellectual and developmental delay, epilepsy, chorea and other progressive and debilitating motor impairments. There is currently no treatment available for this disease. FRRS1 L is a key determinant in the biogenesis of a- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the brain, essential to their maturation and recruitment into synaptic membranes. In the absence of FRRS1 L, AMPA receptors assembly line is compromised, causing impaired excitatory neurotransmission in the central nervous system (CNS) and a range of other neurological deficits. To counter this, the current disclosure provides novel polynucleotides and viral vector based therapeutics. In some aspects, the therapeutic comprises an adeno-associated virus stereotype 9 (AAV9) vector utilizing a JeT promoter to drive the expression of human FRRS1 L gene.

[0200] The polynucleotides and viral vectors (for example, rAAV9) of the disclosure may be delivered to a subject in compositions according to any appropriate methods known in the art. For example, an rAAV, preferably suspended in a physiologically compatible carrier (i.e. , in a composition), may be administered to a subject, i.e. host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a non-human primate (e.g., Macaque). In some aspects a host animal is a human.

[0201] The present disclosure provides for methods of treating, attenuating, and preventing FRRS1 L related disease in a subject in need thereof. In several aspects, a method for treating, attenuating, or preventing FRRS1 L related disease in a subject can include administering to a subject, including a human subject, an effective amount of one or more polynucleotides (for example, rAAV9) molecules encoding FRRS1 L disclosed herein or a nucleic acid encoding an polynucleotides (for example, rAAV9) molecule encoding FRRS1 L as disclosed herein. In several aspects, a method for treating, attenuating, or preventing FRRS1 L related disease in51298854442Atty. Docket No. UTSDP4429WO-1001354027 a subject can include administering to a subject, including a human subject, an effective amount of a nucleic acid encoding FRRS1 L.

[0202] Methods disclosed herein may include treating a subject in need thereof by administrating a therapeutically effective amount of one or more polynucleotides (for example, rAAV9) molecules or a pharmaceutical composition disclosed herein. The subject may be a human subject having or suspected of having, or at risk of having FRRS1 L related disease. Non-limiting examples of FRRS1 L related disease to be treated using the methods disclosed herein may include EDE. In some aspects, the subject benefits with an increase in life expectancy compared to an untreated subject with identical disease condition and predicted outcome. In some other aspects, the treatment improves the subject’s brain function as compared to an untreated subject with identical disease condition and predicted outcome.

[0203] A subject suitable for the FRRS1 L related disease treatment as disclosed herein may be selected based on the subject’s diagnosis. In some aspects, a method of diagnosis may detect one or more serum markers indicative of FRRS1 L related disease. The diagnosis method may also include the evaluation of at least one clinical symptom associated with a FRRS1 L related disease. Non-limiting examples of clinical symptoms associated with a FRRS1 L related disease may include mild to moderate intellectual and developmental delay, epilepsy, chorea and other progressive and debilitating motor impairments. Impairments may be detectable by physiological based tests, biochemical based tests or physical and symptomatic assessments. In some cases, one or more tests may be used to characterize the disease state.

[0204] In some aspects, any of the methods disclosed herein can further include monitoring for an occurrence of one or more adverse effects in the subject. Adverse effects may include, but are not limited to neurologic toxicity, cutaneous toxicity, gastrointestinal toxicity, or a combination thereof. When one or more adverse effects are observed, the methods disclosed herein can further include reducing or increasing the dose of one or more of the treatment regimens depending on the adverse effect or effects in the subject.

[0205] In certain aspects, treatments administered according to the methods disclosed herein can improve patient life expectancy and / or reduce symptoms compared to the life expectancy and / or symptoms an untreated subject with identical disease condition and predicted outcome. As used herein, “patient life expectancy” is defined as the time at which 50 percent of subjects are alive and 50 percent have passed away. In some aspects, patient life expectancy can be indefinite following treatment according to the methods disclosed herein. In other aspects, patient life expectancy can be increased at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater52298854442Atty. Docket No. UTSDP4429WO-1001354027 to at least about 80% or greater, at least about 40% or greater to at least about 60% or greater compared to an untreated subject with identical disease condition and predicted outcome. In some aspects, patient life expectancy can be increased at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, at least about 100% compared to an untreated subject with identical disease condition and predicted outcome. In some aspects, patient life expectancy can be increased at least about 5% or greater to at least about 10% or greater, at least about 10% or greater to at least about 15% or greater, at least about 15% or greater to at least about 20% or greater, at least about 20% or greater to at least about 25% or greater, at least about 25% or greater to at least about 30% or greater, at least about 30% or greater to at least about 35% or greater, at least about 35% or greater to at least about 40% or greater, at least about 40% or greater to at least about 45% or greater, at least about 45% or greater to at least about 50% or greater, at least about 50% or greater to at least about 55% or greater, at least about 55% or greater to at least about 60% or greater, at least about 60% or greater to at least about 65% or greater, at least about 65% or greater to at least about 70% or greater, at least about 70% or greater to at least about 75% or greater, at least about 75% or greater to at least about 80% or greater, at least about 80% or greater to at least about 85% or greater, at least about 85% or greater to at least about 90% or greater, at least about 90% or greater to at least about 95% or greater, at least about 95% or greater to at least about 100% compared to an untreated patient with identical disease condition and predicted outcome.

[0206] In some aspects, treatment of a FRRS1 L related disease, such EDE, according to the methods disclosed herein can result in an attenuating, reducing or a preventing of one or more symptoms of the disease in comparison to the starting symptoms. In some aspects, the one or more symptoms may include intellectual and developmental delay, epilepsy, chorea and other progressive and debilitating motor impairments.VI. Kits and Related Compositions

[0207] The compositions described herein may, in some aspects, be assembled into pharmaceutical or research kits to facilitate their use in therapeutic, diagnostic or research applications. A kit may include one or more containers housing the components of the disclosure and instructions for use. Specifically, such kits may include one or more53298854442Atty. Docket No. UTSDP4429WO-1001354027 compositions described herein, along with instructions describing the intended application and the proper use of these agents. In certain aspects agents in a kit may be in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents.

[0208] Kits for research purposes may contain the components in appropriate concentrations or quantities for running various experiments.EXAMPLES

[0209] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1 : Development and in vitro testing of the scAAV9-JeT-h-FRRS1 L vector

[0210] A novel recombinant adeno-associated virus stereotype 9 (AAV9) vector utilizing a JeT promoter to drive the expression of human FRRS1 L gene was developed. A vector map of the developed scAAV9-JeT-h-FRRS1 L (rAA 9-FRRS1 L) is provided in FIG. 1. The rAAV9- FRRS1 L encodes a codon optimized version of the FRRS1 L gene. T ransfection of the rAAV9- FRRS1 L into HEK293 cells resulted in robust expression of FFRS1 L as shown in FIG. 3B. A sequence alignment of the codon optimized human-FRRS1 L cDNA sequence and its similarity to the mouse sequence is provided in FIG. 2A. The protein sequence expressed because of the translation of the codon optimized gene, in comparison to the wild-type gene is provided in FIG. 2B.Example 2: In vivo studies using the AAV9

[0211] To test the efficacy of the vector in vivo, the assembled vector was administered via intrathecal injections at postnatal day 10-14 in low and high doses to FRRSI L-Knockout (KO) mice (schematic in FIG. 3A), which share multiple phenotypic traits with affected patients. Virus injected KO-mice were examined and compared with phosphate-buffered saline (PBS) injected-KO and Wild-type (WT) controls, assessing the safety and efficacy of this approach. Initial observations (N= 8-11 per group) showed that both doses are well tolerated, followed by promising levels of FRRS1 L gene and protein detection in the CNS.54298854442Atty. Docket No. UTSDP4429WO-1001354027Example 3: Preclinical trails to determine efficacy of using the rAAV9-FRRS1L for therapy

[0212] Preclinical trial were conducted to further investigate possible functional restoration of AMPA receptors and subsequent neurological improvements using the rAAV9-FRRS1 L, through comprehensive biochemical and behavioral evaluations. FIG. 4A provides a schematic of the approach to preclinical trials. Briefly, FRRSI L-Knockout (KO) mice or control mice were intrathecally injected with either low or high doses of the rAAV9-FRRS1 L or PBS. The transgene expression was tested using both relative mRNA expression and RT-qPCR analysis (FIGs. 4B and 4C). Probed with specifically designed primers for human and mouse transgenes, the WT mice showed undetectable expression (Ct-values >37) of human- FRRS1 L, but high expression levels of mouse-FRRS1 L. While the un-treated KO mice showed non-detectable mRNA expression of either mouse or human FRRS1 L, the treated-KO mice exhibited a significant and dose dependent upregulation of Human-FRRS1 L mRNA, with a relative fold-change ranging roughly between 103-104for the low-dose and 104-105for the high-dose group (FIG. 4B).

[0213] A set of mice were sacrificed at 2 months (N=6-8 / group) and analyzed for expression of FRRS1 L in various organs including the spinal cord (FIG. 4D), cerebellum (FIG. 4E) and e kidney (FIG. 4F) and the liver (FIG. 4G). Preliminary results indicate a dose-dependent increase of AMPA receptors on synaptic membranes prepared from whole brain samples (FIG. 4H) and Cerebellum (FIG. 4I). Immunofluorescence for FRRS1 L and AMPA-Rs further confirmed re-expression of FRRS1 L in the cerebellum and hippocampus (red, see FIG. 4J and 4K respectively) and co-localization of AMPA-R (green) with FRRS1 L (red) and subsequent neuronal changes in injected-KO brains (FIG. 4L and FIG. 4M).Example 4: Behavioral studies on rAAV9-FRRS1L transfected mice.

[0214] To further confirm efficacy of treating FRRS1 L related conditions using the rAAV9- FRRS1 L gene therapy, behavioral studies were conducted on the mice to determine improvements in the treated KOs (N=7-9 per dose) compared to untreated ones. Multiple behavioral tests including rotarod, grip strength, and fear-conditioning tasks were used. FIGS. 5A and 5B provide results of a tail suspension test. Results show that at least half of the mice with low dose injection of rAAV9-FRRS1 L had restored responses to tail suspension tasks. The injected mice also performed better than the KO mice in horizontal ladder tasks (FIGS. 5D and 5E), rotarod test (FIGS. 5F and 5G), and grip strength test (FIGS. 5H and 5I). Open filed tests were conducted for 10 minutes per session and total distance, velocity, time within zones and frequency of entry into zones was evaluated. As shown in the FIGS. 5J-5M, in almost all the tests, the mice with high dosage injection performed as well as wildtype or55298854442Atty. Docket No. UTSDP4429WO-1001354027 heterozygous mice. Fear conditioning experiments were used to study freezing responses following cue (tone) and conditioning (shock). Freezing times were recording and are provided in FIG. 50. Here again, the mice injected with low or high doses of rAAV9-FRRS1 L performed better than knock out mice and for high does close to wild-type response could be restored. Finally, Digi-gate analysis was conducted essentially as shown in FIGS. 5P and 5Q. FIG. 5R provides a mapping of the areas of the mice monitored during the analysis. FIG. 5S provides a schematic of the paw area contact metrics used in this study. FIGS. 5T-5X are bar graphs with the results of this analysis. Significant difference was seen between the KO and injected mice in this study in hind paw drag, ataxia, gait symmetry, strides per second and stride / stride velocity.

[0215] EEG recording for mice injected with low and high doses of vector were also compared with the KO mice and WT mice as shown in FIG. 6A. Normal EEG of a wild-type mouse is shown on the top, with uniform distribution of power bands and stable activity. KO-Untreated mice show high variability / amplitude spikes; delta / theta bands reaching 80-100% of relative power with erratic extreme spikes, and abnormal lower-frequency oscillations likely due to epileptiform discharges (poly-spikes) and / or seizures (spike-trains). Low-dose treated KO showed delta and theta bands with notably lower amplitudes, suggesting partial reduction of the pathological neural activity seen in the untreated KO mice. Both treated-KO groups show higher relative contribution of alpha bands (green), which is typically associated with cognitive and sensorimotor processing, allowing for more efficient motor control. The high-dose treated- KOs show a more balanced distribution of power bands over time, offering a better normalization potential.

[0216] Next, brain MRI was conducted at 9 months to test if AAV9 / H-FRRS1 L treatment has the potential to rescue brain atrophy (see FIG. 6B). Consecutive transverse brain scans were processed using a 3D-slicer. Although not statistically significant, a dose-dependent rescue trend (brain volume / surface area) in treated-KOs was seen suggesting a probable treatment effect for minimizing the long-term (cerebellar / cortical) atrophy seen in later stages of the disease.

[0217] Body weight follow-ups were conducted for these mice (see FIG. 6C). The untreated- KO mice exhibit a plateau or slight decline in weight starting roughly around 6-months, staying significantly lower than the WT group. The low-dose treated-KO mice show slower weight gain compared to the high-dose group, but while still outperforming the untreated-KO mice, it remains statically unsignificant. The high-dose treated-KO mice more closely follow the weight trajectory of WT mice, suggesting efficacy of the treatment in counteracting weight loss typically observed in the FRSS1 L- KO mice.56298854442Atty. Docket No. UTSDP4429WO-1001354027

[0218] The results suggest the potential of gene replacement as a promising treatment for children suffering this severe epileptic encephalopathy.57298854442

Claims

Atty. Docket No. UTSDP4429WO-1001354027CLAIMSWhat is claimed is:

1. A polynucleotide sequence comprising a nucleic acid sequence as set forth in SEQ ID NO: 1 , or a sequence at least about 60% identical thereto, and an operably linked promoter sequence.

2. The polynucleotide sequence of claim 1 , wherein the nucleic acid sequence encodes a FRRS1 L protein as set forth in SEQ ID NO: 2 or a sequence at least about 80% identical thereto.

3. The polynucleotide sequence of claim 1 , wherein the promoter sequence is a JeT promoter sequence.

4. The polynucleotide sequence of claim 3, wherein the JeT promoter comprises a sequence as set forth in SEQ ID NO: 3, or a sequence at least about 90% identical thereto.

5. The polynucleotide sequence of any one of claims 1-4, further comprising one or more regulatory sequences operably linked to the nucleic acid sequence.

6. The polynucleotide construct of any one of claims 1-5, wherein the one or more regulatory sequences comprise, enhancers, polyadenylation signals, or terminators, or any combination thereof.

7. The polynucleotide sequence of any one of claims 1-6, wherein the polynucleotide sequence comprises a viral vector, or a plasmid.

8. The polynucleotide sequence of claim 7, wherein the viral vector comprises a lentiviral, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, herpes simplex viral vector, or a chimeric viral vector.

9. The polynucleotide sequence of claim 8, wherein the viral vector is an adeno- associated viral vector serotype 9.

10. The polynucleotide sequence of claim 9, wherein the viral vector is an adeno- associated viral vector serotype 9, comprising a sequence as set forth in SEQ ID NO: 1 , or a sequence at least 60% identical thereto.58298854442Atty. Docket No. UTSDP4429WO-100135402711. A viral vector comprising a polynucleotide sequence, wherein the polynucleotide sequence comprises a nucleic acid sequence as set forth in SEQ ID NO: 1 or a nucleic acid sequence at least 80% identical thereto.

12. The viral vector of claim 11 , wherein the nucleic acid sequence encodes a FRRS1 L protein as set forth in SEQ ID NO: 2 or a sequence at least about 80% identical thereto.

13. The viral vector of claim 11 , wherein the viral vector is an adeno associated virus 9 (AAV9).

14. The viral vector of any one of claims 11-13, wherein the viral vector is neurotropic.

15. A pharmaceutical composition comprising the polynucleotide sequence of any one of claims 1-10, or the viral vector of any one of claims 11-14, and at least one pharmaceutically acceptable excipient.

16. The pharmaceutical composition of claim 15, wherein the composition further comprises a nanoparticle, a liposome, a nanoconjugate, a nanocapsule, a micelle, an exosome, or a polymeric delivery vehicle.

17. The pharmaceutical composition of claim 15 or claim 16, wherein the pharmaceutical composition is formulated for intravenous, intracranial, intrathecal, subcutaneous, intramuscular, intranasal, cranial, transmucosal, trans-nasal, transcranial, or intracerebroventricular delivery.

18. The pharmaceutical composition of any one of claims 15-17, wherein the pharmaceutical composition is formulated to cross the blood-brain barrier.

19. A method of treating an FRRS1 L related disease or disorder in a subject in need thereof, wherein the method comprises administering to the subject an effective amount of the pharmaceutical composition of any one of claims 15-18.

20. The method of claim 19, wherein the FRRS1 L related disease or disorder is epileptic- dyskinetic encephalopathy.21 . The method of claim 19 or claim 20, wherein the subject is a human.

22. The method of claim 21 , wherein the subject is less than 1 year of age.59298854442Atty. Docket No. UTSDP4429WO-100135402723. The method of any one of claims 19-22, wherein the administering of the pharmaceutical composition results in reduction in one or more of epileptic episodes, seizures, infantile spasms, and hyperkinetic movements by at least 25% or more.60298854442

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