Melanopsin variants for vision restoration

Abstract

Provided herein are polynucleotide cassettes, vectors, and recombinant adeno- associated viruses for the expression of a melanopsin and for optogenetic expression of a transgene in a mammalian cell.

Description

MELANOPSIN VARIANTS FOR VISION RESTORATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Application No. 63 / 665,653 filed June 28, 2024, the content of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (627002001640SEQLIST. xml; Size: 44,663 bytes; and Date of Creation: June 25, 2025) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present invention relates to polynucleotide cassettes for the expression of a melanopsin and for optogenetic expression of a transgene in a mammalian cell.BACKGROUND OF THE INVENTION

[0004] Delivery of therapeutic agents using gene delivery vectors is a promising approach for treating and preventing genetic diseases and disorders. Viral vectors are highly efficient gene transfer vehicles and may be useful as gene delivery vectors. In particular, adeno-associated virus (AAV)-based vectors are desirable due to the non-integrating nature of their viral life cycle.

[0005] One significant challenge of designing polynucleotide cassettes and expression vectors for use in gene therapy is obtaining sufficient expression of the transgene in target cells following gene transfer. Therefore, expression cassettes that result in sufficient or high expression of the transgene can be an important part of a therapeutically effective vector and a successful gene therapy method. Thus, there is a need for optimized polynucleotide expression cassettes and vectors for expressing genes in mammalian cells.

[0006] The disclosures of all publications, patents, patent applications and published patent applications referred to herein are hereby incorporated herein by reference in their entirety.BRIEF SUMMARY OF THE INVENTION

[0007] The present application in one aspect provides a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region, (b) a promoter region, (c) a tripartite leader (TPL), (d) an enhanced majorlate promoter (eMLP), (e) a Kozak consensus sequence, (f) a sequence encoding the melanopsin comprising a nucleic acid sequence selected from SEQ ID NOs: 1-6 and 19-25, (g) a second enhancer region, and (h) a polyadenylation site; wherein the sequence encoding the melanopsin is operably linked to the promoter region.

[0008] In some embodiments according to the polynucleotide cassette described above, the first enhancer region comprises the cytomegalovirus (CMV) sequence set forth in SEQ ID NO: 7 or a nucleic acid sequence with at least 85% sequence identity thereto. In some embodiments, the promoter region comprises the CMV promoter sequence set forth in SEQ ID NO: 8 or a nucleic acid sequence with at least 85% sequence identity thereto. In some embodiments, the TPL comprises the nucleic acid sequence of SEQ ID NO: 9 or a nucleic sequence with at least 85% sequence identity thereto. In some embodiments, the eMLP comprises the nucleic acid sequence of SEQ ID NO: 10 or a nucleic sequence with at least 85% sequence identity thereto. In some embodiments, the Kozak consensus sequence comprises the nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence with at least 99% sequence identity thereto. In some embodiments, the second enhancer comprises the full EES sequence set forth in SEQ ID NO: 12 or a nucleic sequence with at least 85% sequence identity thereto. In some embodiments, the polyadenylation site comprises the human growth hormone (hGH) polyadenylation site set forth in SEQ ID NO: 13 or a nucleic sequence with at least 85% sequence identity thereto.

[0009] In some embodiments according to any one of the polynucleotide cassette described above, the polynucleotide cassette further comprises an intron, wherein the intron is between the 3’ end of the eMLP and the 5’ end of the Kozak consensus sequence. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 14 or 15, or a nucleic sequence with at least 85% sequence identity thereto.

[0010] In some embodiments according to any one of the polynucleotide cassette described above, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0011] In some embodiments according to any one of the polynucleotide cassettes described above, the sequence encoding the melanopsin comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 19-25.

[0012] In some embodiments according to any one of the polynucleotide cassette described above, the polynucleotide cassette comprises (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising a nucleic acid sequence of SEQ ID NOs: 3 and 19-25; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13.

[0013] In another aspect, provided herein is a polynucleotide cassette for optogenetic expression of a transgene in a mammalian cell, comprising in 5’ to 3’ order: (a) a promoter region, (b) an intron, (c) a coding sequence encoding the transgene, (d) a polyadenylation site, and flanked by inverted terminal repeats (ITRs). In some embodiments, the promoter region comprises a human synapsin promoter.

[0014] In some embodiments according to the polynucleotide cassette described above, the human synapsin promoter comprises the nucleic acid sequence of SEQ ID NO: 16 or 17, or a nucleic sequence with at least 85% sequence identity thereto. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 14 or 15, or a nucleic sequence with at least 85% sequence identity thereto. In some embodiments, the polyadenylation site is an SV40 or human growth hormone (hGH) polyadenylation site. In some embodiments, an ITR located 5’ of the promoter region is selected from the group consisting of: a singlestranded ITR and a self-complementary ITR.

[0015] In some embodiments according to any of the polynucleotide cassette described above, the polynucleotide cassette further comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or scaffold attachment region (SAR), wherein the WPRE or SAR is located between the coding sequence and the polyadenylation site.

[0016] In some embodiments according to any of the polynucleotide cassette described above, the polynucleotide cassette comprises (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a WPRE; and (f) an SV40 polyadenylation site.

[0017] In some embodiments according to any of the polynucleotide cassette described above, the polynucleotide cassette comprises (a) a self-complementary ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 15; (d) a transgene; and (e) an SV40 polyadenylation site.

[0018] In certain aspects, provided herein is a recombinant adeno-associated virus (rAAV) comprising: (a) a capsid protein, and b) an adeno-associated virus vector genome comprising any one of the polynucleotide cassettes described herein, wherein the polynucleotide cassette is flanked by a 5’ and a 3’ ITR.

[0019] In certain aspects, provided herein is a pharmaceutical composition comprising the rAAV described herein and a pharmaceutically acceptable excipient.

[0020] In certain aspects, provided herein is a method for producing an rAAV comprising: (a) culturing a host cell under a condition that rAAV are produced, wherein the host cell comprises: (i) a polynucleotide encoding a capsid protein; (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide cassette of any one of claims 1-24; and (iv) AAV helper functions; and (b) recovering the rAAV produced by the host cell.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1A depicts a schematic of the Cl 1 backbone used for ubiquitous retinal expression. The Cl 1 backbone comprises in 5’ to 3’ order: inverted terminal repeat (ITR), a CMV promoter, a tripartite leader (TPL), an intron, a Kozak consensus sequence, a transgene, a human scaffold attachment region (hSAR), a human growth hormone (hGH) polyadenylation site, and ITR. All constructs were packaged into the Cl 1 backbone, including codon optimized melanopsin, wild-type (WT) melanopsin, and GFP serves as a reference control. FIG. IB depicts a schematic of how a primer probe can distinguish between AAV genomic DNA and mature mRNA transcript. The black pentagon and chevron arrows represent primer probes that anneal to the sequence once the intron is spliced out. The AAV genome comprises in 5’ to 3’ order: ITR, a CMV promoter, a TPL, a major late promoter (MLP), an intron, a Kozak consensus sequence, a transgene, an hSAR, an hGH polyadenylation site, and ITR. Transcription of the AAV genome sequence generates the pre- mRNA. The intron, which is included in the AAV genome and pre-mRNA, is spliced out in the final mature mRNA transcript. The presence or absence of the intron can be detected using a primer probe.

[0022] FIG. 2 depicts the gene expression fold change (as measured by the AACt value) of a specific Cl 1 construct encoding a codon optimized melanopsin relative to that of GFP in HEK293T cells. The values were normalized to GAPDH. GFP was used to set a baseline gene expression value of 1.0 (horizontal dashed line). Negative qPCR controls included a non-template control (“NTC”), no reverse transcription (“no RT”), and blank samples. Primers specific for the Cl 1 construct (“CH”) and for the reference housekeeping gene (“GAPDH”) were used.

[0023] FIG. 3 depicts the gene expression fold change (as measured by the AACt value) of a specific Cl 1 construct encoding a codon optimized melanopsin relative to that of GFP in ARPE19 cells. The values were normalized to GAPDH. GFP was used to set a baseline gene expression value of 1.0 (horizontal dashed line). Negative qPCR controls included a nontemplate control (“NTC”), no reverse transcription (“no RT”), and blank samples. Primers specific for the Cl 1 construct (“CH”) and for the reference housekeeping gene (“GAPDH”) were used.

[0024] FIG. 4 shows maps of optogenetic retinal AAV expression cassettes comprising a human synapsin (hSyn) promoter. The pADV1723_pTR-hSyn-Cl lintron-GFP-SV40 plasmid comprises in 5’ to 3’ order: an AAV2 self-complementary ITR (scITR), a human synapsin promoter, an intron found in the Cl 1 backbone (synthetic intron eMLP + mouse IgH intron), a Kozak consensus sequence, GFP, an SV40 polyadenylation site, and an AAV4 ITR. The pADV1724_pTR-hSyn-HBB-IgG-GFP-SV40 plasmid comprises in 5’ to 3’ order: an AAV2 scITR, a human synapsin promoter, an HBB-IgG chimeric intron, a Kozak consensus sequence, GFP, an SV40 polyadenylation site, and an AAV4 ITR. pADV1725_pTR-hSyn- CBA / MVM-GFP-SV40 plasmid comprises in 5’ to 3’ order: an AAV2 scITR, a human synapsin promoter, a hybrid CBA / MVM intron, a Kozak consensus sequence, GFP, an SV40 polyadenylation site, and an AAV4 ITR. The pADV1726_pTR-hSyn-MSV-GFP-SV40 plasmid comprises in 5’ to 3’ order: an AAV2 scITR, a human synapsin promoter, an MSV intron, a Kozak consensus sequence, GFP, an SV40 polyadenylation site, and an AAV4 ITR. The pADV1727_pTR-hSyn-SV40-GFP-SV40 plasmid comprises in 5’ to 3’ order: an AAV2 scITR, a human synapsin promoter, an SV40 intron, a Kozak consensus sequence, GFP, an SV40 polyadenylation site, and an AAV4 ITR. The pADV1728_pTR-hSyn-GFP-SV40 plasmid (a control construct with no intron) comprises in 5’ to 3’ order: an AAV2 scITR, a human synapsin promoter, a Kozak consensus sequence, GFP, an SV40 polyadenylation site, and an AAV4 ITR.

[0025] FIG. 5A-5B show agarose gels visualizing amplification products of various cDNAs and the respective plasmids shown in FIG. 4. FIG. 5A shows the cDNA and plasmid amplification products of pADV1723_pTR-hSyn-Cl lintron-GFP-SV40 (“1723”), pADVl 724_pTR-hSyn-HBB-IgG-GFP-SV40 (“1724”), pADVl 725_pTR-hSyn-CBA / MVM- GFP-SV40 (“1725”), pADV1726_pTR-hSyn-MSV-GFP-SV40 (“1726”), pADVl 727_pTR- hSyn-SV40-GFP-SV40 (“1727”), and pADV1728_pTR-hSyn-GFP-SV40 (“1728”) in B35 cells. 1728 is the no intron control. FIG. 5B shows the cDNA and plasmid amplification products of pADV1723_pTR-hSyn-Cl lintron-GFP-SV40 (“1723”), pADVl 724_pTR-hSyn- HBB-IgG-GFP-SV40 (“1724”), pADVl 725_pTR-hSyn-CBA / MVM-GFP-SV40 (“1725”), pADVl 726_pTR-hSyn-MSV-GFP-SV40 (“1726”), pADVl 727_pTR-hSyn-SV40-GFP- SV40 (“1727”), and pADV1728_pTR-hSyn-GFP-SV40 (“1728”) in SH-SY5Y cells.

[0026] FIG. 6A depicts a graph of the TauOFF (s) measured from fluorescent calcium imaging of SH-SY5Y cells transfected with pADV1718-AAV-Cl 1-OPN4, pADVl 721- AAV-C 11 -OPN4 BH3 , pADVl 744-AAV-C 11 -OPN4(419AA-T129S-Q 135N-S 183 A)BH3, pAD VI 745- AAV-C 11 -OPN4(419 AA-T 129S-S 183 A-M226T- Y382D-R386 A-R390D)BH3 , pAD VI 746- AAV-C 11 -OPN4(419 AA-T 129S-S 183 A)BH3 , pAD VI 747- AAV-C 11 - OPN4(419AA-S 183 A)BH3, pAD VI 748-AAV-C 11 -OPN4(419AA-S 183 A-S384D- R386A)BH3, pADV1749-AAV-Cl 1-OPN4(419AA-S183A-S384D-R386A-R390D)BH3, or pADV 1750- AAV-C 11-OPN4(419AA-Y382D-S384D-R386A-R390D)BH3. FIG. 6B depicts a graph of the amplitude (a.u.) measured from fluorescent calcium imaging of SH-SY5Y cells transfected with pADV1718-AAV-Cl 1-OPN4, pADV 1721 -AAV-C 11-OPN4 BH3, pADVl 744-AAV-C 11-OPN4(419AA-T129S-Q135N-S183A)BH3, pADV1745-AAV-Cl 1- OPN4(419 AA-T 129S-S 183 A-M226T- Y382D-R386 A-R390D)BH3 , pADVl 746- AAV-C 11 - OPN4(419AA-T 129S-S 183 A)BH3, pADVl 747- AAV-C 11 -OPN4(419AA-S 183 A)BH3, pADVl 748- AAV-C 11 -OPN4(419 AA-S 183 A-S384D-R386 A)BH3 , pADVl 749- AAV-C 11 - OPN4(419AA-S 183 A-S384D-R386A-R390D)BH3, or pADVl 750-AAV-C 11 - OPN4(419 AA-Y382D-S384D-R386 A-R390D)BH3.

[0027] FIG. 7 depicts images of nuclei staining and GFP fluorescence of SH-SY5Y cell transduced with AAV2.7m8-ssAAV2-hSyn(A)-Cl 1-GFP-SAR (first row), AAV2.7m8- ssAAV2-hSyn(O)-Cl 1-GFP-SAR (second row), AAV2.7m8-scAAV2-hSyn(O)-MSV-GFP- SV40 (third row), AAV2.7m8-scAAV4(DG)-hSyn(O)-MSV-GFP-SV40 (row 4), AAV2.7m8-ssAAV2-hSyn(O)-Cll-GFP-WPRE (row 5), or AAV2.7m8- AAV-C 11 -GFP (row 6), at different multiplicity of infections (MOI), with formulation buffer as a control(row 7). For GFP staining, the MOI from left to right is 1E3, 1E4, and 1E5. For Hoechst nuclear dye, the MOI from left to right is 1E3, 1E4, and 1E5.

[0028] FIG. 8 depicts fluorescent fundus images of retinas from Rdl mice dosed in eyes with AAV2.7m8-scAAV4(DG)-hSyn(O)-MSV-GFP-SV40 (first column), AAV2.7m8-scAAV2- hSyn(O)-MSV-GFP-SV40 (second column), AAV2.7m8-ssAAV2-hSyn(O)-Cl 1-GFP- WPRE (third column), AAV2.7m8-ssAAV2-hSyn(A)-Cl 1-GFP-SAR (fourth column), AAV2.7m8-ssAAV2-hSyn(O)-Cl l-GFP-SAR (fifth column), AAV2.7m8-AAV-Cl l-GFP (sixth column), or a negative control (seventh column).DETAILED DESCRIPTION OF THE INVENTION

[0029] The present application provides novel polynucleotide cassettes that results in similar or better expression of melanopsin in a retinal cell line, compared to a reference cassette. This is advantageous because melanopsin is expressed poorly relative to other transgenes. Hence, these polynucleotide cassettes are potentially useful for gene therapy in eye diseases or disorders. Further, the present application provides novel polynucleotide cassettes that utilizes intron splicing to distinguish DNA from mature mRNA, which is useful for preclinical analysis of the gene therapy to be used in a subject.I. Definitions

[0030] In general, terms used in the claims and the specification are intended to be construed as having the plain meaning understood by a person of ordinary skill in the art. Certain terms are defined below to provide additional clarity. In case of conflict between the plain meaning and the provided definitions, the provided definitions are to be used.

[0031] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a selfreplicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0032] The term “expression vector” or “expression construct” or “cassette” or “plasmid” or simply “vector” can include any type of genetic construct, including AAV or rAAV vectors, containing a nucleic acid or polynucleotide coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed and is adapted for genetherapy. The transcript can be translated into a protein. In some embodiments, the transcript is partially translated or not translated. In certain aspects, expression includes both transcription of a gene and translation of mRNA into a gene product. In other aspects, expression only includes transcription of the nucleic acid encoding genes of interest. An expression vector can also comprise control elements operatively linked to the encoding region to facilitate expression of the protein in target cells. The combination of control elements and a gene or genes to which they are operably linked for expression can sometimes be referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

[0033] The term “polynucleotide” can encompass both naturally occurring and non-naturally occurring nucleic acids, fragments, mutants, derivatives and analogs thereof. For the avoidance of doubt, a “polynucleotide” may be any length greater two nucleic acids.

[0034] The term “polypeptide” can encompass both naturally occurring and non-naturally occurring proteins (e.g., a fusion protein), peptides, fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric, dimeric, trimeric, or polymeric. Further, a polypeptide may comprise a number of different domains, each of which has one or more distinct activities. For the avoidance of doubt, a “polypeptide” may be any length greater two amino acids.

[0035] The term “heterologous” can refer to an entity that is genotypically distinct from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species can be a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked can be a heterologous promoter.

[0036] An “enhancer” as used herein encompasses a cis-acting element that stimulates or inhibits transcription of adjacent genes. An enhancer that inhibits transcription also is termed a “silencer”. Enhancers can function (i.e., can be associated with a coding sequence) in either orientation, over distances of up to several kilobase pairs (kb) from the coding sequence and from a position downstream of a transcribed region.

[0037] A “promoter” as used herein encompasses a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis. Promoters and corresponding protein or polypeptide expression may be ubiquitous, meaning strongly active in a wide range ofcells, tissues and species or cell-type specific, tissue-specific, or species specific. Promoters may be “constitutive,” meaning continually active, or “inducible,” meaning the promoter can be activated or deactivated by the presence or absence of biotic or abiotic factors. Also included in the nucleic acid constructs or vectors of the invention are enhancer sequences that may or may not be contiguous with the promoter sequence. Enhancer sequences influence promoter-dependent gene expression and may be located in the 5' or 3' regions of the native gene.

[0038] As used herein, the term “gene” or “coding sequence” refers to a nucleotide sequence that encodes a gene product in vitro or in vivo. The term “transgene” refers to a coding sequence or a gene that is delivered into a cell by a vector. The coding sequence or gene can encode a peptide or polypeptide molecule.

[0039] A “polyadenylation signal sequence” as used herein encompasses a recognition region necessary for endonuclease cleavage of an RNA transcript that is followed by the polyadenylation consensus sequence AATAAA. A polyadenylation signal sequence provides a “polyA site”, i.e. a site on a RNA transcript to which adenine residues will be added by post-transcriptional polyadenylation.

[0040] “Operatively linked” or “operably linked” or “coupled” can refer to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in an expected manner. For instance, a promoter can be operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

[0041] The term “AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The term “AAV” includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

[0042] An “AAV vector” or “rAAV vector” as used herein refers to an adeno-associated virus (AAV) vector or a recombinant AAV (rAAV) vector comprising a polynucleotide sequence not of AAV origin (e.g., a polynucleotide heterologous to AAV such as a nucleic acid sequence that encodes a therapeutic transgene, e.g., melanopsin) for transduction into a target cell or to a target tissue. In general, the heterologous polynucleotide is flanked by at least one, and generally by two, AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids. An rAAV vector may be either single-stranded (ssAAV) or self-complementary (scAAV).

[0043] An “AAV virus” or “AAV viral particle” or “rAAV vector particle” or “rAAV particle” refers to a viral particle comprising at least one AAV capsid protein and a polynucleotide rAAV vector. In some cases, the at least one AAV capsid protein is from a wild type AAV or is a variant AAV capsid protein (e.g., an AAV capsid protein with an insertion). If the particle comprises a heterologous polynucleotide (e.g., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a target cell or target tissue), it is referred to as a “rAAV particle”, “rAAV vector particle” or a “rAAV vector”. Thus, production of rAAV particles necessarily includes production of a rAAV vector, as such a vector contained within a rAAV particle.

[0044] As used herein, “recombinant” can refer to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids. Thus, for example, a protein synthesized by a microorganism is recombinant, for example, if it is synthesized from an mRNA synthesized from a recombinant gene present in the cell.

[0045] The term “packaging” as used herein can refer to a series of intracellular events that can result in the assembly and encapsidation of a rAAV particle.

[0046] AAV “rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV “packaging genes.”

[0047] The term “host cell”, as used herein refers to a cell that has been transduced, infected, transfected or transformed with a vector. The vector may be a plasmid, a viral particle, a phage, etc. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those skilled in the art. It will be appreciated that the term “host cell” refers to the original transduced, infected, transfected or transformed cell and progeny thereof.

[0048] The term “pharmaceutical composition,” or simply “composition” as used herein, can refer to a biologically active compound, optionally mixed with at least one pharmaceutically acceptable chemical component, such as, though not limited to carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, excipients and the like.

[0049] The term “pharmaceutically acceptable” as used herein, can refer to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of a compound disclosed herein, and is relatively nontoxic (i.e., when the material is administered to an individual it does not cause undesirable biological effects nor does it interact in a deleterious manner with any of the components of the composition in which it is contained).

[0050] As used herein, “percent (%) sequence identity” and “homology” with respect to a nucleic acid sequence are defined as the percentage of nucleic acid bases in a candidate sequence that are identical with the nucleic acid bases in the specific polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0051] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered tohave specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0052] It is understood that embodiments of the invention described herein include “consisting of’ and / or “consisting essentially of’ embodiments.

[0053] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used to treat cancer of type X means the method is used to treat cancer of types other than X.

[0054] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. The term “about X-Y” used herein has the same meaning as “about X to about Y.”

[0055] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.II. Polynucleotide cassette, vector, and recombinant adeno-associated virusPolynucleotide cassette

[0056] One aspect the present application provides a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region, (b) a promoter region, (c) a tripartite leader (TPL), (d) an enhanced major late promoter (eMLP), (e) a Kozak consensus sequence, (f) a sequence encoding the melanopsin comprising a nucleic acid sequence selected from SEQ ID NOs: 1-6 and 19-25, (g) a second enhancer region, and (h) a polyadenylation site; wherein the sequence encoding the melanopsin is operably linked to the promoter region. In some embodiments, the polynucleotide is a DNA. In some embodiments, the polynucleotide is an RNA. In some embodiments, the RNA is an mRNA.

[0057] In some embodiments, the sequence encoding the melanopsin is heterologous to the promoter region to which it is operably linked (z.e., not naturally operably associated with the promoter region). In some embodiments, the coding sequence may be endogenous to the promoter sequence which it is operably linked (z.e., is associated in nature with that promoter).

[0058] In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 1, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 2, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 3, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 3. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 4, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 5, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 6, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 19, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 20, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 21, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequenceidentity to the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 22, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 23, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 24, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 25, or a nucleic acid sequence having at least about 70% (such as at least about one of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) sequence identity to the nucleic acid sequence of SEQ ID NO: 25. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 19-25. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 3. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 19. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 20. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 21. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 22. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 23. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 24. In some embodiments, a sequence encoding the melanopsin comprises a nucleic acid sequence of SEQ ID NO: 25.

[0059] In some embodiments, the polynucleotide cassette comprises one or more untranslated regions (UTRs). The UTR may be involved in various regulatory aspects of gene expression. It should be understood that the UTRs (e.g., the 5’ UTRs and / or the 3’ UTRs) provided herein are examples, and that the polynucleotide may comprise any UTR from any gene. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide synthetic (e.g., artificial UTRs) which arenot variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5’ or 3’, UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5’ UTRs or 3’ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3’ or 5’ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3’ or 5’) comprise a variant UTR.

[0060] In some embodiments, the polynucleotide cassette comprises a 5’ UTR. The 5’ UTRs provided herein may be recognized by the ribosome, thereby allowing the ribosome to bind and initiate translation of the mRNA (e.g., translation of the coding sequence and / or nucleic acid encoding a signal peptide of the mRNA).

[0061] In some embodiments, the polynucleotide cassette comprises a 3’ UTR. The 3’ UTRs provided herein may be involved in translation termination (e.g., translation of the coding sequence), and can also be important for post-transcriptional modifications. In some embodiments, the 3’ UTR immediately follows the translation stop codon of the coding sequence of the polynucleotide cassette. In some embodiments, the polynucleotide cassette comprises one or more stop codons before the 3’ UTR.

[0062] In some embodiments, the polynucleotide cassette comprises a 5’ UTR and a 3’ UTR, such as any of the 5’ UTRs and 3’ UTRs provided herein. In some embodiments, the 5’ UTR and the 3’ UTR are derived from the same species. In some embodiments, the 5’ UTR and the 3’ UTR are not derived from the same species. In some embodiments, the 5’ UTR is synthetic, and the 3’ UTR is not synthetic. In some embodiments, the 5’ UTR is not synthetic, and the 3’ UTR is synthetic.

[0063] In some embodiments, the polynucleotide cassette comprises a 5’ UTR upstream of the sequence encoding the melanopsin. In some embodiments, the 5' UTR does not contain the polynucleotide ATG. Exemplary suitable 5 UTR sequences include but are not limited to sequences selected from i) a promoter region; ii) a tripartite leader (TPL) sequence from adenovirus (see, e.g., Logan and Shenk, Proc Natl Acad Sci USA. 1984;81(12):3655-9); iii) an enhancer element sequence from the adenovirus major late promoter (eMLP) (see, e.g.,Durocher et al., NucL Acids. Res. 2002;30(2):e9); and / or iv) a Kozak consensus sequence. In some embodiments, the 5’ UTR comprises in 5’ to 3’ order: (a) a first enhancer region, (b) a promoter region, (c) a tripartite leader (TPL), (d) an enhanced major late promoter (eMLP), and (e) a Kozak consensus sequence.

[0064] In some embodiments, the polynucleotide cassette comprises one or more enhancers. Enhancers are nucleic acid elements that enhance transcription. In some embodiments, the polynucleotide cassette comprises a first enhancer upstream of the coding sequence and a second enhancer downstream of the coding sequence. Exemplary suitable enhancers include but are not limited to EFla, CMV, the full expression enhancer sequence (EES) or a portion thereof such as the 410-564 EES or 511-810 EES. EES corresponds to a human scaffoldattachment region, or SAR, of human beta-interferon (see, e.g., Agarwal et al., J. Virol. 1998;72(5):3720-3728).

[0065] In some embodiments, the first enhancer region comprises the cytomegalovirus (CMV) sequence set forth in SEQ ID NO: 7 or a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the first enhancer region comprises the cytomegalovirus (CMV) sequence set forth in SEQ ID NO: 7.

[0066] In some embodiments, the sequence encoding the melanopsin is under the transcriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a “ubiquitous” promoter. In some embodiments, the promoter is a “strong” or constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) promoter, or a connexin36 (or “Cx36”) promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, such as retinal cells, to reduce potential toxicity or undesirable effects to non-targeted cells. In some embodiments, several AAV vectors may be generated to allow selection of the most optimal serotype and promoter for use with the melanopsin transgene. In some embodiments, the promoter region comprises the CMV promoter sequence set forth in SEQ ID NO: 8 or a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the CMV promoter sequence set forth in SEQ ID NO: 8.

[0067] In some embodiments, the TPL comprises the nucleic acid sequence of SEQ ID NO: 9 or a nucleic sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the TPL comprises the nucleic acid sequence of SEQ ID NO: 9.

[0068] In some embodiments, the eMLP comprises the nucleic acid sequence of SEQ ID NO: 10 or a nucleic sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the eMLP comprises the nucleic acid sequence of SEQ ID NO: 10.

[0069] In some embodiments, the Kozak consensus sequence comprises the nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence with at least 99% sequence identity thereto. In some embodiments, the Kozak consensus sequence comprises the nucleic acid sequence of SEQ ID NO: 11.

[0070] In some embodiments, the promoter region comprises the CMV promoter sequence set forth in SEQ ID NO: 8 or a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the CMV promoter sequence set forth in SEQ ID NO: 8.

[0071] In some embodiments, the polynucleotide cassette comprises a 3’ UTR downstream of the sequence encoding the melanopsin. In some embodiments, the 3’ UTR comprises in 5’ to 3’ order: (a) a second enhancer region, and (b) a polyadenylation site.

[0072] In some embodiments, the second enhancer comprises the full EES sequence set forth in SEQ ID NO: 12 or a nucleic sequence with at least 85% sequence identity thereto. In some embodiments, the second enhancer comprises the full EES sequence set forth in SEQ ID NO: 12.

[0073] In some embodiments, the polynucleotide cassette comprises a polyadenylation site. The polyadenylation site is the site of cleavage at which a poly (A) tail comprising a poly (A) sequence is added to an mRNA. Poly (A) sequences consist of multiple adenosine monophosphates in succession. In some embodiments, the poly (A) sequence is crucial for translation of the mRNA. In some embodiments, the poly (A) sequence is downstream of the coding sequence of the mRNA. In some embodiments, the poly (A) sequence is downstream of a 3’ UTR of the mRNA. In some embodiments, the polyadenylation site comprises the human growth hormone (hGH) polyadenylation site set forth in SEQ ID NO: 13, or a nucleic sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity thereto. In some embodiments, the polyadenylation site comprises the hGH polyadenylation site set forth in SEQ ID NO: 13.

[0074] In some embodiments, the polynucleotide cassette further comprises an intron, wherein the intron is between the 3’ end of the eMLP and the 5’ end of the Kozak consensus sequence. In some embodiments, the intron comprises a splice donor / acceptor region. Introns are DNA polynucleotides that are transcribed into RNA and removed during mRNA processing through intron splicing. Efficiently spliced introns contain a pre-splice donor, branchpoint, and Py rich region (see, e.g., Senapathy et al., Methods Enzymol. 1990: 183:252- 78; Wu and Krainer, Mol Cell Biol. 1999;19(5):3225-36). 5' introns are generally more efficient compared to introns at the 3' end (see, e.g., Huang and Gorman, Mol Cell Biol.1990; 10(4): 1805- 10). Exemplary intron sequences include but are not limited to actin, elongation factor 1 alpha (EFla), enhancer element from the adenovirus major late promoter (eMLP) and CMVc. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 14 or 15, or a nucleic acid sequence having at least 90f%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 15.

[0075] In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0076] In some embodiments, the polynucleotide cassette described herein comprises in 5’ to 3’ order: (a) a first enhancer region comprising the cytomegalovirus (CMV) sequence set forth in SEQ ID NO: 7; (b) a promoter region comprising the CMV promoter sequence set forth in SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9;(d) an eMLP comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a second enhancer region comprising the full EES sequence set forth in SEQ ID NO: 12; and (g) a polyadenylation site comprising the hGH polyadenylation site set forth in SEQ ID NO: 13 (hereinafter denoted as the “Cl 1 backbone”). See, also, US11352644B2, which is herein incorporated by reference in its entirety for all purposes.

[0077] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 1; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0078] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 2; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0079] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 3; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0080] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 4; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0081] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 5; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0082] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 6; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0083] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 19; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0084] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 20; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0085] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 21; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0086] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 22; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0087] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 23; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0088] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 24; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0089] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7;(b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 25; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0090] In some embodiments, the polynucleotide cassette described herein promotes melanopsin expression at a similar level as a reference cassette in the same mammalian cell type. In some embodiments, the polynucleotide cassette described herein promotes melanopsin expression at a higher expression level relative to a reference cassette in the same mammalian cell type. In some embodiments, the polynucleotide cassette described herein promotes increased melanopsin expression at about 1-fold, about 1.1 -fold, about 1.2-fold, about 1.3 fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 2.1 fold, about 2.2-fold, about 2.3-fold, about 2.4-fold, about 2.5-fold, about 2.6-fold, about 2.7-fold, about 2.8-fold, about 2.9-fold, about 3-fold, about 3.1-fold, about 3.2-fold, about 3.3-fold, about 3.4-fold, about 3.5-fold, about 3.6-fold, about 3.7-fold, about 3.8-fold, about 3.9-fold, or about 4-fold higher than a reference cassette in the same mammalian cell type. In some embodiments, expression in a mammalian cell type of the polynucleotide cassette described herein results in TauOFF measurements of about 3.50 s, about 3.55 s, about 3.60 s, about 3.65 s, about 3.70 s, about 3.75 s, about 3.80 s, about 3.85 s, about 3.90 s, about 3.95 s, about 4.00 seconds (s), about 4.05 s, about 4.10 s, about 4.15 s, about 4.20 s, about 4.25 s, about 4.30 s, about 4.35 s, about 4.40 s, about 4.45 s, about 4.50 s, about 4.55 s, about 4.60 s, about 4.65 s, about 4.70 s, about 4.75 s, about 4.80 s, about 4.85 s, about 4.90 s, about 4.95 s, about 5.00 seconds (s), about 5.05 s, about 5.10 s, about 5.15 s, about 5.20 s, about 5.25 s, about 5.30 s, about 5.35 s, about 5.40 s, about 5.45 s, about 5.50 s, about 5.55 s, about 5.60 s, about 5.65 s, about 5.70 s, about 5.75 s, about 5.80 s, about 5.85 s, about 5.90 s, about 5.95 s, about 6.00 s, about 6.05 s, about 6.10 s, about 6.15 s, about 6.20 s, about 6.25 s, about 6.30 s, about 6.35 s, about 6.40 s, about 6.45 s, about 6.50 s, about 6.55 s, about 6.60 s, about 6.65 s, about 6.70 s, about 6.75 s, about 6.80 s, about 6.85 s, about 6.90 s, about 6.95 s, about 7.00 s, about 7.05 s, about 7.10 s, about 7.15 s, about 7.20 s,about 7.25 s, about 7.30 s, about 7.35 s, about 7.40 s, about 7.45 s, about 7.50 s, about 7.55 s, about 7.60 s, about 7.65 s, about 7.70 s, about 7.75 s, about 7.80 s, about 7.85 s, about 7.90 s, about 7.95 s, about 8.00 s, about 8.05 s, about 8.10 s, about 8.15 s, about 8.20 s, about 8.25 s, about 8.30 s, about 8.35 s, about 8.40 s, about 8.45 s, about 8.50 s, about 8.55 s, about 8.60 s, about 8.65 s, about 8.70 s, about 8.75 s, about 8.80 s, about 8.85 s, about 8.90 s, about 8.95 s, about 9.00 s, about 9.05 s, about 9.10 s, about 9.15 s, about 9.20 s, about 9.25 s, about 9.30 s, about 9.35 s, about 9.40 s, about 9.45 s, or about 9.50 s. In some embodiments, the polynucleotide cassette described herein promotes faster TauOFF (s) at about 1.01-fold, about 1.02-fold, about 1.03-fold, about 1.04-fold, about 1.05-fold, about 1.06-fold, about 1.07-fold, about 1.08-fold, about 1.09-fold, about 1.10-fold, about 1.11-fold, about 1.12-fold, about 1.13-fold, about 1.14-fold, about 1.15-fold, about 1.16-fold, about 1.17-fold, about 1.18-fold, about 1.19-fold, about 1.20-fold, about 1.22-fold, about 1.24-fold, about 1.26-fold, about 1.28-fold, about 1.30-fold, about 1.32-fold, about 1.34-fold, about 1.36-fold, about 1.38-fold, about 1.40-fold, about 1.42-fold, about 1.44-fold, about 1.46-fold, about 1.48-fold, about 1.50-fold, about 1.52-fold, about 1.54-fold, about 1.56-fold, about 1.58-fold, about 1.60-fold, about 1.65-fold, about 1.70-fold, about 1.75-fold, about 1.80-fold, about 1.85-fold, about 1.90-fold, about 1.95-fold, or about 2.00-fold, compared to a reference cassette in the same mammalian cell type. In some embodiments, expression in a mammalian cell type of the polynucleotide cassette described herein results in amplitude measurements of about 1500 arbitrary units (a.u.), about 1550 a.u., about 1600 a.u., about 1650 a.u., about 1700 a.u., about 1750 a.u., about 1800 a.u., about 1850 a.u., about 1900 a.u., about 1950 a.u., about 2000 a.u., about 2050 a.u., about 2100 a.u., about 2150 a.u., about 2200 a.u., about 2250 a.u., about 2300 a.u., about 2350 a.u., about 2400 a.u., about 2450 a.u., about 2500 a.u., about 2550 a.u., about 2600 a.u., about 2650 a.u., about 2700 a.u., about 2750 a.u., about 2800 a.u., about 2850 a.u., about 2900 a.u., about 2950 a.u., about 3000 a.u., about 3050 a.u., about 3100 a.u., about 3150 a.u., about 3200 a.u., about 3250 a.u., about 3300 a.u., about 3350 a.u., about 3400 a.u., about 3450 a.u., about 3500 a.u., about 3550 a.u., about 3600 a.u., about 3650 a.u., about 3700 a.u., about 3750 a.u., about 3800 a.u., about 3850 a.u., about 3900 a.u., about 3950 a.u., or about 4000 a.u.. In some embodiments, the polynucleotide cassette described herein promotes greater amplitude (a.u.) (i.e., improved light sensitivity) at about 1.01-fold, about 1.02-fold, about 1.03-fold, about 1.04-fold, about 1.05-fold, about 1.06-fold, about 1.07-fold, about 1.08-fold, about 1.09-fold, about 1.10-fold, about 1.11-fold, about 1.12-fold, about 1.13-fold, about 1.14-fold, about 1.15-fold, about 1.16-fold, about 1.17-fold, about 1.18-fold, about 1.19-fold, about 1.20-fold, about 1.22-fold, about 1.24-fold, about 1.26-fold,about 1.28-fold, about 1.30-fold, about 1.32-fold, about 1.34-fold, about 1.36-fold, about 1.38-fold, about 1.40-fold, about 1.42-fold, about 1.44-fold, about 1.46-fold, about 1.48-fold, about 1.50-fold, about 1.52-fold, about 1.54-fold, about 1.56-fold, about 1.58-fold, about 1.60-fold, about 1.62-fold, about 1.64-fold, about 1.65-fold, about 1.66-fold, about 1.68-fold, about 1.70-fold, about 1.72-fold, about 1.74-fold, about 1.76-fold, about 1.78-fold, about 1.80-fold, about 1.82-fold, about 1.84-fold, about 1.86-fold, about 1.88-fold, about 1.90-fold, about 1.92-fold, about 1.94-fold, about 1.96-fold, about 1.98-fold, about 2.00-fold, about 2.02-fold, about 2.04-fold, about 2.06-fold, about 2.08-fold, or about 2.10-fold, compared to a reference cassette in the same mammalian cell type. In some embodiments, the mammalian cell includes but is not limited to HEK293T cells, ARPE19 cells, Chinese hamster ovary (CHO) cells, B35 cells, SH-SY5Y cells, retinal ganglion cells, amacrine cells, horizontal cells, bipolar cells, photoreceptor cells, cone cells, rod cells, Muller glial cells, and retinal pigmented epithelium.

[0091] In another aspect, provided herein is a polynucleotide cassette for optogenetic expression of a transgene in a mammalian cell, comprising in 5’ to 3’ order: (a) a promoter region, (b) an intron, c) a coding sequence encoding the transgene, (d) a polyadenylation site, and flanked by inverted terminal repeats (ITRs). In some embodiments, the polynucleotide is a DNA. In some embodiments, the polynucleotide is an RNA. In some embodiments, the RNA is an mRNA.

[0092] In some embodiments, the polynucleotide cassette comprises one or more untranslated regions (UTRs). The UTR may be involved in various regulatory aspects of gene expression. It should be understood that the UTRs (e.g., the 5’ UTRs and / or the 3’ UTRs) provided herein are examples, and that the polynucleotide cassette may comprise any UTR from any gene. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide synthetic (e.g., artificial UTRs) which are not variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5’ or 3’, UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5’ UTRs or 3’ UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3’ or 5’ UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition ofnucleotides. Any of these changes producing an “altered” UTR (whether 3’ or 5’) comprise a variant UTR.

[0093] In some embodiments, the polynucleotide cassette comprises a 5’ UTR. The 5’ UTRs provided herein may be recognized by the ribosome, thereby allowing the ribosome to bind and initiate translation of the mRNA (e.g., translation of the coding sequence and / or nucleic acid encoding a signal peptide of the mRNA).

[0094] In some embodiments, the polynucleotide cassette comprises a 3’ UTR. The 3’ UTRs provided herein may be involved in translation termination (e.g., translation of the coding sequence), and can also be important for post-transcriptional modifications. I. In some embodiments, the 3’ UTR immediately follows the translation stop codon of the coding sequence of the polynucleotide cassette. In some embodiments, the polynucleotide cassette comprises one or more stop codons before the 3’ UTR.

[0095] In some embodiments, the polynucleotide cassette comprises a 5’ UTR and a 3’ UTR, such as any of the 5’ UTRs and 3’ UTRs provided herein. In some embodiments, the 5’ UTR and the 3’ UTR are derived from the same species. In some embodiments, the 5’ UTR and the 3’ UTR are not derived from the same species. In some embodiments, the 5’ UTR is synthetic, and the 3’ UTR is not synthetic. In some embodiments, the 5’ UTR is not synthetic, and the 3’ UTR is synthetic.

[0096] In some embodiments, the polynucleotide cassette comprises a 5’ UTR upstream of the sequence encoding the transgene. In some embodiments, the 5' UTR does not contain the polynucleotide ATG. Exemplary suitable 5 UTR sequences include but are not limited to sequences selected from i) a promoter region and / or ii) an intron. In some embodiments, the 5’ UTR comprises in 5’ to 3’ order: (a) a promoter region and (b) an intron.

[0097] In some embodiments, the sequence encoding the transgene is under the transcriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a “ubiquitous” promoter. In some embodiments, the promoter is a “strong” or constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) promoter, or a connexin36 (or “Cx36”) promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, such as retinal cells, to reduce potential toxicity or undesirable effects to non-targeted cells. In some embodiments, several AAV vectors may be generated to allow selection of the most optimalserotype and promoter for use with the transgene. In some embodiments, the promoter region comprises a human synapsin promoter. In some embodiments, the human synapsin promoter comprises the nucleic acid sequence of SEQ ID NO: 16, or a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the human synapsin promoter comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0098] In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto. In some embodiments, the intron comprises the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto.

[0099] In some embodiments, the polynucleotide cassette comprises a 3’ UTR downstream of the coding sequence encoding the transgene. In some embodiments, the 3’ UTR comprises a polyadenylation site. The polyadenylation site is the site of cleavage at which a poly (A) tail comprising a poly (A) sequence is added to an mRNA. Poly (A) sequences consist of multiple adenosine monophosphates in succession. In some embodiments, the poly (A) sequence is crucial for translation of the mRNA. In some embodiments, the poly (A) sequence is downstream of the coding sequence of the mRNA. In some embodiments, the poly (A) sequence is downstream of a 3’ UTR of the mRNA. In some embodiments, the polyadenylation site comprises an SV40 or human growth hormone (hGH) polyadenylation site.

[0100] In some embodiments, the polynucleotide cassette further comprises an ITR located 5’ of the promoter region selected from the group consisting of: a single-stranded ITR and a self-complementary ITR. In some embodiments, the single-stranded ITR is selected from any suitable AAV serotype including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rhlO, AAV-DJ, and any hybrid or chimeric AAV thereof. In some embodiments, the single-stranded ITR comprises an AAV2 ITR. In some embodiments, the self-complementary ITR is selected from any suitable AAV serotype including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rhlO, AAV-DJ, and any hybrid or chimeric AAV thereof.In some embodiments, the self-complementary ITR comprises an AAV2 or AAV4 ITR. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0101] In some embodiments, the polynucleotide cassette further comprises a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or scaffold attachment region (SAR), wherein the WPRE or SAR is located between the coding sequence and the polyadenylation site.

[0102] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a WPRE; and (f) an SV40 polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0103] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 17; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a SAR; and (f) an hGH polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0104] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a SAR; and (f) an hGH polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0105] In some embodiments, there is provided a polynucleotide cassette comprising in 5’ to 3’ order: (a) a self-complementary ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ IDNO: 15; (d) a transgene; and (e) an SV40 polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0106] In some embodiments, the intron is spliced out in the mature mRNA. In some embodiments, the splicing out of the intron results in detection of the mature mRNA using probes.

[0107] As will be recognized by a person of ordinary skill in the art, the polynucleotide cassettes may optionally contain other elements including, but not limited to restriction sites to facilitate cloning and regulatory elements for a particular gene expression vector. Examples of regulatory sequence include ITRs for AAV vectors, bacterial sequences for plasmid vectors, attP or attB sites for phage integrase vectors, and transposable elements for transposons.Vector

[0108] In some embodiments, the polynucleotide cassettes described herein are used to deliver a transgene to cells of a mammal. Accordingly, the polynucleotide cassette is cloned into a gene delivery vector.

[0109] Any convenient gene delivery vector that finds use delivering polynucleotide sequences to mammalian cells is encompassed by the gene delivery vectors of the present disclosure. For example, the vector may comprise single or double stranded nucleic acid, e.g. single stranded or double stranded DNA. For example, the gene delivery vector may be DNA, e.g., a naked DNA, e.g. a plasmid, or a minicircle, etc. The vector may comprise single-stranded or double-stranded RNA, including modified forms of RNA. In another example, the gene delivery vector may be an RNA, e.g., an mRNA or modified mRNA.

[0110] As another example, the gene delivery vector may be a viral vector derived from a virus, e.g. an adenovirus, an adeno-associated virus (AAV), a lentivirus, a herpes virus, an alpha virus or a retrovirus, e.g., Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) or lentivirus. While embodiments encompassing the use of adeno-associated virus are described in greater detail below, it is expected that a person of ordinary skill in the art will appreciate that similar knowledge and skill in the art can bebrought to bear on non-AAV gene delivery vectors as well. See, e.g., the discussion of retroviral vectors in, e.g., U.S. Pat. Nos. 7,585,676 and 8,900,858, and the discussion of adenoviral vectors in, e.g. U.S. Pat. No. 7,858,367, each of which is expressly incorporated by reference herein.Recombinant adeno-associated virus (rAA V)[OHl] In some embodiments, the polynucleotide cassette and vector encoding the polynucleotide cassette described herein are packaged into an adeno-associated virus (AAV).

[0112] In some embodiments, the AAV has been altered so that it is replication-defective in the subject (e.g., a human or a non-human primate). In some embodiments, the adeno- associated virus (AAV) is a recombinant AAV (rAAV).

[0113] AAV or rAAV are small non-enveloped single-stranded DNA viruses. rAAVs are non-pathogenic human parvoviruses and can be made to be dependent on helper viruses, including adenovirus, herpes simplex virus, vaccinia virus and CMV, for replication.

[0114] Exposure to wild type (wt) AAV is not associated or known to cause any human pathologies and is common in the general population, making AAV or rAAV a suitable delivery system for gene therapy. AAV and rAAV used for gene therapy for delivery of an anti-VEGF agent, e.g., aflibercept, can be of any serotype. In some embodiments, the methods of the disclosure provide for use of any suitable AAV serotype, including AAV1, AAV2, AAV2.5, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, rhlO, AAV-DJ, and any hybrid or chimeric AAV thereof. In some embodiments, the serotype used is based on tropism of the virus, or infectivity of a target cell of interest. In some embodiments, several AAV vectors may be generated to allow selection of the most optimal serotype for use with a transgene (e.g., melanopsin transgene).

[0115] In some embodiments, the AAV is a pseudotyped AAV. Pseudotyped AAV particles comprise AAV genome inverted terminal repeats (ITRs) of one AAV serotype encapsidated by an AAV capsid of another AAV serotype. Typically, pseudotyped AAV is designated as “AAV# / #”, where the first “#” indicates the AAV ITR serotype and the second “#” indicates capsid serotype. For example, an AAV particle comprising AAV2 ITRs and an AAV1 capsid would be designated “AAV2 / 1.”

[0116] In some embodiments, the rAAV particles comprise a nucleic acid, e.g., a heterologous nucleic acid. In some embodiments, the nucleic acid encodes a transgene, e.g., melanopsin. In some embodiments, the encoded transgene, e.g., melanopsin, is under thetranscriptional control of a promoter that initiates transcription of the nucleic acid. In some embodiments, the promoter is a “ubiquitous” promoter. In some embodiments, the promoter is a “strong” or constitutively active promoter, e.g., a cytomegalovirus (CMV) promoter, an elongation factor 1 alpha (EFla) promoter, a glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) promoter, or a connexin36 (or “Cx36”) promoter. In some embodiments, the promoter is a tissue-specific promoter that is activated in specific tissues or cells, such as retinal cells, to reduce potential toxicity or undesirable effects to non-targeted cells. In some embodiments, several AAV vectors may be generated to allow selection of the most optimal serotype and promoter for use with the transgene (e.g., melanopsin). In some embodiments, the nucleic acid is flanked by AAV ITRs.

[0117] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 1; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0118] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 2; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette furthercomprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0119] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 3; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0120] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 4; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0121] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprisingthe nucleic acid sequence of SEQ ID NO: 5; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0122] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 6; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0123] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 19; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0124] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPLcomprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 20; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0125] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 21; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0126] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 22; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0127] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 23; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0128] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 24; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0129] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order: (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising the nucleic acid sequence of SEQ ID NO: 25; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleicacid sequence of SEQ ID NO: 13. In some embodiments, the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

[0130] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for optogenetic expression of a transgene in a mammalian cell, comprising in 5’ to 3’ order: (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a WPRE; and (f) an SV40 polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0131] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for optogenetic expression of a transgene in a mammalian cell, comprising in 5’ to 3’ order: (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 17; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a SAR; and (f) an hGH polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0132] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for optogenetic expression of a transgene in a mammalian cell, comprising in 5’ to 3’ order: (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a SAR; and (f) an hGH polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.

[0133] In some embodiments, the rAAV comprises a vector comprising a polynucleotide cassette for optogenetic expression of a transgene in a mammalian cell, comprising in 5’ to 3’ order: (a) a self-complementary ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 15; (d) a transgene; and (e) an SV40 polyadenylation site. In some embodiments, the polynucleotide cassette further comprises an ITR located 3’ of the polyadenylation site selected from the group consisting of: a full-length AAV2 ITR and a full-length AAV4 ITR.III. Methods of making rAAV particles comprising polynucleotide cassettes

[0134] Numerous methods are known in the art for production of recombinant AA V virions, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus-AAV hybrids, herpesvirus-AAV hybrids (Conway, JE et al., (1997) J Virology 71(11):8780-8789) and baculovirus-AAV hybrids. rAAV production cultures for the production ofrAAV virus particles all require; 1) suitable host cells, 2) suitable helper virus function, 3) AAV rep and cap genes; 4) a nucleic acid (such as a sequence encoding a gene product) flanked by at least one AAV ITR sequence (e.g., an oversized rAAV vector genome); and 5) suitable media and media components to support rAA V production. In some embodiments, the suitable host cell is a primate host cell. In some embodiments, the suitable host cell is a human-derived cell line such as HeLa, A549, 293, or Perc.6 cells.

[0135] In some embodiments, the suitable helper function is provided by wild-type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus (HSV), baculovirus, or a plasmid construct providing helper functions. In some embodiments, the AAV helper functions are provided by adenovirus or HSV. In some embodiments, the AAV helper functions are provided by baculovirus and the host cell is an insect cell (e.g., Spodopterafrugiperda (Sf9) cells).

[0136] In some embodiments, the AA V virion described herein was produced in an insect cell. In some embodiments, the AA V virion described herein was produced in a Spodoptera frugiperda (Sf9) cell. In some embodiments, the AA V virion described herein was produced in a human-derived cell line. In some embodiments, the AAV virion described herein was produced in a HeLa, A549, 293, or Perc.6 cell. In some embodiments, the AAV virion described herein was produced in a HEK-293 cell.

[0137] In some embodiments, the AA V rep gene may be from any AA V serotype. In general, but not obligatory, the AAV rep gene is of the same serotype as the ITRs of the rAAV vector genome as long as the rep protein encoded by the rep gene may function to replicate and package the rAAV genome.

[0138] Suitable media known in the art may be used for the production of rAA V vectors. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), custom formulations such as those described in U.S. Patent No. 6,566,118, and Sf-900 IISFM media as described in U.S. Patent No. 6,723,551, each of which is incorporated herein by reference in its entirety, particularly with respect to custom media formulations for use in production of recombinant AAV vectors.

[0139] One method for producing rAAV virions is the triple transfection method. Briefly, a plasmid containing a rep gene and a capsid gene, such as a polynucleotide encoding a variant AAV capsid protein disclosed herein, along with a helper adenoviral plasmid, may be transfected (e.g., using the calcium phosphate method) into a cell line (e.g., HEK-293 cells), and virions may be collected and optionally purified. As such, in some embodiments, the rAAV virion was produced by triple transfection of a vector genome, a nucleic acid encoding AAV rep and a cap, such as a polynucleotide encoding a variant capsid protein disclosed herein, and a nucleic acid encoding AAV helper functions into a host cell, wherein the transfection of the nucleic acids to the host cells generates a host cell capable of producing rAAV virions.

[0140] In some embodiments, rAAV virions may be produced by a producer cell line method, such as the exemplary producer cell line method described in Martin et al., (2013) Human Gene Therapy Methods 24:253-269; U.S. Pub. No. US2004 / 0224411; and Liu, X.L. et al. (1999) Gene Ther. 6:293-299). Briefly, a cell line (e.g., a HeLa, 293, A549, or Perc.6 cell line) may be stably transfected with a plasmid containing a rep gene, a capsid gene, such as a polynucleotide encoding a variant capsid protein disclosed herein, and a vector genome. Cell lines may be screened to select a lead clone for rAAV production, which may then be expanded to a production bioreactor and infected with a helper virus (e.g., an adenovirus or HSV) to initiate rAAV production. Virions may subsequently be harvested, adenovirus may be inactivated (e.g., by heat) and / or removed, and the rAAV virions may be purified. As such, in some embodiments, the rAAV virion was produced by a producer cell line comprising one or more of a nucleic acid encoding the rAAV genome, a nucleic acid encoding AAV rep, a polynucleotide encoding a variant capsid protein disclosed herein, and a nucleic acid encoding AAV helper functions.

[0141] In some embodiments, the nucleic acid encoding the AAV rep gene, the AAV cap gene, and / or the rAAV genome are stably maintained in the producer cell line. In some embodiments, the nucleic acid encoding the AAV rep gene, the AAV cap gene, and / or the rAAV genome is introduced on one or more plasmids into a cell line to generate a producer cell line. In some embodiments, the AAV rep gene, the AAV cap gene, and rAAV genome are introduced into a cell on the same plasmid. In other embodiments, the AAV rep, theAAV cap, and rAAV genome are introduced into a cell on two or more different plasmids. In some embodiments, a cell line stably transfected with a plasmid maintains the plasmid for multiple passages of the cell line (e.g., 5, 10, 20, 30, 40, 50 or more than 50 passages of the cell line). For example, the plasmid(s) may replicate as the cell replicates, or the plasmid(s) may integrate into the cell genome. A variety of sequences that enable a plasmid to replicate autonomously in a cell (e.g., a human cell) have been identified (see, e.g., Krysan, P.J. et al. (1989) Mol. Cell Biol. 9:1026-1033). In some embodiments, the plasmid(s) may contain a selectable marker (e.g., an antibiotic resistance marker) that allows for selection of cells maintaining the plasmid. Selectable markers commonly used in mammalian cells include without limitation blasticidin, G418, hygromycin B, zeocin, puromycin, and derivatives thereof. Methods for introducing nucleic acids into a cell are known in the art and include without limitation viral transduction, cationic transfection (e.g., using a cationic polymer such as DEAE-dextran or a cationic lipid such as lipofectamine), calcium phosphate transfection, microinjection, particle bombardment, electroporation, and nanoparticle transfection (for more details, see, e.g., Kim, T.K. and Eberwine, J.H. (2010) Anal. BioanaL Chem. 397:3173- 3178).

[0142] In some embodiments, the nucleic acids encoding the AAV rep gene, the AAV cap gene, and / or the rAAV genome are stably integrated into the genome of the producer cell line. In some embodiments, the nucleic acids encoding the AAV rep gene, the AAV cap gene, and / or the rAAV genome are introduced on one or more plasmids into a cell line to generate a producer cell line. In some embodiments, the AAV rep gene, the AAV cap gene, and the rAAV genome are introduced into a cell on the same plasmid. In other embodiments, the AAV rep gene, the AAV cap gene, and the rAAV genome are introduced into a cell on two or more different plasmids. In some embodiments, the plasmid(s) may contain a selectable marker (e.g., an antibiotic resistance marker) that allows for selection of cells maintaining the plasmid. Methods for stable integration of nucleic acids into a variety of host cell lines are known. For example, repeated selection (e.g., through use of a selectable marker) may be used to select for cells that have integrated a nucleic acid containing a selectable marker (and the AAV cap gene, the AAV rep gene, and / or a rAAV genome). In other embodiments, nucleic acids may be integrated in a site-specific manner into a cell line to generate a producer cell line. Several site-specific recombination systems are known in the art, such as FLP / FRT (see, e.g., O’Gorman, S. et al. (1991) Science 251 : 1351-1355),Cre / loxP (see, e.g., Sauer, B. and Henderson, N. (1988) Proc. Natl. Acad. Sci. 85:5166-5170), and phi C31-att (see, e.g., Groth, A.C. et al. (2000) Proc. Natl. Acad. Sci. 97:5995-6000).

[0143] In some embodiments, the producer cell line is derived from a primate cell line (e.g., a non-human primate cell line, such as a Vero or FRhL-2 cell line). In some embodiments, the cell line is derived from a human cell line. In some embodiments, the producer cell line is derived from HeLa, 293, A549, or PERC.6® (Crucell) cells. For example, prior to introduction and / or stable maintenance / integration of nucleic acids encoding the AAV rep gene, the AAV cap gene, and / or the rAAV genome into a cell line to generate a producer cell line, the cell line is a HeLa, 293, A549, or PERC.6® (Crucell) cell line, or a derivative thereof.

[0144] In some embodiments, the producer cell line is adapted for growth in suspension. As is known, anchorage-dependent cells are typically not able to grow in suspension without a substrate, such as microcarrier beads. Adapting a cell line to grow in suspension may include, for example, growing the cell line in a spinner culture with a stirring paddle, using a culture medium that lacks calcium and magnesium ions to prevent clumping (and optionally an antifoaming agent), using a culture vessel coated with a siliconizing compound, and selecting cells in the culture (rather than in large clumps or on the sides of the vessel) at each passage. For further description, see, e.g., ATCC frequently asked questions document (available at atcc[dot]org / Global / FAQs / 9 / l / Adapting%20a%20monolayer%20cell%201ine%20to%20susp ension-40[dot]aspx) and references cited therein.

[0145] In some aspects, a method is provided for producing a rAAV virion as disclosed herein comprising (a) culturing a host cell under a condition that rAAV virions are produced, wherein the host cell comprises (i) a polynucleotide encoding a variant AAV capsid protein disclosed herein; (ii) a polynucleotide encoding a rep protein; (iii) a polynucleotide cassette comprising a sequence that encodes a gene product, e.g., a therapeutic gene product flanked by at least one AAV ITR; and (iv) AAV helper functions; and (b) recovering the rAAV virion produced by the host cell. In some embodiments, said at least one AAV ITR is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, nonprimate AAV, or ovine AAV serotype ITRs or the like. In some embodiments, the method further comprises purifying the rAAV virion.

[0146] Suitable rAAV production culture media of the present invention may be supplemented with serum or serum-derived recombinant proteins at a level of 0.5%-20% (v / v or w / v). Alternatively, as is known in the art, rAAV virions may be produced in serum-free conditions which may also be referred to as media with no animal-derived products. One of ordinary skill in the art may appreciate that commercial or custom media designed to support production of rAAV virions may also be supplemented with one or more cell culture components know in the art, including without limitation glucose, vitamins, amino acids, and or growth factors, in order to increase the titer of rAAV in production cultures.

[0147] rAAV production cultures can be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, rAAV production cultures include attachmentdependent cultures which can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized- bed bioreactors. rAAV production cultures may also include suspension-adapted host cells such as HeLa, 293, and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank bioreactors, and disposable systems such as the Wave bag system.IV. Pharmaceutical Compositions

[0148] The present invention also provides pharmaceutical compositions comprising i) any of the vectors (e.g., viral vector) comprising the polynucleotide cassettes described herein or rAAV comprising such polynucleotide cassettes; and ii) an optional pharmaceutically acceptable carrier.

[0149] Suitable formulations (e.g., pharmaceutical composition) of the vectors (e.g., viral vector) comprising the polynucleotide cassettes described herein or rAAV comprising such polynucleotide cassettes, can be obtained by mixing the vectors (e.g., viral vector) comprising the polynucleotide cassettes described herein or rAAV comprising such polynucleotide cassettes having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).

[0150] In some embodiments, the pharmaceutical composition further comprises additional ingredients. Additional ingredients include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington’s Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, Pa.), which is incorporated herein by reference. Additional excipients include agents which can serve as one or more of the following: (1) bulking agents, (2) solubility enhancers, (3) stabilizers and (4) and agents preventing denaturation or adherence to the container wall (e.g., surfactant, such as polysorbate (e.g., polysorbate 80) or poloxamer). The excipient is different depending on whether a pharmaceutical composition comprises a protein, nucleic acid, or vector (e.g., viral vector). Those skilled in the art can determine the appropriate excipient for a pharmaceutical composition.

[0151] For example, acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as olyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). In some embodiments, lyophilized formulations are provided.

[0152] Pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, e.g., filtration through sterile filtration membranes. The pharmaceutical compositions herein generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0153] The pharmaceutical compositions may be prepared by any method known or hereafter developed in the art of pharmacology. Preparations include but are not limited to, bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit. The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3- butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0154] Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for parenteral, intraperitoneal, intramuscular, intradermal, subcutaneous, or intravenous routes of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

[0155] A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. A unit dose is 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 be administered to an individual or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.EXAMPLES

[0156] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. The examples below are intended to be purely exemplary of the application and should therefore not be considered to limit the application in any way.Example 1: Improved Expression of Melanopsin through Codon Optimization

[0157] Codon optimized constructs encoding melanopsin were designed and tested to identify the construct with improved melanopsin expression. This example identified the codon optimized melanopsin constructs with an equivalent or increased expression compared to a reference construct.

[0158] Melanopsin has been found to express poorly relative to other transgenes. Codon optimization is a gene engineering approach that uses synonymous codon changes to improve protein production. Six constructs encoding different codon compositions for melanopsin without altering the amino acid sequence were generated to identify constructs with improved expression.Plasmids encoding codon optimized melanopsin

[0159] The following six codon optimized constructs encoding melanopsin were synthesized: pADV1729-AAV-Cl 1-OPN4_A3 (comprising SEQ ID NO: 4), pADV1730-AAV-Cl 1- OPN4 A4 (comprising SEQ ID NO: 5), pADV1731-AAV-Cl l-OPN4_A5 (comprising SEQ ID NO: 6), pADV1719-AAV-Cl 1-OPN4 BH1 (comprising SEQ ID NO: 1), pADV1720- AAV-C11-OPN4 BH2 (comprising SEQ ID NO: 2), and pADV1721-AAV-Cl 1-OPN4 BH3 (comprising SEQ ID NO: 3). Control vectors encoding wild-type melanopsin (pADV1718- AAV-C11-OPN4) and a reference control GFP (pADV1717-AAV-Cl 1-GFP) were also synthesized. All transgenes were cloned into the Cl 1 backbone for ubiquitous retinal expression (FIG. 1A).Assessment of Expression

[0160] mRNA expression experiments were performed to determine which codon optimized version of melanopsin expressed at equivalent or increased levels compared to the referencecontrol GFP. Expression was tested in a human retinal pigment epithelial cell line (e.g., ARPE-19) and a human epithelial-like cell line derived from a kidney (e.g., HEK293T).

[0161] Eight plasmids (six codon optimized versions of melanopsin, WT melanopsin, and GFP) were transfected (FugeneHD) into HEK293T cells and separately transfected into ARPE-19 cells. After 72 hours, cells were imaged under a fluorescent microscope, and fluorescence was verified in cells transfected with GFP. Cells were then harvested to extract mRNA (Qiagen RNeasy kit) and to generate cDNA (Invitrogen SuperScript IV Vilo). qPCR was performed to quantify expression of various transcripts in the transfected cells.

[0162] cDNA from HEK293T cells was assayed using qPCR primer probes for Cl 1 and GAPDH (FIG. 2). The fold change in gene expression in FIG. 2 was calculated by finding the ACt of each sample relative to GAPDH and then calculating the AACt relative to the first GFP sample (pADV1717-AAV-Cl 1-GFP-l). The fold change relative to the first GFP sample was then calculated (2'AACt). The biological GFP replicate had a similar expression to the first GFP sample (~1; FIG. 2). The WT melanopsin samples had lower expression than the GFP reference control. Three of the codon optimized constructs, pADV1729-AAV-Cl 1- OPN4 A3, pADV1730-AAV-Cl 1-OPN4_A4, and pADV1731-AAV-Cl l-OPN4_A5 had much lower expression, compared to the reference control. The other three codon optimized constructs, pADV1719-AAV-Cl 1-OPN4 BH1, pADV1720-AAV-Cl 1-OPN4 BH2, and pADV1721-AAV-Cl 1-OPN4 BH3, had better expression than wild-type melanopsin. pADV1721-AAV-Cl 1-OPN4 BH3 exhibited the highest melanopsin expression and has improved expression levels, relative to that of WT melanopsin, the GFP reference control, and other codon optimized constructs. Hence, AAV-C11-OPN4 BH3 also produces mRNA at a similar or higher rate than the reference control in the HEK293T cell model.

[0163] The following codon optimized melanopsin constructs were transfected into ARPE-19 cells: pADV1719-AAV-Cl 1-OPN4 BH1, pADV1720-AAV-Cl 1-OPN4 BH2, pADV1721- AAV-C11-OPN4 BH3, and pADV1731-AAV-Cl l-OPN4_A5. cDNA from ARPE-19 cells was assayed using qPCR primer probes for Cl 1 and GAPDH (FIG. 3). When comparing the fold change in expression among samples in ARPE-19 cells, a similar pattern of relative expression was seen in ARPE-19 cells as in the HEK293T cells. The second GFP plasmid had a similar expression to its biological duplicate (FIG. 3). The WT melanopsin sample had lower mRNA expression than the GFP reference control. One of the constructs, pADV1731- AAV-C11-OPN4 A5, had lower expression, compared to the reference control GFP. Codon optimized pADV1719-AAV-Cl 1-OPN4 BH1, pADV1720-AAV-Cl 1-OPN4 BH2, and pADV1721-AAV-Cl 1-OPN4 BH3 had higher expression than WT melanopsin and the GFPreference control, with pADVl 721 -AAV-C 11-OPN4 BH3 exhibiting the highest mRNA expression among the tested codon optimized constructs (FIG. 3).Example 2: Selection of Optogenetic Retinal AAV Expression Cassettes

[0164] Optogenetic retinal AAV expression cassettes were designed to include introns that allow DNA and mature mRNA to be distinguished through primer probes that span the intron (FIG. IB). Once the intron is spliced out, the probes can anneal to the sequence allowing for identification of the mature mRNA, while excluding the DNA. This example identifies the constructs with successful intron splicing for the proper identification of mature mRNA. Plasmid Constructs

[0165] Plasmids encoding AAV expression cassettes comprising 5’ and 3’ ITRs, an hSyn promoter, an intron, a Kozak consensus sequence, a GFP transgene, and an SV40 polyadenylation site, were designed and constructed (FIG. 4). The hSyn promoter restricts expression to neural cells. Five constructs with different introns were designed and tested (Table 1). All the introns were cloned behind the hSyn promoter in an AAV packaging plasmid driving the expression of a GFP transgene to test how well the introns were spliced.Table 1: Optogenetic retinal AAV expression cassettes comprising intronsAssessment of Intron Splicing

[0166] The splicing of these introns and as well as proper transgene transcription and translation was confirmed by expression of these plasmids in cells a rat neuroblastoma cell line (e.g., B35 cells) and a human neuroblastoma cell line (e.g., SH-SY5Y cells), which are cell lines that can drive transgene expression from the hSyn promoter. Cells were transfected with plasmids listed in Table 1. After 72 hours, the cells were imaged with a fluorescent microscope to verify GFP fluorescence and then harvested to extract mRNA (Qiagen RNeasyKit). The extracted mRNA was reverse transcribed into cDNA (Invitrogen Superscript IV VILO). Primers flanking the intronic area were used to amplify the sequence in both the cDNA and the plasmid from each construct. The products amplified from the cDNA were expected to be smaller in size than the plasmid amplification products if the intron was spliced out. The PCR products were run on an agarose gel (FIGs. 5A and 5B), then extracted and purified for sequence analysis.

[0167] As shown FIG. 5A, the cDNA band was smaller for pADV1723_pTR-hSyn- C1 lintron-GFP-SV40, pADV1724_pTR-hSyn-HBB-IgG-GFP-SV40, pADV1725_pTR- hSyn-CBA / MVM-GFP-SV40, and pADV1726_pTR-hSyn-MSV-GFP-SV40, indicating successful intron splicing in B35 cells. Successful splicing was observed for the same constructs in SH-SY5Y cells (FIG. 5B). However, due to failed splicing, pADV1727_pTR- hSyn-SV40-GFP-SV40 led to multiple bands in the B35 preparation and a smear when extracted from SH-SY5Y cells (FIGs. 5A and 5B).Example 3: Codon optimized melanopsin mutants have superior kinetics (smaller TauOFF) and light sensitivity (greater amplitude) compared to wild-type (WT) melanopsin and WT codon optimized melanopsin

[0168] The following seven codon optimized melanopsin constructs encoding melanopsin were synthesized: pADVl 744-AAV-C 11 -OPN4(419AA-T129S-Q 135N-S 183 A)BH3 (comprising SEQ ID NO: 19), pADV1745-AAV-Cl l-OPN4(419AA-T129S-S183A-M226T- Y382D-R386A-R390D)BH3 (comprising SEQ ID NO: 20), p ADV 1746- AAV-C11- OPN4(419AA-T129S-S183A)BH3 (comprising SEQ ID NO: 21), pADV1747-AAV-Cl l- OPN4(419AA-S183A)BH3 (comprising SEQ ID NO: 22), pADV1748-AAV-Cl l- OPN4(419AA-S183A-S384D-R386A)BH3 (comprising SEQ ID NO: 23), pADV1749-AAV- C11-OPN4(419AA-S183A-S384D-R386A-R390D)BH3 (comprising SEQ ID NO: 24), and pADV1750-AAV-Cl 1-OPN4(419AA-Y382D-S384D-R386A-R390D)BH3 (comprising SEQ ID NO: 25). Control vectors encoding wild-type (WT) melanopsin (pADV1718-AAV-Cl 1- OPN4; comprising SEQ ID NO: 18) and WT codon optimized melanopsin (pADV1721- AAV-C11-OPN4 BH3; comprising SEQ ID NO: 3) were also synthesized. All transgenes were cloned into the Cl 1 backbone for ubiquitous retinal expression.

[0169] The nine plasmids were transfected with opsin and RGECO (calcium indicator) into SH-SY5Y cells. Cells were briefly pulsed with 488 nm light to initiate the light response then recorded at 595 nm light to measure the fluorescence from RGECO coupled with calcium. TauOFF was calculated from the decay of fluorescence overtime. The amplitude wascalculated from the peak of fluorescence from SH-SY5Y cells transfected with opsin and GCaMP6s. Cells were imaged at 488 nm light to both stimulate the cells and recorded from fluorescence generated by GCaMP6s coupled with calcium.Table 2: Melanopsin and melanopsin mutants TauOFF (s) and amplitude (a.u.) measurements from fluorescent calcium imaging

[0170] As show in FIG. 6A, the seven codon optimized melanopsin constructs have lower TauOFF, compared to the WT and WT codon optimized melanopsins (pADV1718-AAV- C11-OPN4 and pADV1721-AAV-Cl 1-OPN4_BH3, respectively), indicating that the codon optimized melanopsin constructs have superior kinetics. Additionally, all of the codon optimized melanopsin constructs have greater amplitudes than the WT melanopsin, while all codon optimized melanopsin constructs except pADV1750-AAV-Cl 1-OPN4(419AA- Y382D-S384D-R386A-R390D)BH3 have greater amplitudes than the WT codon optimized melanopsin, demonstrating superior light sensitivity, compared to the controls (FIG. 6B). The numerical values of the measured TauOFF and amplitude for each construct from FIGs. 6 A and 6B, respectively, are listed in Table 2.Example 4: Functionality of expression cassettes in vitro

[0171] SH-SY5Y cells, comprising expression cassettes comprising an hSyn promoter, were plated on an optical 96-well plate and transduced with recombinant adeno-associated virus(rAAVs) comprising one of the expression cassettes disclosed herein: AAV2.7m8-ssAAV2- hSyn(A)-Cl 1-GFP-SAR; AAV2.7m8-ssAAV2-hSyn(O)-Cl 1-GFP-SAR; AAV2.7m8- scAAV2-hSyn(O)-MSV-GFP-SV40; AAV2.7m8-scAAV4(DG)-hSyn(O)-MSV-GFP-SV40; AAV2.7m8-ssAAV2-hSyn(O)-Cl 1-GFP-WPRE; or AAV2.7m8-AAV-Cl 1-GFP (positive control) at different multiplicity of infections (MOIs) of 1E5, 1E4, and 1E3. Negative control cells were exposed to formulation buffer only. After 3 days, Hoechst nuclear dye was added to the cells to label nuclei, and the cells in the 96-well plate were imaged using ImageExpress Micro (Molecular Devices). Cells were imaged with the 10X objective using the DAPI channel to image the Hoechst nuclear stain and using the FITC channel to image GFP fluorescence. The same light exposure settings were used for all images per channel.

[0172] All the wells transduced with AAVs produced GFP fluorescence. In comparison, no fluorescence was detected in the formulation buffer negative control wells. In wells transduced with the highest MOIs, the cell nuclei (visible due to Hoechst nuclei staining) were less dense than that of wells transduced with lower MOIs. High MOIs can be potentially toxic, and this may explain the lower cell density in those wells. The strongest GFP expression was detected in the cells transduced with the positive control vector AAV2.7m8- AAV-C11-GFP. This was due to strong CMV promotor driving expression. For cells transduced with rAVV with an hSyn backbone, the strongest GFP expression was detected in cells transduced with AAV2.7m8-scAAV2-hSyn(O)-MSV-GFP (FIG. 7). This expression was much stronger than GFP expression in cells transduced with rAAV, ssAAV, or the other scAAV using the AAV4ITR. The highest expression from an ssAAV came from the AAV2.7m8-ssAAV2-hSyn(O)-GFP-WPRE, suggesting that the WPRE promotes stronger expression than the SAR. No noticeable difference was detected between the ssAAV vectors carrying the hSyn(O) and hSyn(A) promoters.Example 5: Functionality of expression cassettes in vivo

[0173] Rdl retinitis pigmentosa mice, with a mutation in phosphodiesterase beta (PDEP) which causes rod photoreceptor degeneration, were dosed with 1E10 vg / eye (2 uL) of rAAVs comprising one of the expression cassettes disclosed herein: AAV2.7m8-ssAAV2-hSyn(A)- C11-GFP-SAR; AAV2.7m8-ssAAV2-hSyn(O)-Cl 1-GFP-SAR; AAV2.7m8-scAAV2- hSyn(O)-MSV-GFP-SV40; AAV2.7m8-scAAV4(DG)-hSyn(O)-MSV-GFP-SV40; AAV2.7m8-ssAAV2-hSyn(O)-Cl 1-GFP-WPRE; or AAV2.7m8-AAV-Cl 1-GFP (positive control). Three weeks post dosing, the retinas of the mice were imaged in vivo by fluorescent fundus photography to observe GFP expression and general retinal health with a Micron IIFluorescent Fundus Camera (Phoenix Micron). All fluorescent images were taken with the same intensity of light.

[0174] Positive fluorescence data was detected for all rAAVs tested (FIG. 8). The strongest GFP fluorescence was observed with AAV2.7m8-scAAV2-hSyn(O)-MSV-GFP-SV40. The second strongest GFP fluorescence was observed with the AAV2.7m8-ssAAV2-hSyn(O)- C11-GFP-WPRE vector. Mice transfected with AAV2.7m8-AAV-Cl 1-GFP did not produce fluorescence as bright as AAV2.7m8-scAAV2-hSyn(O)-MSV-GFP-SV40. The GFP expression from the hSyn vectors was more concentrated near the optic near head, while the CMV vector had more widespread or pan retinal expression.SEQUENCE LISTING

Claims

CLAIMS1. A polynucleotide cassette for expression of a melanopsin in a mammalian cell, comprising in 5’ to 3’ order:(a) a first enhancer region,(b) a promoter region,(c) a tripartite leader (TPL),(d) an enhanced major late promoter (eMLP),(e) a Kozak consensus sequence,(f) a sequence encoding the melanopsin comprising a nucleic acid sequence selected from SEQ ID NOs: 1-6 and 19-25,(g) a second enhancer region, and(h) a polyadenylation site; wherein the sequence encoding the melanopsin is operably linked to the promoter region.

2. The polynucleotide cassette of claim 1, wherein the first enhancer region comprises the cytomegalovirus (CMV) sequence set forth in SEQ ID NO: 7 or a nucleic acid sequence with at least 85% sequence identity thereto.

3. The polynucleotide cassette of claim 1 or 2, wherein the promoter region comprises the CMV promoter sequence set forth in SEQ ID NO: 8 or a nucleic acid sequence with at least 85% sequence identity thereto.

4. The polynucleotide cassette of any one of claims 1-3, wherein the TPL comprises the nucleic acid sequence of SEQ ID NO: 9 or a nucleic sequence with at least 85% sequence identity thereto.

5. The polynucleotide cassette of any one of claims 1-4, wherein the eMLP comprises the nucleic acid sequence of SEQ ID NO: 10 or a nucleic sequence with at least 85% sequence identity thereto.

6. The polynucleotide cassette of any one of claims 1-5, wherein the Kozak consensus sequence comprises the nucleic acid sequence of SEQ ID NO: 11 or a nucleic acid sequence with at least 99% sequence identity thereto.

7. The polynucleotide cassette of claims any one of claims 1-6, wherein the second enhancer comprises the full EES sequence set forth in SEQ ID NO: 12 or a nucleic sequence with at least 85% sequence identity thereto.

8. The polynucleotide cassette of claims any one of claims 1-7, wherein the polyadenylation site comprises the human growth hormone (hGH) polyadenylation site set forth in SEQ ID NO: 13 or a nucleic sequence with at least 85% sequence identity thereto.

9. The polynucleotide cassette of any one of claims 1-8, wherein the polynucleotide cassette further comprises an intron, wherein the intron is between the 3’ end of the eMLP and the 5’ end of the Kozak consensus sequence.

10. The polynucleotide cassette of claim 9, wherein the intron comprises the nucleic acid sequence of SEQ ID NO: 14 or 15, or a nucleic sequence with at least 85% sequence identity thereto.

11. The polynucleotide cassette of any one of claims 1-10, wherein the polynucleotide cassette further comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR, wherein the 5’ ITR is at the 5’ end of the first enhancer region and the 3’ ITR is at the 3’ end of the polyadenylation site.

12. The polynucleotide cassette of claim any one of claims 1-11, wherein the sequence encoding the melanopsin comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 19-25.

13. The polynucleotide cassette of any one of claims 1-12, wherein the polynucleotide cassette comprises (a) a first enhancer region comprising the nucleic acid sequence of SEQ ID NO: 7; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 8; (c) a TPL comprising the nucleic acid sequence of SEQ ID NO: 9; (d) an eMLP sequence comprising the nucleic acid sequence of SEQ ID NO: 10; (e) a Kozak consensus sequence comprising the nucleic acid sequence of SEQ ID NO: 11; (f) a sequence encoding the melanopsin comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 19-25; (g) a second enhancer region comprising the nucleic acid sequence of SEQ ID NO: 12; (h) a polyadenylation site comprising the nucleic acid sequence of SEQ ID NO: 13.

14. A polynucleotide cassette for optogenetic expression of a transgene in a mammalian cell, comprising in 5’ to 3’ order:(a) a promoter region,(b) an intron,(c) a coding sequence encoding the transgene,(d) a polyadenylation site, and flanked by inverted terminal repeats (ITRs).

15. The polynucleotide cassette of claim 14, wherein the promoter region comprises a human synapsin promoter.

16. The polynucleotide cassette of claim 15, wherein the human synapsin promoter comprises the nucleic acid sequence of SEQ ID NO: 16 or 17, or a nucleic sequence with at least 85% sequence identity thereto.

17. The polynucleotide cassette of any one of claims 14-16, wherein the intron comprises the nucleic acid sequence of SEQ ID NO: 14 or 15, or a nucleic sequence with at least 85% sequence identity thereto.

18. The polynucleotide cassette of any one of claims 14-17, wherein the polyadenylation site is an SV40 or human growth hormone (hGH) polyadenylation site.

19. The polynucleotide cassette of any one of claims 14-18, wherein an ITR located 5’ of the promoter region is selected from the group consisting of: a single-stranded ITR and a self- complementary ITR.

20. The polynucleotide cassette of any one of claims 14-19, further comprising a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) or scaffold attachment region (SAR), wherein the WPRE or SAR is located between the coding sequence and the polyadenylation site.

21. The polynucleotide cassette of any one of claims 14-20, wherein the polynucleotide cassette comprises (a) a single-stranded ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 14; (d) a transgene; (e) a WPRE; and (f) an SV40 polyadenylation site.

22. The polynucleotide cassette of any one of claims 14-20, wherein the polynucleotide cassette comprises (a) a self-complementary ITR; (b) a promoter region comprising the nucleic acid sequence of SEQ ID NO: 16; (c) an intron comprising the nucleic acid sequence of SEQ ID NO: 15; (d) a transgene; and (e) an SV40 polyadenylation site.

23. A recombinant adeno-associated virus (rAAV) comprising:(a) a capsid protein, and b) an adeno-associated virus vector genome comprising the polynucleotide cassette of any one of claims 1-22, wherein the polynucleotide cassette is flanked by a 5’ and a 3’ ITR.

24. A pharmaceutical composition comprising the rAAV of claim 23 and a pharmaceutically acceptable excipient.

25. A method for producing an rAAV comprising:(a) culturing a host cell under a condition that rAAV are produced, wherein the host cell comprises:(i) a polynucleotide encoding a capsid protein;(ii) a polynucleotide encoding a rep protein;(iii) a polynucleotide cassette of any one of claims 1-22; and(iv) AAV helper functions; and(b) recovering the rAAV produced by the host cell.

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