Elovl2 constructs for gene therapy

Abstract

Disclosed herein are therapeutic agents capable of increasing the expression level of ELOVL2. Also described herein are therapeutic agents that reduce or slow an aging phenotype. Methods for treating age-related eye diseases or conditions are also provided. Methods for treating an age-related eye disease or condition in a subject by administering one or more therapeutic agents are provided.

Description

VGENX.005WO PATENT ELOVL2 CONSTRUCTS FOR GENE THERAPY INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 536,013, filed August 31, 2023, which is hereby incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING

[0002] The present application is filed with a Sequence Listing in Electronic format. The Sequence Listing is provided as a file entitled “VGENX.005WOSequenceListing.xml,” created August 14, 2024, which is approximately 31 kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety. BACKGROUND Field

[0003] The present disclosure relates to the fields of biochemistry and medicine. More particularly, the present disclosure relates to methods and compositions useful for ELOVL2 gene therapy to treat eye diseases. Description of the Related Art

[0004] Long chain and very long chain polyunsaturated fatty acids (LC-PUFA and VLC-PUFA respectively) have been shown to be important in multiple biological processes. They are widely accepted to act in multiple tissues as: 1) energy sources, 2) structural membrane components, and 3) key mediators in signal transduction pathways.

[0005] LC-PUFA cannot be synthesized de novo by humans and dietary sources of shorter chain fatty acid precursors are necessary for LC-PUFA tissue biosynthesis. The shorter chain fatty acids are “elongated” in a process where 2 carbons are added in a step-by-step manner by the action of various elongase enzymes. One such elongase, ELOVL2, has been shown to play an essential role in the conversion of docosapentanoic acid (DPA) (22:5n-3) to DHA (22:6n-3).

[0006] ELOVL fatty acid elongase 2 (ELOVL2) is a gene that encodes a transmembrane protein of the same name involved in catalyzing the rate-limiting step of the long-chain fatty acids elongation cycle. Studies in ELOVL2 knockdown mice suggest that ELOVL2 maintains homeostasis of LC-PUFA and VLC-PUFA. ELOVL2 genetic knock- down and knock-out animals show pathologies including reproductive sterility, ocular abnormalities, metabolic dysfunction, cognitive impairment and increased cellular aging. SUMMARY

[0007] In one aspect, disclosed herein are compositions for treating eye diseases. In some embodiments, a composition includes an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll-like Receptor 9 (anti-TLR9) sequence, or a bovine growth hormone polyadenylation signal (bGH poly-A) sequence.

[0008] In some embodiments, the ELOVL2 coding sequence includes a sequence exhibiting at least 95% identity to SEQ ID NO: 2 or to SEQ ID NO: 3. In some embodiments, the ELOVL2 coding sequence exhibits at least 95% identity to SEQ ID NO: 3.

[0009] In some embodiments, the composition includes a CB7 promotor sequence. In some embodiments, the CB7 promotor sequence exhibits at least 95% identity to SEQ ID NO: 4. In some embodiments, the anti-TLR9 sequence exhibits at least 95% identity to SEQ ID NO: 5. In some embodiments, the bGH poly-A sequence exhibits at least 95% identity to SEQ ID NO: 6.

[0010] In some embodiments, the composition further includes an enhancer, an inverted terminal repeat (ITR), a left polyadenylation signal, a signal sequence, a stuffer sequence, or a combination thereof. In some embodiments, the composition includes an adenovirus tripartite leader (ATL) sequence, a synthetic intron, a human ELOVL2 enhancer sequence, a woodchuck hepatitis posttranscriptional regulatory element (wpre element), or a human growth hormone (hGH) poly-A sequence.

[0011] In some embodiments, the composition comprises a DNA minicircle or synthetic AAV genome. In some embodiments, the composition comprises a viral capsid or envelope or a non-viral delivery system.

[0012] In some embodiments, the composition comprises an adeno-associated virus, adenovirus, alphavirus, herpesvirus, retrovirus, lentivirus, or a vaccinia virus capsid orenvelope. In some embodiments, the composition comprises an adeno-associated virus (AAV) based capsid. In some embodiments, the adeno-associated virus (AAV) based capsid comprises a AAV serotype 1 (AAV1) based, AAV-serotype 2 (AAV2) based, AAV serotype 3 (AAV3) based, AAV serotype 4 (AAV4) based, AAV serotype 5 (AAV5) based, AAV serotype 6 (AAV6) based, AAV serotype 7 (AAV7) based, AAV serotype 8 (AAV8) based, AAV serotype 9 (AAV9), or humanized AAV-based. In some embodiments, the AAV based capsid comprises a AAV-serotype 8 (AAV8) based capsid. In some embodiments, the composition comprises an AAV based capsid having a nucleotide sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0013] In some embodiments, the composition further includes an additional therapeutic agent selected from a corticosteroid, an anti-inflammation agent, a demethylating agent, and a combination thereof. In some embodiments, the composition is formulated for intravitreal, subretinal, subconjunctival, subtenon, suprachoroidal, or posterior juxtascleral administration.

[0014] In another aspect, disclosed herein is a composition comprising an ELOVL2 coding sequence and a CB7 promotor sequence. In some embodiments, the ELOVL2 coding sequence comprises a sequence exhibiting at least 95% identity to SEQ ID NO: 3. In some embodiments, the CB7 promotor sequence exhibits at least 95% identity to SEQ ID NO: 4.

[0015] In some embodiments, the composition further comprises an anti-Toll-like Receptor 9 (anti-TLR9) sequence or a bovine growth hormone polyadenylation signal (bGH poly-A) sequence. In some embodiments, the anti-TLR9 sequence exhibits at least 95% identity to SEQ ID NO: 5. In some embodiments, the bgh poly-A sequence exhibits at least 95% identity to SEQ ID NO: 6.

[0016] In some embodiments, the composition further comprises an enhancer, an inverted terminal repeat (ITR), a left polyadenylation signal, a signal sequence, a stuffer sequence, or a combination thereof. In some embodiments, the composition comprises an adenovirus tripartite leader (ATL) sequence, a synthetic intron, a human ELOVL2 enhancer sequence, a woodchuck hepatitis posttranscriptional regulatory element (wpre element), or a human growth hormone (hGH) poly-A sequence.

[0017] In some embodiments, the composition comprises a DNA minicircle or synthetic AAV genome. In some embodiments, the composition comprises a viral capsid orenvelope or a non-viral delivery system. In some embodiments, the composition comprises an adeno-associated virus, adenovirus, alphavirus, herpesvirus, retrovirus, lentivirus, or a vaccinia virus capsid or envelope. In some embodiments, the composition comprises an adeno- associated virus (AAV) based capsid. In some embodiments, the adeno-associated virus (AAV) based capsid comprises a AAV serotype 1 (AAV1) based, AAV-serotype 2 (AAV2) based, AAV serotype 3 (AAV3) based, AAV serotype 4 (AAV4) based, AAV serotype 5 (AAV5) based, AAV serotype 6 (AAV6) based, AAV serotype 7 (AAV7) based, AAV serotype 8 (AAV8) based, AAV serotype 9 (AAV9) , or humanized AAV-based. In some embodiments, the AAV based capsid comprises a AAV-serotype 8 (AAV8) based capsid. In some embodiments, the composition comprises an AAV based capsid having a nucleotide sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0018] In some embodiments, the composition further includes an additional therapeutic agent selected from a corticosteroid, an anti-inflammation agent, a demethylating agent, and a combination thereof. In some embodiments, the composition is formulated for intravitreal, subretinal, subconjunctival, subtenon, suprachoroidal, or posterior juxtascleral administration.

[0019] In a further aspect, disclosed herein are methods of treating, ameliorating or preventing an age-related eye disease or condition in a subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of the composition as described herein. For example, the method may include administering to the subject a therapeutically effective amount of a composition including an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll- like Receptor 9 (anti-TLR9) sequence, and a bovine growth hormone polyadenylation signal (bGH poly-A) sequence. For example, the method may include administering to the subject a therapeutically effective amount of a composition comprising an ELOVL2 coding sequence and a CB7 promotor sequence.

[0020] In some embodiments, the age-related eye disease comprises age-related macular degeneration (AMD), diabetic eye disease, glaucoma, low vision or dry eye. In some embodiments, the age-related eye disease comprises age-related macular degeneration (AMD).In some embodiments, administering comprises intravitreal, subretinal, subconjunctival, subtenon, suprachoroidal, or posterior juxtascleral administration. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Features of examples of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, though perhaps not identical, components. For the sake of brevity, reference numerals or features having a previously described function may or may not be described in connection with other drawings in which they appear. In addition to the features described herein, additional features and variations will be readily apparent from the following descriptions of the drawings and exemplary embodiments. It is to be understood that these drawings depict typical embodiments, and are not intended to be limiting in scope.

[0022] FIG. 1 is a line graph which presents the results of intraocular pressure (IOP) measurements using different AAV constructs.

[0023] FIG.2 is a set of line graphs, having Panels a-e which present the results of slit lamp examination using different AAV constructs and which measured inflammation- related data.

[0024] FIG.3 shows line graphs which present the results of ffERG measurements of a scotopic 2.51 a-wave amplitude (Panel a) and scotopic 0.158 b-wave amplitude (Panel b) for various AAV constructs.

[0025] FIG. 4 shows line graphs which present the results of OCT measurements of average thickness (Panel a) and average volume (Panel b) for various AAV constructs.

[0026] FIG. 5 is a schematic identifying locations of tissue sample punches for qPCR and lipid analysis.

[0027] FIG. 6 is a bar chart which presents the results of qPCR measurement of human ELOVL2-CV-4 transgene expression relative to African Green Monkey (AGM) endogenous ELOVL2 expression.

[0028] FIG.7 graphically presents the results of retinal LC-PUFA and VLC-PUFA measurements.DETAILED DESCRIPTION

[0029] The foregoing and other aspects of the present disclosure will now be described in more detail with respect to the description and methodologies provided herein. This description is not intended to be a detailed catalogue of all the ways in which the embodiments of the present disclosure may be implemented, or of all the features that may be added to the present disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein, which do not depart from the instant disclosure, will be apparent to those skilled in the art in light of the instant detailed description, figures and claims. Hence, the following specification is intended to illustrate some particular embodiments, and not to exhaustively specify all permutations, combinations and variations thereof.

[0030] All patents, patent applications, and other publications, including all sequences disclosed within these references, referred to herein are expressly incorporated herein by reference, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. All documents cited are, in relevant part, incorporated herein by reference in their entireties for the purposes indicated by the context of their citation herein. However, the citation of any document is not to be construed as an admission that it is prior art with respect to the present disclosure.

[0031] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

[0032] Embodiments of the disclosure relate to methods and compositions for increasing ELOVL2 enzyme concentration in tissues where ELOVL2 gene expression has declined. In some embodiments, the disclosure relates to methods for increasing ELOVL2 gene expression using ELOVL2 gene therapy within the eye. In some embodiments, the methods represent an improved therapeutic strategy. In some embodiments, methods for treating vision loss and other aging related dysfunctions using ELOVL2 gene therapy are provided herein.

[0033] In some embodiments, genetic constructs optimized for expression in the target tissue are provided herein. In some embodiments, the gene constructs are optimized to express more highly in human retina cells as compared to expression of the wild-type gene in retina cells. In some embodiments, the compositions disclosed herein include an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti- Toll-like Receptor 9 (anti-TLR9) sequence, and a bovine growth hormone polyadenylation signal (bGH poly-A) sequence. In some embodiments, the compositions include an ELOVL2 coding sequence and a CB7 promotor sequence. Definitions

[0034] Although the following terms are believed to be well understood by one of skill in the art, the following definitions are set forth to facilitate understanding of the presently disclosed subject matter.

[0035] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent techniques that would be apparent to one of skill in the art.

[0036] As used herein, the terms “a” or “an” or “the” may refer to one or more than one. For example, “a” marker can mean one marker or a plurality of markers.

[0037] As used herein, the term “about,” when used in reference to a measurable value such as an amount of mass, dose, time, temperature, and the like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0038] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0039] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0040] As used herein, the term “consists essentially of” (and grammatical variants thereof), as applied to the compositions and methods of the present disclosure, means that the compositions / methods may contain additional components so long as the additional components do not materially alter the composition / method. The term “materially alter,” as applied to a composition / method, refers to an increase or decrease in the effectiveness of the composition / method of at least about 20% or more. For example, a component added to a composition of the present disclosure would “materially alter” the composition if it increases or decreases the composition's ability to inhibit tumor growth by at least 20%.

[0041] In some embodiments, the “purity” of any given agent (e.g., antibody, polypeptide binding agent) in a composition may be specifically defined. For embodiments, certain compositions may comprise an agent that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, including all decimals in between, as measured, for example and by no means limiting, by high pressure liquid chromatography (HPLC), a well-known form of column chromatography used frequently in biochemistry and analytical chemistry to separate, identify, and quantify compounds.

[0042] As used herein, the terms “function” and “functional” and the like refer to a biological, enzymatic, or therapeutic function.

[0043] The term “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated cell” or “isolated population of cells” as used herein, includes a cell or population of cells that has been purified from sample material, including other cells, debris, or extraneous sample material from its naturally-occurring state, Alternatively, an “isolated cell” or “isolated population of cells” and the like, as used herein, includes the in vitro, extracorporeal, or other isolation and / or purification of a cell or population of cells from its natural environment, and from associationwith other components of the sample or material in which it occurs. In some embodiments, isolated means that the component is not significantly associated with in vivo substances.

[0044] As used herein, “subject” means a human or a non-human mammal, e.g., a dog, a cat, a mouse, a rat, a cow, a sheep, a pig, a goat, a non-human primate or a bird, e.g., a chicken, as well as any other vertebrate or invertebrate.

[0045] The term “mammal” is used in its usual biological sense. Thus, it specifically includes, but is not limited to, primates, including simians (chimpanzees, apes, monkeys) and humans, cattle, horses, sheep, goats, swine, rabbits, dogs, cats, rodents, rats, mice, guinea pigs, or the like.

[0046] An “effective amount” or a “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent that is effective to relieve, to some extent, or to reduce the likelihood of onset of, one or more of the symptoms of a disease or condition, and includes curing a disease or condition. “Curing” means that the symptoms of a disease or condition are eliminated; however, certain long-term or permanent effects may exist even after a cure is obtained (such as extensive tissue damage).

[0047] As used herein, “treat,” “treatment,” or “treating” refers to administering a compound or pharmaceutical formulation to a subject for prophylactic and / or therapeutic purposes. The term “prophylactic treatment” refers to treating a subject who does not yet exhibit symptoms of a disease or condition, but who is susceptible to, or otherwise at risk of, a particular disease or condition, whereby the treatment reduces the likelihood that the patient will develop the disease or condition. The term “therapeutic treatment” also refers to administering treatment to a subject already suffering from a disease or condition.

[0048] As used herein “administration” or “administering” refers to routes of introducing a compound or composition provided herein to an individual to perform its intended function. For example, “administration” means both intravitreal injection and injection via non intra-vitreal routes. Non-intravitreal routes can include subconjunctiva injection, sub-retinal injection, sub-tenon injection, retrobulbar injection and suprachoroidal injection. Additional examples also include gene therapy delivery with or without a delivery device. Other non-intravitreal routes include topical application to the eye and injections at other regions of the body intravenous and subcutaneous injection.

[0049] As used herein, “co-administration” and similar terms are broad terms, and are to be given their ordinary and customary meaning to a person of ordinary skill in the art (and are not to be limited to a special or customized meaning), and refer without limitation to administration of the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time. In some embodiments, the compounds disclosed herein are co-administered.

[0050] As used herein a “pharmaceutical formulation” means a bio-compatible aqueous or non-aqueous solution, suspension, dispersion or other physical form that includes an optimized polynucleotide sequence wherein the optimized polynucleotide sequence is at a concentration suitable for administering an effective amount to a mammalian subject.

[0051] As used herein, an “optimized” polynucleotide generally refers to nucleotide sequences that have been optimized for a particular host species by replacing any nucleotides from the wild-type so that the nucleotide sequence performs better in a heterologous environment. In one embodiment, optimized polynucleotide sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl. Acids Res. 17:477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence.

[0052] As used herein, a “stuffer sequence” generally refers to a stretch of nucleotides which is used to modulate the length of an AAV genome. For example, a stuffer sequence may be of a defined length. For example, a stuffer sequence may be between a polyA sequence and the right AAV2 ITR.

[0053] As used herein, a “vector” generally refers to a particle (for example, an AAV genome) used as a vehicle to artificially carry a foreign nucleic acid sequence into a cell, where the vector can be replicated and / or expressed. For example, a vector may include a viral vector, which may include a viral genome with disease-causing genes removed and replaced by a gene of interest.Compositions

[0054] In one aspect, compositions are described herein. In some embodiments, the composition is formulated as a composition for upregulating ELOVL2 expression.

[0055] In some embodiments, a composition as described herein includes one or more polynucleotides. For example, in some embodiments, the one or more polynucleotides comprise an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll-like Receptor 9 (anti-TLR9) sequence, a bovine growth hormone polyadenylation signal (bGH poly-A) sequence, and a stuffer sequence, as described herein. For example, in some embodiments, the one or more polynucleotides comprise an ELOVL2 coding sequence and a CB7 promotor sequence, as described herein.

[0056] In some embodiments, a composition as described herein comprises a therapeutic agent. For example, in some embodiments, a therapeutic agent comprises an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll-like Receptor 9 (anti-TLR9) sequence, a bovine growth hormone polyadenylation signal (bGH poly-A) sequence, and a stuffer sequence, as described herein. For example, in some embodiments, a therapeutic agent comprises an ELOVL2 coding sequence and a CB7 promotor sequence, as described herein.

[0057] In some embodiments, a composition as described herein includes one or more vectors. For example, in some embodiments, the one or more vectors comprise an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll-like Receptor 9 (anti-TLR9) sequence, a bovine growth hormone polyadenylation signal (bGH poly-A) sequence, and a stuffer sequence, as described herein. For example, in some embodiments, the one or more vectors comprise an ELOVL2 coding sequence and a CB7 promotor sequence, as described herein.

[0058] In some embodiments, the ELOVL2 coding sequence comprises an optimized polynucleotide sequence that when contacted with a tissue, can increase the tissue levels of ELOVL2. In some embodiments, the ELOVL2 coding sequence comprises a sequence exhibiting at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, or to SEQ ID NO: 3. In some embodiments, the ELOVL2 coding sequence exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 3.

[0059] In some embodiments, the composition comprises a CB7 promotor sequence. In some embodiments, the CB7 promotor sequence exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 4.

[0060] In some embodiments, the composition comprises anti-TLR9 sequence. In some embodiments, the anti-TLR9 sequence exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 5.

[0061] In some embodiments, the composition comprises a bGH poly-A sequence. In some embodiments, the bGH poly-A sequence exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 6.

[0062] In some embodiments, the composition comprises one or more stuffer sequences. In some embodiments, a stuffer sequence is a stuffer DNA sequence. In some embodiments, a stuffer sequence is located between two different sequence elements in a polynucleotide sequence. For example, in some embodiments, a stuffer sequence is located between a left ITR sequence and an enhancer sequence, such as an ELOVL2 enhancer sequence. In some embodiments, a stuffer sequence is located between an enhancer sequence, such as an ELOVL2 enhancer sequence, and a promoter sequence, such as a CB7 promoter sequence. In some embodiments, a stuffer sequence is located between a promoter sequence, such as a CB7 promoter sequence, and a ELOVL2 coding sequence. In some embodiments, a stuffer sequence is located between an a ELOVL2 coding sequence and a WPRE element sequence. In some embodiments, a stuffer sequence is located between a WPRE element sequence and a forward poly-A sequence, such as an hGH poly-A sequence. In some embodiments, a stuffer sequence is located between a forward poly-A sequence, such as an hGH poly-A sequence, and an anti-TLR9 sequence. In some embodiments, a stuffer sequence is located between an anti-TLR9 sequence and a reverse poly-A sequence, such as a bGH poly- A sequence. In some embodiments, a stuffer sequence is located between a reverse poly-A sequence, such as a bGH poly-A sequence, and a right ITR sequence.

[0063] In some embodiments, the composition further comprises an enhancer, an inverted terminal repeat (ITR), a left polyadenylation signal, a signal sequence, a stuffer sequence, or a combination thereof. In some embodiments, the composition comprises an adenovirus tripartite leader (ATL) sequence, a synthetic intron, a human ELOVL2 enhancer sequence, a woodchuck hepatitis posttranscriptional regulatory element (wpre element), or ahuman growth hormone (hGH) poly-A sequence. In some embodiments, the composition comprises a nucleotide sequence of at least one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0064] In some embodiments, the composition comprises a viral capsid or envelope or a non-viral delivery system. In some embodiments, the composition comprises an adeno- associated virus, adenovirus, alphavirus, herpesvirus, retrovirus, lentivirus, or a vaccinia virus capsid or envelope. In some embodiments, the composition comprises an adeno-associated virus (AAV) based capsid. In some embodiments, the adeno-associated virus (AAV) based capsid comprises a AAV serotype 1 (AAV1) based, AAV-serotype 2 (AAV2) based, AAV serotype 3 (AAV3) based, AAV serotype 4 (AAV4) based, AAV serotype 5 (AAV5) based, AAV serotype 6 (AAV6) based, AAV serotype 7 (AAV7) based, AAV serotype 8 (AAV8) based, AAV serotype 9 (AAV9) , or humanized AAV-based. In some embodiments, the AAV based capsid comprises a AAV-serotype 8 (AAV8) based capsid. In some embodiments, the composition comprises an AAV based capsid having a nucleotide sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18.

[0065] In some embodiments, the composition comprises a synthetic AAV genome. For example, the composition may comprise a synthetic AAV genome as described in PCT / US2023 / 066873, which is hereby incorporated by reference in its entirety. For example, an expression cassette used in a synthetic AAV genome may comprise an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti- Toll-like Receptor 9 (anti-TLR9) sequence, a bovine growth hormone polyadenylation signal (bGH poly-A) sequence, and / or a stuffer sequence, as described herein, as well as other sequences as described herein.

[0066] In some embodiments, the composition comprises a DNA minicircle. In some embodiments, the composition is administered without a viral capsid or envelope. (i.e. a non-viral delivery system). In some embodiments the non-viral delivery system is based on synthetic polymers that encapsulate polynucleotides of the present disclosure. For example, a non-viral delivery system may include synthetic polymeric micro-particles which are associated with or which encapsulate polynucleotides of the present disclosure. In some embodiments, a DNA minicircle format is utilized for transferring polynucleotides of the present disclosure. A “minicircle DNA vector” may be referred to as “minicircle vector” or“minicircle” is a small (usually in the range of 3-4 kb, approximately 3-4 kb or usually no larger than 10 kb) circular, episomal plasmid derivative wherein all prokaryotic vector parts (for example, bacterial origin of replication, genes associated with bacterial propagation of plasmids) have been removed. Since minicircle vectors contain no prokaryotic DNA sequences, they are less likely to be perceived as foreign and destroyed when they are employed as vehicles for transferring transgenes into target mammalian cells. In embodiments, a minicircle DNA vector is a minicircle carrying a transgene expression cassette. In examples, a minicircle DNA vector is a minicircle carrying a transgene expression cassette and does not contain an empty vector without an insert. In one or more embodiments, the nucleotide sequence of a minicircle containing the transgene expression cassette comprises an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti- Toll-like Receptor 9 (anti-TLR9) sequence, a bovine growth hormone polyadenylation signal (bGH poly-A) sequence, and a stuffer sequence, as described herein.

[0067] For example, minicircle vectors may be prepared using a two-step procedure. Firstly, a full-size parental plasmid containing bacterial sequences and transgene is produced in, for example, Escherichia coli. While the parental plasmid is still inside the E. coli host, the expression of a site-specific recombinase is induced and the prokaryotic or bacterial bone is excised by the enzyme at the recombinase recognition sites. Examples of site- specific recombinases include Tyr- and Ser-recombinases such as Cre recombinase, Flp recombinase, ParA resolvase and PhiC31 integrase. The resulting minicircle vector is recovered by capillary gel electrophoresis. An example of suitable materials, techniques, approaches, and methods are described in U.S. Pat. No. 8,236,548 which is hereby incorporated by reference in its entirety. Further description may be found in Kay et al, A Robust System for Production of Minicircle DNA Vectors, Nature Biotechnology, 2010 28:1287-1289, which is hereby incorporated by reference in its entirety.

[0068] In some embodiments, the sequences of the polynucleotides of the present disclosure may be further modified for expression in a particular organism, depending on that organism's biological constraints. In some embodiments, a polynucleotide may comprise a codon optimized for translation in a human cell. For example, the ELOVL2 coding sequence may comprise sequences that have been optimized for increased ELOVL2 expression. In some embodiments, the polynucleotide sequences of the present disclosure have been optimized forexpression in a human cell. In some embodiments, the polynucleotide sequences of the present disclosure may further comprise additional mutations resulting in a conservative amino acid substitution and or improved activity or stability of mRNA or the encoded polypeptide. In some embodiments, the polynucleotide sequences of the present disclosure include additional mutations resulting in improved stability of the gene or encoded protein in an eye of a subject.

[0069] In some embodiments, the composition comprises a polynucleotide having a sequence which exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14. Pharmaceutical Formulations

[0070] In an aspect, the compositions described herein comprise a pharmaceutical formulation.

[0071] In some embodiments, the pharmaceutical formulation (for example, a composition comprising an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-TLR9 sequence, a bGH poly-A sequence, and a stuffer sequence, as described above) may be administered to a subject by multiple administration routes, including but not limited to intravitreal, subretinal, subconjunctival, subtenon, posterior juxtascleral route, intravenous, subcutaneous, intramuscular), oral, intranasal, buccal, rectal, or transdermal administration routes. In some embodiments, the composition may be formulated for sub-retinal administration.

[0072] In some embodiments, the pharmaceutical formulations include, but are not limited to, aqueous solutions or liquid dispersions, self-emulsifying dispersions, solid solutions, suspensions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (for example, nanoparticle formulations), and mixed immediate and controlled release formulations.

[0073] In some embodiments, the pharmaceutical formulations include buffering, tonicity, stabilizing, suspending, stabilizing, solubilizing, whetting, carrier materials, pH adjusting agents, and the like selected on the basis of compatibility with the composition disclosed herein, and the profile properties of the desired dosage form. Carrier agents mayinclude, but are not limited to, acacia, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerine, magnesium silicate, polyvinylpyrrollidone (PVP), cholesterol, cholesterol esters, fatty acids, poloxamers, dextrans, polyethylene glycols, sodium caseinate, soy lecithin, taurocholic acid, phosphotidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995), Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975, Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N. Y., 1980, and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999).

[0074] In some embodiments, the pharmaceutical formulations further include pH adjusting agents or buffering agents which include acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris- hydroxymethylaminomethane, and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0075] In some embodiments, the pharmaceutical formulation includes one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0076] In some embodiments, the pharmaceutical formulations include, but are not limited to, sugars like trehalose, sucrose, mannitol, maltose, glucose, or salts like potassium phosphate, sodium citrate, ammonium sulfate and / or other agents to increase solubility and in vivo stability. In some embodiments, the pharmaceutical formulations further include a diluent. Salts dissolved in buffered solutions (which also can provide pH control or maintenance) are utilized as diluents in the art, including, but not limited to a phosphate buffered saline solution.

[0077] In some embodiments, the composition further comprises an additional therapeutic agent selected from a corticosteroid, an anti-inflammation agent, a demethylating agent, and a combination thereof. In some embodiments, the additional therapeutic agent may be a C18-C34 polyunsaturated fatty acid. In some embodiments, the additional therapeutic agent may be an anti-inflammatory agent. In some embodiments, the additional therapeutic agent may be a steroid. In some embodiments, the steroid may be a corticosteroid. In some embodiments, the additional therapeutic agent may be a demethylating agent. In some embodiments, the demthylating agent may be decitabine. In some embodiments, the additional therapeutic agent may be decitabine-PLGA. In some embodiments, the additional therapeutic agent may be selected from bevacizumab, ranibizumab, afibercept, lucentis, eylea, beovu, brolucizumab, macugen, ranibizumab, visudyne, aflibercept, vertecporfin, pegaptanib, or a combination thereof.

[0078] In some embodiments, the composition may be formulated as a composition for upregulating the expression of one or more additional genes. For example, the composition may comprise a polynucleotide encoding the one or more additional genes or a functionally- active fragment thereof. In some embodiments, composition may be formulated as a therapeutic agent for upregulating the expression of ELOVL2 in combination with one or more of additional genes selected from Slc6a4, Sst, Hdac4, Nefm, Calbl , I14U, Grin2c, Chga, Grm2, Neurodl, Ardbl, Dio3, Ghsr, Avprla, Cadps2, Gria2, Irs2, Smad2, Htr7, Sypl2, Madlll, Vgf, or a combination thereof. Methods of Use

[0079] In some aspects, disclosed herein are methods to modify, maintain, or increase tissue levels of ELOVL2 enzyme in a subject in need thereof. In some embodiments, the disclosure relates to methods for increasing ELOVL2 gene expression within the eye. In some embodiments, the disclosure relates to methods for maintaining ELOVL2 gene expression within the eye. In some embodiments, the disclosure relates to modifying ELOVL2 gene expression within the eye. In some embodiments, the methods include administering the subject a composition comprising an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll-like Receptor 9 (anti-TLR9) sequence, a bovine growth hormone polyadenylation signal (bGH poly-A) sequence, and a stuffersequence, as described herein. In some embodiments, the method includes administering the subject a composition comprising an ELOVL2 coding sequence and a CB7 promotor sequence, as described herein. For example, in some embodiments, the composition includes a delivery vector. In some embodiments, the delivery vector comprises an adeno associated virus vector, adenovirus-based vector, an alphavirus-based vector, a herpesvirus-based vector, a retrovirus- based vector, a lentivirus-based vector, or a vaccinia virus-based vector. In some embodiments, polynucleotides of the present disclosure may be in a minicircle format or synthetic AAV genome format. In some embodiments, the composition comprises a polynucleotide having a sequence which exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.

[0080] In some aspects, a method of reducing or slowing-down an aging phenotype in a subject in need thereof may be described herein. In some cases, the aging phenotype comprises a decrease in photoreceptors in an eye, retinal function, oxidative stress, age-related macular degeneration, diabetic eye disease, or dry eyes. In some embodiments, the methods include administering the subject a composition comprising an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll-like Receptor 9 (anti-TLR9) sequence, a bovine growth hormone polyadenylation signal (bGH poly-A) sequence, and a stuffer sequence, as described herein. In some embodiments, the method includes administering the subject a composition comprising an ELOVL2 coding sequence and a CB7 promotor sequence, as described herein. For example, in some embodiments, the composition includes a delivery vector. In some embodiments, the delivery vector comprises an adeno associated virus vector, adenovirus-based vector, an alphavirus-based vector, a herpesvirus-based vector, a retrovirus-based vector, a lentivirus-based vector, or a vaccinia virus-based vector. In some embodiments, polynucleotides of the present disclosure may be in a minicircle format or synthetic AAV genome format. In some embodiments, the composition comprises a polynucleotide having a sequence which exhibits at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 14.

[0081] In some embodiments, the aging-related disease or condition may be age- related macular degeneration (AMD). In some embodiments, a composition comprising a vector as described above is administered to treat AMD. In other cases, a composition comprising a vector described above reduces and / or slows-downs the progression of AMD. Insome embodiments, the aging-related disease or condition may be cataracts. In some embodiments, the aging-related disease or condition may be glaucoma. In some embodiments, the aging-related disease or condition may be dry eye syndrome. In some embodiments, the aging-related disease or condition may be low vision.

[0082] In some embodiments, the aging-related disease or indication may be a metabolic disease or condition. In some embodiments, the age-related disease or indication may be Alzheimer ’s disease. In some embodiments, the age-related disease or indication may be non-alcoholic fatty liver disease. In some embodiments, the age-related disease or indication may be cancer. In some embodiments, the age-related disease or indication may be retinitis pigmentosa. In some embodiments, the age-related disease or indication may be a corneal disease. In such embodiments, the metabolic disease or condition may be diabetes (diabetes mellitus, DM). In some cases, diabetes is type 1 diabetes, type 2 diabetes, type 3 diabetes, type 4 diabetes, double diabetes, latent autoimmune diabetes (LAD), gestational diabetes, neonatal diabetes mellitus (NDM), maturity onset diabetes of the young (MODY), Wolfram syndrome, Alstrom syndrome, prediabetes, or diabetes insipidus. Type 2 diabetes, also called non-insulin dependent diabetes, is the most common type of diabetes accounting for 95% of all diabetes cases. In some cases, type 2 diabetes is caused by a combination of factors, including insulin resistance due to pancreatic beta cell dysfunction, which in turn leads to high blood glucose levels. In some cases, increased glucagon levels stimulate the liver to produce an abnormal amount of unneeded glucose, which contributes to high blood glucose levels.

[0083] In some embodiments, the methods include administering the composition by sub-retinal injection. In some embodiments, the methods include administering the composition by intravitreal administration. In some embodiments, the methods include administering the composition by suprachoroidal administration. In some embodiments, the methods include administering the composition by intravenous administration. In some embodiments, administration may be via a narrow gauge needle or a cannula type device for delivering the composition.

[0084] In some embodiments, the methods described herein include administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent may be a C18-C28 polyunsaturated fatty acid. In some embodiments, the additional therapeutic agent may be an anti-inflammatory agent. In some embodiments, the additional therapeuticagent may be a steroid. In some embodiments, the steroid may be a corticosteroid. In some embodiments, the additional therapeutic agent may be a demethylating agent. In some embodiments, the demthylating agent may be decitabine. In some embodiments, the additional therapeutic agent may be decitabine-PLGA. In some embodiments, the additional therapeutic agent may be selected from bevacizumab, ranibizumab, afibercept, lucentis, eylea, beovu, brolucizumab, macugen, ranibizumab, visudyne, aflibercept, vertecporfin, pegaptanib, or a combination thereof.

[0085] In some embodiments, the method comprises treating a subject in need thereof, which comprises administering to the subject a composition comprising the vector comprising a polynucleotide encoding one or more additional genes or a functionally-active fragment thereof. In some embodiments, the therapeutic agent may be formulated as a composition for upregulating the expression of ELOVL2 in combination with one or more of additional genes selected from Slc6a4, Sst, Hdac4, Nefm, Calbl , I14U, Grin2c, Chga, Grm2, Neurodl, Ardbl, Dio3, Ghsr, Avprla, Cadps2, Gria2, Irs2, Smad2, Htr7, Sypl2, Madlll, Vgf, or a combination thereof. Therapeutic Regimens

[0086] The present disclosure provides pharmaceutical formulations, and kits for use in the treatment of a subject. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the formulations provide a therapeutic benefit to a subject in need of treatment for an eye disease.

[0087] In some aspects, a formulation as described herein is provided to a subject to treat, prevent, or ameliorate a disease or condition associated with vision loss. In some embodiments, the vision loss may be caused by a retinal disorder. In some embodiments, the retinal disorder may be age-related macular degeneration. In some embodiments, the formulation further comprises a pharmaceutically acceptable aqueous or non-aqueous medium. In some embodiments, the administration may be a sub-retinal injection.

[0088] In some aspects, the formulation as described herein results in increased levels of ELOVL2 enzyme in in the eye of a subject. In some embodiments, the formulation further comprises a pharmaceutically acceptable aqueous or non-aqueous medium. In some embodiments, the administration may be a sub-retinal injection. In some embodiments, theformulation may be administered in a volume from about 10 μL to about 200 μL. In some embodiments, the volume is about 150 μL. In some embodiments, the compositions as described herein are administered subretinally to an eye. In some embodiments, a single administration of the compositions as described herein is sufficient for therapeutic treatment. In some embodiments repeat administrations are required for therapeutic treatment. While not intended to be limiting, in some embodiments the therapeutic agent described herein is administered at intervals of 3 months, six months, 12 months, 18, months, 24 months, 30 months, 36 months 42 months, 48 months, 54 months, 60 months, 66 months, 72 months or combinations thereof. In some embodiments, the composition is described herein is administered after administration of a corticosteroid. In some embodiments, a corticosteroid is administered to a subject, followed by administration of a composition as described herein, followed by administration of a corticosteroid.

[0089] In some embodiments, toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50. Treatments exhibiting high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used in formulating a range of dosages for use in humans. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized. EXAMPLES

[0090] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the disclosure, as it is described herein above and in the claims. Example 1

[0091] In the following example, an AAV8-CB7-CV4 construct, including a CB7 promoter having the sequence of SEQ ID NO: 4 and an ELOVL2 coding sequence of SEQ IDNO: 3 (ELOVL2 Codon Variant 4 (CV4)), was administered by a single subretinal injection in healthy African Green Monkeys, and the effects were measured over a 56 day study. The injection location was in the superior retina, creating a subretinal fluid bleb.

[0092] 11 male and female monkeys were used for the study. 3 dosages were investigated in the study: Low (4E9), Mid (4E10), High (4E11). A vehicle control group was also used. The treatment groups are described in the table below. Table 1 2(4 eyes) ControlOne eye from each(3 eyes) Lipid analysis and 3 (6 eyes) Low group used for (5 eyes) N = 11 gene expression by 3 (6 eyes) Mid histopathology (5 eyes) qPCR 3 (6 eyes) High and RNA scope (5 eyes)

[0093] In life measures included ffERG, OCT, tonometry, color fundus imaging, fluorescence imaging, fluorescense angiography, and cSLO IR and AF imaging. Laboratory measures included gene expression qPCR, gene expression RNA scope, retinal LC and VLC PUFA content, and histopathology.

[0094] The results of intraocular pressure (IOP) measurements are presented in FIG. 1. As shown in FIG. 1, IOPs were considered to have remained relatively stable throughout the study period with no dose-dependent changes.

[0095] FIG. 2 presents the results of slit lamp examination which measured inflammation. As shown in FIG. 2, ocular inflammation ranged from absent to mild, as revealed by a) total clinical score, b) fibrin strands, c) iris hyperemia, d) vitreous cells, and e) keratic precipitates. In FIG. 2, the mean score is shown, ± SEM.

[0096] The results of Full-Field electroretinogram (ffERG) measurements are presented in FIG.3. ERG is considered to be a highly sensitive indicator of toxicity. The ERG a-wave reflects photoreceptor response. ERG b-wave reflects activity of bi-polar cells. As shown in FIG. 3, a modest decline in a-wave amplitude (less negative) was observed in mid and high dose groups as compared to vehicle control. However, there was no difference in a- wave amplitude between low dose and vehicle. A modest decline in the amplitude of the b- wave (less positive) was also observed in the high dose group compared to control. However, there were no significant differences in b-wave amplitude between mid and low dose comparedto control. For both a-wave and b-wave, low dose showed no difference from control amplitudes at day 56 suggesting the low dose was well tolerated as measured by the highly sensitive ERG metric.

[0097] FIG. 4 presents the results of Optical Coherence Tomography (OCT) measurements. As depicted in FIG.4, Panels a and b, and consistent with ERG findings, OCT showed small but measurable declines in both thickness and volume at high and mid dose exposures compared to control. However, no difference in either OCT thickness or volume was observed between vehicle and low dose suggesting the low dose was well tolerated.

[0098] Based on the data shown in FIGS. 1-4, it was concluded that the mid and high dose exposures resulted in measurable dose related changes in ERG and OCT, but that low dose exposure was well tolerated. From a toxicological perspective, based on the findings from ERG and OCT tests, the low dose could be considered a no adverse effect level.

[0099] FIG. 5 is a schematic identifying locations of tissue sample punches for qPCR and lipid analysis. Larger (12 mm punches) in superior and inferior retina were used for lipid analysis. Smaller (3 mm punches) were used for qPCR analysis.

[0100] The results of qPCR measurement of CV4 transgene expression are shown in FIG.6 from the 3 mm sub-punch 2 shown in FIG.5. In the graph of FIG.6, numbers above bars reflect the fold increase in transgene ELOVL2 over endogenous African Green Monkey (AGM) ELOVL2. As illustrated in FIG.6, the low dose expressed at levels 5 – 18 fold greater than endogenous ELOVL2. With increasing dose there was greater spread from the injection location, as evident by dose related increase in expression in inferior retina (subretinal injection was in superior retina).

[0101] FIG. 7 graphically illustrates Long Chain / Very-Long-Chain Polyunsaturated Fatty Acids (LC / VLC-PUFA) measurements. Total fatty acids (both free and esterified) are shown from Punch 1 taken from the superior retina as shown in FIG. 5. As shown in FIG. 7, low dose and mid dose significantly increased VLC-PUFA. DHA (C22:6) was unchanged in low dose but significantly decreased in the mid and high dose groups. This may have resulted from a “re-directing” of fatty acid C24:6 towards the VLC-PUFA pathway. In FIG. 7, key functional fatty acids (22:6, 32:6, 34:6, 24:6, 26:6) and precursors to the functional fatty acids (22:5 (2), 24:5) are highlighted by shading. As shown in FIG.7, low dose resulted in a significant increase in LC / VLC-PUFA with carbon chains of C24, C26, C32 andC34. Of note, these specific fatty acids were markedly lower in in retina obtained from human donors with AMD, compared to age-matched donors without disease. See Liu et al., Long- chain and very long-chain polyunsaturated fatty acids in ocular aging and age-related macular degeneration, J. Lipid Res. (2010), vol.51 at 3217-3229.

[0102] Based on these in life and laboratory results, it was concluded that AAV8- CB7-CV4 demonstrated a high degree of transgene expression in the retina (dose related) following subretinal injection, that tolerability was dose dependent, that low dose exposure (4E9) is likely a No Observed Adverse Effect Level “NOAEL”, and that significant increases in the target VLC-PUFA were observed at the low dose. Other Considerations

[0103] Conditional language used herein, such as, among others, “can,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that certain embodiments include, while other embodiments do not include, certain features, elements and / or states. Thus, such conditional language is not generally intended to imply that features, elements and / or states are in any way required for one or more embodiments or that one or more embodiments necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or states are included or are to be performed in any particular embodiment. The terms “comprising,” “including,” “having,” “involving,” and the like are synonymous and are used inclusively, in an open-ended fashion, and do not exclude additional elements, features, acts, operations, and so forth. Also, the term “or” is used in its inclusive sense (and not in its exclusive sense) so that when used, for example, to connect a list of elements, the term “or” means one, some, or all of the elements in the list.

[0104] Disjunctive language such as the phrase “at least one of X, Y or Z,” unless specifically stated otherwise, is otherwise understood with the context as used in general to present that an item, term, etc., may be either X, Y or Z, or any combination thereof (such as X, Y and / or Z). Thus, such disjunctive language is not generally intended to, and should not, imply that certain embodiments require at least one of X, at least one of Y or at least one of Z to each be present.

[0105] The terms “about” or “approximate” and the like are synonymous and are used to indicate that the value modified by the term has an understood range associated with it, where the range can be ±20%, ±15%, ±10%, ±5%, or ±1%. The term “substantially” is used to indicate that a result (such as a measurement value) is close to a targeted value, where close can mean, for example, the result is within 80% of the value, within 90% of the value, within 95% of the value, or within 99% of the value.

[0106] Unless otherwise explicitly stated, articles such as “a” or “an” should generally be interpreted to include one or more described items.

[0107] While the above detailed description has shown, described, and pointed out novel features as applied to illustrative embodiments, it will be understood that various omissions, substitutions, and changes in the form and details of the devices or algorithms illustrated can be made without departing from the spirit of the disclosure. As will be recognized, certain embodiments described herein can be embodied within a form that does not provide all of the features and benefits set forth herein, as some features can be used or practiced separately from others. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope.

[0108] It should be appreciated that all combinations of the foregoing concepts (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

[0109] The scope of the present disclosure is not intended to be limited by the specific disclosures of examples in this section or elsewhere in this specification, and may be defined by claims as presented in this section or elsewhere in this specification or as presented in the future. The language of the claims is to be interpreted broadly based on the language employed in the claims and not limited to the examples described in the present specification or during the prosecution of the application, which examples are to be construed as non- exclusive.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an ELOVL2 coding sequence and one or more sequences selected from a CB7 promotor sequence, an anti-Toll-like Receptor 9 (anti-TLR9) sequence, and a bovine growth hormone polyadenylation signal (bGH poly-A) sequence.

2. The composition of claim 1, wherein the ELOVL2 coding sequence comprises a sequence exhibiting at least 95% identity to SEQ ID NO: 2 or to SEQ ID NO:

3.

3. The composition of claim 1 or claim 2, wherein the ELOVL2 coding sequence exhibits at least 95% identity to SEQ ID NO:

3.

4. The composition of any of claims 1-3, wherein the composition comprises a CB7 promotor sequence.

5. The composition of any of claims 1-4, wherein the CB7 promotor sequence exhibits at least 95% identity to SEQ ID NO:

4.

6. The composition of any of claims 1-5, wherein the anti-TLR9 sequence exhibits at least 95% identity to SEQ ID NO:

5.

7. The composition of any of claims 1-6, wherein the bGH poly-A sequence exhibits at least 95% identity to SEQ ID NO:

6.

8. The composition of any of claims 1-7, wherein the composition further comprises an enhancer, an inverted terminal repeat (ITR), a left polyadenylation signal, a signal sequence, a stuffer sequence, or a combination thereof.

9. The composition of any of claims 1-8, wherein the composition comprises an adenovirus tripartite leader (ATL) sequence, a synthetic intron, a human ELOVL2 enhancer sequence, a woodchuck hepatitis posttranscriptional regulatory element (wpre element), or a human growth hormone (hGH) poly-A sequence.

10. The composition of any of claims 1-9, wherein the composition comprises a DNA minicircle or synthetic AAV genome.

11. The composition of any of claims 1-10, wherein the composition comprises a viral capsid or envelope or a non-viral delivery system.

12. The composition of claim 11, wherein the composition comprises an adeno- associated virus, adenovirus, alphavirus, herpesvirus, retrovirus, lentivirus, or a vaccinia virus capsid or envelope.

13. The composition of claim 11 or claim 12, wherein the composition comprises an adeno-associated virus (AAV) based capsid.

14. The composition of claim 13, wherein the adeno-associated virus (AAV) based capsid comprises a AAV serotype 1 (AAV1) based, AAV-serotype 2 (AAV2) based, AAV serotype 3 (AAV3) based, AAV serotype 4 (AAV4) based, AAV serotype 5 (AAV5) based, AAV serotype 6 (AAV6) based, AAV serotype 7 (AAV7) based, AAV serotype 8 (AAV8) based, AAV serotype 9 (AAV9) , or humanized AAV-based.

15. The composition of claim 13 or claim 14, wherein the AAV based capsid comprises a AAV-serotype 8 (AAV8) based capsid.

16. The composition of any of claims 1-15, wherein the composition comprises an AAV based capsid having a nucleotide sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO:

18.

17. The composition of any of claims 1-16, further comprising an additional therapeutic agent selected from a corticosteroid, an anti-inflammation agent, a demethylating agent, and a combination thereof.

18. The composition of any of claims 1-17, wherein the composition is formulated for intravitreal, subretinal, subconjunctival, subtenon, suprachoroidal, or posterior juxtascleral administration.

19. A composition comprising an ELOVL2 coding sequence and a CB7 promotor sequence.

20. The composition of claim 19, wherein the ELOVL2 coding sequence comprises a sequence exhibiting at least 95% identity to SEQ ID NO:

3.

21. The composition of claim 19 or claim 20, wherein the CB7 promotor sequence exhibits at least 95% identity to SEQ ID NO:

4.

22. The composition of any of claims 19-21, wherein the composition further comprises an anti-Toll-like Receptor 9 (anti-TLR9) sequence or a bovine growth hormone polyadenylation signal (bGH poly-A) sequence.

23. The composition of claim 22, wherein the anti-TLR9 sequence exhibits at least 95% identity to SEQ ID NO:

5.

24. The composition of claim 22 or claim 23, wherein the bgh poly-A sequence exhibits at least 95% identity to SEQ ID NO: 6.

25. The composition of any of claims 19-24, wherein the composition further comprises an enhancer, an inverted terminal repeat (ITR), a left polyadenylation signal, a signal sequence, a stuffer sequence, or a combination thereof.

26. The composition of any of claims 19-25, wherein the composition comprises an adenovirus tripartite leader (ATL) sequence, a synthetic intron, a human ELOVL2 enhancer sequence, a woodchuck hepatitis posttranscriptional regulatory element (wpre element), or a human growth hormone (hGH) poly-A sequence.

27. The composition of any of claims 19-26, wherein the composition comprises a DNA minicircle or synthetic AAV genome.

28. The composition of any of claims 19-27, wherein the composition comprises a viral capsid or envelope or a non-viral delivery system.

29. The composition of claim 28, wherein the composition comprises an adeno- associated virus, adenovirus, alphavirus, herpesvirus, retrovirus, lentivirus, or a vaccinia virus capsid or envelope.

30. The composition of claim 28 or claim 29, wherein the composition comprises an adeno-associated virus (AAV) based capsid.

31. The composition of claim 30, wherein the adeno-associated virus (AAV) based capsid comprises a AAV serotype 1 (AAV1) based, AAV-serotype 2 (AAV2) based, AAV serotype 3 (AAV3) based, AAV serotype 4 (AAV4) based, AAV serotype 5 (AAV5) based, AAV serotype 6 (AAV6) based, AAV serotype 7 (AAV7) based, AAV serotype 8 (AAV8) based, AAV serotype 9 (AAV9) , or humanized AAV-based.

32. The composition of claim 30 or claim 31, wherein the AAV based capsid comprises a AAV-serotype 8 (AAV8) based capsid.

33. The composition of any of claims 19-32, wherein the composition comprises an AAV based capsid having a nucleotide sequence selected from SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO:

18.

34. The composition of any of claims 19-33, further comprising an additional therapeutic agent selected from a corticosteroid, an anti-inflammation agent, a demethylating agent, and a combination thereof.

35. The composition of any of claims 19-34, wherein the composition is formulated for intravitreal, subretinal, subconjunctival, subtenon, suprachoroidal, or posterior juxtascleral administration.

36. A method of treating, ameliorating or preventing an age-related eye disease or condition in a subject, comprising: administering to the subject a therapeutically effective amount of the composition of any of claims 1-35.

37. The method of claim 36, wherein the age-related eye disease comprises age- related macular degeneration (AMD), diabetic eye disease, glaucoma, low vision or dry eye.

38. The method of claim 37, wherein the age-related eye disease comprises age- related macular degeneration (AMD).

39. The method of any of claims 36-38, wherein administering comprises intravitreal, subretinal, subconjunctival, subtenon, suprachoroidal, or posterior juxtascleral administration.

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