Patent Publication Number: US-2022213494-A1

Title: Dual leucine zipper kinase inhibitors for gene therapy

Description:
FIELD OF THE INVENTION 
     The present disclosure relates generally to cellular or gene therapy using an engineered inhibitor of Dual Leucine Zipper Kinase, including for neurodegenerative disorders such as glaucoma. 
     RELATED APPLICATIONS 
     This application is the National Stage of International Application No. PCT/US2020/018161, filed Feb. 13, 2020, which claims priority to U.S. Provisional Patent Appl. No. 62/806,213, filed Feb. 15, 2019, and U.S. Provisional Patent Appl. No. 62/806,210, filed Feb. 15, 2019, each of which is incorporated herein by reference in its entirety. 
     SEQUENCE LISTING 
     The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 7, 2022, is named “EXHA-002_O1US_SeqList.txt” and is about ˜41 kilobytes in size. 
     BACKGROUND OF THE INVENTION 
     Dual Leucine Zipper Kinase (DLK) a member of the serine/threonine protein kinase family encoded, in humans, by the DLK gene, also known as Mitogen-Activated Protein Kinase Kinase Kinase 12 or MAP3K12. This kinase contains a leucine-zipper domain and is predominately expressed in neuronal cells. The phosphorylation state of this kinase in synaptic terminals was shown to be regulated by membrane depolarization via calcineurin. This kinase forms homodimers, as well as heterodimers with leucine zipper containing transcription factors, such as cAMP responsive element binding protein (CREB) and MYC, and thus may play a regulatory role in PKA or retinoic acid induced neuronal differentiation. Alternatively spliced transcript variants encoding different proteins have been described. Expression of DLK with a K152A point mutation in the active site of the kinase results has a dominant negative affect on endogenous DLK. Xu et al. Mol Cell Biol. 21(14):4713-24 (2001). 
     DLK has been shown to be an essential component of neuronal response to axon damage. DLK protein is present in axons, and protein levels are increased in response to axonal injury (Xiong X, et al. J. Cell Biol, 2010; 191(1):211-223). DLK initiates a transcriptional program that couples apoptotic and regenerative responses to axonal injury (Watkins et al., Proc Natl Acad Sci, 2013; 110(10): 4039-4044). 
     Optic neuropathies are a group of vision diseases related to neuronal cell death, specifically the death of projection neurons called retinal ganglion cells (RGCs). Among the optic neuropathies, the U.S. spends $1.9 billion per annum to treat glaucoma. 
     Thus, there is a long-felt yet unmet need for compositions and methods for treating diseases or disorders related to DLK and for compositions and methods for cellular or gene therapy for glaucoma. The disclosure provides such novel compositions and methods to address and solve this need. 
     SUMMARY OF THE INVENTION 
     The present disclosure provides various embodiments of inhibitors of DLK, e.g., inhibitors of DLK that act in a dominant negative fashion by forming inactive heterodimers with endogenous DLK, called Dominant-Negative Dual Leucine Zipper Kinases (DN-DLKs). 
     In one aspect, the disclosure provides compositions and methods useful for treating diseases or disorders related to DLK and to retinal cells. In particular, the disclosure provides vectors (including viral vectors) for delivery of DN-DLKs, methods of using such vectors in treatment, and methods of inhibiting DLK activity in cells. 
     In some embodiments, the disclosure provides inhibitors that comprises a sequence at least 95% identical to the leucine zipper domain of DLK (SEQ ID NO: 3) with substitutions in at least one of positions 391 and 393, using the numbering of full-length, human DLK (SEQ ID NO: 1). 
     The disclosure also provides recombinant virus vectors, including recombinant adeno-associated virus (rAAV) vectors, comprising an expression cassette whose polynucleotide sequence encodes one of the inhibitors of the disclosure. 
     The disclosure also provides methods of inhibiting DLK activity in a cell using the vectors, as well as pharmaceutical compositions and unit doses that include vector(s) of the disclosure. The disclosure further provides methods of treatment for conditions including, without limitation, diseases or disorders related to dysfunction in retinal cells is selected from the group consisting of glaucoma, wet macular degeneration, dry macular degeneration, geographic atrophy, retinal detachment, and retinal dystrophy, such as Usher&#39;s syndrome, Stargardt&#39;s disease, Leber&#39;s congenital amaurosis, or retinitis pigmentosa. In yet other embodiments, the disease is related to retinopathy, such as diabetic retinopathy, myopic maculopathy. In other embodiments, the disease is an optic neuropathy, such as optic neuritis, non-arteritic anterior ischemic optic neuropathy (NAION), arteritic anterior ischemic optic neuropathy (AION), traumatic optic neuropathy, Leber&#39;s optic neuropathy, dominant optic atrophy, recessive optic atrophy, or radiation optic neuropathy. In other embodiments, the disease may include one or more of amyotrophic lateral sclerosis (ALS), Parkinson Disease, Alzheimer Disease, prion-related neurodegenerative disease, Huntington&#39;s disease, spinocerebellar ataxia, spinal muscular atrophy, and multiple system atrophy. 
     In another aspect, the disclosure provides vectors that comprise a polynucleotide encoding an inhibitor of Dual Leucine Zipper Kinase (DLK). The polynucleotide may be operatively linked to a promoter. The inhibitor is capable of binding either (i) an endogenous DLK protein, or (ii) an endogenous mRNA encoding an endogenous DLK. In some embodiments, the inhibitor is capable of reducing the expression, signaling activity, or kinase activity of the endogenous DLK in a retinal cell. The disclosure also provides methods of treating diseases or disorders related to retinal cells that involve administering such vectors to the retina of a subject. Diseases or disorders that can be treated include, with limitation, diseases or disorders related to dysfunction in retinal cells. Examples include glaucoma, retinopathy, retinal detachment, optic neuropathies, and retinal dystrophy. 
     The disclosure also provides a genetically modified retinal cell that includes the types of polynucleotides already described, ones encoding an inhibitor of Dual Leucine Zipper Kinase (DLK). The disclosure provides methods of making such cells, including contacting them with a viral vector. Pharmaceutical compositions are also provided herein. 
     The foregoing paragraphs are not intended to define every aspect of the invention, and additional aspects are described in other sections, such as the Detailed Description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. The invention includes, as an additional aspect, all embodiments of the invention narrower in scope in any way than the variations defined by specific paragraphs above. For example, where certain aspects of the invention that are described as a genus, it should be understood that every member of a genus is, individually, an aspect of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIGS. 1A-1B  depict the leucine zipper domain of dual leucine zipper kinase (DLK).  FIG. 1A  shows a schematic depiction of the dimerization of a single heptad repeat of a leucine zipper. Amino acid residues within the heptad on one monomer are represented by letters (a) through (g) in N-terminal to C-terminal direction. The equivalent positions on the other monomer are represented by letters (a′) through (g′). The pairs formed between e on one strand of the dimer, and g′ on the opposite strand (called e-g′ or e′-g pairs) can form electrostatic interactions and are important in determining the stability of the leucine zipper dimer.  FIG. 1B  shows the amino-acid sequence of residues 387 to 467 of human DLK (also known as MAP3K12), according to isoform 1 numbering (UniProt Q12852). The eight heptad repeats are labeled L1 through L8, and each residues with in the repeat is labeled a through g, following the convention shown in  FIG. 1A . 
         FIGS. 2A-2H  depict the eight heptad repeats L1 ( FIG. 2A ), L2 ( FIG. 2B ), L3 ( FIG. 2C ), L4 ( FIG. 2D ), L5 ( FIG. 2E ), L6 ( FIG. 2F ), L7 ( FIG. 2G ), L8 ( FIG. 2H ). In the native homodimer of DLK each of the heptad repeats homodimerizes with the equivalent heptad (L1 with L1, L2 with L2, etc.). In each panel, the dimerization of one heptad is shown in the standard helical wheel representation as shown in  FIG. 1A . Standard one-letter abbreviation are used in for each amino acid position. The character of the amino acid at key positions are indicated as positive (+), negative (−), or hydrophobic (hydro). The g-e′ and e-g′ interactions are indicated by blocking arrows (repulsive or charged-hydrophobic interactions) or pointing arrows (charge-charge attractive forces) at the center top or center bottom of each panel. 
         FIG. 3  depicts a designed dominant-negative DLK (DN-DLK) construct. Substitutions with respect to native DLK are shown underlined and bold. 
         FIG. 4A-4C  depicts the homo- or hetero-dimerization interactions of DLK and the DN-DLN of  FIG. 3 . Only g and e residues are shown to highlight g-e′ and g′-e interactions. Electrostatic interactions are depicts by lines. Interactions between opposite charges are shown as bolded solid lines to indicate attractive force. Interactions between like charges are shown as dashed lines to indicate repulsive force.  FIG. 4A  shows a homodimer of DLK. 
         FIG. 4B  shows a heterodimer of DLK and DN-DLK.  FIG. 4C  shows a homodimer of DN-DLK. 
     
    
    
     DETAILED DESCRIPTION 
     1. Overview 
     The disclosure provides compositions and methods useful for treating diseases or disorders related to retinal cells. In particular, the invention provides cells and vectors comprising a polynucleotide encoding an inhibitor of Dual Leucine Zipper Kinase (DLK), and method of use thereof. Advantageously, the vector is capable of reducing the expression, signaling activity, or kinase activity of the endogenous DLK in a cell, which is, in some embodiments, a retinal cell. 
     2. Definitions 
     All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world. 
     In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%. It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. The term “and/or” should be understood to mean either one, or both of the alternatives. As used herein, the terms “include” and “comprise” are used synonymously. 
     As used in this application, the terms “about” and “approximately” are used as equivalents. Any numerals used in this application with or without about/approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). 
     “Decrease” or “reduce” refers to a decrease or a reduction in a particular value of at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% as compared to a reference value. A decrease or reduction in a particular value may also be represented as a fold-change in the value compared to a reference value, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold, or more, decrease as compared to a reference value. 
     “Increase” refers to an increase in a particular value of at least 5%, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100, 200, 300, 400, 500% or more as compared to a reference value. An increase in a particular value may also be represented as a fold-change in the value compared to a reference value, for example, at least 1-fold, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000-fold or more, increase as compared to the level of a reference value. 
     “Complementary” refers to the capacity for pairing, through base stacking and specific hydrogen bonding, between two sequences comprising naturally or non-naturally occurring (e.g., modified as described above) bases (nucleosides) or analogs thereof. For example, if a base at one position of a nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a target, then the bases are considered to be complementary to each other at that position. Nucleic acids can comprise universal bases, or inert abasic spacers that provide no positive or negative contribution to hydrogen bonding. Base pairings may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as such as 3-nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Nichols et al., Nature, 1994; 369:492-493 and Loakes et al., Nucleic Acids Res., 1994; 22:4039-4043. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U, or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33 (19): 6258-6267. 
     The term “subject” includes animals, such as e.g. mammals. In some embodiments, the mammal is a primate. In some embodiments, the mammal is a human. In some embodiments, subjects are livestock such as cattle, sheep, goats, cows, swine, and the like; or domesticated animals such as dogs and cats. In some embodiments (e.g., particularly in research contexts) subjects are rodents (e.g., mice, rats, hamsters), rabbits, primates, or swine such as inbred pigs and the like. The terms “subject” and “patient” are used interchangeably herein. 
     “Administration” refers herein to introducing an agent or composition into a subject. 
     “Treating” as used herein refers to delivering an agent or composition to a subject to affect a physiologic outcome. In some embodiments, treating refers to the treatment of a disease in a subject, e.g., in a human, including (a) inhibiting the disease, e.g., arresting disease development or preventing disease progression; (b) relieving the disease, e.g., causing regression of the disease state; (c) curing the disease; and (d) preventing onset of disease, e.g., arresting disease development in an asymptomatic subject identified as a carrier of a genetic defect. In one aspect, treatment excludes prophylaxis or prevention. 
     The term “effective amount” or “therapeutically effective amount” refer to the minimum amount of an agent or composition required to result in a particular physiological effect (e.g., an amount required to increase, activate, or enhance a particular physiological effect). The effective amount or therapeutically effective amount of a particular agent may be represented in a variety of ways based on the nature of the agent, such as mass/volume, # of cells/volume, particles/volume, (mass of the agent)/(mass of the subject), # of cells/(mass of subject), or particles/(mass of subject). The effective amount or therapeutically effective amount of a particular agent may also be expressed as the half-maximal effective concentration (EC 50 ), which refers to the concentration of an agent that results in a magnitude of a particular physiological response that is half-way between a reference level and a maximum response level. 
     “Population” of cells refers to any number of cells greater than 1, but is preferably at least 1×10 3  cells, at least 1×10 4  cells, at least at least 1×10 5  cells, at least 1×10 6  cells, at least 1×10 7  cells, at least 1×10 8  cells, at least 1×10 9  cells, at least 1×10 10  cells, or more cells. A population of cells may refer to an in vitro population (e.g., a population of cells in culture) or an in vivo population (e.g., a population of cells residing in a particular tissue). 
     The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. 
     As used herein “pharmaceutically acceptable carrier, diluent or excipient” includes without limitation any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans and/or domestic animals. 
     As used herein “vector” refers to a nucleic acid molecule capable transferring or transporting a nucleic acid molecule to cell, along with, in a viral vector, one or more viral proteins, such as for encapsulated viruses the capsid of the virus. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication or reverse transcription in a cell, or may include sequences sufficient to allow integration into host cell DNA. “Vectors” include gene therapy vectors. As used herein, the term “gene therapy vector” refers to a vector capable of use in performing gene therapy, e.g., delivering a polynucleotide sequence encoding a therapeutic polypeptide to a subject. Gene therapy vectors may comprise a polynucleotide (“transgene”) encoding a protein. 
     As used herein, the term “expression cassette” refers to a DNA segment that is capable in an appropriate setting of driving the expression of a polynucleotide (e.g., a transgene) encoding a protein that is incorporated in said expression cassette. When introduced into a host cell, an expression cassette inter alia is capable of directing the cell&#39;s machinery to transcribe the transgene into RNA, which is then usually further processed and finally translated into the therapeutically active polypeptide. The gene therapy vector can comprise, or consist essentially of expression cassette. The term expression cassette excludes polynucleotide sequences 5′ to the 5′ ITR and 3′ to the 3′ ITR. Provided herein are host cells comprising, or consisting essentially of, or yet further consisting of the vector of this disclosure. The cells can be of any appropriate species, e.g., mammalian cells. 
     As used herein, the phrases “operably linked” or “under the transcriptional control” with respect to a polynucleotide refers, interchangeably, to a configuration of the promoter and the polynucleotide that enables the polynucleotide to be transcribed by a polymerase capable of binding to the promoter. 
     The term “sequence identity” refers to the percentage of bases or amino acids between two polynucleotide or polypeptide sequences that are the same, and in the same relative position. As such one polynucleotide or polypeptide sequence has a certain percentage of sequence identity compared to another polynucleotide or polypeptide sequence. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. The term “reference sequence” refers to a molecule to which a test sequence is compared. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to a reference sequence means that, when aligned, that percentage of bases (or amino acids) at each position in the test sequence are identical to the base (or amino acid) at the same position in the reference sequence. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov/blast/Blast.cgi. 
     An “equivalent” or “functional equivalent” of a polypeptide or protein is one that has a certain sequence identity to that reference polypeptide or protein, (for example, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to the reference) and retains similar activity or function compared to the reference polypeptide or protein. 
     “Comprising” or “comprises” is intended to mean that the compositions, for example media, and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. “Consisting of” shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure. 
     General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley &amp; Sons 1999); Protein Methods (Bollag et al., John Wiley &amp; Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift &amp; Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle &amp; Griffiths, John Wiley &amp; Sons 1998), the disclosures of which are incorporated herein by reference. 
     The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. 
     3.1 Vectors 
     Various embodiments of the invention include viral vectors and non-viral vectors. Viral vectors include adeno-associated virus, adenovirus, lentivirus, Sleeping Beauty, herpesvirus, and other viruses known in the art. It is within the skill of the ordinary artisan to adapt the polynucleotides of the invention to various viral vectors. Non-viral vectors include liposomes, lipid-like particles, and cationic polymers, as well as electroporation of naked DNA in plasmid, bacmid, linear, mincircular or other form. The polynucleotides of the disclosure may be delivered as either DNA or RNA. Modified polynucleotides are known in the art and are included methods and compositions of the present disclosure. The disclosure of particular types of viral and non-viral vectors in particular embodiments described herein is not intended as limiting the invention. 
     As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus or a recombinant vector thereof. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. General information and reviews of AAV can be found in, for example, Carter, 1989 , Handbook of Parvoviruses , Vol. 1, pp. 169-228, and Berns, 1990 , Virology , pp. 1743-1764, Raven Press, (New York). It is fully expected that the same principles described in these reviews will be applicable to additional AAV serotypes characterized after the publication dates of the reviews because it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of  Parvoviruses and Human Disease , J. R. Pattison, ed.; and Rose,  Comprehensive Virology  3: 1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control. 
     As used herein, an “AAV vector” or “rAAV vector” refers to a recombinant vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products. 
     As used herein, an “AAV virion” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. As used herein, if the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector.” Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle. 
     Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including two 145 nucleotide inverted terminal repeat (ITRs). There are multiple known variants of AAV, also sometimes called serotypes when classified by antigenic epitopes. The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV-3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_—001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV-9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol. Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). The sequence of the AAV rh.74 genome is provided in U.S. Pat. No. 9,434,928, incorporated herein by reference. The sequence of ancestral AAVs including AAV.Anc80, AAV.Anc80L65 and their derivatives are described in WO2015054653A2 and Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS One. 2017 Aug. 1; 12(8):e0182473. 
     AAV vectors may refer to either naturally occurring AAVs, such as AAV-1 to AAV-10, or non-naturally occurring vectors, such as those that have been engineered through rational design, directed evolution, and the like. Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and p9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka, Current Topics in Microbiology and Immunology, 158: 97-129 (1992). 
     AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy. AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic. Moreover, AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo. Moreover, AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element). The AAV proviral genome is inserted as cloned DNA in plasmids, which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication and genome encapsidation are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. To generate AAV vectors, the rep and cap proteins may be provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56° to 65° C. for several hours), making cold preservation of AAV less critical. AAV may even be lyophilized. Finally, AAV-infected cells are not resistant to superinfection. 
     3.2 AAV Serotypes and Genomes 
     AAV DNA in the rAAV genomes may be from any AAV variant or serotype for which a recombinant virus can be derived including, but not limited to, AAV variants or serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and Anc80L65. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). The nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote eye-specific expression, AAV6, AAV8 or AAV9 may be used. 
     In some cases, the rAAV comprises a self-complementary genome. As defined herein, an rAAV comprising a “self-complementary” or “double stranded” genome refers to an rAAV which has been engineered such that the coding region of the rAAV is configure to form an intra-molecular double-stranded DNA template, as described in McCarty et al. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis.  Gene Therapy.  8 (16): 1248-54 (2001). The present disclosure contemplates the use, in some cases, of an rAAV comprising a self-complementary genome because upon infection (such transduction), rather than waiting for cell mediated synthesis of the second strand of the rAAV genome, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. It will be understood that instead of the full coding capacity found in rAAV (4.7-6 kb), rAAV comprising a self-complementary genome can only hold about half of that amount (≈2.4 kb). 
     In other cases, the rAAV vector comprises a single stranded genome. As defined herein, a “single standard” genome refers to a genome that is not self-complementary. In most cases, non-recombinant AAVs are have singled stranded DNA genomes. There has been some indications that rAAVs should be scAAVs to achieve efficient transduction of cells, such as ocular cells. The present disclosure contemplates, however, rAAV vectors that maybe have singled stranded genomes, rather than self-complementary genomes, with the understanding that other genetic modifications of the rAAV vector may be beneficial to obtain optimal gene transcription in target cells. In some cases, the present disclosure relates to single-stranded rAAV vectors capable of achieving efficient gene transfer to anterior segment in the mouse eye. See Wang et al. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS ONE 12(8): e0182473 (2017). 
     In some cases, the rAAV vector is of the serotype AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV2.7m8, or Anc80L65. Anc80L65 is described in Sharma et al. Transduction efficiency of AAV 2/6, 2/8 and 2/9 vectors for delivering genes in human corneal fibroblasts. PLoS ONE 12(8): e0182473 (2017). Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic et al., Molecular Therapy, 22(11): 1900-1909 (2014). In some cases, the rAAV vector is of the serotype AAV9. In some embodiments, said rAAV vector is of serotype AAV9 and comprises a single stranded genome. In some embodiments, said rAAV vector is of serotype AAV9 and comprises a self-complementary genome. In some embodiments, a rAAV vector comprises the inverted terminal repeat (ITR) sequences of AAV2. In some embodiments, the rAAV vector comprises an AAV2 genome, such that the rAAV vector is an AAV-2/9 vector, an AAV-2/6 vector, or an AAV-2/8 vector. Other combinations of genome and serotype are contemplated by the present disclosure, including, without limitation, non-naturally occurring variants made by rational design, directed evolution, and the like, and those described in.  Brain Res Bull.  2010 Feb. 15; 81(2-3): 273; Castle et al. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids.  Methods Mol Biol.  2016; 1382: 133-149; and Kay et al. Targeting Photoreceptors via Intravitreal Delivery Using Novel, Capsid-Mutated AAV Vectors. PLoS One. 2013 Apr. 26; 8(4):e62097. 
     3.3 Promoters 
     In some cases, a polynucleotide sequence encoding a transgene is operatively linked to an inducible promoter. A polynucleotide sequence operatively linked to an inducible promoters may be configured to cause the polynucleotide sequence to be transcriptionally expressed or not transcriptionally expressed in response to addition or accumulation of an agent or in response to removal, degradation, or dilution of an agent. The agent may be a drug. The agent may be tetracycline or one of its derivatives, including, without limitation, doxycycline. In some cases, the inducible promoter is a tet-on promoter, a tet-off promoter, a chemically-regulated promoter, a physically-regulated promoter (i.e. a promoter that responds to presence or absence of light or to low or high temperature). This list of inducible promoters is non-limiting. 
     In some embodiments, the polynucleotide sequence encoding the transgene is operably linked to a CMV promoter. The present disclosure further contemplates the use of other promoter sequences. Promoters useful in embodiments of the present disclosure include, without limitation, cytomegalovirus (CMV) and murine stem cell virus (MSCV), phosphoglycerate kinase (PGK), a promoter sequence comprised of the CMV enhancer and portions of the chicken beta-actin promoter and the rabbit beta-globin gene (CAG), a CBh promoter, a promoter sequence comprised of portions of the SV40 promoter and CD43 promoter (SV40/CD43), and a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (MND). In some cases, the promoter may be a synthetic promoter. Exemplary synthetic promoters are provided by Schlabach et al. Synthetic design of strong promoters. Proc Natl Acad Sci USA. 2010 Feb. 9; 107(6): 2538-2543. 
     In some embodiments, the polynucleotide sequence encoding the transgene is operably linked to a Nefh promoter, as described in Hanlon et al.  Front Neurosci.  11:521 (2017) (PMID: 28983234). In some embodiments, the polynucleotide sequence encoding the transgene is operably linked to a DCX promoter, as described in Smith et al. Sci. Rep. 8:1490 (2018) (PMID: 29367685). In some embodiments, the polynucleotide sequence encoding the transgene is operably linked to a Thy1 promoter, a rhodopsin promoter, a rhodopsin kinase promoter, a pR2.1 promoter, or a MNTC promoter. Promoters provided by this disclosure include those described in US20180100165A1, US20180066022A1, US20180258446A1, US20150259395A1, US20140248701A1, US20120278912A1, WO2006108201A1, the disclosures of each of which are incorporated by reference herein in their entireties. 
     3.4 Dual-Leucine Zipper Kinase (DLK) 
     As used herein, the terms “dual-leucine zipper kinase” or “DLK” refers to the kinase, also known in the art as Mitogen-activated protein kinase kinase kinase 12 (MAP3K12), encoded by the DLK gene. DLK is a member of the Mixed Lineage Kinase (MLK). MLKs were identified initially as signaling molecules in the nervous system. They were also shown to play a role in the cell cycle. Like other MLKs, DLK has a kinase catalytic domain followed C-terminally by two leucine/isoleucine zippers separated by a short space sequence. Mata et al.  J. Bio. Chem.  271, 16888-16896 (1996). 
     DLK is predominately expressed in neuronal cells. The phosphorylation state of this kinase in synaptic terminals was shown to be regulated by membrane depolarization via calcineurin. This kinase forms heterodimers with leucine zipper containing transcription factors, such as cAMP responsive element binding protein (CREB) and MYC, and thus may play a regulatory role in PKA or retinoic acid induced neuronal differentiation. 
     Table 1 provides the native protein sequence of human DLK, isoform 1 and isoform 2. DLKs from other species may be used in the present invention. Through the disclosure, the numbering of human isoform 1 is used. Table 1 also provides other protein sequences. Table 2 provides corresponding polynucleotide sequences, including expression cassettes and vectors. The polynucleotides disclosed can be used in various viral and non-viral vectors. In particular, the AAV genome provided as SEQ ID NO: 22 may be provided either as a plasmid for use in generating a viral particle or as a constituent of a pre-formed viral particle. It is well within the skill of those in the art to modify these sequences, such as by codon optimization, removal of spurious start or stop codons, reducing G/C content, removal of CpG dinucleotides or CpG islands, substitutions of promoter or other genetic elements, use of lentiviral LTRs rather than AAV ITRs, etc. Such polynucleotides are themselves embodiments of this disclosure. 
     The AAV genome disclosed as SEQ ID NO: 22 has a total length of 4099 bp. In some embodiments, SEQ ID NO: 22 is modified by insert of other polynucleotides between the flanking ITRs to increase the length of the sequence to about 4.2 to about 4.5 kilobases, in line with the expectation that this size is optimal in some contexts for AAV vectors, provided modification of SEQ ID NO: 22 does not disrupt the function of the polynucleotide sequence as an AAV genome. The disclosure contemplates substitution of various coding regions for DLK inhibitors into SEQ ID NO: 22, or into other viral genomes, including into other AAV genomes. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Exemplary Dual-Leucine Zipper Kinase Sequences 
               
            
           
           
               
               
               
            
               
                 Sequence 
                   
                 SEQ 
               
               
                 description 
                 Sequence 
                 ID NO 
               
               
                   
               
            
           
           
               
               
               
            
               
                 DLK/Map3k12 
                 MACLHETRTPSPSFGGFVSTLSEASMRKLDPDTSDCTPE 
                 1 
               
               
                 (variant 2, aka 
                 KDLTPTHVLQLHEQDAGGPGGAAGSPESRASRVRADEV 
                   
               
               
                 isoform 1) 
                 RLQCQSGSGFLEGLFGCLRPVWTMIGKAYSTEHKQQQE 
                   
               
               
                   
                 DLWEVPFEEILDLQWVGSGAQGAVFLGRFHGEEVAVKK 
                   
               
               
                   
                 VRDLKETDIKHLRKLKHPNIITFKGVCTQAPCYCILMEFC 
                   
               
               
                   
                 AQGQLYEVLRAGRPVTPSLLVDWSMGIAGGMNYLHLH 
                   
               
               
                   
                 KIIHRDLKSPNMLITYDDVVKISDFGTSKELSDKSTKMSF 
                   
               
               
                   
                 AGTVAWMAPEVIRNEPVSEKVDIWSFGVVLWELLTGEIP 
                   
               
               
                   
                 YKDVDSSAIIWGVGSNSLHLPVPSSCPDGFKILLRQCWN 
                   
               
               
                   
                 SKPRNRPSFRQILLHLDIASADVLSTPQETYFKSQAEWRE 
                   
               
               
                   
                 EVKLHFEKIKSEGTCLHRLEEELVMRRREELRHALDIRE 
                   
               
               
                   
                 HYERKLERANNLYMELNALMLQLELKERELLRREQALE 
                   
               
               
                   
                 RRCPGLLKPHPSRGLLHGNTMEKLIKKRNVPQKLSPHSK 
                   
               
               
                   
                 RPDILKTESLLPKLDAALSGVGLPGCPKGPPSPGRSRRGK 
                   
               
               
                   
                 TRHRKASAKGSCGDLPGLRTAVPPHEPGGPGSPGGLGG 
                   
               
               
                   
                 GPSAWEACPPALRGLHHDLLLRKMSSSSPDLLSAALGSR 
                   
               
               
                   
                 GRGATGGAGDPGSPPPARGDTPPSEGSAPGSTSPDSPGG 
                   
               
               
                   
                 AKGEPPPPVGPGEGVGLLGTGREGTSGRGGSRAGSQHLT 
                   
               
               
                   
                 PAALLYRAAVTRSQKRGISSEEEEGEVDSEVELTSSQRW 
                   
               
               
                   
                 PQSLNMRQSLSTFSSENPSDGEEGTASEPSPSGTPEVGST 
                   
               
               
                   
                 NTDERPDERSDDMCSQGSEIPLDPPPSEVIPGPEPSSLPIPH 
                   
               
               
                   
                 QELLRERGPPNSEDSDCDSTELDNSNSVDALRPPASLPP* 
                   
               
               
                   
               
               
                 DLK/Map3k12 
                 MACLHETRTPSPSFGGFVSTLSEASMRKLDPDTSDCTPE 
                 2 
               
               
                 (isoform 2) 
                 KDLTPTQCVLRDVVPLGGQGGGGPSPSPGGEPPPEPFAN 
                   
               
               
                   
                 SVLQLHEQDAGGPGGAAGSPESRASRVRADEVRLQCQS 
                   
               
               
                   
                 GSGFLEGLFGCLRPVWTMIGKAYSTEHKQQQEDLWEVP 
                   
               
               
                   
                 FEEILDLQWVGSGAQGAVFLGRFHGEEVAVKKVRDLKE 
                   
               
               
                   
                 TDIKHLRKLKHPNIITFKGVCTQAPCYCILMEFCAQGQLY 
                   
               
               
                   
                 EVLRAGRPVTPSLLVDWSMGIAGGMNYLHLHKIIHRDL 
                   
               
               
                   
                 KSPNMLITYDDVVKISDFGTSKELSDKSTKMSFAGTVAW 
                   
               
               
                   
                 MAPEVIRNEPVSEKVDIWSFGVVLWELLTGEIPYKDVDS 
                   
               
               
                   
                 SAIIWGVGSNSLHLPVPSSCPDGFKILLRQCWNSKPRNRP 
                   
               
               
                   
                 SFRQILLHLDIASADVLSTPQETYFKSQAEWREEVKLHFE 
                   
               
               
                   
                 KIKSEGTCLHRLEEELVMRRREELRHALDIREHYERKLE 
                   
               
               
                   
                 RANNLYMELNALMLQLELKERELLRREQALERRCPGLL 
                   
               
               
                   
                 KPHPSRGLLHGNTMEKLIKKRNVPQKLSPHSKRPDILKT 
                   
               
               
                   
                 ESLLPKLDAALSGVGLPGCPKGPPSPGRSRRGKTRHRKA 
                   
               
               
                   
                 SAKGSCGDLPGLRTAVPPHEPGGPGSPGGLGGGPSAWE 
                   
               
               
                   
                 ACPPALRGLHHDLLLRKMSSSSPDLLSAALGSRGRGATG 
                   
               
               
                   
                 GAGDPGSPPPARGDTPPSEGSAPGSTSPDSPGGAKGEPPP 
                   
               
               
                   
                 PVGPGEGVGLLGTGREGTSGRGGSRAGSQHLTPAALLY 
                   
               
               
                   
                 RAAVTRSQKRGISSEEEEGEVDSEVELTSSQRWPQSLNM 
                   
               
               
                   
                 RQSLSTFSSENPSDGEEGTASEPSPSGTPEVGSTNTDERPD 
                   
               
               
                   
                 ERSDDMCSQGSEIPLDPPPSEVIPGPEPSSLPIPHQELLRER 
                   
               
               
                   
                 GPPNSEDSDCDSTELDNSNSVDALRPPASLPP 
                   
               
               
                   
               
               
                 DLK leucine 
                 LSTPQETYFKSQAEW REEVKLHFEKIKSEGTCLHRLEEEL   
                 3 
               
               
                 zipper domain 
                 
                   VMR 
                   RREELRHALDIREHYERKLERANNL 
                   YMELNALMLQL 
                 
                   
               
               
                 fragment (Nihalani 
                   ELKERELLRREQALERR CPGLLKPHPSRGLLHGNTME 
                   
               
               
                 et al.  J. Bio Chem.   
                   
                   
               
               
                 275, 7273-7279 
                   
                   
               
               
                 (2000)) 
                   
                   
               
               
                   
               
               
                 Zipper 1, bridge, 
                 
                   REEVKLHFEKIKSEGTCLHRLEEELVMR 
                   RREELRHALDIR 
                 
                 4 
               
               
                 and zipper 2 from 
                 
                   EHYERKLERANNL 
                   YMELNALMLQLELKERELLRREQALE 
                 
                   
               
               
                 DLK 
                 
                   RR 
                 
                   
               
               
                   
               
               
                 Kinase domain 
                 ILDLQWVGSGAQGAVFLGRFHGEEVAVKKVRDLKETDI 
                 5 
               
               
                 from DLK 
                 KHLRKLKHPNIITFKGVCTQAPCYCILMEFCAQGQLYEV 
                   
               
               
                   
                 LRAGRPVTPSLLVDWSMGIAGGMNYLHLHKIIHRDLKSP 
                   
               
               
                   
                 NMLITYDDVVKISDFGTSKELSDKSTKMSFAGTVAWMA 
                   
               
               
                   
                 PEVIRNEPVSEKVDIWSFGVVLWELLTGEIPYKDVDSSAII 
                   
               
               
                   
                 WGVGSNSLHLPVPSSCPDGFKILLRQCWNSKPRNRPSFR 
                   
               
               
                   
                 QILLHLDI 
                   
               
               
                   
               
               
                 Modified DLK 
                 
                   REEV E L E FEKI R SEGTCL E R E EEEL E M E   
                   RREELRHALDIRE 
                 
                 6 
               
               
                 Zipper 1, bridge, 
                 
                   HYERKLERANNL 
                   YMELNALMLQLEL R EREL E R E EQALER 
                 
                   
               
               
                 and zipper 2. 
                 
                   R 
                 
                   
               
               
                 Modifications in 
                   
                   
               
               
                 bold enhance 
                   
                   
               
               
                 heterodimerization 
                   
                   
               
               
                 with DLK, 
                   
                   
               
               
                 compared to DLK 
                   
                   
               
               
                 homodimers or 
                   
                   
               
               
                 LZ-LZ 
                   
                   
               
               
                 homodimers 
                   
                   
               
               
                   
               
               
                 Longer LZ domain 
                 LSTPQETYFKSQAEW REEV E L E FEKI R SEGTCL E R E EEEL   
                 7 
               
               
                 with same 
                 
                     E M E   
                   RREELRHALDIREHYERKLERANNL 
                   YMELNALMLQL 
                 
                   
               
               
                 modifications 
                   EL R EREL E R E EQALERR CPGLLKPHPSRGLLHGNTME 
                   
               
               
                   
               
               
                 Kinase domain of 
                 ILDLQWVGSGAQGAVFLGRFHGEEVAV   A   KVRDLKETDI 
                 8 
               
               
                 kinase-dead DLK 
                 KHLRKLKHPNIITFKGVCTQAPCYCILMEFCAQGQLYEV 
                   
               
               
                 (K152A) 
                 LRAGRPVTPSLLVDWSMGIAGGMNYLHLHKIIHRDLKSP 
                   
               
               
                   
                 NMLITYDDVVKISDFGTSKELSDKSTKMSFAGTVAWMA 
                   
               
               
                   
                 PEVIRNEPVSEKVDIWSFGVVLWELLTGEIPYKDVDSSAII 
                   
               
               
                   
                 WGVGSNSLHLPVPSSCPDGFKILLRQCWNSKPRNRPSFR 
                   
               
               
                   
                 QILLHLDI 
                   
               
               
                   
               
               
                 Zipper1 
                 REEV X L X FEKI Y SEGTCL X R X EEELXMX 
                 9 
               
               
                   
                 X = E or D 
                   
               
               
                   
                 Y = R, H, or K 
                   
               
               
                   
               
               
                 Zipper2 
                 YMELNALMLQLEL Y EREL X R X EQALERR 
                 10 
               
               
                   
                 X = E or D 
                   
               
               
                   
                 Y = R, H, or K 
                   
               
               
                   
               
               
                 Bridge 
                 RREELRHALDIREHYERKLERANNL 
                 11 
               
               
                   
               
               
                 Zipper1 - K391D, 
                 REEV D L D FEKI Y SEGTCL X R X EEELXMX 
                 12 
               
               
                 H393D 
                 X = E or D 
                   
               
               
                   
                 Y = R, H, or K 
                   
               
               
                   
               
               
                 Zipper1 - K391E, 
                 REEV E L D FEKI Y SEGTCL X R X EEELXMX 
                 13 
               
               
                 H393D 
                 X = E or D 
                   
               
               
                   
                 Y = R, H, or K 
                   
               
               
                   
               
               
                 Zipper1 - K391D, 
                 REEV D L E FEKI Y SEGTCL X R X EEELXMX 
                 14 
               
               
                 H393E 
                 X = E or D 
                   
               
               
                   
                 Y = R, H, or K 
                   
               
               
                   
               
               
                 Zipper1 - K391E, 
                 REEV E L E FEKI Y SEGTCL X R X EEELXMX 
                 15 
               
               
                 H393E 
                 X = E or D 
                   
               
               
                   
                 Y = R, H, or K 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Exemplary Polynucleotide Sequences 
               
            
           
           
               
               
               
            
               
                 Sequence 
                   
                 SEQ 
               
               
                 description 
                 Sequence 
                 ID NO 
               
               
                   
               
               
                 Zipper-bridge- 
                 GTAAAACTGCACTTTGAAAAGATTAAGTCAGAAGGGA 
                 16 
               
               
                 zipper of native 
                 CCTGTCTGCACCGCCTAGAAGAGGAACTGGTGATGAG 
                   
               
               
                 DLK 
                 GAGGAGGGAGGAGCTCAGACACGCCCTGGACATCAG 
                   
               
               
                   
                 GGAGCACTATGAAAGGAAGCTGGAGAGAGCCAACAA 
                   
               
               
                   
                 CCTGTATATGGAACTTAATGCCCTCATGTTGCAGCTGG 
                   
               
               
                   
                 AACTCAAGGAGAGGGAGCTGCTCAGGCGAGAGCAAG 
                   
               
               
                   
                 CTTTA 
                   
               
               
                   
               
               
                 Zipper-bridge- 
                 CTCTCCACACCCCAGGAGACTTACTTTAAGTCCCAGGC 
                 17 
               
               
                 zipper of native 
                 AGAGTGGCGGGAAGAA GTAAAACTGCACTTTGAAAA   
                   
               
               
                 DLK with flanking 
                 
                   GATTAAGTCAGAAGGGACCTGTCTGCACCGCCTAGAA 
                 
                   
               
               
                 sequence 
                 
                   GAGGAACTGGTGATGAGGAGGAGGGAGGAGCTCAGA 
                 
                   
               
               
                   
                 
                   CACGCCCTGGACATCAGGGAGCACTATGAAAGGAAGC 
                 
                   
               
               
                   
                 
                   TGGAGAGAGCCAACAACCTGTATATGGAACTTAATGC 
                 
                   
               
               
                   
                 
                   CCTCATGTTGCAGCTGGAACTCAAGGAGAGGGAGCTG 
                 
                   
               
               
                   
                   CTCAGGCGAGAGCAAGCTTTA GAGCGGAGGTGCCCAG 
                   
               
               
                   
                 GCCTGCTGAAGCCACACCCTTCCCGGGGCCTCCTGCA 
                   
               
               
                   
                 TGGAAACACAATGGAG 
                   
               
               
                   
               
               
                 Zipper-bridge- 
                 CTCTCCACACCCCAGGAGACTTACTTTAAGTCCCAGGC 
                 18 
               
               
                 zipper of native 
                 AGAGTGGCGGGAAGAA GTA GAA CTG GAA TTTGAAAA   
                   
               
               
                 DLK with flanking 
                 
                   GATTAAGTCAGAAGGGACCTGTCTGCACCGCCTAGAA 
                 
                   
               
               
                 sequence with 
                 
                   GAGGAACTGGTGATGAGGAGGAGGGAGGAGCTCAGA 
                 
                   
               
               
                 codons 
                 
                   CACGCCCTGGACATCAGGGAGCACTATGAAAGGAAGC 
                 
                   
               
               
                 corresponding to 
                 
                   TGGAGAGAGCCAACAACCTGTATATGGAACTTAATGC 
                 
                   
               
               
                 substituted bases in 
                 
                   CCTCATGTTGCAGCTGGAACTCAAGGAGAGGGAGCTG 
                 
                   
               
               
                 bold for K391E 
                   CTCAGGCGAGAGCAAGCTTTA GAGCGGAGGTGCCCAG 
                   
               
               
                 and H393E 
                 GCCTGCTGAAGCCACACCCTTCCCGGGGCCTCCTGCA 
                   
               
               
                   
                 TGGAAACACAATGGAG 
                   
               
               
                   
               
               
                 Zipper-bridge- 
                 
                   CTCTCCACACCCCAGGAGACTTACTTTAAGTCCCAGGC 
                 
                 19 
               
               
                 zipper of native 
                 
                   AGAGTGGCGGGAAGAAGTA GAA CTG GAA TTTGAAAA 
                 
                   
               
               
                 DLK with flanking 
                 
                   GATT CGT TCAGAAGGGACCTGTCTG GAA CGC GAA GA 
                 
                   
               
               
                 sequence with 
                 
                   AGAGGAACTG GAA ATG GAA AGGAGGGAGGAGCTCAG 
                 
                   
               
               
                 codons 
                 
                   ACACGCCCTGGACATCAGGGAGCACTATGAAAGGAA 
                 
                   
               
               
                 corresponding to 
                 
                   GCTGGAGAGAGCCAACAACCTGTATATGGAACTTAAT 
                 
                   
               
               
                 substituted bases in 
                 
                   GCCCTCATGTTGCAGCTGGAACTC CGT GAGAGGGAGC 
                 
                   
               
               
                 bold for K391E, 
                 
                   TG GAA AGG GAA GAGCAAGCTTTAGAGCGGAGGTGCC 
                 
                   
               
               
                 H393E, K398R, 
                 
                   CAGGCCTGCTGAAGCCACACCCTTCCCGGGGCCTCCT 
                 
                   
               
               
                 H405E, L407E, 
                 
                   GCATGGAAACACAATGGAG 
                 
                   
               
               
                 V412E, R414E, 
                   
                   
               
               
                 K453R, L458E, 
                   
                   
               
               
                 R460E 
                   
                   
               
               
                   
               
               
                 Above sequence 
                   GCCGCCATGGCG CTCTCCACACCCCAGGAGACTTACT 
                 20 
               
               
                 with Kozak and 
                 TTAAGTCCCAGGCAGAGTGGCGGGAAGAAGTAGAACT 
                   
               
               
                 stop codon 
                 GGAATTTGAAAAGATTCGTTCAGAAGGGACCTGTCTG 
                   
               
               
                   
                 GAACGCGAAGAAGAGGAACTGGAAATGGAAAGGAGG 
                   
               
               
                   
                 GAGGAGCTCAGACACGCCCTGGACATCAGGGAGCACT 
                   
               
               
                   
                 ATGAAAGGAAGCTGGAGAGAGCCAACAACCTGTATAT 
                   
               
               
                   
                 GGAACTTAATGCCCTCATGTTGCAGCTGGAACTCCGT 
                   
               
               
                   
                 GAGAGGGAGCTGGAAAGGGAAGAGCAAGCTTTAGAG 
                   
               
               
                   
                 CGGAGGTGCCCAGGCCTGCTGAAGCCACACCCTTCCC 
                   
               
               
                   
                 GGGGCCTCCTGCATGGAAACACAATGGAG TAA   
                   
               
               
                   
               
               
                 Expression 
                 CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGA 
                 21 
               
               
                 cassette including 
                 CCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA 
                   
               
               
                 above sequence 
                 CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT 
                   
               
               
                 with Cbh 
                 TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCC 
                   
               
               
                 promoter, hybrid 
                 ACTTGGCAGTACATCAAGTGTATCATATGCCAAGTAC 
                   
               
               
                 intron, bGH polyA 
                 GCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCC 
                   
               
               
                 tail, and WPRE 
                 TGGCATTATGCCCAGTACATGACCTTATGGGACTTTCC 
                   
               
               
                   
                 TACTTGGCAGTACATCTACGTATTAGTCATCGCTATTA 
                   
               
               
                   
                 CCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTC 
                   
               
               
                   
                 CCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTT 
                   
               
               
                   
                 ATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGG 
                   
               
               
                   
                 GGGGGGGGGGGGGGCGGGGCGAGGGGCGGGGCGGG 
                   
               
               
                   
                 GCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGC 
                   
               
               
                   
                 GGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGC 
                   
               
               
                   
                 GGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC 
                   
               
               
                   
                 GGGCGGGAGTCGCTGCGACGCTGCCTTCGCCCCGTGC 
                   
               
               
                   
                 CCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCT 
                   
               
               
                   
                 CTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGG 
                   
               
               
                   
                 GACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGT 
                   
               
               
                   
                 TTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAA 
                   
               
               
                   
                 GCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGG 
                   
               
               
                   
                 GAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTG 
                   
               
               
                   
                 GGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCT 
                   
               
               
                   
                 GTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCT 
                   
               
               
                   
                 CCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGG 
                   
               
               
                   
                 TGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAA 
                   
               
               
                   
                 AGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCA 
                   
               
               
                   
                 GGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCC 
                   
               
               
                   
                 TGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGC 
                   
               
               
                   
                 TTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGG 
                   
               
               
                   
                 CTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGG 
                   
               
               
                   
                 GTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAG 
                   
               
               
                   
                 GGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCC 
                   
               
               
                   
                 GGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCC 
                   
               
               
                   
                 TTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCC 
                   
               
               
                   
                 TTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGG 
                   
               
               
                   
                 CGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGC 
                   
               
               
                   
                 GGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGG 
                   
               
               
                   
                 GCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCT 
                   
               
               
                   
                 CTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCC 
                   
               
               
                   
                 TTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTG 
                   
               
               
                   
                 GCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCAT 
                   
               
               
                   
                 GTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAA 
                   
               
               
                   
                 CGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAG 
                   
               
               
                   
                 AATTCGAGCGGCCGCCAGTGTGAGTTGGACCGGTGCC 
                   
               
               
                   
                 GCCATGGCGCTCTCCACACCCCAGGAGACTTACTTTA 
                   
               
               
                   
                 AGTCCCAGGCAGAGTGGCGGGAAGAAGTAGAACTGG 
                   
               
               
                   
                 AATTTGAAAAGATTCGTTCAGAAGGGACCTGTCTGGA 
                   
               
               
                   
                 ACGCGAAGAAGAGGAACTGGAAATGGAAAGGAGGGA 
                   
               
               
                   
                 GGAGCTCAGACACGCCCTGGACATCAGGGAGCACTAT 
                   
               
               
                   
                 GAAAGGAAGCTGGAGAGAGCCAACAACCTGTATATG 
                   
               
               
                   
                 GAACTTAATGCCCTCATGTTGCAGCTGGAACTCCGTG 
                   
               
               
                   
                 AGAGGGAGCTGGAAAGGGAAGAGCAAGCTTTAGAGC 
                   
               
               
                   
                 GGAGGTGCCCAGGCCTGCTGAAGCCACACCCTTCCCG 
                   
               
               
                   
                 GGGCCTCCTGCATGGAAACACAATGGAGTAATGAGGC 
                   
               
               
                   
                 ATGCTTCTATATTATTTTCTAAAAGATTTAAAGTTTTG 
                   
               
               
                   
                 CCTTCTCCATTTAGACTTATAATTCACTGGAATTTTTTT 
                   
               
               
                   
                 GTGTGTATGGTATGACATATGGGTTCCCTTTTATTTTTT 
                   
               
               
                   
                 ACATATAAATATATTTCCCTGTTTTTCTAAAAAAGACC 
                   
               
               
                   
                 TAGGAAAACTGTCTTCATAATCAACCTCTGGATTACA 
                   
               
               
                   
                 AAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT 
                   
               
               
                   
                 GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCC 
                   
               
               
                   
                 TTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTT 
                   
               
               
                   
                 CTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGA 
                   
               
               
                   
                 GGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTG 
                   
               
               
                   
                 TGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGG 
                   
               
               
                   
                 CATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCG 
                   
               
               
                   
                 CTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCC 
                   
               
               
                   
                 GCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTT 
                   
               
               
                   
                 GGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCA 
                   
               
               
                   
                 TCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTG 
                   
               
               
                   
                 GATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGG 
                   
               
               
                   
                 CCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTG 
                   
               
               
                   
                 CCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCC 
                   
               
               
                   
                 TCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC 
                   
               
               
                   
                 ATCGATACCGAGCGCTGCTAGAGAGATCGATCTGCCT 
                   
               
               
                   
                 CAACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGC 
                   
               
               
                   
                 CCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCAC 
                   
               
               
                   
                 TCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT 
                   
               
               
                   
                 CGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGT 
                   
               
               
                   
                 GGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAA 
                   
               
               
                   
                 GAGAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA 
                   
               
               
                   
                 TGGGTACCCAGGTGCTGAAGAATTGACCCGGTTCCTC 
                   
               
               
                   
                 CTGGG 
                   
               
               
                   
               
               
                 Expression 
                 ATGTCCTACAGCGCGCTCGCTCGCTCACTGAGGCCC 
                 22 
               
               
                 cassette above with 
                 CCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGC 
                   
               
               
                 flanking AAV 
                 CCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGA 
                   
               
               
                 ITRs 
                 GTGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAAT 
                   
               
               
                   
                 GATTAACCTCTGGAGACCGCCATGCTACTTATCTACCA 
                   
               
               
                   
                 GGGTAATGGGGATCCTCTAGAACTATAGCGTTACATA 
                   
               
               
                   
                 ACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC 
                   
               
               
                   
                 GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCC 
                   
               
               
                   
                 CATAGTAACGCCAATAGGGACTTTCCATTGACGTCAA 
                   
               
               
                   
                 TGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAG 
                   
               
               
                   
                 TACATCAAGTGTATCATATGCCAAGTACGCCCCCTATT 
                   
               
               
                   
                 GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG 
                   
               
               
                   
                 CCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAG 
                   
               
               
                   
                 TACATCTACGTATTAGTCATCGCTATTACCATGGTCGA 
                   
               
               
                   
                 GGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCC 
                   
               
               
                   
                 CCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTT 
                   
               
               
                   
                 AATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGG 
                   
               
               
                   
                 GGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAG 
                   
               
               
                   
                 AGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGA 
                   
               
               
                   
                 AAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGC 
                   
               
               
                   
                 CCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCG 
                   
               
               
                   
                 CTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGC 
                   
               
               
                   
                 CGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCG 
                   
               
               
                   
                 TTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTC 
                   
               
               
                   
                 CTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTT 
                   
               
               
                   
                 GTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCT 
                   
               
               
                   
                 CCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGG 
                   
               
               
                   
                 GGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGCGT 
                   
               
               
                   
                 GCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGG 
                   
               
               
                   
                 GCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGC 
                   
               
               
                   
                 GAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGC 
                   
               
               
                   
                 GGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGG 
                   
               
               
                   
                 GGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGC 
                   
               
               
                   
                 GCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTC 
                   
               
               
                   
                 CCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGG 
                   
               
               
                   
                 GCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCG 
                   
               
               
                   
                 GGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGG 
                   
               
               
                   
                 GCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAG 
                   
               
               
                   
                 GGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGA 
                   
               
               
                   
                 GGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAAT 
                   
               
               
                   
                 CGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAAT 
                   
               
               
                   
                 CTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACC 
                   
               
               
                   
                 CCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCG 
                   
               
               
                   
                 GCAGGAAGGAAATGGGCGGGGAGGGCCTTCGTGCGT 
                   
               
               
                   
                 CGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGG 
                   
               
               
                   
                 GGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGAC 
                   
               
               
                   
                 GGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGG 
                   
               
               
                   
                 CGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCT 
                   
               
               
                   
                 TCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATT 
                   
               
               
                   
                 GTGCTGTCTCATCATTTTGGCAAAGAATTCGAGCGGCC 
                   
               
               
                   
                 GCCAGTGTGAGTTGGACCGGTGCCGCCATGGCGCTCT 
                   
               
               
                   
                 CCACACCCCAGGAGACTTACTTTAAGTCCCAGGCAGA 
                   
               
               
                   
                 GTGGCGGGAAGAAGTAGAACTGGAATTTGAAAAGATT 
                   
               
               
                   
                 CGTTCAGAAGGGACCTGTCTGGAACGCGAAGAAGAG 
                   
               
               
                   
                 GAACTGGAAATGGAAAGGAGGGAGGAGCTCAGACAC 
                   
               
               
                   
                 GCCCTGGACATCAGGGAGCACTATGAAAGGAAGCTGG 
                   
               
               
                   
                 AGAGAGCCAACAACCTGTATATGGAACTTAATGCCCT 
                   
               
               
                   
                 CATGTTGCAGCTGGAACTCCGTGAGAGGGAGCTGGAA 
                   
               
               
                   
                 AGGGAAGAGCAAGCTTTAGAGCGGAGGTGCCCAGGC 
                   
               
               
                   
                 CTGCTGAAGCCACACCCTTCCCGGGGCCTCCTGCATG 
                   
               
               
                   
                 GAAACACAATGGAGTAATGAGGCATGCTTCTATATTA 
                   
               
               
                   
                 TTTTCTAAAAGATTTAAAGTTTTGCCTTCTCCATTTAG 
                   
               
               
                   
                 ACTTATAATTCACTGGAATTTTTTTGTGTGTATGGTAT 
                   
               
               
                   
                 GACATATGGGTTCCCTTTTATTTTTTACATATAAATAT 
                   
               
               
                   
                 ATTTCCCTGTTTTTCTAAAAAAGACCTAGGAAAACTGT 
                   
               
               
                   
                 CTTCATAATCAACCTCTGGATTACAAAATTTGTGAAAG 
                   
               
               
                   
                 ATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCT 
                   
               
               
                   
                 ATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTA 
                   
               
               
                   
                 TTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATA 
                   
               
               
                   
                 AATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCC 
                   
               
               
                   
                 GTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGC 
                   
               
               
                   
                 TGACGCAACCCCCACTGGTTGGGGCATTGCCACCACC 
                   
               
               
                   
                 TGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCT 
                   
               
               
                   
                 ATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCC 
                   
               
               
                   
                 GCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAA 
                   
               
               
                   
                 TTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTT 
                   
               
               
                   
                 GGCTGCTCGCCTATGTTGCCACCTGGATTCTGCGCGGG 
                   
               
               
                   
                 ACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGC 
                   
               
               
                   
                 GGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGC 
                   
               
               
                   
                 CTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGG 
                   
               
               
                   
                 ATCTCCCTTTGGGCCGCCTCCCCGCATCGATACCGAGC 
                   
               
               
                   
                 GCTGCTAGAGAGATCGATCTGCCTCAACTGTGCCTTCT 
                   
               
               
                   
                 AGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCC 
                   
               
               
                   
                 TTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTT 
                   
               
               
                   
                 CCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAG 
                   
               
               
                   
                 TAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAG 
                   
               
               
                   
                 GACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGG 
                   
               
               
                   
                 CATGCTGGGGATGCGGTGGGCTCTATGGGTACCCAGG 
                   
               
               
                   
                 TGCTGAAGAATTGACCCGGTTCCTCCTGGGGAAGCAA 
                   
               
               
                   
                 TTCGTTGATCTGAATTTCGACCACCGATAATACCTATT 
                   
               
               
                   
                 ACCCTGGTAGATAAGTAGCACGGCGGGTTAATGATTA 
                   
               
               
                   
                 ACTACAGCAATTCGTTGATCTGAATTTCGACCACCCAT 
                   
               
               
                   
                 AATAGATCTCCCATTACCCTGGTAGATAAGTAGCATG 
                   
               
               
                   
                 GCGGGACAATTAAGTACCTCAAAGAACTATTCTTGTTT 
                   
               
               
                   
                 GCCTTATTCCTATGTAAATAACTGAAATCTTTGTTTTT 
                   
               
               
                   
                 CTTCCTAAAAGGGGTGATGTTGATTTTTACTTACAATG 
                   
               
               
                   
                 TATTTTAAGTTTGTCACTCTAAATGGTTATGAGCAAGT 
                   
               
               
                   
                 TTAAGAAAAATCTTCAGCAAATACTACCTTAGATTAT 
                   
               
               
                   
                 GACCCCAAAACACATTTACTTATGATTATGTTGAAAA 
                   
               
               
                   
                 CATAGGGTCTGGGGAAAAAGGGATTTAAATTAAGAAG 
                   
               
               
                   
                 AAAAAGAAGACTTCGGACTTAAAAAGTCTTTTAGAGG 
                   
               
               
                   
                 CCAGCTCACCAACAACACAACACCGAGTCTGTGTTGC 
                   
               
               
                   
                 ACAATATGTTACTTAGGTATAAATCAAGGATTCATGT 
                   
               
               
                   
                 AATTTTGTCATTCCTTGCGTGATATTTTAAAAAACATT 
                   
               
               
                   
                 CTGTGTAAGGTATTTATAAAGCTCTCTTCTAAAAATAC 
                   
               
               
                   
                 AAAAATTTGTGTCATTAATCAACAGTCAGGTTAATCAT 
                   
               
               
                   
                 TAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACT 
                   
               
               
                   
                 CCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGC 
                   
               
               
                   
                 GACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGC 
                   
               
               
                   
                 GGCCTCAGTGAGCGAGCGAGCGCGCACTGTCA 
               
               
                   
               
            
           
         
       
     
     It has been shown the DLK is a target for at least one microRNA, miR-130a. Ye et al. MicroRNA-130a Targets MAP3K12 to Modulate Diabetic Endothelial Progenitor Cell Function.  Cell Physiol Biochem.  2015; 36(2):712-26 (2015). Other examples of microRNA inhibitors of DLK are hsa-miR-10a-3p, hsa-miR-155-5p, and hsa-miR-30a-5p. Hu et al.  Oncology Lett.  15:467-474 (2018). In some embodiments the inhibitor comprises a microRNA from Table 3 or a functional equivalent thereof. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Exemplary microRNA sequences 
               
            
           
           
               
               
               
               
            
               
                 Sequence 
                   
                   
                 SEQ 
               
               
                 description 
                 Sequence 
                 miRBase Accession 
                 ID NO 
               
               
                   
               
               
                 hsa-mir-130a 
                 UGCUGCUGGCCAGAGCUCUUUUCAC 
                 MI0000448 
                 19 
               
               
                   
                 AUUGUGCUACUGUCUGCACCUGUCA 
                   
                   
               
               
                   
                 CUAGCAGUGCAAUGUUAAAAGGGC 
                   
                   
               
               
                   
                 AUUGGCCGUGUAGUG 
                   
                   
               
               
                   
               
               
                 hsa-miR-10a-5p 
                 UACCCUGUAGAUCCGAAUUUGUG 
                 MIMAT0000253 
                 20 
               
               
                   
               
               
                 hsa-miR-155-5p 
                 UUAAUGCUAAUCGUGAUAGGGGUU 
                 MIMAT0000646 
                 21 
               
               
                   
               
               
                 hsa-mir-30a 
                 GCGACUGUAAACAUCCUCGACUGGA 
                 MI0000088 
                 22 
               
               
                   
                 AGCUGUGAAGCCACAGAUGGGCUU 
                   
                   
               
               
                   
                 UCAGUCGGAUGUUUGCAGCUGC 
               
               
                   
               
            
           
         
       
     
     3.5 In Vivo and In Vitro Assays 
     The present disclosure further relates to assessment of efficacy and safety of gene therapy vectors in in vitro assay systems. Expression levels can be measured by immunohistochemistry of target cells or tissues. In some embodiments the vectors of the disclosure comprise nucleic acids encoding fluorescent proteins or other marker proteins used to assess transduction efficiency or gene expression levels. Functional assays include standard test of vision as well as inspection of the subjects&#39; eye. 
     3.6 Therapeutic Compositions and Methods 
     As used herein, the term “patient in need” or “subject in need” refers to a patient or subject at risk of, or suffering from, a disease, disorder or condition that is amenable to treatment or amelioration with a vector as provided herein. A patient or subject in need may, for instance, be a patient or subject diagnosed with a disease associated retinal cells. “Subject” and “patient” are used interchangeably herein. 
     The subject treated by the methods described herein may be a mammal. In some cases, a subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a rabbit, a mouse or a rat. A subject may be a human female or a human male. Subjects may range in age. Thus, the present disclosure contemplates administering any of the vectors of the disclosure to a subject suffering optic neuropathy. 
     Combination therapies are also contemplated by the invention. Combination as used herein includes simultaneous treatment or sequential treatment. Combinations of methods of the invention with standard medical treatments (e.g., corticosteroids or topical pressure reducing medications) are specifically contemplated, as are combinations with novel therapies. In some cases, a subject may be treated with a steroid to prevent or to reduce an immune response to administration of a vector described herein. In certain cases, a subject may receive topical pressure reducing medications before, during, or after administrating of a vector described herein. In certain cases, a subject may receive a medication capable of causing the pupil of the eye to dilate (e.g., tropicamide and/or phenylephrine). In certain cases, the subject may receive a moisturizing gel during recovery to prevent corneal dehydration. 
     A therapeutically effective amount of the rAAV vector is a dose of rAAV ranging from about 1e8 to about 1e13 vector genomes (vg), more specifically 1e10-1e12 vg or Tell vg. 
     In some cases, the therapeutic composition comprises more than about 1e9, 1e10, or 1e11 genomes of the rAAV vector per volume of therapeutic composition injected. In some cases, the therapeutic composition comprises more than approximately 1e10, Tell, 1e12, or 1e13 genomes of the rAAV vector per mL. 
     3.7 Administration of Compositions 
     Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intracameral inoculation, intravitreal inoculation, subretinal inoculation, suprachroidal inoculation, canaloplasty, or episcleral vein-mediated delivery. Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the invention may be chosen and/or matched by those skilled in the art taking into account the disease state being treated and the target cells/tissue(s). 
     The disclosure provides for local administration and systemic administration of an effective dose of rAAV and compositions of the invention. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parental administration through injection, infusion or implantation or intravenous administration. 
     In particular, actual administration of rAAV of the present invention may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration according to the invention includes, but is not limited to, injection into the bloodstream and/or directly into the eye. Simply resuspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for eye expression, and there are no known restrictions on the carriers or other components that can be co-administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV). 
     Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as eye. See, for example, WO 02/053703, the disclosure of which is incorporated by reference herein. Pharmaceutical compositions can be prepared as injectable formulations or as topical formulations to be delivered to the eyes by administration of eye drops or otherwise. Additionally, when a tetracycline-inducible promoter is used to control transgene expression, it may be advantageous to co-administer doxycycline via eyedrops. Numerous formulations of rAAV have been previously developed and can be used in the practice of the invention. The rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling. 
     For purposes of injection, various solutions can be employed, such as sterile aqueous solutions. Such aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose. Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose. A dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art. 
     The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin. 
     Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof. 
     Transduction with rAAV may also be carried out in vitro. In one embodiment, desired target cells are removed from the subject, transduced with rAAV and reintroduced into the subject. Alternatively, syngeneic or xenogeneic ocular cells can be used where those cells will not generate an inappropriate immune response in the subject. 
     Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art. In one embodiment, cells can be transduced in vitro by combining rAAV with cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers. Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intracameral inoculation, intravitreal inoculation, subretinal inoculation, canaloplasty, intrathecal delivery, sterotactic delivery to brain, or episcleral vein-mediated delivery. In some embodiments, transduction of cells with rAAV of the invention results in sustained expression of the inhibitor of DLK for more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, more than 6 months, more than 9 months, or more than 12 months. The present invention thus provides methods of administering/delivering rAAV which express an inhibitor of DLK to a mammalian subject, preferably a human being. These methods include transducing tissues (including the tissues of the eye, motor neurons, or brain) with one or more rAAV of the present invention. Transduction may be carried out with gene cassettes comprising tissue specific control elements. For example, one embodiment of the invention provides methods of transducing eye cells and eye tissues directed by eye specific control elements, including, promoters described herein. 
     4.1 Inhibitors 
     In some embodiments, the disclosure provides an inhibitor, comprising a protein comprising a leucine zipper domain that shares at least 95% identity to SEQ ID NO: 3, wherein the leucine zipper domain comprises at least one or at least two two substitutions at positions 391 and 393, relative to SEQ ID NO: 1. In some embodiments, said substitutions comprise K391 to D or K391 to E, and H393 to D or H393 to E. In some embodiments, said substitutions, individually or collectively, either (i) increase the binding affinity of the leucine zipper domain for endogenous DLK or (ii) decrease affinity of said inhibitor for itself, compared to the corresponding protein without said substitutions. 
     In some embodiments, the leucine zipper domain further comprises one or more additional substitutions at one or more positions, related to SEQ ID NO: 1 selected from one or more of K398, L407, H405, V412, R414, K453, L458, and R460. In some embodiments, the one or more substations comprise K398 to R if the one or more positions comprise K398; L407 to D or L407 to E if the one or more positions comprise L407; H405 to D or H405 to E if the one or more positions comprise H405; V412 to D or V412 to E if the one or more positions comprise V412; R414 to D or R414 to E if the one or more positions comprise R414; K453 to R if the one or more positions comprise K453; L458 to D or L458 to E if the one or more positions comprise L458; and R460 to D or R460 to E if the one or more positions comprise R460. 
     In some embodiments, the inhibitory domain comprises, in N terminal to C terminal order a first polypeptide having sequence REEVXLXFEKIYSEGTCLXRXEEELXMX (SEQ ID NO: 9); a polypeptide bridge comprising 20 to 30 amino-acid residues, optionally SEQ ID NO: 11; and a second polypeptide having sequence YMELNALMLQLELYERELXR XEQALERR (SEQ ID NO: 10); wherein X=E or D and Y=R, H, or K. 
     In some embodiments, the first polypeptide is selected from the group consisting of a polypeptide having sequence REEVDLDFEKIYSEGTCLXRXEEELXMX (SEQ ID NO: 12); a polypeptide having sequence REEVELDFEKIYSEGTCLXRXEEELXMX (SEQ ID NO: 13); a polypeptide having sequence REEVDLEFEKIYSEGTCLXRXEEELXMX (SEQ ID NO: 14); and a polypeptide having sequence REEVELEFEKIYSEGTCLXRXEEELXMX (SEQ ID NO: 15); wherein X=E or D and Y=R, H, or K. 
     In some embodiments, the leucine zipper domain comprises a polypeptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to one of SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the leucine zipper domain comprises a polypeptide identical to one of SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the substitution either (i) increases affinity of the leucine zipper domain for endogenous DLK, or (ii) decreases affinity of the leucine zipper domain for itself; each of (i) and (ii) as compared to the same leucine zipper domain having the native residue at the position of the substitution. In some embodiments, the leucine zipper preferentially forms heterodimers with endogenous DLK compared to either one of (i) homodimers of the inhibitor or (ii) DLK homodimers. 
     In some embodiments, the inhibitor comprises a DLK kinase domain having at least 95% identity to SEQ ID NO: 5, wherein the DLK kinase domain comprises a substitution that decreases the kinase activity of the DLK kinase domain. In some embodiments, the substitution that decreases the kinase activity of the DLK kinase domain is a substitution of lysine (K) at position 152 relative to SEQ ID NO: 1, from lysine (K) to alanine (A). In some embodiments, the DLK kinase domain comprises SEQ ID NO. 8. 
     In some embodiments, the inhibitor comprises a functional fragment of LZK. In some embodiments, the inhibitor comprises a functional fragment a neurotrophic factor, optionally BDNF, GDNF, NT4, TrkB, RdCVF, or CNTF, or a functional fragment thereof. In some embodiments, the inhibitor further comprises a functional fragment capable of inhibiting cAMP-mediated signaling, optionally cAMP-mediated signalling through mAKAP. In some embodiments, the inhibitor comprises a second domain, and the inhibitor is capable of having neurotrophic activity in the cell. 
     4.2 Vectors 
     The disclosure provides a vector, comprising a polynucleotide encoding an inhibitor of Dual Leucine Zipper Kinase (DLK), the polynucleotide operatively linked to a promoter, wherein the inhibitor is capable of binding either (i) an endogenous DLK protein, or (ii) an endogenous mRNA encoding an endogenous DLK; and wherein the inhibitor is capable of reducing the expression, signaling activity, or kinase activity of the endogenous DLK in a retinal cell. As already stated, the vector may be viral or non-viral. In some embodiments, the vector is recombinant AAV. 
     In some embodiments, the promoter is a ubiquitous promoter. As used herein a “ubiquitous promoter” is a promoter that is capable of mediating expression of a polynucleotide in many, if not most, cells of a target organism. Generally ubiquitous promoters in one eukaryotic organism are capable of mediating expression in others. Ubiquitous promoters include mammalian promoters. In some embodiments, the ubiquitous promoter comprises a CMV promoter, a CAG promoter, CBA promoter, or a CBh promoter. In some embodiments, the promoter is a retinal cell-type specific. In some embodiments, the promoter is an RGC-specific promoter. Exemplary RGC-specific promoters, e.g. the Nefh promoter, are provided by Hanlon et al.  Front Neurosci.  2017 Sep. 21; 11:521. 
     In some embodiments, the promoter is a Thy1 promoter, a Nefh promoter, or a DCX promoter. The DCX promoter is described in Smith et al. Sci Rep. 8:1490 (2018); and US20140248701A1. In some embodiments, the promoter is a minipromoter. In some embodiments, said minipromoter is derived from POU4F1, TUBB3, NEFL, or NEFM. The NEFL promoter is described in Simpson et al. New MiniPromoter P1e345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina. Hum Gene Ther. (2018). In some embodiments, the promoter is a rod photoreceptor-specific promoter. In some embodiments, the promoter is a rhodopsin promoter or an RK promoter. In some embodiments, the promoter is a cone photoreceptor-specific promoter. In some embodiments, the promoter is a pR2.1 promoter or a MNTC promoter, as disclosed in U.S. Pat. No. 10,000,741, which is incorporated here in its entirety. 
     In some embodiments, the inhibitor is an RNA capable of binding an endogenous mRNA encoding an endogenous DLK, and wherein the inhibitor is capable of reducing the expression of the endogenous DLK in the cell. In some embodiments, the inhibitor is an shRNA or an siRNA. In some embodiments, the inhibitor is a microRNA. In some embodiments, the microRNA is miR-130a, miR-122-5p, miR-30a-5p, or miR-10-3p. In some embodiments, inhibitor is a protein capable of binding an endogenous DLK, and In some embodiments, wherein the inhibitor is capable of reducing the signaling activity or the kinase activity of the endogenous DLK in the cell. 
     In some embodiments, the protein comprises an inhibitory domain, the inhibitory domain comprising a leucine zipper domain. In some embodiments, the inhibitory domain is capable of binding the leucine zipper domain of endogenous DLK, thereby competitively inhibiting homodimerization of said endogenous DLK or competitively inhibiting heterodimerization of said endogenous DLK and endogenous Leucine Zipper Kinase (LZK). In some embodiments, the inhibitory domain comprises, consists essentially of, or consists of a polypeptide at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 3 or SEQ ID NO: 4. 
     In some embodiments, the leucine zipper domain of the inhibitory domain comprises at least one amino acid substitution relative to SEQ ID NO: 1, and In some embodiments, wherein the substitution increases affinity of the leucine zipper domain for endogenous DLK or decreases affinity of the leucine zipper domain for itself. In some embodiments, the inhibitory domain contains a substitution selected from those listed in Table 4. 
     In some embodiments, the inhibitory domain comprises, in N terminal to C terminal order, a polypeptide having sequence REEVXLXFEKIYSEGTCLXRXEEELXMX (SEQ ID NO: 9), a polypeptide bridge comprising 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino-acid residues, optionally SEQ ID NO: 11, and a polypeptide having sequence YMELNALMLQLELYERELXRXEQALERR (SEQ ID NO: 10), wherein X=E or D and Y=R, H, or K. 
     In some embodiments, the inhibitory domain comprises a polypeptide that shares at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identity to one of SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the inhibitory zipper domain comprises a polypeptide that is identical to one of SEQ ID NO. 6 or SEQ ID NO. 7. In some embodiments, the substitution increases affinity of the leucine zipper domain for endogenous DLK or decreases affinity of the leucine zipper domain for itself compared to the same leucine zipper domain having the native residue at the position of the substitution. In some embodiments, the leucine zipper preferentially forms heterodimers with endogenous DLK compared to either one of (i) inhibitor homodimers or (ii) DLK homodimers. 
     In some embodiments, the inhibitor comprises a DLK kinase domain having at least 75% identity to SEQ ID NO: 5, wherein the DLK kinase domain comprises a substitution that decreases the kinase activity of the DLK kinase domain. In some embodiments, the inhibitor is an inactive form of DLK. In some embodiments, said inhibitor contains a substitution at position 152 relative to SEQ ID NO: 1 from lysine (K) to alanine (A). In some embodiments, the DLK kinase domain comprises SEQ ID NO. 8. In some embodiments, the inhibitor comprises a functional fragment of LZK. In some embodiments, the inhibitor comprises a functional fragment a neurotrophic factor, optionally BDNF, GDNF, NT4, TrkB, RdCVF, or CNTF, or a functional fragment thereof. In some embodiments, the inhibitor comprises a functional fragment capable of inhibiting cAMP-mediated signaling, optionally cAMP-mediated signaling through mAKAP. In some embodiments, the inhibitor comprises a second domain, and wherein the inhibitor is capable of having neurotrophic activity in the cell. 
     In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the vector is a non-viral vector. 
     In some embodiments, the disclosure provides, a recombinant virus vector, comprising an expression cassette comprising a polynucleotide encoding the inhibitor of claim  1  operably linked to a promoter. 
     In some embodiments, the virus is a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the expression cassette is flanked by 5′ ITR and 3′ ITR. In some embodiments, the vector comprises a genome between 3.2 kb and 5.0 kb, 3.4 kb and 4.9 kb, 3.6 kb and 4.8 kb, 3.8 kb and 4.7 kb, 3.8 kb and 4.6 kb, 4.0 kb and 4.6 kb, 4.2 kb and 4.6 kb, 4.2 kb and 4.4 kb, or 4.4 kb and 4.6 kb. In some embodiments, the vector comprises a genome between 3.2 kb and 3.4 kb, 3.4 kb and 3.6 kb, 3.6 kb and 3.8 kb, 3.8 kb and 4.0 kb, 4.0 kb and 4.2 kb, 4.2 kb and 4.4 kb, 4.4 kb and 4.6 kb, 4.6 kb and 4.8 kb, or 4.8 kb and 5.0 kb. In some embodiments, the vector comprises a genome of about 3.2 kb, 3.3 kb, 3.4 kb, 3.5 kb, 3.6 kb, 3.7 kb, 3.8 kb, 3.9 kb, 4.0 kb, 4.1 kb, 4.2 kb, 4.3 kb, 4.4 kb, 4.5 kb, 4.6 kb, 4.7 kb, or 4.8 kb. In some embodiments, the rAAV comprises a deletion of one or more non-essential genes. In some embodiments, the rAAV comprises a deletion of one or more essential genes, provided in trans during production of the rAAV. In some embodiments, the rAAV comprises a deletion of rep, cap, and rep or cap. In some embodiments, the rAAV is a self-complementary AAV. In some embodiments, the rAAV comprises a deletion in the D region of either or both of the 5′ ITR and 3′ ITR. In some embodiments, the rAAV is a hybrid AAV. In some embodiments, the rAAV comprises a stuffer sequence. In some embodiments, the rAAV comprises the 5′ ITR and 3′ ITR of AAV2 and the capsid proteins of an AAV serotype other than AAV2. In some embodiments, the rAAV comprises 5′ ITR and 3′ ITR from different serotypes. In some embodiments the rAAV is modified by one or more of transcapsidation, adsorption modification, mosaic capsid, or chimeric capsid (e.g. as described in Choi et al.  Curr Gene Ther.  2005 June; 5(3): 299-310). 
     In some embodiments, the vector comprises a capsid of serotype AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, or AAV2/2(7m8). In some embodiments, the rAAV selectively infects a cell type, such as neurons, retinal cells, or RGCs. Serotypes useful in the vectors of the present disclosure include those described in Lebherz et al.  J Gene Med.  2008 Apr.; 10(4): 375-382; Hanlon et al.  Front Neurosci.  2017; 11: 521; and Hickey et al.  Gene Therapy  24:787-800 (2017). 
     In some embodiments, the promoter is a ubiquitous promoter. As used herein a “ubiquitous promoter” is a promoter that is capable of mediating expression of a polynucleotide in many, if not most, cells of a target organism. Generally ubiquitous promoters in one eukaryotic organism are capable of mediating expression in others. Ubiquitous promoters include mammalian promoters. In some embodiments, the ubiquitous promoter comprises a CMV promoter, a CAG promoter, CBA promoter, or a CBh promoter. In some embodiments, the promoter is a retinal cell-type specific. In some embodiments, the promoter is an RGC-specific promoter. Exemplary RGC-specific promoters, e.g. the Nefh promoter, are provided by Hanlon et al.  Front Neurosci.  2017 Sep. 21; 11:521. 
     In some embodiments, the promoter is a Thy1 promoter, a Nefh promoter, or a DCX promoter. The DCS promoter is described in Smith et al. Sci Rep. 8:1490 (2018); and US20140248701A1. In some embodiments, the promoter is a minipromoter. In some embodiments, said minipromoter is derived from POU4F1, TUBB3, NEFL, or NEFM. The NEFL promoter is described in Simpson et al. New MiniPromoter P1e345 (NEFL) Drives Strong and Specific Expression in Retinal Ganglion Cells of Mouse and Primate Retina. Hum Gene Ther. (2018). In some embodiments, the promoter is a rod photoreceptor-specific promoter. In some embodiments, the promoter is a rhodopsin promoter or an RK promoter. In some embodiments, the promoter is a cone photoreceptor-specific promoter. In some embodiments, the promoter is a pR2.1 promoter or a MNTC promoter. 
     4.3 Methods of Treatment 
     In some embodiments, the disclosure provides a method of treating a disease or disorder, comprising contacting a cell with any of the vectors of the disclosure. In some embodiments, the cell is a retinal cell. In some embodiments, the retinal cell is a retinal ganglion cell (RGC), a photoreceptor cell, or a retinal neuron (e.g. a bipolar cell, an amacrine cell, or a horizontal cell). In some embodiments, the cell is a dopaminergic neuron, a dorsal striatum neuron, or a basal forebrain cholinergic neuron. In some embodiments, the vector is administered by injection of a volume of solution of about 10 to 100 μL, preferably 50 μL. In some embodiments, the disease or disorder is related to dysfunction in retinal cells. In some embodiments, the disease or disorder related to dysfunction in retinal cells is selected from the group consisting of glaucoma, wet macular degeneration, dry macular degeneration, geographic atrophy, retinal detachment, and retinal dystrophy, such as Usher&#39;s syndrome, Stargardt&#39;s disease, Leber&#39;s congenital amaurosis, or retinitis pigmentosa. In yet other embodiments, the disease is related to retinopathy, such as diabetic retinopathy, myopic maculopathy. In other embodiments, the disease is an optic neuropathy, such as optic neuritis, non-arteritic anterior ischemic optic neuropathy (NAION), arteritic anterior ischemic optic neuropathy (AION), traumatic optic neuropathy, Leber&#39;s optic neuropathy, dominant optic atrophy, recessive optic atrophy, or radiation optic neuropathy. 
     In some embodiments, the disclosure provides a method of treating a disease or disorder related to retinal cells, comprising administering to the retina of the subject any of the vectors of the disclosure. In some embodiments, the retinal cell is a retinal ganglion cell (RGC), a photoreceptor cell, or a retinal neuron—such as a bipolar cell, an amacrine cell, or a horizontal cell. In some embodiments, the vector is administered by injection of a volume of solution of about 10 to 100 μL, preferably 50 μL. In some embodiments, the disease or disorder related to dysfunction in retinal cells is selected from the group consisting of glaucoma, wet macular degeneration, dry macular degeneration, geographic atrophy, retinal detachment, and retinal dystrophy, such as Usher&#39;s syndrome, Stargardt&#39;s disease, Leber&#39;s congenital amaurosis, or retinitis pigmentosa. In yet other embodiments, the disease is related to retinopathy, such as diabetic retinopathy, myopic maculopathy. In other embodiments, the disease is an optic neuropathy, such as optic neuritis, non-arteritic anterior ischemic optic neuropathy (NAION), arteritic anterior ischemic optic neuropathy (AION), traumatic optic neuropathy, Leber&#39;s optic neuropathy, dominant optic atrophy, recessive optic atrophy, or radiation optic neuropathy. 
     4.4 Retinal Cells 
     In some embodiments, the disclosure provides genetically modified retinal cells, comprising a polynucleotide encoding an inhibitor of Dual Leucine Zipper Kinase (DLK), the polynucleotide operatively linked to a promoter. In some embodiments, the inhibitor is capable of binding either (i) an endogenous DLK protein, or (ii) an endogenous mRNA encoding an endogenous DLK. In some embodiments, wherein the inhibitor is capable of reducing the expression, signaling activity, or kinase activity of the endogenous DLK in the retinal cell. Any of the embodiments of features of the polynucleotide described above with respect to vectors can be incorporated in the retinal cells of the disclosure. Indeed it is contemplated that the retinal cells disclosed with have been generated directly or indirectly using the vectors of the disclosure. 
     In some embodiments, the disclosure provides a method of generating a genetically modified retinal cell, comprising contacting a retinal cell with any vector of the disclosure. The disclosure further provides pharmaceutical compositions comprises any of the vectors or retinal cells of the disclosure. 
     4.5 Pharmaceutical Compositions and Methods of Treatment 
     In some embodiments, the disclosure provides a pharmaceutical composition comprising the vector of any one of claims  18  to  33  and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises at least 1e9-1e13 vector genomes (vg) of the vector. In some embodiments, the disclosure provides a unit dose, comprising any of the vectors of the disclosure, wherein the unit does comprises 1e9-1e13 vg of vector in a total volume of 10-200 μL, in some embodiments 50-100 μL. 
     In some embodiments, the disclosure provides a method of treating a condition in subject in need thereof, comprising administering an effective amount of a pharmaceutical composition of the disclosure to the subject. In some embodiments, the condition is glaucoma, optic neuropathy, retinopathy, retinal detachment, retinitis pigmentosa, retinal dystrophy, macular degeneration, amyotrophic lateral sclerosis (ALS), Parkinson Disease, Alzheimer Disease, prior-related neurodegenerative disease, Huntington&#39;s disease, spinocerebellar ataxia, spinal muscular atrophy, multiple system atrophy. In some embodiments, the glaucoma is primary open angle (e.g. chronic glaucoma), normal tension glaucoma, angle closure glaucoma (e.g. acute glaucoma), secondary glaucoma, inherited glaucoma (e.g. pigmentary glaucoma), or myocilin (MYOC) glaucoma. In some embodiments, the neuropathy is Leber hereditary optic neuropathy (LHON) or autosomal dominant optic atrophy (ADOA or DOA). In some embodiments, the retinopathy is retinopathy of prematurity (ROP) or diabetic retinopathy. 
     In some embodiments, the retinitis pigmentosa is related to Leber congenital amaurosis. The retinitis pigmentosa may be caused by any of the genotypes known to have causative relationship with the condition. Mutations in more than 60 genes are known to cause nonsyndromic retinitis pigmentosa. More than 20 of these genes are associated with the autosomal dominant form of the disorder. Mutations in the RHO gene are the most common cause of autosomal dominant retinitis pigmentosa, accounting for 20 to 30 percent of all cases. At least 35 genes have been associated with the autosomal recessive form of the disorder. The most common of these is USH2A; mutations in this gene are responsible for 10 to 15 percent of all cases of autosomal recessive retinitis pigmentosa. Changes in at least six genes are thought to cause the X-linked form of the disorder. Together, mutations in the RPGR and RP2 genes account for most cases of X-linked retinitis pigmentosa. In some embodiments the retinitis pigmentosa is associated with inheritance of alleles of one or more of genes selected from the group consisting of C2ORF71, C8ORF37, CA4, CERKL, CLRN1, CNGA1, CNGB1, CRB1, CRX, DHDDS, EYS, FAM161A, FSCN2, GUCA1B, IDH3B, IMPDH1, IMPG2, KLHL7, LRAT, MAK, MERTK, NR2E3, NRL, OFD1, PDE6A, PDE6B, PDE6G, PRCD, PROM1, PRPF3, PRPF6, PRPF8, PRPF31, PRPH2, RBP3, RDH12, RGR, RHO, RLBP 1, ROM1, RP1, RP2, RP9, RPE65, RPGR, SAG, SEMA4A, SNRNP200, SPATA7, TOPORS, TTC8, TULP1, USH2A, and ZNF513, as described in Daiger et al.  Clin Genet.  84:132-141 (2013). 
     In some embodiments, the retinal dystrophy is Stargardt macular degeneration, rod-cone dystrophy, or cone-rod dystrophy. 
     EXAMPLES 
     The invention is further described in the following Examples, which do not limit the scope of the invention described in the claims. 
     Example 1: Engineering of Improved Dominant-Negative DLK (DN-DLK) 
     Expression of the leucine zipper domain of DLK has been shown to have a dominant negative effect of DLK function. Nihalani et al.  J. Bio Chem.  275, 7273-7279 (2000). To attempt to generate improved dominant negative DLKs (DN-DLKs), Applicants identified mutations in the leucine zipper domain intended to (1) increase the affinity of the DLK/DN-DLK heterodimeric interaction and/or (2) decrease the affinity of the DN-DLK/DN-DLK homodimeric interaction, with the aim of favoring disruption of DLK/DLK homodimers in cells. 
     Using the known pinwheel structure of leucine zippers ( FIG. 1A ), the sequence DLK ( FIG. 1B ; SEQ ID NO: 4) was modelled as eight pinwheels ( FIG. 2A-2H ). Amino-acid substitutions identified by analysis of these models are listed in Table 4. 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Substitutions to Enhance DN-DLKs 
               
            
           
           
               
               
               
               
            
               
                   
                 Repeat 
                 Substitutions 
                 Effect 
               
               
                   
                   
               
               
                   
                 L1 
                 Lysine (K) 391 to 
                 Create H-E pair 
               
               
                   
                   
                 glutamic acid (E) 
                 Stabilize heterodimer 
               
               
                   
                   
                 Lysine (K) 391 to 
                 Create H-D pair 
               
               
                   
                   
                 aspartic acid (D) 
                 Stabilize heterodimer 
               
               
                   
                   
                 Histidine (H) 393 to 
                 Create K-E pair 
               
               
                   
                   
                 glutamic acid (E) 
                 Stabilize heterodimer 
               
               
                   
                   
                 Histidine (H) 393 to 
                 Create K-D pair 
               
               
                   
                   
                 aspartic acid (D) 
                 Stabilize heterodimer 
               
               
                   
                 L2 
                 Lysine (K) 398 to 
                 Create R-E pair 
               
               
                   
                   
                 glutamic acid (E) 
                 Stabilize heterodimer 
               
               
                   
                   
                 Lysine (K) 398 to 
                 Create R-D pair 
               
               
                   
                   
                 aspartic acid (D) 
                 Stabilize heterodimer 
               
               
                   
                 L3 
                 Lysine (K) 407 to 
                 Create H-E pair 
               
               
                   
                   
                 glutamic acid (E) 
                 Stabilize heterodimer 
               
               
                   
                   
                 Lysine (K) 407 to 
                 Create H-D pair 
               
               
                   
                   
                 aspartic acid (D) 
                 Stabilize heterodimer 
               
               
                   
                 L3 
                 Histidine (H) 405 to 
                 Create L-E pair 
               
               
                   
                   
                 glutamic acid (E) 
                 Destabilize DN-DLK 
               
               
                   
                   
                   
                 homodimer 
               
               
                   
                   
                 Histidine (H) 405 to 
                 Create L-D pair 
               
               
                   
                   
                 aspartic acid (D) 
                 Destabilize DN-DLK 
               
               
                   
                   
                   
                 homodimer 
               
               
                   
                 L4 
                 Valine (V) 412 to 
                 Create E-R pair 
               
               
                   
                   
                 glutamic acid (E) 
                 Stabilize heterodimer 
               
               
                   
                   
                 Valine (V) 412 to 
                 Create D-R pair 
               
               
                   
                   
                 aspartic acid 
                 Stabilize heterodimer 
               
               
                   
                 L4 
                 Arginine (R) 414 to 
                 Create E-R pair 
               
               
                   
                   
                 glutamic acid (E) 
                 Destabilize DN-DLK 
               
               
                   
                   
                   
                 homodimer 
               
               
                   
                   
                 Arginine (R) 414 to 
                 Create D-R pair 
               
               
                   
                   
                 aspartic acid (D) 
                 Destabilize DN-DLK 
               
               
                   
                   
                   
                 homodimer 
               
               
                   
                 L5 
                 N/A 
                 N/A 
               
               
                   
                 L6 
                 Lysine (K) 407 to 
                 Create R-E pair 
               
               
                   
                   
                 glutamic acid (E) 
               
               
                   
                   
                 Lysine (K) 407 to 
                 Create R-D pair 
               
               
                   
                   
                 aspartic acid (D) 
               
               
                   
                 L7 
                 Leucine (L) 458 to 
                 Create L-E pair, 
               
               
                   
                   
                 glutamic acid (E) 
                 Destabilize DN-DLK 
               
               
                   
                   
                   
                 homodimer 
               
               
                   
                   
                 Leucine (L) 458 to 
                 Create L-D pair 
               
               
                   
                   
                 aspartic acid (D) 
                 Destabilize DN-DLK 
               
               
                   
                   
                   
                 homodimer 
               
               
                   
                 L7 
                 Glutamic acid (E) 
                 Strengthen 
               
               
                   
                   
                 454 to A, V, I, L, 
                 hydrophobic core 
               
               
                   
                   
                 M, F, W 
               
               
                   
                   
                 Glutamic acid (E) 
                 Create electrostatic 
               
               
                   
                   
                 454 to R, H, K 
                 interaction. 
               
               
                   
                 L8 
                 Glutamic acid (E) 
                 Strengthen 
               
               
                   
                   
                 461 to A, V, I, L, 
                 hydrophobic core 
               
               
                   
                   
                 M, F, W 
               
               
                   
                   
                 Glutamic acid (E) 
                 Create electrostatic 
               
               
                   
                   
                 461 to R, H, K 
                 interaction. 
               
               
                   
                   
               
            
           
         
       
     
     Polynucleotides encoding DLK fragments with various combinations of the substitutions listed in Table 5 are generated. Polynucleotides encoding DLK fragments with combinations of two or more of the substitutions listed in Table 5 are generated. A polynucleotide encoding a DLK fragment with ten substitutions is generated (SEQ ID NO: 6). A polynucleotide encoding a larger DLK fragment with the same ten substitutions is generated (SEQ ID NO: 7). These designs are tested by expression as isolated DLK fragments and as mutations in full-length DLK. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Exemplary Dominant-Negative Dual-Leucine Zipper Kinase Sequences 
               
            
           
           
               
               
               
            
               
                 Sequence 
                   
                 SEQ 
               
               
                 description 
                 Sequence 
                 ID NO 
               
               
                   
               
               
                 Modified DLK 
                 
                   REEV E L E FEKI R SEGTCL E R E EEEL E M E   
                   RREELRHALDIRE 
                 
                 6 
               
               
                 Zipper 1, bridge, 
                 
                   HYERKLERANNL 
                   YMELNALMLQLEL R EREL E R E EQALER 
                 
                   
               
               
                 and zipper 2. 
                 
                   R 
                 
                   
               
               
                 Modifications in 
                   
                   
               
               
                 bold enhance 
                   
                   
               
               
                 heterodimerization 
                   
                   
               
               
                 with DLK, 
                   
                   
               
               
                 compared to DLK 
                   
                   
               
               
                 homodimers or 
                   
                   
               
               
                 LZ-LZ 
                   
                   
               
               
                 homodimers 
                   
                   
               
               
                   
               
               
                 Longer LZ domain 
                 LSTPQETYFKSQAEW REEV E L E FEKI R SEGTCL E R E EEEL   
                 7 
               
               
                 with same 
                 
                     E M E   
                   RREELRHALDIREHYERKLERANNL 
                   YMELNALMLQL 
                 
                   
               
               
                 modifications 
                   EL R EREL E R E EQALERR CPGLLKPHPSRGLLHGNTME 
                   
               
               
                   
               
               
                 Kinase domain of 
                 ILDLQWVGSGAQGAVFLGRFHGEEVAV   A   KVRDLKETDI 
                 8 
               
               
                 kinase-dead DLK 
                 KHLRKLKHPNIITFKGVCTQAPCYCILMEFCAQGQLYEV 
                   
               
               
                 (K152A) 
                 LRAGRPVTPSLLVDWSMGIAGGMNYLHLHKIIHRDLKSP 
                   
               
               
                   
                 NMLITYDDVVKISDFGTSKELSDKSTKMSFAGTVAWMA 
                   
               
               
                   
                 PEVIRNEPVSEKVDIWSFGVVLWELLTGEIPYKDVDSSAII 
                   
               
               
                   
                 WGVGSNSLHLPVPSSCPDGFKILLRQCWNSKPRNRPSFR 
                   
               
               
                   
                 QILLHLDI 
               
               
                   
               
            
           
         
       
     
     Alternatively or in conjunction with any of these mutations to the leucine zipper domain, the kinase domain of DLK may be either deleted or replaced with a kinase-dead kinase domain. One exemplary kinase-dead kinase domain is a kinase domain with the K152A substitution, which replaces lysine K152 with alanine (A), as shown in SEQ ID NO: 8. 
     The various constructs are tested for in vitro competition with native DLK and for in vivo inhibition of DLK kinase activity or downstream signaling. 
     All, documents, patents, patent applications, publications, product descriptions, and protocols which are cited throughout this application are incorporated herein by reference in their entireties for all purposes. 
     The embodiments illustrated and discussed in this specification are intended only to teach those skilled in the art the best way known to the inventors to make and use the invention. Modifications and variation of the above-described embodiments of the invention are possible without departing from the invention, as appreciated by those skilled in the art in light of the above teachings. It is therefore understood that, within the scope of the claims and their equivalents, the invention may be practiced otherwise than as specifically described.