Gene Therapy for Eye Disease

Abstract

Methods and compositions for gene therapy of retinal degeneration associated with mutations in the MER protooncogene tyrosine kinase (MERTK).

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 437,346, filed January 5, 2023, and U.S. Provisional Application No. 63 / 497,850, filed April 24, 2023, both of which are incorporated by reference in their entireties. [Background technology]

[0002] The MER proto-oncogene tyrosine kinase (MERTK) is a transmembrane protein that is part of the MER / AXL / TYRO3 receptor kinase family. MERTK is involved in vision, and mutations in the MERTK gene are associated with multiple eye diseases.

[0003] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Summary of the Invention

[0004] Provided herein are methods of treating a subject with an ocular disease or disorder, the methods comprising administering to the subject a nucleic acid comprising a nucleotide sequence at least 90% identical to any one of SEQ ID NOS:3-6. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 90% identical to SEQ ID NOS:3. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 95% identical to SEQ ID NOS:3. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NOS:3. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 90% identical to SEQ ID NOS:4. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 95% identical to SEQ ID NOS:4. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NOS:4. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 90% identical to SEQ ID NOS:5. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 95% identical to SEQ ID NOS:5. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NOS:5. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 90% identical to SEQ ID NOS:6. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 95% identical to SEQ ID NOS:6. In some embodiments, the nucleic acid comprises the nucleotide sequence of SEQ ID NOS:6. In some embodiments, the nucleotide sequence is comprised in a vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, AAV2 / 5, AAV2tYF, and AAV2.7m8. In some embodiments, the vector comprises an AAV capsid protein. In some embodiments, the AAV capsid protein is selected from the group consisting of AAV2 capsid protein, AAV2tYF capsid protein, AAV5 capsid protein, AAV8 capsid protein, AAV9 capsid protein, and AAV2.7m8 capsid protein. In some embodiments, the vector further comprises 1) a 5' AAV ITR, and 2) a 3' AAV ITR.In some embodiments, the vector comprises an AAV expression cassette. In some embodiments, the vector further comprises a rhodopsin (RHO) promoter. In some embodiments, the vector further comprises a CBA promoter. In some embodiments, the vector further comprises a VMD2 promoter. In some embodiments, the vector further comprises a human rhodopsin kinase (hGRK1) promoter. In some embodiments, the vector further comprises a CASI promoter. In some embodiments, the disease or disorder comprises a MERTK-mediated disease or disorder. In some embodiments, the MERTK-mediated disease comprises retinitis pigmentosa. In some embodiments, the disease or disorder comprises vision loss. In some embodiments, administration occurs before the onset of the disease or disorder. In some embodiments, administration occurs after the onset of the disease or disorder. In some embodiments, administration occurs in at least one eye of the subject. In some embodiments, administration occurs by subretinal injection, intravitreal injection, or suprachoroidal injection. In some embodiments, administration occurs after at least 10 days. 9 In some embodiments, administration results in at least a 10% improvement in vision loss as measured using visual field testing. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0005] Also provided herein is a functional MERTK nucleic acid sequence, or a fragment thereof, comprising a nucleic acid sequence at least 90%, 95%, or 99% identical to any one of SEQ ID NOs: 3-6. Also provided herein is a composition comprising a functional MERTK nucleic acid sequence, or a fragment thereof. In some embodiments, the composition further comprises a promoter that expresses a product of the functional MERTK nucleic acid sequence in a plurality of photoreceptor cells or retinal pigment epithelial cells. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 95% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 4. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 90% identical to SEQ ID NO: 5. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 95% identical to SEQ ID NO:5. In some embodiments, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:5. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 90% identical to SEQ ID NO:6. In some embodiments, the nucleic acid sequence comprises a nucleotide sequence at least 95% identical to SEQ ID NO:6. In some embodiments, the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:6. In some embodiments, the nucleic acid sequence is comprised in a vector. In some embodiments, the vector is an adeno-associated virus (AAV) vector. In some embodiments, the AAV vector is a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, AAV2 / 5, AAV2tYF, and AAV2.7m8. In some embodiments, the vector comprises AAV capsid proteins.In some embodiments, the AAV capsid protein is selected from the group consisting of AAV2 capsid protein, AAV2tYF capsid protein, AAV5 capsid protein, AAV8 capsid protein, AAV9 capsid protein, and AAV2.7m8 capsid protein. In some embodiments, the vector further comprises 1) a 5' AAV ITR, and 2) a 3' AAV ITR. In some embodiments, the vector comprises an AAV expression cassette. In some embodiments, the vector further comprises a rhodopsin (RHO) promoter. In some embodiments, the vector further comprises a CBA promoter. In some embodiments, the vector further comprises a human rhodopsin kinase (hGRK1) promoter. In some embodiments, the vector further comprises a CASI promoter. In some embodiments, the vector further comprises a VMD2 promoter. In some embodiments, the composition is for use in treating a disease or disorder. In some embodiments, the disease or disorder comprises a MERTK-mediated disease or disorder. In some embodiments, the MERTK-mediated disease comprises retinitis pigmentosa. In some embodiments, the disease or disorder comprises vision loss. In some embodiments, the composition is administered to the subject before the onset of the disease or disorder. In some embodiments, the composition is administered to the subject after the onset of the disease or disorder. In some embodiments, the composition is administered to at least one eye of the subject. In some embodiments, the composition is administered to the subject by subretinal injection, intravitreal injection, or suprachoroidal injection. In some embodiments, the composition is administered to the subject by at least about 10 minutes. 9 In some embodiments, the composition, when administered to a subject, restores at least partial vision to the subject. In some embodiments, the composition, when administered to a subject, improves the subject's vision loss by at least 10%, as measured using a visual field test.

[0006] Also provided herein is the use of a functional MERTK nucleic acid sequence or a fragment thereof described herein for the manufacture of a medicament. Also provided herein is a kit comprising a functional MERTK nucleic acid sequence or a fragment thereof described herein, and optionally instructions for use. [Brief explanation of the drawings]

[0007] [Figure 1A] 1 illustrates an exemplary MERTK gene construct. [Figure 1B] 1 illustrates an exemplary plasmid containing a MERTK gene construct. [Figure 2] Illustrates an overview of mouse experiments utilizing subretinal delivery of AAV2-VMD2-hMERTK. [Figure 3] Exemplary MERTK plasmids are illustrated. Panel A of Figure 3 illustrates VMD2-MERTKwt, a MERTK plasmid containing the vitelloid macular dystrophy-2 (VMD2) (bestrophin-1 (BEST1)) promoter with an unmodified wild-type (wt) MERTK sequence, resulting in a 4336 base pair (bp) AAV transduction region. Panel B of Figure 3 illustrates CBA-MERTKwt, a MERTK plasmid containing the cytomegalovirus (CMV) early enhancer and chicken β-actin (CBA) promoter and an unmodified wt MERTK sequence, resulting in a 4374 bp AAV transduction region. Panel C of Figure 3 illustrates VMD2-MERTKopt, a MERTK plasmid containing the VMD2 promoter with a codon-optimized (codop) MERTK sequence, resulting in a 4336 bp AAV transduction region. Figure 3, panel D, illustrates CBA-MERTKopt, a MERTK plasmid containing the CBA promoter with the CMV enhancer and codopMERTK sequence, resulting in a 4374 bp AAV transduction region. [Figure 4] FIG. 1 is a schematic diagram of an exemplary MERTK plasmid experiment. [Figure 5]MERTK gene expression measured using dPCR with 1 nanogram (ng) cDNA input. Gene of interest (GOI) (MERTKwt or MERTKcodop) copies / uL were normalized to the loading control RPP30 copies / uL run in the same experiment with the same cDNA input. n=3, p<0.01, ns: not significant compared to the respective control. [Figure 6A] Representative immunoblots showing MERTK from whole cell lysates of 293T cells after 48 hours of incubation and 72 hours after reverse transfection are shown. GAPDH was used as a loading control. Sample "A" uses the plasmid from panel A of Figure 3, sample "B" uses the plasmid from panel B of Figure 3, sample "C" uses the plasmid from panel C of Figure 3, and sample "D" uses the plasmid from panel D of Figure 3. [Figure 6B] Representative immunoblots showing MERTK from whole cell lysates of ARPE-19 cells after 48 hours of incubation and 72 hours after reverse transfection are shown. GAPDH was used as a loading control. Sample "A" uses the plasmid from panel A of Figure 3, sample "B" uses the plasmid from panel B of Figure 3, sample "C" uses the plasmid from panel C of Figure 3, and sample "D" uses the plasmid from panel D of Figure 3. [Figure 7A] Percent cell death visualized by phase contrast microscopy of ARPE19 cells is shown. Sample "A" uses the plasmid from Figure 3, panel A, sample "B" uses the plasmid from Figure 3, panel B, sample "C" uses the plasmid from Figure 3, panel C, and sample "D" uses the plasmid from Figure 3, panel D. [Figure 7B] 3 shows cell density visualized by phase contrast microscopy of ARPE19 cells. Sample "A" uses the plasmid from panel A of FIG. 3, sample "B" uses the plasmid from panel B of FIG. 3, sample "C" uses the plasmid from panel C of FIG. 3, and sample "D" uses the plasmid from panel D of FIG. 3. [Figure 8] Figure 1 shows the results of an ARPE-19 phagocytosis assay in which transfected ARPE-19 cells were treated with bPOS-FITC and analyzed using flow cytometry to determine the percentage of FITC+ cells as a measure of MERTK-driven phagocytic activity. n=3, p<0.00001 compared to control. [Figure 9] Representative immunoblots showing rhodopsin internalization (top) or total rhodopsin (bottom) from cell lysates of ARPE-19 cells after 30, 60, or 120 minutes of incubation with bPOS(+). Sample "A" uses the plasmid from panel A of Figure 3, sample "B" uses the plasmid from panel B of Figure 3, sample "C" uses the plasmid from panel C of Figure 3, and sample "D" uses the plasmid from panel D of Figure 3. [Figure 10] Figure 1 shows the relative MERTK gene expression in cell culture models of RPE cells: iPSC-RPE, ARPE19, and differentiated ARPE19 cells. 293T cells were used as a control cell line. [Figure 11] Figure 1 shows the relative native MERTK and codon-optimized MERTK gene expression in ARPE19 cells in response to AAV2-VMD2-MERTKwt and AAV2-VMD2-MERTK codop vectors, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0008] The present disclosure employs, unless otherwise indicated, conventional molecular biology techniques within the skill of the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0009] definition Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and is not to be construed as an inflexible limitation on the scope of any embodiment. Accordingly, unless the context clearly dictates otherwise, a description of a range should be construed to include all possible subranges specifically disclosed, to the nearest tenth of the unit of the lower limit of each numerical value within that range. For example, a description of a range such as 1 to 6 should be construed to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values ​​within that range, e.g., 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0010] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise. It will be further understood that the terms "comprise" and / or "comprising," as used herein, specify the presence of stated features, elements, steps, operations, elements, components, and / or groups, but do not exclude the presence or addition of one or more other features, elements, steps, operations, elements, components, and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0011] As used herein, unless otherwise stated or clear from the context, the term "about" when referring to a number or range of numbers is understood to mean the stated number and a number plus or minus 10% thereof, or a number 10% below the recited lower limit and 10% above the recited upper limit for the recited values ​​for a range.

[0012] As used herein, unless otherwise specified, the term "nucleic acid" encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in 5' to 3' direction unless otherwise specified. The methods described herein provide for the production of isolated nucleic acids. The methods described herein further provide for the production of isolated and purified nucleic acids. As used herein, a "nucleic acid" may contain at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length. Nucleic acids provided herein may include RNA, DNA, DNA / RNA hybrids, nucleic acid analogs, chemically modified nucleic acids, chimeras composed of two or more nucleic acids or nucleic acid analogs, or any combination thereof. Nucleic acids containing DNA can be transcribed from DNA to RNA. A nucleic acid comprising DNA can be transcribed from the DNA into RNA when administered to a subject. The nucleic acids provided herein can comprise one or more nucleic acid sequences.

[0013] Further provided herein are methods for synthesizing nucleotide sequences encoding any number of polypeptide segments, including sequences encoding nonribosomal peptides (NRPs), sequences encoding nonribosomal peptide synthetase (NRPS) modules and synthetic variants, polypeptide segments of other regulatory proteins such as antibodies, polypeptide segments from other protein families, and non-coding DNA or RNA, including regulatory sequences such as promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nuclear RNA derived from microRNA, or any functional or structural DNA or RNA entity of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci defined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNA representation of mRNA, usually obtained by reverse transcription or amplification of messenger RNA (mRNA), DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, modified nucleic acid sequences, synthetic nucleic acid sequences, and primers. cDNA encoding the gene or gene fragment referred to herein may contain at least one region encoding an exon sequence without an intervening intron sequence in the genomic equivalent.

[0014] The terms "percent identity," "sequence identity," "percent sequence identity," or "percent identity" refer to a quantitative measure of similarity between two sequences (nucleic acid or amino acid).

[0015] "Percent identity," "sequence identity," "percent sequence identity," or "percent identity" between a query nucleic acid sequence and a subject nucleic acid sequence is an "identity" value expressed as a percentage, calculated using an appropriate algorithm or software, such as BLASTN, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign, over the entire length of the query sequence after performing a pairwise global sequence alignment using an appropriate algorithm or software, such as BLASTN, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene. Importantly, the query nucleic acid sequence may be described by a nucleic acid sequence identified in one or more claims herein.

[0016] The "percent identity," "sequence identity," "percent sequence identity," or "percent identity" between a query amino acid sequence and a subject amino acid sequence is an "identity" value expressed as a percentage, calculated using an appropriate algorithm or software, such as BLASTP, FASTA, DNASTAR Lasergene, GeneDoc, Bioedit, EMBOSS needle, or EMBOSS infoalign, over the entire length of the query sequence after performing a pairwise global sequence alignment using an appropriate algorithm / software, such as BLASTP, FASTA, ClustalW, MUSCLE, MAFFT, EMBOSS Needle, T-Coffee, and DNASTAR Lasergene. Importantly, the query amino acid sequence may be described by an amino acid sequence identified in one or more claims herein. The query sequence may be 100% identical to the subject sequence, or may contain up to a specified integer number of amino acid or nucleotide modifications compared to the subject sequence such that the percent identity is less than 100%. For example, the query sequence is at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% identical to the subject sequence. Such alterations include at least one amino acid deletion, substitution (including conservative and non-conservative substitution), or insertion, which may occur at the amino- or carboxy-terminal positions of the query sequence, or anywhere between these terminal positions, and which may be interspersed between amino acids or nucleotides of the query sequence, either individually, or in one or more adjacent groups within the query sequence.

[0017] In some embodiments, the length of sequence identity comparison can be over the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500-5000 nucleotides. In some embodiments, identity over smaller fragments, e.g., at least about 9 nucleotides, at least about 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 or more nucleotides, can also be desired.

[0018] A "subject" in need thereof refers to an individual who has a disease, a symptom of a disease, or a predisposition to a disease, and the purpose is to treat, cure, alleviate, relieve, improve, ameliorate, or affect the disease, a symptom of the disease, or a predisposition to the disease.

[0019] As used herein, the terms "treat," "treating," or "treatment," and their grammatical equivalents, can include alleviating, ameliorating, or improving at least one symptom of a disease or condition; preventing additional symptoms; inhibiting a disease or condition, e.g., delaying, reducing, suppressing, attenuating, diminishing, arresting, or stabilizing the onset or progression of a disease or condition; relieving a disease or condition; regressing a disease or condition; alleviating symptoms caused by a disease or condition; reducing the severity of a disease; or prophylactically and / or therapeutically arresting symptoms of a disease or condition. "Treating" also includes reducing the frequency or severity of any symptoms or other pathological effects associated with a disease or condition, and / or the occurrence or recurrence of side effects associated with a disease or condition. "Treating" does not necessarily require a therapeutic result. It will be understood that treating a disorder or condition does not require, although not excluded, the complete elimination of the disorder, condition, or symptoms associated therewith. The term "treating" encompasses the concept of "managing," which refers to reducing the severity of or delaying the recurrence of a particular disease or disorder in a patient, for example, extending the period of remission in a patient suffering from the disease. "Treating" can refer to the application or administration of a composition to a subject after the onset or suspected onset of a disease or condition.

[0020] The term "treating" further encompasses the concepts of "prevent," "preventing," and "prevention." As used herein, the terms "prevent," "preventing," and "prevention" refer to a reduction in the occurrence of pathology of a condition in a subject who does not have the disease or condition, but who is at risk of or susceptible to developing the condition. Prevention may be complete (e.g., no pathology of the condition is present in the subject). Prevention may also be partial, such that the occurrence of pathology of the condition in the subject is less than would have occurred in the absence of the present disclosure.

[0021] As used herein, "administering" and its grammatical equivalents may refer to providing a pharmaceutical composition described herein to a subject or patient. Depending on the type of disease or disease site being treated, the composition can be administered to a subject using conventional methods known to those skilled in the pharmaceutical arts. For example, the composition can be administered orally, parenterally, topically, or via an implanted reservoir. In some embodiments, the composition is administered via injection suitable for delivery to the eye (e.g., via intravitreal, subretinal, or suprachoroidal injection). One or more such routes may be used.

[0022] MERTK gene therapy MERTK is a receptor tyrosine kinase that transduces signals from the extracellular matrix to the cytoplasm by binding to several ligands (e.g., LGALS3, TUB, TULP1, or GAS6). MERTK regulates many physiological processes, including cell survival, migration, differentiation, and phagocytosis of apoptotic cells (efferocytosis). Ligand binding at the cell surface induces autophosphorylation of MERTK at its intracellular domain, which provides a docking site for downstream signaling molecules. After ligand activation, MERTK interacts with GRB2 or PLCG2 and induces phosphorylation of MAPK1, MAPK2, FAK / PTK2, or RAC1. MERTK signaling plays a role in various processes, such as macrophage clearance of apoptotic cells, platelet aggregation, cytoskeletal reorganization, and engulfment. The MERTK gene is expressed in several hematopoietic tissues (e.g., macrophages), epithelial tissues (e.g., retinal pigment epithelium), and reproductive tissues.

[0023] In the eye, MERTK functions in the retinal pigment epithelium (RPE) as a regulator of photoreceptor outer segment phagocytosis (e.g., rod outer segment fragment phagocytosis). Photoreceptor outer segment (POS) phagocytosis involves three major steps: 1) POS binding to integrins, 2) this binding is transduced into intracellular signals via an increase in intracellular inositol 1,4,5-triphosphate (InsP3), resulting in 3) POS ingestion promoted by the macrophage receptor CD36 and signal transduction by MERTK and focal adhesion kinase (FAK). MERTK also plays an important role in inhibiting Toll-like receptor (TLR)-mediated innate immune responses by activating STAT1, which selectively induces the production of inhibitors of SOCS1 and SOCS3 cytokine signaling. MERTK mutations can be associated with disruption of the retinal pigment epithelium (RPE) phagocytic pathway and the development of vision-related diseases and disorders, such as autosomal recessive retinitis pigmentosa (RP).

[0024] The method includes the use of a sequence encoding human MERTK, or a biologically active fragment thereof. In some embodiments, the MERTK-encoding sequence comprises SEQ ID NO: 1, or a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 1. In some embodiments, the MERTK-encoding sequence comprises SEQ ID NO: 2, or a sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 2. Table 1 provides exemplary sequences of MERTK.

[0025] In some embodiments, the MERTK encoding sequence may be wild-type. In some embodiments, the MERTK encoding sequence may be codon-optimized so that it can be more efficiently translated into amino acid sequence. In some embodiments, the MERTK sequence may take the form of Figure 1A.

[0026] In one aspect, a codon-optimized engineered nucleic acid is provided, comprising a nucleic acid sequence encoding human MERTK. In some embodiments, the MERTK-encoding nucleic acid sequence is codon-optimized for expression in humans. In some embodiments, the codon-optimized MERTK coding sequence has less than about 80% identity, preferably about 76% or less identity, to the wild-type or native MERTK coding sequence (SEQ ID NO: 1). In some embodiments, the codon-optimized MERTK coding sequence has about 76% identity to the native MERTK coding sequence of SEQ ID NO: 1. In some embodiments, the codon-optimized MERTK coding sequence has about 64% identity to the native MERTK coding sequence of SEQ ID NO: 1. In some embodiments, the codon-optimized MERTK coding sequence is characterized by an improved translation rate compared to wild-type or native MERTK after delivery in a plasmid or recombinant viral vector (e.g., rAAV). In some embodiments, the codon-optimized MERTK coding sequence shares about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or less identity with the wild-type MERTK coding sequence of SEQ ID NO: 1. In some embodiments, the codon-optimized nucleic acid sequence comprises any one of SEQ ID NOs: 3-6. In some embodiments, the codon-optimized nucleic acid sequence comprises SEQ ID NO: 3. In some embodiments, the codon-optimized nucleic acid sequence comprises SEQ ID NO: 4. In some embodiments, the codon-optimized nucleic acid sequence comprises SEQ ID NO: 5. In some embodiments, the codon-optimized nucleic acid sequence comprises SEQ ID NO: 6. In some embodiments, the codon-optimized nucleic acid sequence comprises a variant of SEQ ID NOs: 3-6.In some embodiments, the codon-optimized nucleic acid sequence is a sequence that shares about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or more identity to any one of SEQ ID NOs:3-6.

[0027] In some embodiments, the nucleic acid comprises a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NOs: 3-6. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:3. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 4. In some embodiments, the nucleic acid comprises a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:5. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:6.

[0028] In some embodiments, the codon-optimized MERTK comprises an amino acid sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:2.

[0029] In some embodiments, the MERTK construct comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:7. In some embodiments, the MERTK construct comprises a sequence that is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:8. In some embodiments, the MERTK construct comprises a sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:9. In some embodiments, the MERTK construct comprises a sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:10.

[0030] In some embodiments, a nucleic acid comprising a MERTK-encoding nucleic acid sequence may comprise a non-coding sequence or a regulatory sequence. In some embodiments, the MERTK-encoding nucleic acid sequence further comprises a non-coding sequence or a regulatory sequence. In some embodiments, the non-coding sequence or regulatory sequence may comprise a 5'-untranslated region (5'-UTR), a 3'-untranslated region (3'-UTR), a promoter, an enhancer, a polyadenylation (poly-A) sequence, or a cis-regulatory element. In some embodiments, the cis-regulatory element comprises a silencer, an operator, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), or a minute virus of mice (MVM) intron. In some embodiments, the non-coding sequence or regulatory sequence may comprise a portion of an exon or a portion of an intron. In some embodiments, the polyadenylation sequence comprises a bovine growth hormone polyadenylation signal (bGHpA).

[0031] In some embodiments, the non-coding or regulatory sequence may include a promoter. In some cases, the promoter may include, but is not limited to, a cytomegalovirus (CMV) promoter, a hybrid CMV enhancer / chicken beta-action (CBA) promoter, a CAG promoter, a rhodopsin (RHO) promoter (e.g., a human rhodopsin (hRHO) promoter, a Rhol94 promoter), a CASI promoter, a rhodopsin kinase promoter (e.g., a GRK1 promoter), an interphotoreceptor retinoid-binding protein (IRBP) promoter, a red opsin promoter, a vitelloid macular dystrophy (VMD2) promoter, and a cadherin promoter (e.g., a CDH5 promoter or a CD144 promoter). The vector may include a promoter that drives expression in many cell types (e.g., a CAG, CMV, or CASI promoter). Alternatively, the vector can include a promoter that drives expression in a specific cell type, such as photoreceptor cells (RHO, rhodopsin kinase (GRK1), cone arrestin (CAR)) or RPE cells (e.g., a promoter for an RPE-specific protein, such as VMD2, RPE65, RLBP1, RGR, or TIMP3). In some embodiments, the promoter can be CAG, CAGGS, CASI, CBh, GFAP, TRE3G, SMVP, Spc512, H1 / T0, CK7-miniCMV, pICAM2, HCRhAATp, or Tight promoter. Synthetic promoters ProC1 and ProD5 can also be used. In some embodiments, the promoter can be a hybrid promoter. For example, the promoter can be a CASI promoter, which includes a CMV enhancer and a CBA promoter. In some cases, the CASI promoter further includes a ubiquitin (UBC) enhancer.

[0032] In some embodiments, the non-coding or regulatory sequence may comprise an enhancer or a portion thereof, including, but not limited to, a CMV enhancer, a ubiquitin enhancer (e.g., a UBC enhancer), a CBA / CAG enhancer, a RHO enhancer, a rhodopsin kinase enhancer, an IRBP enhancer, a red opsin enhancer, a VMD2 enhancer, a CASI enhancer, and a cadherin enhancer. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11]

[0033] In some aspects, provided herein is a functional MERTK nucleic acid sequence comprising a nucleotide sequence at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any one of SEQ ID NOs: 3-6. In some embodiments, the functional nucleic acid sequence comprises a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:3. In some embodiments, a functional nucleic acid sequence comprises a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 4. In some embodiments, a functional nucleic acid sequence comprises a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 5. In some embodiments, a functional nucleic acid sequence comprises a nucleic acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 6.

[0034] The human MERTK protein may contain one or more mutations, for example, mutations in up to about 1%, up to about 2%, up to about 3%, up to about 4%, up to about 5%, up to about 10%, up to about 15%, up to about 20%, or more of the residues. Such variants can retain the activity of the wild-type protein. In some embodiments, the mutations are conservative substitutions. Such changes may include, but are not limited to, substitutions of any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids, substitutions of aspartic acid (D) for glutamic acid (E) and vice versa, substitutions of glutamine (Q) for asparagine (N) and vice versa, and substitutions of serine (S) for threonine (T) and vice versa. Other substitutions may also be considered conservative depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can often be interchangeable, as can alanine (A) and valine (V). Methionine (M) is relatively hydrophobic and can frequently be interchanged with leucine and isoleucine, and occasionally with valine. Lysine (K) and arginine (R) are often interchangeable at positions where the important feature of the amino acid residue is its charge and the difference in pK between these two amino acid residues is not significant.

[0035] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison purposes). In some cases, the length of a reference sequence that is aligned for comparison purposes is at least about 70%, at least about 75%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the length of the reference sequence.

[0036] The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0037] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated into the GAP program in the GCG software package (available on the World Wide Web at gcg.com) using default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0038] vector Described herein are targeted expression vectors for delivery, transfection, and expression of polynucleotides encoding the MERTK polypeptides described herein. In some embodiments, expression is targeted to retinal cells (e.g., inner retinal cells or retinal pigment epithelial (RPE) cells). In some embodiments, expression is targeted to photoreceptor cells (e.g., rod cells or cone cells). Such component expression constructs can be administered in any effective carrier, e.g., any formulation or composition that can effectively deliver the component genes to cells.

[0039] One approach for in vivo introduction of nucleic acids into cells is by using viral vectors containing nucleic acids, e.g., cDNA. Infecting cells with viral vectors has the advantage that a large proportion of target cells can receive the nucleic acid. Furthermore, molecules encoded within the viral vector, e.g., by cDNA contained in the viral vector, are efficiently expressed in cells that have taken up the viral vector nucleic acid.

[0040] Viral vectors can be used as recombinant gene delivery systems to introduce exogenous genes in vivo, especially into humans. These vectors provide efficient delivery of genes into cells, and in some cases, the introduced nucleic acid is stably integrated into the host's chromosomal DNA. Protocols for producing recombinant viruses and infecting cells with such viruses in vitro or in vivo can be found in Ausubel, et al., eds., Gene Therapy Protocols Volume 1: Production and In Vivo Applications of Gene Transfer Vectors, Humana Press, (2008), pp. 1-32, and other standard laboratory manuals.

[0041] Viral vectors may include, but are not limited to, recombinant retroviruses, adenoviruses, adeno-associated viruses (AAV), lentiviruses, herpes simplex virus type 1, alphaviruses, vaccinia viruses, or recombinant bacterial or eukaryotic plasmids. The vector may be a single-stranded or double-stranded vector. The vector may be self-complementary. The vector may be linear. Alternatively, the vector may be circularized. In some cases, the vector may be a plasmid. In some cases, the vector may be the pAAV.VMD2.hMERTK.bGH / SV40 vector of FIG. 1B. In some embodiments, the vector may comprise any one of the vectors of FIG. 3. In some embodiments, the vector may comprise, from the 5' to 3' end, a 5' ITR, a CMV enhancer, a CBA promoter, a codon-optimized MERTK-encoding nucleic acid sequence, a bovine growth hormone polyadenylation signal (bGHpA), and a 3' ITR. In some embodiments, the vector may comprise, from the 5' to the 3' end, a 5' ITR, a CMV enhancer, a CBA promoter, a wild-type MERTK-encoding nucleic acid sequence, a bovine growth hormone polyadenylation signal (bGHpA), and a 3' ITR. In some embodiments, the vector may comprise, from the 5' to the 3' end, a 5' ITR, a VMD2 promoter, a codon-optimized MERTK-encoding nucleic acid sequence, a bovine growth hormone polyadenylation signal (bGHpA), and a 3' ITR. In some embodiments, the vector may comprise, from the 5' to the 3' end, a 5' ITR, a VMD2 promoter, a wild-type MERTK-encoding nucleic acid sequence, a bovine growth hormone polyadenylation signal (bGHpA), and a 3' ITR. In some embodiments, the vector comprises a bGH polyA signal but does not comprise an SV40 polyA region.

[0042] Viral vectors may include adeno-associated viral vectors (AAV vectors). Adeno-associated viruses are naturally occurring defective viruses that require another virus, such as adenovirus or herpesvirus, as a helper virus for efficient replication and a productive life cycle. AAV vectors can efficiently transduce various cell types and result in long-term transgene expression in vivo. While the AAV vector genome can persist intracellularly as an episome, vector integration has been observed. AAV vectors, particularly AAV2, have been widely used for gene augmentation or replacement and have demonstrated therapeutic efficacy in various animal models and clinical settings. AAV vectors containing as little as 300 base pairs of AAV can be packaged and can express recombinant proteins. Space for exogenous DNA is limited to approximately 4.5 kb. AAV can be an AAV variant or serotype. The AAV serotype may be, but is not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV13. AAV variants may include genetically engineered AAV (e.g., AAV2.7m8 or AAV2tYF). The vector may include all of AAV. Alternatively, or in addition, the vector may include a portion of AAV (e.g., AAV capsid). The vector may include amino acids from one AAV serotype in another AAV serotype (e.g., AAV2 / 5). The AAV vector may be a recombinant AAV (rAAV) vector.

[0043] The vector may comprise an AAV capsid protein. The AAV capsid protein is a protein shell enclosure produced by AAV. The AAV capsid protein comprises a viral protein. The AAV capsid protein may comprise at least one, at least two, at least three, at least four, at least five, or more viral proteins. The AAV capsid may be, but is not limited to, an AAV1 capsid protein, an AAV2 capsid protein, an AAV2tYF capsid protein, an AAV2.7m8 capsid protein, an AAV3 capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV9 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, or an AAV13 capsid protein.

[0044] AAVs can be specialized for use in the ocular environment (e.g., AAV2, AAV4, AAV5, or AAV8). AAV vectors can be used to introduce DNA into the retina, for example, photoreceptors (e.g., rod cells or cone cells), inner retinal cells, or retinal pigment epithelial (RPE) cells.

[0045] Viral vectors can transfect cells directly, and plasmid DNA can be delivered naked or with the aid of, for example, cationic liposomes (lipofectamine) or derivatized (e.g., antibody conjugates), cationic dendrimers, inorganic vectors (e.g., iron oxide magnetofection), lipidoids, lipid nanoparticles (LNPs) (e.g., solid lipid nanoparticles, polymeric lipid nanoparticles), cell-penetrating peptides, pamam dendrimers, phosphorylcholines (e.g., methylphosphorylcholine), cyclodextrin polymers (CDPs), polylysine conjugates, gramacidin S, artificial viral envelopes, or other such intracellular carriers. Alternatively, plasmid DNA can be delivered by direct injection of the gene construct, electroporation, or the use of CaPO precipitation.

[0046] LNPs can be used to deliver AAV vectors. LNPs can contain cationic lipids, sterols, neutral lipids, and / or polyethylene glycol (PEG). The size of the LNPs can be at least about 5 nm, at least about 10 nm, at least about 15 nm, at least about 20 nm, at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, at least about 100 nm, at least about 110 nm, at least about 120 nm, at least about 130 nm, at least about 140 nm, at least about 150 nm, at least about 160 nm, at least about 170 nm, at least about 180 nm, at least about 190 nm, at least about 200 nm, at least about 250 nm, at least about 300 nm, or larger. The size of the LNP can be at most about 300 nm, at most about 250 nm, at most about 200 nm, at most about 190 nm, at most about 180 nm, at most about 170 nm, at most about 160 nm, at most about 150 nm, at most about 140 nm, at most about 130 nm, at most about 120 nm, at most about 110 nm, at most about 100 nm, at most about 90 nm, at most about 80 nm, at most about 70 nm, at most about 60 nm, at most about 50 nm, at most about 40 nm, at most about 30 nm, at most about 20 nm, at most about 15 nm, at most about 10 nm, at most about 5 nm, or less. In some cases, the AAV vector or LNP is in a liquid (e.g., an emulsion or solution). In some cases, the LNP is a solid LNP or a polymer LNP. Polymer LNPs can be, but are not limited to, polylactic-coglycolic acid (PLGA) nanoparticles or poly-β-amino-ester (PβAE) nanoparticles. In some cases, LNPs are sorted to obtain a population of LNPs of approximately the same size. Alternatively, LNPs can be of different sizes.

[0047] In some embodiments, the AAV vector can include one or more helper elements derived from a helper virus such as adenovirus (Ad), herpes simplex virus (HSV), or human papillomavirus (HPV). Optionally, the one or more helper elements include the E1A, E1B, VA, E2A DNA-binding protein (DBP), or E4 region from an adenovirus. Optionally, the AAV vector includes the E4 gene. Optionally, the AAV vector includes the E2 gene. Optionally, the AAV vector includes the VA gene. Optionally, the AAV vector includes two open reading frames. Optionally, the AAV vector includes at least one, at least two, at least three, at least four, at least five, at least six, or more open reading frames. Optionally, the AAV vector includes up to six, up to five, up to four, up to three, up to two, or up to one open reading frame.

[0048] In some cases, the AAV contains non-coding or regulatory sequences capable of regulating the expression of a gene of interest. The regulatory sequences can be derived from eukaryotic or non-eukaryotic genomes (e.g., viral or bacterial genomes). The non-coding or regulatory sequences can include a 5'-untranslated region (5'-UTR), a 3'-untranslated region (3'-UTR), a promoter, an enhancer, a polyadenylation (poly-A) sequence, or a cis-regulatory element. In some embodiments, the cis-regulatory element includes a silencer, an operator, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), or a minute virus of mice (MVM) intron. In some embodiments, the non-coding or regulatory sequence can include a portion of an exon or a portion of an intron. In some embodiments, the regulatory sequence includes a polyadenylation sequence (polyA). The polyA sequence can include a bovine growth hormone polyadenylation sequence (e.g., bGH polyA) or an SV40 polyA sequence. In some embodiments, the polyA sequence includes bGH polyA and SV40 polyA sequences. In some embodiments, the poly A sequence comprises a bGH poly A and does not comprise an SV40 poly A sequence. In some embodiments, the regulatory sequence comprises a Kozak sequence.

[0049] In some cases, the AAV contains a promoter sequence. In some cases, the promoter may include, but is not limited to, a cytomegalovirus (CMV) promoter, a hybrid CMV enhancer / chicken beta-action (CBA) promoter, a CAG promoter, a rhodopsin (RHO) promoter (e.g., a human rhodopsin (hRHO) promoter, a Rhol94 promoter), a CASI promoter, a rhodopsin kinase promoter (e.g., a GRK1 promoter), an interphotoreceptor retinoid-binding protein (IRBP) promoter, a red opsin promoter, a vitelloid macular dystrophy (VMD2) promoter, and a cadherin promoter (e.g., a CDH5 promoter or a CD144 promoter). The vector may contain a promoter that drives expression in many cell types (e.g., CAG, CMV, or CASI). Alternatively, the vector can contain a promoter that drives expression in a specific cell type, such as photoreceptor cells (RHO, rhodopsin kinase (GRK1), cone arrestin (CAR)) or RPE cells (e.g., a promoter for an RPE-specific protein such as VMD2, RPE65, RLBP1, RGR, or TIMP3). The synthetic promoters ProC1 and ProD5 can also be used.

[0050] In some cases, the AAV contains an inverted terminal repeat (ITR) sequence. The ITR sequence may be at the 3' end of the vector. Alternatively or additionally, the ITR sequence may be at the 5' end of the vector. The ITR may fold to create double-stranded DNA from single-stranded DNA.

[0051] In some cases, the AAV contains an enhancer, including, but not limited to, a CMV enhancer, a CBA / CAG enhancer, a ubiquitin enhancer, a RHO enhancer, a rhodopsin kinase enhancer, an IRBP enhancer, a red opsin enhancer, a VMD2 enhancer, a CASI enhancer, and a cadherin enhancer. Alternatively or additionally, the AAV may contain other types of cis-regulatory elements (e.g., a silencer, an operator, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), or a minute virus of mice (MVM) intron).

[0052] In some cases, the AAV contains an expression cassette. The expression cassette may comprise a recombinant DNA molecule containing the desired coding sequence of a gene of interest (e.g., MERTK) and appropriate nucleic acid sequences required for expression of the operably linked coding sequence in a particular host organism. The expression cassette may contain at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more genes. The expression cassette may contain up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 gene. In some embodiments, the vector provided herein comprises an AAV expression cassette containing a MERTK coding sequence.

[0053] In some embodiments, the gene encoding MERTK is encapsulated in a liposome (e.g., lipofectin) that has a positive charge on its surface, and the liposome can be tagged with an antibody against a cell surface antigen of target tissue.In some cases, the gene encoding MERTK is encapsulated in a liposome that carries a negative charge on its surface.In some cases, the gene encoding MERTK is encapsulated in a liposome that carries a zwitterionic charge on its surface.In some cases, the gene encoding MERTK is encapsulated in a liposome that does not carry an ionic moiety on its surface.

[0054] The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system (viral vector and any associated agents, e.g., helper virus, proteins, lipids, etc.) in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[0055] The vector may comprise a pharmaceutically acceptable carrier, which may be, but is not limited to, an excipient, adhesive, disintegrant, lubricant, stabilizer, diluent, injection solvent, fatty base, tactile modifier, surfactant, polymer, thickening gelling agent, solvent, propellant, antioxidant, reducing agent, oxidizing agent, chelating agent, acid, alkali, powder, inorganic salt, water, unsaturated monomer, polyalcohol, polymer additive, auxiliary agent, wetting agent, thickener, or viscosity-increasing material.

[0056] In some aspects, the present disclosure provides compositions comprising any one of the nucleic acids and / or nucleic acid sequences encoding human MERTK provided herein. The compositions may comprise any one of the functional MERTK nucleic acid sequences provided herein. In some embodiments, the compositions comprise a functional MERTK nucleic acid sequence that is at least 90%, 95%, or 99% identical to any one of SEQ ID NOs: 3-6. The compositions may further comprise a non-coding nucleic acid sequence and / or a regulatory nucleic acid sequence provided herein. In some embodiments, the compositions further comprise a promoter that expresses a product of the functional MERTK nucleic acid sequence in a plurality of photoreceptor cells or retinal pigment epithelial cells. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier.

[0057] In some aspects, the present disclosure provides a composition comprising any one of the vectors provided herein. In some embodiments, the composition comprises a recombinant AAV (rAAV) vector provided herein. In some embodiments, the composition comprises an rAAV selected from the group consisting of AAV2, AAV5, AAV8, AAV9, AAV2 / 5, AAV 2tYF, and AAV2.7m8. In some embodiments, the rAAV is rAAV2. In some embodiments, the rAAV is rAAV5. In some embodiments, the rAAV is rAAV4.

[0058] Treatment method MERTK may be essential for maintaining proper vision. The gene therapy and other methods disclosed herein can be used to correct ocular diseases and disorders. Ocular diseases and disorders include, but are not limited to, uveitis, glaucoma, macular edema, diabetic macular edema, retinopathy, age-related macular degeneration (e.g., wet AMD or dry AMD), scleritis, optic nerve degeneration, geographic atrophy, macular dystrophy, choroidal disease, ocular sarcoidosis, optic neuritis, choroidal neovascularization, ocular cancer, genetic diseases, autoimmune diseases affecting the posterior segment of the eye, retinitis (e.g., cytomegalovirus retinitis, retinitis pigmentosa), and corneal ulcers. In some embodiments, the ocular disease or disorder comprises a MERTK-mediated disease or disorder. In some embodiments, the MERTK-mediated disease or disorder comprises retinitis pigmentosa.

[0059] In some embodiments herein, gene therapy can treat retinitis pigmentosa. Retinitis pigmentosa (RP) is a group of rare eye diseases (e.g., inherited retinal dystrophies) that affect the retina (the light-sensitive layer of tissue at the back of the eye). RP slowly degrades retinal cells over time, causing vision loss and can be characterized by abnormalities in the photoreceptors (rods and / or cones) of the retinal pigment epithelium (RPE). Early symptoms of RP include loss of night vision and peripheral vision. Other symptoms of RP may include, but are not limited to, reduced visual field, night blindness, attenuation of retinal blood vessels, optic disc pallor, autofluorescent macula, macular atrophy, and bone spicule pigments. Retinitis pigmentosa is a progressive disease that can eventually cause partial or total blindness. In some embodiments herein, the retinitis pigmentosa is autosomal recessive retinitis pigmentosa. In some embodiments herein, the retinitis pigmentosa is autosomal dominant retinitis pigmentosa. In some embodiments herein, the retinitis pigmentosa is retinitis pigmentosa 38 (RP38). In some embodiments herein, the retinitis pigmentosa is rod-cone dystrophy.

[0060] The therapeutic method may include administering a MERTK gene (e.g., a wild-type gene or a codon-optimized gene) to a subject. The therapeutic method may include administering a composition provided herein to a subject. In some embodiments, the therapeutic method includes administering a nucleic acid sequence encoding a MERTK gene (e.g., a wild-type gene or a codon-optimized gene) to a subject. In some embodiments, the therapeutic method includes administering an rAAV comprising a MERTK gene. Administration may be topical (e.g., eye drops, ointment), oral (e.g., tablet, capsule, liquid), or by injection. Intraocular injection of a therapeutic agent may be intravitreal, subretinal, or suprachoroidal. In some embodiments, administering a MERTK gene to a subject occurs before the onset of an ocular disease or disorder. In some embodiments, administering a MERTK gene to a subject occurs after the onset of an ocular disease or disorder.

[0061] Gene therapy is at least about 10 7 , at least about 10 8 , at least about 10 9 , at least about 10 10 , at least about 10 11 , at least about 10 12 , or at least about 10 13 The gene therapy may be administered in amounts of up to about 10 viral particles / mL. 13 , up to about 10 12 , up to about 10 11 , up to about 10 10 , up to about 10 9 , up to about 10 8 , or up to about 10 7 The gene therapy may be administered in an amount of viral particles / mL. The gene therapy may be administered to one eye of the subject. Alternatively, the gene therapy may be administered to both eyes of the subject. In some embodiments, the administration of the MERTK gene is at least 10 9 In some embodiments, the administration of the MERTK gene is carried out in an amount of at least 10 viral particles / mL. 10 It is performed in viral particles / mL.

[0062] Administration of the therapy may improve vision loss. In some cases, administration of the therapy may at least partially restore the subject's vision. In some cases, administration of the therapy may completely restore the subject's vision. By administering the therapy, visual acuity loss may be improved by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more, as measured using a visual field test. With therapy, vision loss, as measured using visual field testing, can be up to about 95%, up to about 90%, up to about 85%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, up to about 50%, up to about 45%, up to about 40%, up to about 35%, up to about 30%, up to about 25%, up to about 20% , may be improved by up to about 19%, up to about 18%, up to about 17%, up to about 16%, up to about 15%, up to about 14%, up to about 13%, up to about 12%, up to about 11%, up to about 10%, up to about 9%, up to about 8%, up to about 7%, up to about 6%, up to about 5%, up to about 4%, up to about 3%, up to about 2%, up to about 1%, or less.

[0063] Therapy can improve the phagocytic activity of the retinal pigment epithelium (RPE). The ability of the RPE to ingest photoreceptor outer segments (POS) can be measured via a phagocytosis assay. For example, POS can be labeled with a fluorescent dye, and RPE phagocytosis of the labeled POS can be measured by fluorescence microscopy or flow cytometry. In some embodiments, the therapy increases the internalization of POS by the RPE. The therapy can increase the internalization of POS by the RPE by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, or at least about 190%, as measured using a phagocytosis assay. The increase may be at least about 190%, at least about 200%, at least about 200%, at least about 210%, at least about 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or more.

[0064] The subject may be an animal such as a monkey, ape (e.g., chimpanzee, gorilla), horse, cow, pig, sheep, goat, dog, cat, rabbit, guinea pig, gerbil, hamster, rat, mouse, camillid, bird, reptile, or amphibian. The subject may be a mammal. The subject may be a human. The subject may be a human with an eye disease or disorder. Alternatively, or in addition, the subject may be a human with a genetic susceptibility or genetic predisposition to an eye disease or disorder.

[0065] Another aspect provides a kit comprising a composition comprising a MERTK-encoding nucleic acid sequence provided herein. In some embodiments, the kit further comprises a second therapeutic agent. The second therapeutic agent can be a small molecule drug, a protein, a nucleic acid, a virus, or a combination thereof. In some embodiments, the kit further comprises a device for administering the composition provided herein, such as a tube suitable for ocular administration. In some embodiments, the kit further comprises instructions for use (IFU). The IFU can include instructions for administering the composition provided herein to the eye of a subject. [Example]

[0066] The following examples are presented for the purpose of illustrating various embodiments of the present disclosure and are not intended to limit the disclosure in any way. The examples, together with the methods described herein, represent preferred embodiments herein and are exemplary, not intended to limit the scope of the disclosure. Modifications thereof and other uses encompassed within the spirit of the disclosure as defined by the claims will occur to those skilled in the art.

[0067] Example 1: MERTK gene therapy in iPSC-RPE cells In this study, we designed and screened rAAV2-hMERTK constructs to verify their ability to restore MERTK expression and function in MERTK-based RP patient-derived iPSC-RPE cell lines. An exemplary gene construct is shown in Figure 1A, and an exemplary vector is shown in Figure 1B.

[0068] method AAV-hMERTK plasmids were designed with either a hybrid cytomegalovirus-chicken β-actin (CBA) promoter or an RPE-specific vitelloid macular dystrophy 2 (VMD2) promoter and two different MERTK sequences (native, wt, and codon-optimized, codop) (Figure 3). MERTK plasmids were generated on a pUC-kanamycin backbone containing 5' and 3' inverted terminal repeats (ITRs) and a bovine growth hormone polyadenylation signal (bGHpA) using either a variant of the selected promoter or gene of the selected sequence of interest. MERTK plasmid sequences can be found in Table 3.

[0069] MERTK expression was measured in multiple RPE cell models by digital PCR using the MERTKwt primer set listed in Table 4. Cell lines tested included ARPE19 cells (human spontaneous RPE cells), ARPE19 cells differentiated in MEM-nicotinamide medium for 1 and 2 weeks, JBWT11 p28 (induced pluripotent stem cells (iPSCs) (non-RPE cells)), iPSC-RPE cells (human iPSC-derived differentiated RPE cells), and 293T cells (human embryonic kidney cells (non-RPE cells)).

[0070] ARPE19, 293T, and iPSC-RPE (proband and parental) cells were transfected with four MERTK constructs: VMD2-hMERTKwt, VMD2-hMERTKcodop, CBA-hMERTKwt, and CBA-hMERTKcodop, and MERTK expression was quantified by digital PCR and Western blot (Figure 4). Cells were transfected using 500 ng of plasmid per 400,000 cells using Lipofectamine™ reagent. Mock (Lipofectamine + nuclease-free water) transfected cells served as a control. Cells were harvested for protein and RNA isolation at both 48 and 72 hours.

[0071] RPE cells were infected with a MERTK construct packaged in AAV2 serotype. MERTK expression (protein and RNA) and phagocytic activity were measured. The effect of MERTK mutations on global gene expression in iPSC-RPE cells (parental and proband) was also characterized by RNA-seq. Digital PCR was performed using the QIAcuity2-Plex system. MERTK primer sequences are listed in Table 4. For Western blots, SDS-PAGE was performed on 10% gels using whole cell lysates, and the blots were probed with MERTK antibody. Transfected ARPE19 cells were treated with FITC-POS, washed, trypsinized, and cell lysates were collected at 30, 60, and 120 minutes. The blots were probed with rhodopsin antibody to examine the time course of POS internalization. GAPDH was used as a loading control. For phagocytosis, transfected ARPE19 cells were incubated with bPOS-FITC (10 bPOS per cell) for 3 hours. Cells were washed, trypsinized, stained with DRAQ5, and then assayed by flow cytometry to determine the percentage of DRAQ5+ and FITC+ cells, which represent the percentage of cells in which POS was internalized.

[0072] The viability of transfected cells was measured. ARPE19 cells were treated with Cell Titer Glo 2.0 48 hours after transfection and luminescence was measured.

[0073] One-way analysis of variance with Dunnett's test was performed for multiple comparisons, and values ​​were considered statistically significant at p<0.05.

[0074] ARPE19 cells were transduced with AAV2-VMD2-MERTKwt and AAV2-VMD2-MERTKcodop at a multiplicity of infection (MOI) according to Table 2 for 48 hours. Cells were washed, trypsinized, and pelleted. RNA was isolated from the cells, followed by cDNA preparation. Digital PCR was performed (QIAcuity One Plex) with 10 ng of cDNA input per well. Primers specific for native MERTK (MERTKwt) were used to detect MERTK expression in cells transduced with AAV2-VMD2-MERTKwt, and primers specific for codon-optimized MERTK (MERTKcodop) were used to detect MERTKcodop expression in cells transduced with AAV2-VMD2-MERTKcodop. Primers (VIC fluorophore) for the housekeeping gene RPP30 (Taqman ID: Hs01124518_m1) were used to normalize MERTK expression in each reaction. Diluent was used as a control. The relative expression level of the gene of interest (GOI, MERTK) was calculated using the following formula and plotted as a function of MOI:

[0075] Relative GOI = (copies / ng-MERTK) / (copies / ng-RPP30) [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] Table 3-7 Table 3-8 Table 3-9 Table 3-10 Table 3-11 Table 3-12 Table 3-13 Table 3-14 Table 3-15 Table 3-16 Table 4

[0076] result ARPE19 cells expressed very low levels of MERTK (approximately 21 copies / ng cDNA). Transfection with CBA-hMERTKwt, VMD2-hMERTKwt, and VMD2-hMERTKcodop significantly upregulated MERTK expression compared with control-transfected cells (700-fold, 250-fold, and 3-fold, respectively). Consistent with the gene expression data, ARPE19 cells transfected with CBA-hMERTKwt, CBA-hMERTKcodop, and VMD2-hMERTKcodop plasmids showed significant upregulation of MERTK protein expression. 293T cells showed a 100-fold upregulation of MERTK expression as a result of transfection with CBA-hMERTKwt, but as expected, there was no significant induction with VMD2-hMERTKwt. See Figure 5.

[0077] The gene expression results showed that MERTK mRNA expression driven by the CBA promoter was relatively higher than that driven by the VMD2 promoter (Figure 5 and Table 5). Western blot results showed that MERTK protein expression driven by the CBA promoter was relatively higher than that driven by the VMD2 promoter (Figures 6A-6B). [Table 5]

[0078] The effect of MERTK overexpression was assessed by measuring cell viability of ARPE19 cells after transfection. Plasmid transfection with the MERTK constructs resulted in moderate cytotoxicity, which was not significantly different from control-treated cells (Figures 7A-7B). ARPE-19 cells were reverse transfected with each plasmid in duplicate and then incubated for 48 hours. After treating the cells with CellTiter Glo 2.0, cell death was calculated by subtracting the normalized RLU from 100 (100 - [RLU / control RLU] x 100) (Figure 7A). Cell density was visualized by phase-contrast microscopy (Figure 7B).

[0079] Because MERTK is an essential regulator of POS phagocytosis by RPE cells, we used a flow cytometry-based assay to measure phagocytosis of FITC-labeled POS by ARPE19 cells. Transfected ARPE-19 cells treated with bPOS-FITC were analyzed using flow cytometry to determine the percentage of FITC+ cells as a measure of MERTK-driven phagocytic activity. Treatment with all four constructs increased POS phagocytosis compared to baseline control-transfected ARPE19 cells (Figure 8). Transfection with all tested plasmids resulted in a significant upregulation of POS internalization compared to baseline. The CBA-MERTKopt plasmid showed the greatest upregulation of phagocytosis.

[0080] Furthermore, we assessed POS internalization by transfected ARPE19 cells using a Western blot-based assay measuring rhodopsin internalization. A time-dependent increase in rhodopsin internalization was observed in transfected ARPE19 cells at 60 and 120 minutes after incubation with bovine POS (Figure 9). All four constructs demonstrated upregulation of rhodopsin internalization in ARPE19 cells, suggesting a consequent functional enhancement of MERTK protein expression. Treatment with all four constructs increased POS phagocytosis by RPE cells compared to baseline control-transfected ARPE19 cells.

[0081] Rhodopsin internalization from ARPE-19 cell lysates was assayed using cells that were washed twice, trypsinized, and lysed after two washes. Transfected ARPE-19 cells were incubated with bPOS (bPOS(+)), whereas control cells were not incubated with bPOS (bPOS(-)). Five micrograms of protein was used for SDS-PAGE followed by immunoblotting with rhodopsin (approximately 36 kDa) and the loading control GAPDH (approximately 36 kDa). Total rhodopsin from ARPE-19 cell lysates was also assayed using cells that were washed twice and then directly lysed without trypsinization. Five micrograms of protein was used for SDS-PAGE followed by immunoblotting with rhodopsin (approximately 36 kDa) and the loading control GAPDH (approximately 36 kDa) (Figure 9).

[0082] MERTK expression was quantified in an RPE cell culture model, as depicted in Figure 10. ARPE19 cells were observed to have relatively lower baseline expression than iPSC-RPE cells.

[0083] Plasmid constructs driving expression of the codon-optimized MERTK sequence under the control of the CBA promoter significantly increased MERTK mRNA and protein expression compared with constructs with the VMD2 promoter. ARPE19 cells exhibited low baseline phagocytic activity, which was significantly upregulated by all four constructs. Furthermore, all four constructs showed increased internalization of bPOS compared with controls, as measured by rhodopsin immunoblotting.

[0084] Induction of MERTK expression was evaluated in ARPE19 cells 48 hours after transduction with AAV2-VMD2-MERTKwt and AAV2-VMD2-MERTKcodop. As shown in Figure 11, transduction with both AAVs induced the expression of their respective MERTK sequences (native and codop) in ARPE19 cells. We also provide in vitro proof-of-concept data supporting the efficacy of AAV vectors in inducing the expression of native and codop MERTK in ARPE19 cells.

[0085] These results showed that the rAAV2-hMERTK construct containing the CBA promoter and codon-optimized sequence induced higher expression than the VMD2 promoter and wild-type sequence, but showed equivalent rescue of POS internalization in ARPE19 cells. Example 2: Administration of MERTK gene therapy in mice

[0086] The plasmid constructs from Example 1 will be placed into recombinant AAV vectors and tested for their ability to induce RPE-specific transgene expression and rescue retinal degradation in MERTK+ mice.

[0087] Mice were bred and maintained in an animal care facility, where they were fed a 4% fat rodent diet and water ad libitum and housed on a 12-hour light / dark cycle. Tissue biopsies were prepared for polymerase chain reaction (PCR) using Allele-In-One Mouse Tail Direct Lysis Buffer according to the manufacturer's instructions. PCR was performed to amplify a region of MERTK. A 20 μL PCR reaction contained a final concentration of 200 μmol / L of each primer, 200 nmol / L of each dNTP (dATP, dGTP, dTTP, and dCTP), 2 mmol / L MgCl2, and 1 unit of Hot FirePol DNA polymerase. The thermocycling protocol was 95°C for 14 minutes; 30 cycles of 95°C for 45 seconds, 53°C for 45 seconds, and 72°C for 30 seconds; and 72°C for 7 minutes. The amplified products are subjected to Sanger sequencing and electropherograms are analyzed at c.25 to identify each mouse as wild-type, heterozygous, or homozygous for the MERTK mutation.

[0088] Preparation of DNA constructs and AAV vectors Codon-optimized human MERTK cDNA is synthesized into a gBlock gene fragment and incorporated into a construct, which is then packaged into a recombinant AAV viral vector.

[0089] Plasmids containing the complete constructs are generated using standard endotoxin-free molecular cloning techniques and verified by sequencing MERTK and the region across the ligation site.

[0090] AAV is prepared and the purified virus is collected in a final buffer containing 1x PBS, 35 mM NaCl, and 0.001% Pluronic F68 surfactant, and the purified virus is titered. If necessary, the virus is further diluted using the same buffer to achieve the target dose. To aid in the injection procedure, less than 0.25% fluorescein (AK-Fluor, Akorn, Lake Forest, IL) is mixed into the working solution as a tracer.

[0091] For general anesthesia, a mixture of ketamine and xylazine is delivered intraperitoneally. Two-week-old mice receive a dose of 37.5 mg / kg ketamine and 3.8 mg / kg xylazine, while adult mice receive a dose of 100 mg / kg ketamine and 20 mg / kg xylazine. To prevent the formation of permanent anesthesia-induced corneal opacity, a 2 mg / kg dose of Yohimbine HCl is administered subcutaneously immediately after each recovery procedure using ketamine / xylazine (i.e., AAV injection, in vivo imaging, ERG). For neonatal mice, general anesthesia via hypothermia was induced by indirect exposure to ice.

[0092] In 2-week-old mice, a Micro4 microinjection pump with an RPE kit was used to deliver viral reagents into either the subretinal space or the vitreous. Pupils were dilated using either tropicamide (1%) or a 1 / 2 mixture of tropicamide (0.25%), phenylephrine hydrochloride (0.25%), and cyclpentalate (1%). Mice were deeply anesthetized with ketamine / xylazine, and topical anesthesia was administered using proparacaine hydrochloride (0.5%). The eye was then proptotically punctured using a 30 g needle through the superior temporal sclera and retina, just after the episcleral vessels at the limbus, to create an access point for a blunt-tipped 33 g cannula.

[0093] For subretinal injection, the cannula's longitudinal passage through the vitreous cavity is visualized through the dilated pupil via a dissecting microscope, and the tip of the cannula is positioned in the subretinal space posterior to the inferior nasal quadrant of the eye. Four consecutive 185.5 nL reagent boluses (total 0.75 μL) are injected, and bleb formation is confirmed by visualization enhanced with fluorescein tracer. To avoid reagent backflow, the cannula is held in place for approximately 3 seconds after injection and then gently removed from the eye. Finally, the puncture wound is tamponaded with a cotton swab. The eye is then moistened with artificial tears, and the mouse is allowed to recover from anesthesia on a heating pad.

[0094] The procedure for intravitreal injection in 2-week-old mice was the same except that the tip of the cannula was placed in the center of the vitreous cavity during injection.

[0095] Subretinal injections are performed on neonates using a FemtoJet 4i microinjection system (Eppendorf, Hamburg, Germany). While the mouse is anesthetized on ice, the tip of a 30 g hypodermic needle is used to separate the upper and lower eyelids. The eye is protruded, and a custom-made beveled glass needle (catalog no. C060609, Orgio, Trumbull, CT) is inserted directly through the sclera and placed into the underlying subretinal space. A single bolus of 0.5 μL of reagent is administered at a pressure of 330 hPa for 6 seconds, after which the needle is held in place for approximately 3 seconds to avoid backflow and then gently removed. The mouse is allowed to recover from anesthesia on a heating pad.

[0096] En face and cross-sectional images of the retina are acquired using fundus photography and spectral domain optical coherence tomography (OCT). Photoreceptor layer thickness is measured using InVivoVue OCT software, taking four caliper measurements at approximately equal intervals from the outer plexiform layer to the retinal pigment epithelium.

[0097] Full-field flash electroretinograms are collected from mice. Briefly, mice are dark-adapted overnight, and rod and mixed rod / cone responses are generated using broadband light stimuli of 0.01 cd.s / m² (scotopic) and 10 cd.s / m² (scotopic), respectively. Mice are then light-adapted by a 10-minute exposure to a steady broadband light of 30 cd / m² (phototopic), which is left on in the background while cone-segregated responses to a 20 cd.s / m² (phototopic) broadband light stimulus are acquired.

[0098] Statistical analysis is completed in Prism. For the time courses of OCT and electroretinogram (ERG), a two-way analysis of variance using a mixed-effects regression model is performed. When analyzing the effect of treatment, the inferior retina of the uninjected eye is used as a negative control, and the means of all other measurements are compared to this negative control. Dunnett's post-hoc test is used to account for Type I errors generated from multiple comparison tests.

[0099] Additional data will be collected from RPE-choroid flat mounts (showing tight junctions, morphology, and subretinal immune cells), retinal cross sections (showing MERTK / GFP expression), and tunneling electron microscopy (TEM) (showing RPE morphology, cellular vacuoles, outer segment rosettes, and sub-RPE deposits).

[0100] Example 3: Administration of MERTK gene therapy in humans In this example, a codon-optimized MERTK sequence is injected into the eye to treat retinitis pigmentosa in a human subject.

[0101] Generate adeno-associated virus proviral plasmids containing codon-optimized MERTK cDNA (e.g., SEQ ID NOs: 3-6). In these proviral plasmids, the hMERTK cDNA is driven by the VMD2 promoter. For example, a dose of the AAV2-VMD2-hMERTK codop vector (e.g., SEQ ID NO: 9) can be administered to the eye of a human subject to treat retinitis pigmentosa.

[0102] The rAAV plasmids are stored at 10 10 Viral particles are administered via subretinal injection. Expression of codon-optimized hMERTK in transduced cells or the retina is assessed by retinal and visual function.

[0103] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be used in practicing the present disclosure. It is intended that the following claims define the scope of the disclosure, and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method of treating a subject with an eye disease or disorder, comprising administering to the subject a nucleic acid comprising a nucleotide sequence that is at least 90% identical to any one of SEQ ID NOs: 3-6.

2. 2. The method of claim 1, wherein the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

3.

3. 3. The method of claim 1 or 2, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

3.

4. The method of any one of claims 1 to 3, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:

3.

5. 2. The method of claim 1, wherein the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

6. The method of claim 1 or 5, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

4.

7. The method of any one of claims 1 or 5-6, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:

4.

8. 2. The method of claim 1, wherein the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

5.

9. 10. The method of claim 1 or 8, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

5.

10. The method of any one of claims 1 or 8-9, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:

5.

11. 2. The method of claim 1, wherein the nucleic acid comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

6.

12. 12. The method of claim 1 or 11, wherein the nucleic acid comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

6.

13. The method of any one of claims 1 or 11-12, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID NO:

6.

14. The method of any one of claims 1 to 13, wherein the nucleotide sequence is contained in a vector.

15. 15. The method of claim 14, wherein the vector is an adeno-associated virus (AAV) vector.

16. 16. The method of claim 15, wherein the AAV vector is a recombinant AAV (rAAV) vector.

17. 17. The method of claim 16, wherein the rAAV vector is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, AAV2 / 5, AAV2tYF, and AAV2.7m8.

18. The method of any one of claims 14 to 17, wherein the vector comprises an AAV capsid protein.

19. 19. The method of claim 18, wherein the AAV capsid protein is selected from the group consisting of AAV2 capsid protein, AAV2tYF capsid protein, AAV5 capsid protein, AAV8 capsid protein, AAV9 capsid protein, and AAV2.7m8 capsid protein.

20. 20. The method of any one of claims 14 to 19, wherein the vector further comprises 1) a 5' AAV ITR, and 2) a 3' AAV ITR.

21. The method of any one of claims 14 to 20, wherein the vector comprises an AAV expression cassette.

22. The method of any one of claims 14 to 21, wherein the vector further comprises a rhodopsin (RHO) promoter.

23. The method of any one of claims 14 to 22, wherein the vector further comprises a vitelloid macular dystrophy (VMD2) promoter.

24. The method of any one of claims 14 to 23, wherein the vector further comprises a human rhodopsin kinase (hGRK1) promoter.

25. The method of any one of claims 14 to 24, wherein the vector further comprises a CBA promoter or a CASI promoter.

26. The method of any one of claims 1 to 25, wherein the disease or disorder comprises a MERTK-mediated disease or disorder.

27. 27. The method of claim 26, wherein the MERTK-mediated disease comprises retinitis pigmentosa.

28. The method of any one of claims 1 to 27, wherein the disease or disorder comprises vision loss.

29. The method of any one of claims 1 to 28, wherein the administration occurs before the onset of the disease or disorder.

30. 30. The method of any one of claims 1 to 29, wherein the administration occurs after onset of the disease or disorder.

31. The method of any one of claims 1 to 30, wherein the administration is to at least one eye of the subject.

32. 32. The method of any one of claims 1 to 31, wherein the administration is performed by subretinal, intravitreal, or suprachoroidal injection.

33. The administration is at least 10 ９ 33. The method of any one of claims 1 to 32, carried out in an amount of virus particles / mL.

34. 34. The method of any one of claims 1 to 33, wherein said administering restores at least partial vision to said subject.

35. 35. The method of any one of claims 1-34, wherein said administration results in at least a 10% improvement in visual acuity loss as measured using visual field testing.

36. The method of any one of claims 1 to 35, wherein the subject is a mammal.

37. The method of any one of claims 1 to 36, wherein the subject is a human.

38. A functional MERTK nucleic acid sequence comprising a nucleic acid sequence at least 90%, 95%, or 99% identical to any one of SEQ ID NOs: 3-6.

39. 39. A composition comprising the functional MERTK nucleic acid sequence of claim 38.

40. 40. The composition of claim 39, further comprising a promoter that expresses the product of the functional MERTK nucleic acid sequence in a plurality of photoreceptor cells or retinal pigment epithelial cells.

41. 41. The composition of any one of claims 39 or 40, further comprising a pharmaceutically acceptable carrier.

42. The composition of any one of claims 39 to 41, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

3.

43. 43. The composition of any one of claims 39 to 42, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

3.

44. The composition of any one of claims 39 to 43, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:

3.

45. The composition of any one of claims 39 to 41, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

4.

46. 46. ​​The composition of any one of claims 39 to 41 or 45, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

4.

47. 47. The composition of any one of claims 39-41 or 45-46, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:

4.

48. The composition of any one of claims 39 to 41, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

5.

49. 49. The composition of any one of claims 39 to 41 or 48, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

5.

50. 50. The composition of any one of claims 39-41 or 48-49, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:

5.

51. The composition of any one of claims 39 to 41, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

6.

52. 52. The composition of any one of claims 39 to 41 or 51, wherein the nucleic acid sequence comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO:

6.

53. 53. The composition of any one of claims 39 to 41 or 51 to 52, wherein the nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO:

6.

54. The composition of any one of claims 39 to 53, wherein the nucleic acid sequence is contained in a vector.

55. 55. The composition of claim 54, wherein the vector is an adeno-associated virus (AAV) vector.

56. 56. The composition of claim 55, wherein the AAV vector is a recombinant AAV (rAAV) vector.

57. 57. The composition of claim 56, wherein the rAAV vector is selected from the group consisting of AAV2, AAV5, AAV8, AAV9, AAV2 / 5, AAV2tYF, and AAV2.7m8.

58. 58. The composition of any one of claims 54 to 57, wherein the vector comprises an AAV capsid protein.

59. 59. The composition of claim 58, wherein the AAV capsid protein is selected from the group consisting of AAV2 capsid protein, AAV2tYF capsid protein, AAV5 capsid protein, AAV8 capsid protein, AAV9 capsid protein, and AAV2.7m8 capsid protein.

60. 60. The composition of any one of claims 54 to 59, wherein the vector further comprises 1) a 5' AAV ITR, and 2) a 3' AAV ITR.

61. 61. The composition of any one of claims 54 to 60, wherein the vector comprises an AAV expression cassette.

62. 62. The composition of any one of claims 54 to 61, wherein the vector further comprises a rhodopsin (RHO) promoter.

63. 63. The composition of any one of claims 54 to 62, wherein the vector further comprises a vitelloid macular dystrophy (VMD2) promoter.

64. The composition of any one of claims 54 to 63, wherein the vector further comprises a human rhodopsin kinase (hGRK1) promoter.

65. 65. The composition of any one of claims 54 to 64, wherein the vector further comprises a CBA or CASI promoter.

66. 66. The composition of any one of claims 42 to 65, for use in treating a disease or disorder.

67. 67. The composition of claim 66, wherein the disease or disorder comprises a MERTK-mediated disease or disorder.

68. 68. The composition of claim 67, wherein the MERTK-mediated disease comprises retinitis pigmentosa.

69. 69. The composition of any one of claims 66 to 68, wherein the disease or disorder comprises vision loss.

70. 70. The composition of any one of claims 66 to 69, which is administered to a subject before the onset of the disease or disorder.

71. 71. The composition of any one of claims 66 to 70, administered to a subject after the onset of the disease or disorder.

72. 72. The composition of any one of claims 39 to 71, administered to at least one eye of a subject.

73. 73. The composition of any one of claims 39 to 72, which is administered to a subject by subretinal, intravitreal, or suprachoroidal injection.

74. At least about 10 ９ 74. The composition of any one of claims 39 to 73, comprising virus particles / mL.

75. 75. The composition of any one of claims 39 to 74, which, when administered to a subject, restores at least partial vision to said subject.

76. 76. The composition of any one of claims 39 to 75, which, when administered to a subject, improves the subject's vision loss by at least 10% as measured using a visual field test.

77. Use of a functional MERTK nucleic acid sequence according to claim 38 or a composition according to any one of claims 39 to 76 for the manufacture of a medicament.

78. 77. A kit comprising a functional MERTK nucleic acid sequence according to claim 38 or a composition according to any one of claims 39 to 76, and optionally instructions for use.

79. 38. The method of any one of claims 1 to 37, wherein said administration increases internalization of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) as measured using a phagocytosis assay.

80. 80. The method of claim 79, wherein said administration increases internalization of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) by at least 50%, as measured using a phagocytosis assay.

81. 77. The composition of any one of claims 39 to 76, which, when administered to a subject, increases internalization of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) as measured using a phagocytosis assay.

82. 82. The composition of claim 81, which, when administered to a subject, increases internalization of photoreceptor outer segments (POS) by the retinal pigment epithelium (RPE) by at least 50% as measured using a phagocytosis assay.

Images