Compositions and methods for gene therapy

Abstract

The present disclosure provides pharmaceutical compositions containing nucleic acid molecules encoding PEDF which are codon-optimized, as well as methods for increasing the level of PEDF in the treatment or prophylaxis of retinal degenerative diseases, such as age-related macular degeneration (AMD), or other neurodegenerative diseases.

Description

[0001] 01001 / 012745-WQ0

[0002] COMPOSITIONS AND METHODS FOR GENE THERAPY

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority to U.S. Provisional Patent Application No. 63 / 704,796 (filed on October 8, 2024), which is incorporated herein by reference in its entirety.

[0005] FIELD OF THE INVENTION

[0006] The present disclosure relates to compositions and methods for treating age-related diseases including neurodegenerative disorders.

[0007] BACKGROUND OF THE DISCLOSURE

[0008] Age-related macular degeneration (AMD) is a disease that causes vision impairment and is expected to impact nearly 300 million people by 2040 (Vyawahare et al., Age-related macular degeneration: epidemiology, pathophysiology, diagnosis, and treatment, Cureus. 2022; 14(9): e295383). There are 2 forms of AMD: dry and wet or neovascular.

[0009] Dry AMD is characterized by the build-up of cell debris and lipids between the basement membrane of the retinal pigment epithelium (RPE) and Bruch’s membrane (Bakeri et al., Geographic atrophy: mechanism of disease, pathophysiology, and role of the complement system, J. Manag. Care Spec. Pharm., 2023; 25 (5-aSuppl)). Extensive build-up of subretinal debris can cause chronic inflammation of the retina, which may result in irreversible death of RPE and photoreceptor cells. Geographic atrophy (GA) occurs in advanced cases of dry AMD where progressive degeneration of retina can lead to blindness. Color fundus photography (CFP) and optical coherence tomography (OCT) imaging modalities can be used to examine and diagnose GA. There is currently no treatment for dry AMD. Syfovre, also known as pegcetacoplan, is a peptide therapeutic that targets the overactivation of the complement pathway and was approved by the FDA in 2023 to treat GA.

[0010] Wet / neovascular AMD results in acute vision loss from leakage of overgrown blood vessels into the macula of the eye. Several disease-modifying therapies exist for wet AMD where anti-angiogenic drugs targeting vascular endothelial growth factor (VEGF) have been developed. Despite the availability of anti- VEGF therapeutics for wet AMD, the need for recurrent intravitreal injections poses a significant burden to patients, resulting in reduced treatment 01001 / 012745-WQ0 adherence and loss of vision over time.

[0011] Gene therapy offers the potential of a cure through continuous endogenous production of therapeutic proteins following a single administration of vectors. Adeno-associated virus (AAV) is a small, replication-defective, non-enveloped animal virus that infects humans and some other primate species. Several features of AAV make this virus an attractive vehicle for delivery of therapeutic proteins by gene therapy, including, for example, that AAV is not known to cause human disease and induces a mild immune response, and that AAV vectors can infect both dividing and quiescent cells without integrating into the host cell genome. Gene therapies are under investigation for wet AMD, as one-time injection would reduce the burden of recurrent intravitreal injections of anti-VEGF drugs (Vyawahare et al., Age-related macular degeneration: epidemiology, pathophysiology, diagnosis, and treatment, Cureus. 2022; 14(9): e295383).

[0012] Pigment epithelium-derived factor (PEDF), known to be depleted in the retina of individuals with AMD, is a glycoprotein encoded by the SERPINF1 gene (Wang et al., Pigment epithelium-derived growth factor and its role in microvascular-related diseases, Biochemie. 2022; 200: pp. 153-171). PEDF is a secreted factor that has known neurotrophic and anti- angiogenic / anti-VEGF properties.

[0013] Therapies to augment levels of PEDF have been explored in rodent models of retinal degeneration and various cancers. PEDF therapies have also been explored in clinical trials to treat wet AMD (Wang et al., Pigment epithelium-derived growth factor and its role in microvascular-related diseases, Biochemie. 2022; 200: pp. 153-171). PEDF levels decline in many tissues with aging, and PEDF has appreciable roles in the inhibition of cellular senescence (Abooshahab et al., The biological relevance of pigment epithelium-derived factor on the path from aging to age-related disease, Meeh. Ageing Dev. 2021; 196: 111478). As such, the present compositions and methods can provide needed therapeutic avenues for individuals with AMD and have broader regenerative utility in other retinal conditions or age-related diseases.

[0014] SUMMARY

[0015] The present disclosure provides for a nucleic acid molecule encoding human pigment epithelium-derived factor (PEDF). The nucleic acid molecule may comprise nucleotides 31-42 and 79-90 of the wildtype PEDF coding sequence which encode a stem-loop hairpin in a messenger RNA (mRNA) transcript of the nucleic acid molecule, where nucleotides 31-42 and 01001 / 012745-WQ0

[0016] 79-90 of the wildtype PEDF coding sequence have the nucleotide sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively. The nucleic acid molecule may be human codon optimized. The nucleic acid molecule may be no greater than 95%, no greater than 90%, or no greater than 85%, identical to the wildtype PEDF coding sequence set forth in SEQ ID NO:1.

[0017] The present nucleic acid molecule may comprise a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:5. The nucleic acid molecule may comprise a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0018] The present nucleic acid molecule may encode a wildtype PEDF protein comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0019] The present nucleic acid molecule may further comprise AAV 5’ and 3’ inverted terminal repeat (ITR) sequences. In certain embodiments, the nucleic acid molecule may further comprise AAV2 5’ and 3’ inverted terminal repeat (ITR) sequences.

[0020] The present nucleic acid molecule may further comprise a promoter element. In certain embodiments, the promoter element comprises a chicken beta-actin (CB) promoter. In certain embodiments, the promoter element comprises a cytomegalovirus (CMV) early promoter sequence, a chicken beta-actin (CB) promoter sequence, a truncated chicken beta-actin intron sequence, and a minute virus of mouse (MVM) intron sequence. In certain embodiments, the promoter element comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:6.

[0021] The present nucleic acid molecule may further comprise a post-transcriptional regulatory element. In certain embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In certain embodiments, the post-transcriptional regulatory element comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:8.

[0022] The nucleic acid molecule may further comprise a polyadenylation signal sequence. In certain embodiments, the polyadenylation signal sequence is a bovine growth hormone polyadenylation signal sequence. In certain embodiments, the polyadenylation signal sequence comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:9.

[0023] The nucleic acid molecule may comprise (or may be contained in) a recombinant adeno- 01001 / 012745-WQ0 associated virus (AAV) vector, such as AAV9.

[0024] The nucleic acid molecule may comprise a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 12.

[0025] The present disclosure provides for a vector comprising the present nucleic acid molecule.

[0026] Also encompassed by the present disclosure is a cell comprising the present nucleic acid molecule, or a cell comprising the vector. Further encompassed by the present disclosure is a cell expressing the present nucleic acid molecule, or a cell expressing the vector.

[0027] The present disclosure provides for a pharmaceutical composition comprising the nucleic acid molecule, the vector, or the cell.

[0028] The present disclosure also provides for a method of treating a disorder in a subject. The method may comprise administering to the subject the present pharmaceutical composition, the nucleic acid molecule, the vector, or the cell.

[0029] The disorder may be an ocular degenerative disease or a retinal degenerative disease. The ocular’ degenerative disease may be age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), or glaucoma.

[0030] The disorder may be a neurodegenerative disease. The neurodegenerative disease may be amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, Batten disease, or a prion disease.

[0031] The disorder may be cancer.

[0032] The disorder may be a cardiovascular disease.

[0033] The pharmaceutical composition may be administered by intravitreal injection or subretinal injection. The pharmaceutical composition may be administered to the central nervous system (CNS) of the subject. The pharmaceutical composition may be administered to the spinal cord of the subject. The pharmaceutical composition may be administered by intrathecal injection. The pharmaceutical composition may be administered orally, intravenously, intramuscularly, topically, arterially, or subcutaneously.

[0034] The subject may be a mammal, such as a human, a rodent, or a simian. In certain embodiments, the mammal is a human. 01001 / 012745-WQ0

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 is a schematic depicting several embodiments of AAV vectors that include an expression construct encoding SERPINF1 (PEDF). Constructs contain either the wild-type PEDF open reading frame (ORF), or a codon-optimized version of PEDF ORF that either preserves (CO2, CO4) or does not preserve (CO1, CO3) the stem- loop hairpin sequences from nucleotides 31-42 and 79-90 of the wildtype PEDF coding nucleotide sequence. All constructs contain AAV2 ITRs, a CBh promoter element, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), and a bovine growth hormone (bGH) polyA signal.

[0037] Figure 2 shows ELISA analyses of PEDF concentrations from supernatants of ARPE-19 retinal pigment epithelium cultures transfected with plasmids containing the constructs listed in Figure 1. Means are presented. Error bars refer to standard error of the mean (SEM).

[0038] Figure 3 shows ELISA analyses of PEDF concentrations from whole mouse retinas 1 week after intravitreal injection of either AAV9-EGFP control, AAV9-PEDF (wildtype or WT), AAV9-CO2, or AAV9-CO4. Means are presented. Error bars are SEM. *p<0.05; **p<0.01, by two-tailed t-test with Welch’s correction.

[0039] Figure 4 shows representative data of optical coherence tomography (OCT) images of mouse retina. Shown are untreated control, sodium iodate (Sl)-treated (35 mg / kg I.P. injection), and SI treated animals that were also injected intravitreally with AAV9-CO4 1 week prior to SI treatment (“SI + CO4”). OCT images were captured 4 weeks after SI treatment. Means are presented. Error bars are SEM. ****p<0.0001, by two-tailed t-test with Welch’s correction.

[0040] 01001 / 012745-WQ0

[0041] DETAILED DESCRIPTION

[0042] The present disclosure provides for pharmaceutical compositions containing nucleic acid molecules or vectors encoding pigment epithelium-derived factor (PEDF) which are codon- optimized, as well as gene therapies for increasing the level of PEDF in the treatment or prophylaxis of retinal degenerative diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), and glaucoma, or other neurodegenerative diseases.

[0043] The present disclosure provides for a nucleic acid molecule encoding human PEDF where the nucleic acid molecule is human codon-optimized. Also provided is a vector comprising the nucleic acid molecule.

[0044] The present method of treating a disorder (e.g., a neurodegenerative disease) in a subject may comprise administering to the subject an effective amount of a nucleic acid molecule encoding PEDF (or the vector comprising the nucleic acid molecule).

[0045] The nucleic acid molecule encoding PEDF may comprise (or may be contained in) a viral vector, e.g., a recombinant adeno-associated vims (AAV) vector. The AAV vector may be replication deficient.

[0046] Also provided herein is a nucleic acid molecule comprising a codon-optimized coding sequence of PEDF. The codon-optimized coding sequence of PEDF may be used in such vectors as recombinant adeno-associated virus (AAV) vectors to achieve long term expression of PEDF. In one embodiment, the AAV vector also includes one or more of the following: an enhancer; inverted terminal repeat (ITR) sequences; a promoter; a post- transcriptional regulatory element; an intron; and a polyadenylation (polyA) signal.

[0047] In one embodiment, the PEDF transgene is contained in an AAV vector which comprises (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) a promoter element; (c) a codon- optimized PEDF coding sequence, or a wildtype PEDF coding sequence, or a variant of the wildtype PEDF coding sequence; (d) a regulatory element; (e) a polyA signal; and (d) an AAV 3' ITR.

[0048] The present disclosure provides for a nucleic acid molecule encoding human pigment epithelium-derived factor (PEDF). The nucleic acid molecule comprises nucleotides 31-42 (set forth in SEQ ID NO: 13) and nucleotide 79-90 (set forth in SEQ ID NO: 14) of the wildtype 01001 / 012745-WQ0

[0049] PEDF coding sequence (set forth in SEQ ID NO:1). Nucleotides 31 -42 and 79-90 of the wildtype PEDF coding sequence encode a stem-loop hairpin in a messenger RNA (mRNA) transcript of the nucleic acid molecule.

[0050] The nucleic acid molecule may be human codon-optimized, where the nucleic acid molecule is no greater than 95%, no greater than 94%, no greater than 93%, no greater than 92%, no greater than 91%, no greater than 90%, no greater than 89%, no greater than 88%, no greater than 87%, no greater than 86%, no greater than 85%, no greater than 84%, no greater than 83%, no greater than 82%, no greater than 81%, or no greater than 80%, identical to the wildtype PEDF coding sequence set forth in SEQ ID NO:1.

[0051] In certain embodiments, the codon-optimized PEDF coding sequence comprises (or consists essentially of, or consists of) a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the nucleotide sequence set forth in SEQ ID NO:5.

[0052] In certain embodiments, the codon-optimized PEDF coding sequence comprises (or consists essentially of, or consists of) a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the nucleotide sequence set forth in SEQ ID NO:3.

[0053] In certain embodiments, the codon-optimized PEDF coding sequence comprises (or consists essentially of, or consists of) a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at 01001 / 012745-WQ0 least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the nucleotide sequence set forth in SEQ ID NO:2.

[0054] In certain embodiments, the codon-optimized PEDF coding sequence comprises (or consists essentially of, or consists of) a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 4.

[0055] In another aspect, provided herein is an isolated nucleic acid molecule comprising (or consisting essentially of, or consisting of a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO: 5, SEQ ID NO: 3, SEQ ID NO: 2, or SEQ ID NO: 4.

[0056] In certain embodiments, the AAV 5' ITR and / or AAV 3' ITR may be from a heterologous AAV pseudotype. The ITR sequences may be derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In a specific embodiment, the ITR sequences are derived from AAV2.

[0057] The promoter element may be a synthetic promoter sequence. In one embodiment, the promoter element is a CBh promoter, comprising a cytomegalovirus (CMV) early promoter sequence, a chicken beta-actin (CB) promoter sequence, and a truncated chicken P-actin intron 01001 / 012745-WQ0 and minute virus of mouse (MVM) intron sequences.

[0058] In certain embodiments, the promoter clement comprises (or consists essentially of, or consists of) a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the nucleotide sequence set forth in SEQ ID NO:6.

[0059] The regulatory element may be a post-transcriptional regulatory element. The post- transcriptional regulatory element may be woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

[0060] In certain embodiments, the regulatory element comprises (or consists essentially of, or consists of) a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the nucleotide sequence set forth in SEQ ID NO:8.

[0061] The nucleic acid molecule may further comprise a polyadenylation (polyA) signal sequence. The polyadenylation signal sequence may be a bovine growth hormone (bGH) polyadenylation signal.

[0062] In certain embodiments, the poly adenylation signal sequence comprises (or consists essentially of, or consists of) a nucleotide sequence at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 95%, at least or about 99%, at least or about 81%, at least or about 82%, at least or about 83%, at least or about 84%, at least or about 85%, at least or about 86%, at least or about 87%, at least or about 88%, at least or about 89%, at least or about 90%, at least or about 91%, at least or about 92%, at 01001 / 012745-WQ0 least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or about 100% identical to the nucleotide sequence set forth in SEQ ID NO:9.

[0063] The nucleic acid molecule may comprise an intron. In certain embodiments, the intron is a composite truncated chicken -actin intron and minute virus of mouse (MVM) intron sequences. In certain embodiments, the intron is a composite globin / AlAT intron sequence.

[0064] The present nucleic acid molecule encodes a functional PEDF. In one embodiment, the functional PEDF is human PEDF. In one embodiment, the coding sequence for PEDF is codon- optimized for expression in humans. Such codon-optimized sequence may share no greater than 99%, no greater than 98%, no greater than 97%, no greater than 96%, no greater than 95%, no greater than 94%, no greater than 93%, no greater than 92%, no greater than 91%, no greater than 90%, no greater than 89%, no greater than 88%, no greater than 87%, no greater than 86%, no greater than 85%, no greater than 84%, no greater than 83%, no greater than 82%, no greater than 81%, or no greater than 80%, identity to the wildtype / native hPEDF coding sequence (SEQ ID NO: 1).

[0065] In certain embodiments, the codon optimized PEDF nucleic acid molecule has a G / C content of less than 65%, less than 60%, or less than 58%.

[0066] The present disclosure provides for a vector comprising the present nucleic acid molecule. Such a vector may be a viral vector or a non-viral vector. The vector may be an adeno- associated viral (AAV) vector.

[0067] In aspects where gene transfer is mediated by a DNA viral vector, such as an adenovirus or adeno-associated virus (AAV), a vector may refer to the polynucleotide comprising the viral genome or part thereof, and a transgene.

[0068] The present disclosure provides for a cell or host comprising (or expressing) the nucleic acid molecule or vector. The present disclosure also provides for a pharmaceutical composition comprising the nucleic acid molecule or vector, or a pharmaceutical composition comprising the cell or host.

[0069] In another aspect, provided herein is a pharmaceutical composition comprising (or consisting essentially of, or consisting of) the present nucleic acid molecule, the present vector (e.g., an AAV vector), or the present AAV particle. In another aspect, provided herein is an 01001 / 012745-WQ0 immunogenic composition comprising the present nucleic acid molecule, the present vector, or the present AAV particle. In another aspect, provided here is a vaccine comprising the present nucleic acid molecule, the present vector, or the present AAV particle.

[0070] The present disclosure provides for a method for treating or preventing a disorder in a subject, the method comprising administering to the subject an effective amount of the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition. The effective amount may be a therapeutically effective amount. As used herein, the term “effective amount” refers to an amount sufficient to effect beneficial or desirable biological and / or clinical results. A “therapeutically effective amount” may be an amount of the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition that, after administration, results in the expression of functional PEDF in a level sufficient to at least partially or fully ameliorate the symptoms of the disorder. The terms “therapeutically effective amount”, “therapeutically effective dose” may refer to an amount of the present nucleic acid molecules or vectors that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A therapeutically effective dose may refer to that amount of the nucleic acid molecule, vector, cell, host, or pharmaceutical composition sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity. The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may prevent or delay onset or amelioration of symptoms of the condition in a subject or an attainment of a desired biological outcome, such as correction of neuropathology, e.g., cellular pathology associated with a neurodegenerative disease.

[0071] The disorder may be a retinal degenerative disease, such as age-related macular degeneration (including dry AMD and wet AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), or glaucoma. The disorder may be a neurodegenerative disease, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), multiple system atrophy, Batten disease, Lewy body dementia, or a prion disease. 01001 / 012745-WQ0

[0072] The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may result in a decrease in the ncurodcgcncration, the degeneration of neurons, the loss of neurons, neuronal cell death, etc. of the subject, where the neurodegeneration, the degeneration of neurons, the loss of neurons, neuronal cell death, etc. of the subject affected by the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition is no greater than 90%, no greater than 85%, no greater than 80%, no greater than 75%, no greater than 70%, no greater than 65%, no greater than 60%, no greater than 55%, no greater than 50%, no greater than 45%, no greater than 40%, no greater than 35%, no greater than 30%, no greater than 25%, no greater than 20%, no greater than 15%, no greater than 10%, about 10% to about 90%, about 15% to about 80%, about 20% to about 70%, about 25% to about 60%, about 30% to about 50%, about 30% to about 40%, about 25% to about 40%, about 20% to about 30%, about 25% to about 35%, about 10% to about 30%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 20% to about 50%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, about 1% to about 100%, about 5% to about 90%, about 10% to about 80%, about 5% to about 70%, about 5% to about 60%, about 10% to about 50%, about 15% to about 40%, about 5% to about 20%, about 1% to about 20%, about 10% to about 30%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 10% to about 90%, about 12.5% to about 80%, about 20% to about 70%, about 25% to about 60%, or about 25% to about 50%, of the neurodegeneration, the degeneration of neurons, the loss of neurons, neuronal cell death, etc. of the subject in the absence of the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition.

[0073] The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may ameliorate the symptoms of a neurodegenerative disease or disorder in a subject. The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may result in at least partial correction of neuropathology, and / or alleviation and / or prevention and / or stabilization and / or slowing of disease progression, and / or progression of the symptoms of a neurodegenerative disease or disorder. The present method and composition may prevent neuron death, and / or delay the onset of paralysis and death.

[0074] The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may 01001 / 012745-WQ0 be used in vitro or administered to a subject. The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered via subrctinal or intravitrcal injection. The administration may be topical, intravenous, intranasal, or any other suitable route as described herein. The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered intrathecally, orally, intravenously, intramuscularly, topically, arterially, or subcutaneously. The routes of administration of the nucleic acid molecule, vector, cell, host, or pharmaceutical composition include oral, intravenous, subcutaneous, intramuscular, inhalation, or intranasal administration. Additionally, specifically targeted delivery of the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition could be delivered by targeted liposome, nanoparticle or other suitable means.

[0075] The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered to the central nervous system (CNS) of the subject. The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered to the spinal cord or brain (e.g., the brainstem region) of the subject. The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered by intrathecal, intraventricular (known also as intracerebroventricular or ICV), intracranial, or intramuscular’ administration (e.g., injection). The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered to a particular ventricle, e.g., to the lateral ventricles or to the fourth ventricle of the brain. The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered by stereotaxic microinjection.

[0076] The present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be used for prophylaxis as well as treating a disease as described herein (such as a neurodegenerative disease).

[0077] For prophylaxis, the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be administered to a subject in order to prevent the onset of one or more symptoms of a disorder (e.g., a neurodegenerative disease). In one embodiment, the subject is asymptomatic. A prophy tactically effective amount of the nucleic acid molecule, vector, cell, host, or pharmaceutical composition is administered to such a subject. A prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the disorder (e.g., a neurodegenerative disease).

[0078] The administration regimen may depend on several factors, including the serum or tissue 01001 / 012745-WQ0 turnover rate of the composition, the level of symptoms, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient composition to effect improvement in the target disease state, while simultaneously minimizing undesired side effects.

[0079] A further aspect provided herein is a method of expressing a protein in a cell, or in a subject, the method comprising administering to the cell or subject the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition, thereby expressing the encoded PEDF protein in the cell or subject.

[0080] The present nucleic acid molecules or vectors may be used as a research tool to study the role of SERPINF1 / PEDF in aging, cancer, or regenerative medicine.

[0081] Increasing Protein Expression, including Codon Optimization

[0082] To increase the protein expression levels, strategies including codon optimization, introducing one or more introns, and / or reconfiguring the number and positioning of enhancers may be used.

[0083] For optimal expression of a recombinant protein, it may be beneficial to employ coding sequences that produce mRNA with codons preferentially used by the host cell. Thus, proper expression of transgenes can require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is being expressed.

[0084] “Codon optimization” or “codon optimized” may refer to changes made in the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the noncodon optimized sequence. Codon optimization does not change the amino acid for which each codon encodes.

[0085] To conduct codon optimization, desired mRNA sequence(s) may be determined and incorporated into a DNA template using standard methods. For example, starting from a desired amino acid sequence, a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons. The G / C content may be optimized to achieve the highest possible G / C content on one hand, taking into account the frequency of the tRNAs according to codon usage on the other hand. The optimized RNA sequence can be established and compared with the original (wildtype) sequence. A secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, 01001 / 012745-WQ0 regions of the RNA.

[0086] The nucleotide sequence encoding the PEDF protein can be modified to improve expression efficiency of the protein. The methods that can be used to improve the transcription and / or translation of a gene herein are not particularly limited. For example, the nucleotide sequence can be modified to better reflect host codon usage to increase gene expression (e.g., protein production) in the host (e.g., a subject such as a mammal). As another non-limiting example for the modification, one or more of the splice donors and / or splice acceptors in the nucleotide sequence of PEDF is modified to reduce the potential for extraneous splicing. As another non-limiting example for the modification, one or more introns can be inserted within or adjacent to the nucleotide sequence of PEDF to optimize AAV vector packaging and enhance expression.

[0087] In certain embodiments, the nucleotide sequence coding for a functional PEDF has an improved codon usage bias for the human cell as compared to naturally occurring (wildtype, non-codon optimized) nucleotide sequence.

[0088] In certain embodiments, the codon-optimized nucleic acid molecule encodes a functional PEDF protein which has the functionality of wild type PEDF. In certain embodiments, the nucleic acid molecule, when expressed in a suitable system (e.g., a host cell, a subject such as a mammal), produces a functional PEDF protein at a relatively high level.

[0089] Generally, codon optimization does not change the amino acid for which each codon encodes. It simply changes the nucleotide sequence so that it is more likely to be expressed at a relatively high level compared to the non-codon optimized sequence. This means that the nucleotide sequences of the nucleic acid molecules provided herein, for example, SEQ ID NO: 5 (or SEQ ID NO: 2-4), may be different from the wildtype PEDF coding sequence, but when they are translated, the amino acid sequence of the protein that is produced is the same.

[0090] The nucleotide sequence of SEQ ID NO:5, also referenced as codon-optimized variant 4 (CO4), is a codon-optimized human PEDF nucleic acid sequence which is based on the sequence of the wild-type human PEDF nucleotide sequence (SEQ ID NO:1). The nucleotide sequence of SEQ ID NO:5 is a PEDF sequence that has been codon optimized using Genscript GenSmart codon optimization tool.

[0091] The nucleotide sequence of SEQ ID NOG, also referenced as codon-optimized variant 2 (CO2), is a codon-optimized human PEDF nucleic acid sequence which is based on the sequence 01001 / 012745-WQ0 of the wild-type human PEDF nucleotide sequence (SEQ ID NO:1). The nucleotide sequence of SEQ ID NO:3 is a PEDF sequence that has been codon optimized using Integrated DNA Technologies codon optimization tool.

[0092] The nucleotide sequence of SEQ ID NO:2, also referenced as codon-optimized variant 1 (CO1), is a codon-optimized human PEDF nucleic acid sequence which is based on the sequence of the wild-type human PEDF nucleotide sequence (SEQ ID NO:1). The nucleotide sequence of SEQ ID NO:2 is a PEDF sequence that has been codon optimized using Integrated DNA Technologies codon optimization tool.

[0093] The nucleotide sequence of SEQ ID NO:4, also referenced as codon-optimized variant 3 (CO3), is a codon-optimized human PEDF nucleic acid sequence which is based on the sequence of the wild-type human PEDF nucleotide sequence (SEQ ID NO:1). The nucleotide sequence of SEQ ID NO:4 is a PEDF sequence that has been codon optimized using Genscript GenSmart codon optimization tool.

[0094] In certain embodiments, the nucleic acid molecule may have at least or about 100, at least or about 120, at least or about 150, at least or about 170, at least or about 200, at least or about 220, at least or about 250, at least or about 270, at least or about 300, at least or about 310, at least or about 320, at least or about 330, at least or about 331, at least or about 332, or at least or about 333, of all codons coding for the functional PEDF being identical to the codons (in corresponding positions) in SEQ ID NO: 1.

[0095] In certain embodiments, the nucleic acid molecule has at least or about 30, at least or about 40, at least or about 50, at least or about 60, at least or about 70, at least or about 80, at least or about 81, at least or about 82, at least or about 83, at least or about 84, at least or about 85, at least or about 86, at least or about 87, or at least or about 88, codon changes as compared to the wild-type PEDF coding sequence (e.g., SEQ ID NO:1).

[0096] In some embodiments, the vector or the nucleic acid molecule comprises one or more introns. The introns may facilitate processing of the RNA transcript in mammalian host cells, increase expression of the protein of interest (e.g., PEDF), and / or optimize packaging of the vector into AAV particles, compared with expression in the absence of the intron element (see e.g. Kurachi et al., 1995, J Biol Chem. 1995 Mar. 10; 270(10):5276-81). Non-limiting examples of such an intron include, a p-actin intron (e.g., truncated or full-length chicken -actin intron), a minute virus of mouse (MVM) intron, a P-globin intron, Al AT intron and / or hPAH intron. In 01001 / 012745-WQ0 some embodiments, the intron is a synthetic intron. The location and size of the intron in the vector can vary. In some embodiments, the intron is located between the promoter and the sequence encoding the protein of interest. In some embodiments, the intron is located within the promoter. In some embodiments, the intron includes an enhancer element. In some embodiments, the intron is located within the sequence encoding the protein of interest, e.g., between exons of the sequence encoding the protein of interest. In some embodiments, the intron may comprise all or a portion of a naturally occurring intron within the vector.

[0097] Methods of Gene Delivery

[0098] The present nucleic acid molecule or the present vector may be delivered into cells or a subject by a suitable method. Methods of delivery may include transfection (e.g., using reagents such as liposomes or nanoparticles); electroporation; or viral transduction.

[0099] The present vector may be a viral vector. Alternatively, the present nucleic acid molecule or vector may be delivered by a non- viral system.

[0100] Vectors according to the present disclosure can be transformed, transfected or otherwise introduced into a wide variety of host cells. Numerous methods of transfection are known to the ordinarily skilled artisan, for example, lipofectamine, calcium phosphate co-precipitation, electroporation, DEAE-dextran treatment, microinjection, viral transduction, and other methods known in the ait. Transduction refers to entry of a virus into the cell and expression (e.g., transcription and / or translation) of the sequence(s) delivered by the viral vector genome. In the case of a recombinant vector, “transduction” generally refers to entry of the recombinant viral vector into the cell and expression of a nucleic acid of interest delivered by the vector genome.

[0101] Viral vectors include, but are not limited to, lentiviral vectors, adenoviral vectors, herpes viral vectors, retroviral vectors, alphavirus-based vectors, parvoviral vectors, adenoviral vectors, herpes simplex viral vectors, and adeno-associated viral (AAV) vectors.

[0102] Non-viral systems include, but are not limited to, nanoparticle -based nucleic acid delivery systems, lipid-based nucleic acid delivery systems, gene guns, hydrodynamic delivery, electroporation, sonoporation, nucleofection microinjection, and biolistics. Non-viral systems also include using naked DNA (with or without chromatin attachment regions) or conjugated DNA that is introduced into cells by various transfection methods such as lipids or electroporation. Various gene delivery methods are discussed in detail by Nayerossadat et al. 01001 / 012745-WQ0

[0103] (Adv Biomed Res. 2012; 1 : 27) and Ibraheem et al. (Int J Pharm. 2014;459(l -2):70-83).

[0104] In certain embodiments, hybrid methods may be used to deliver a nucleic acid that combines two or more techniques.

[0105] AAV Vectors

[0106] An AAV vector may be a nucleic acid molecule, either single- stranded or doublestranded, having an AAV 5 ' inverted terminal repeat (ITR) sequence (which may or may not be modified), and an AAV 3' ITR (which may or may not be modified), flanking a protein-coding sequence (e.g., encoding PEDF or variants thereof) operably linked to transcription regulatory elements that are heterologous to the AAV viral genome, i.e., one or more promoters and / or enhancers and, optionally, a poly adenylation sequence and / or one or more introns. A singlestranded AAV vector may be a nucleic acid molecule that is present in the genome of an AAV virus particle, and can be either the sense strand or the anti-sense strand of the nucleic acid sequences disclosed herein. A double-stranded AAV vector may be a nucleic acid molecule that is present in the DNA of plasmids, e.g., pUC19, or genome of a double-stranded virus, e.g., baculovirus, used to express or transfer the AAV vector nucleic acids.

[0107] In one embodiment, the AAV vector comprises a nucleic acid encoding a functionally active PEDF protein. The PEDF coding sequence may be wild-type, codon optimized, or a variant. In certain embodiments, wildtype PEDF has / comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0108] Other embodiments provided herein are directed to vectors encoding a functional PEDF polypeptide, wherein the vectors comprise one or more of the elements described herein, in one or more different orientation(s). Another embodiment provided herein is directed to the abovedescribed vectors in an opposite orientation. In another embodiment, provided are recombinant AAV virus particles comprising the herein described AAV PEDF vectors.

[0109] The AAV vector may be less than about 7.0 kb, less than 6.5 kb, less than 6.4 kb, less than 6.3 kb, less than 6.2 kb, less than 6.0 kb, less than 5.8 kb, less than 5.6 kb, less than 5.5 kb, less than 5.4 kb, less than 5.3 kb, less than 5.2 kb, less than 5.0 kb, less than 4.8 kb, less than 4.6 kb, less than 4.5 kb, less than 4.4 kb, less than 4.3 kb, less than 4.2 kb, less than 4.1 kb, less than 4.0 kb, less than 3.9 kb, less than 3.8 kb, less than 3.7 kb, less than 3.6 kb, less than 3.5 kb, less than 3.4 kb, less than 3.3 kb, less than 3.2 kb, less than 3.1 kb, less than 3.0 kb, less than 2.9 kb, 01001 / 012745-WQ0 less than 2.8 kb, less than 2.7 kb, or less than 2.6 kb in length. The AAV vectors may range from about 5.0 kb to about 6.5 kb, from about 4.8 kb to about 5.2 kb, from 4.8 kb to 5.3 kb, from about 4.9 kb to about 5.5 kb, from about 4.8 kb to about 6.0 kb, from about 5.0 kb to 6.2 kb, from about 5.1 kb to about 6.3 kb, from about 5.2 kb to about 6.4 kb, from about 5.5 kb to about

[0110] 6.5 kb, from about 4.0 kb to about 5.0 kb, from about 3.8 kb to about 4.8 kb, from about 3.6 kb to 4.6 kb, from about 3.4 kb to about 4.4 kb, from about 3.2 kb to about 4.2 kb, from about 3.0 kb to 4.0 kb, from about 2.9 kb to about 3.9 kb, from about 2.8 kb to about 3.8 kb, from about

[0111] 2.6 kb to about 3.6 kb, from about 4.5 kb to about 5 kb, from about 4 kb to about 4.5 kb, from about 3.5 kb to about 4.0 kb, from about 3.0 kb to about 3.5 kb, or from 2.5 kb to 3.0 kb in length.

[0112] Also provided is a recombinant viral particle comprising any of the AAV vectors provided herein.

[0113] In some embodiments, the AAV vector comprises a 5' inverted terminal repeat (ITR) of AAV and a 3' AAV ITR, a promoter, a polynucleotide encoding PEDF, and a post-transcriptional regulatory element, where the promoter, the polynucleotide encoding PEDF and the posttranscription regulatory element are located downstream of the 5' AAV ITR and upstream of the 3' AAV ITR.

[0114] AAV of any serotype can be used. The serotype of the viral vector used in certain embodiments of the disclosure may be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 (see, e.g., Gao et al. (2002) PNAS, 99:11854-11859; and Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). Other serotypes besides those listed herein can be used. Furthermore, pseudotyped AAV vectors may also be utilized. Pseudotyped AAV vectors are those which contain the genome of one AAV serotype in the capsid of a second AAV serotype; for example, an AAV vector that contains the AAV2 capsid and the AAV 1 genome or an AAV vector that contains the AAV5 capsid and the AAV 2 genome (Auricchio et al., (2001) Hum. Mol. Genet., 10(26):3075-81).

[0115] Promoters

[0116] Various promoters can be operably linked with a nucleic acid comprising the coding region of the protein of interest (e.g., PEDF) in the vector disclosed herein. In some embodiments, the promoter can drive the expression of the protein of interest in a cell infected 01001 / 012745-WQ0 with the viral vector (or a virus derived from the viral vector), such as a target cell. The promoter can be naturally occurring or non-naturally occurring. In some embodiments the promoter is a synthetic promoter. In one embodiment, the synthetic promoter comprises sequences that do not exist in nature and which are designed to regulate the activity of an operably linked gene. In another embodiment, the synthetic promoter comprises fragments of natural promoters to form new stretches of DNA sequence that do not exist in nature. Synthetic promoters may contain regulatory elements, promoters, enhancers, introns, splice donors and / or acceptors that are designed to produce enhanced expression. Examples of promoters include, but are not limited to, viral promoters, plant promoters and mammalian promoters.

[0117] The promoter may be constitutive, regulatable, or inducible, cell type specific, tissuespecific, or species- specific. In addition to the sequence sufficient to direct transcription, a promoter can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, Kozak sequences and introns).

[0118] Promoter / regulatory sequences useful for driving constitutive expression of a gene include, but are not limited to, CMV (cytomegalovirus promoter), EFla (human elongation factor 1 alpha promoter), SV40 (simian vacuolating virus 40 promoter), PGK (mammalian phosphoglycerate kinase promoter), Ubc (human ubiquitin C promoter), human beta- actin promoter, rodent betaactin promoter, CBh (chicken beta-actin promoter), CAG (hybrid promoter contains CMV enhancer, chicken beta-actin promoter, and rabbit beta-globin splice acceptor), TRE (Tetracycline response element promoter), Hl (human polymerase III RNA promoter), U6 (human U6 small nuclear promoter), and the like. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.

[0119] Inducible and tissue-specific expression of an RNA, transmembrane proteins, or other proteins can be accomplished by placing the nucleic acid encoding such a molecule under the control of an inducible or tissue specific promoter / regulatory sequence. Examples of tissuespecific or inducible promoter / regulatory sequences include, but are not limited to, the rhodopsin promoter, the MMTV LTR inducible promoter, the SV40 late enhancer / promoter, synapsin 1 promoter, ET hepatocyte promoter, GS glutamine synthase promoter and many others. Thus, the 01001 / 012745-WQ0 present disclosure includes the use of any promoter / regulatory sequence known in the art that is capable of driving expression of the desired protein operably linked thereto.

[0120] In addition, promoters that can be induced in response to inducing agents such as metals, glucocorticoids, tetracycline, hormones, and the like, are also contemplated. Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Examples of inducible promoters regulated by exogenously supplied promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98 / 10088); the ecdysone insect promoter (No et al., Proc. Natl. Acad. Sci. USA, 93:3346-3351 (1996)), the tetracycline- repressible system (Gossen et al. (1992) Proc. Natl. Acad. Sci. USA, 89:5547-5551), the tetracycline-inducible system (Gossen etal. (1995) Science, 268:1766-1769, see also Harvey et al. (1998) Curr. Opin. Chem. Biol., 2:512-518), the RU486-inducible system (Wang et al. (199T) Nat. Biotech., 15:239-243 and Wang et al. (1997) Gene Ther., 4:432-441) and the rapamycin-inducible system (Magari et al. (1997) J. Clin. Invest., 100:2865-2872). Still other types of inducible promoters which may be useful in this context are those which are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.

[0121] In some embodiments, the regulatory sequences impart tissue-specific gene expression capabilities. In some cases, the tissue- specific regulatory sequences bind tissue-specific transcription factors that induce transcription in a tissue specific manner. Such tissue-specific regulatory sequences (e.g., promoters, enhancers) are well known in the art. Exemplary tissuespecific regulatory sequences include but are not limited to the following tissue specific promoters: neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al. (1993) Cell. Mol. Neurobiol., 13:503-15), neurofilament light-chain gene promoter (Piccioli etal. (1991) Frac. Natl. Acad. Sci. USA, 88:5611-5), and the neuron- specific vgf gene promoter (Piccioli et al. (1995) Neuron, 15:373-84). In some embodiments, the tissue- specific promoter is a promoter of a gene selected from: neuronal nuclei (NeuN), glial fibrillary acidic protein (GFAP), adenomatous polyposis coli (APC), and ionized calcium-binding adapter molecule 1 (Iba-1). In some embodiments, the promoter is a chicken Beta-actin promoter. 01001 / 012745-WQ0

[0122] In some embodiments, the promoter comprises the human alpha- 1 anti-trypsin (hAAT) promoter complex. In some embodiments, the promoter comprises at least a portion of the hAAT promoter.

[0123] In some embodiments, the promoter can include a nucleic acid sequence having at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99%, or more, sequence identity to SEQ ID NO: 6.

[0124] In some embodiments, the promoter is operably linked with a polynucleotide encoding one or more proteins of interest. In some embodiments, the promoter is operably linked with a polynucleotide encoding the PEDF protein.

[0125] The size of the promoter can vary. For example, in some embodiments the promoter is at most or about 1.5 kb, at most or about 1.4 kb, at most or about 1.35 kb, at most or about 1.3 kb, at most or about 1.25 kb, at most or about 1.2 kb, at most or about 1.15 kb, at most or about 1.1 kb, at most or about 1.05 kb, at most or about 1 kb, at most or about 800 base pairs, at most or about 600 base pairs, at most or about 400 base pairs, at most or about 200 base pairs, or at most or about 100 base pairs.

[0126] In certain embodiments, the native promoter, or fragment thereof, for the transgene will be used. The native promoter may be used when it is desired that expression of the transgene should mimic the native expression. The native promoter may be used when expression of the transgene needs to be regulated temporally or developmentally, or in a tissue-specific manner, or in response to specific transcriptional stimuli. In a further embodiment, other native expression control elements, such as enhancer elements, polyadenylation sites or Kozak consensus sequences may also be used to mimic the native expression.

[0127] Regulatory Elements

[0128] Various additional regulatory elements can be used in the vector, for example enhancers to further increase expression level of the protein of interest in a host cell, a poly adenylation signal, a ribosome binding sequence, and / or a consensus splice acceptor or splice donor site. In some embodiments, the regulatory element can facilitate maintenance of the recombinant DNA molecule extrachromosomally in a host cell and / or improve vector potency (e.g. scaffold / matrix attachment regions (S / MARs)). 01001 / 012745-WQ0

[0129] The vectors may include regulatory elements such as a transcription initiation region and / or a transcriptional termination region. Examples of a transcription termination region include, but are not limited to, polyadenylation signal sequences. Examples of polyadenylation signal sequences include, but are not limited to, bovine growth hormone (bGH) poly(A), SV40 late poly(A), rabbit beta-globin (rBG) poly(A), thymidine kinase (TK) poly(A) sequences, and any variants thereof. In some embodiments, the transcriptional termination region is located downstream of the post-transcriptional regulatory element. In some embodiments, the transcriptional termination region is a polyadenylation signal sequence. In some embodiments, the transcriptional termination region is bGH poly(A) sequence.

[0130] In some embodiments, the vector may include additional transcription and translation initiation sequences, and / or additional transcription and translation terminators.

[0131] The vector may include conventional control elements which are operably linked to the transgene in a manner which permits its transcription, translation and / or expression in a cell transfected with the vector or infected with the vims. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.

[0132] As used herein, a polynucleotide sequence and regulatory sequences are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription of the polynucleotide sequence under the influence or control of the regulatory sequences. If it is desired that the polynucleotide sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably linked to a nucleic acid sequence if the promoter region were 01001 / 012745-WQ0 capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide. Similarly, two or more coding regions arc operably linked when they are linked in such a way that their transcription from a common promoter results in the expression of two or more proteins having been translated in frame.

[0133] In certain embodiments, a polyadenylation sequence is inserted following the transgene sequences and before the 3' AAV ITR sequence.

[0134] An AAV vector may also contain an intron, e.g., located between the promoter / enhancer sequence and the transgene. One possible intron sequence is derived from SV-40, and is referred to as the SV-40 T intron sequence.

[0135] Another vector element that may be used is an internal ribosome entry site (IRES). An IRES sequence may be used to produce more than one polypeptide from a single gene transcript. An IRES sequence may be used to produce a protein that contains more than one polypeptide chain. In some circumstances, a foot and mouth disease vims 2A sequence may be included.

[0136] Polynucleotides and polypeptides including modified forms can be made using various standard cloning, recombinant DNA technology, via cell expression or in vitro translation and chemical synthesis techniques known to those of skill in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition).

[0137] In accordance with the present disclosure, there may be numerous tools and techniques within the skill of the art, such as those commonly used in molecular immunology, cellular immunology, pharmacology, and microbiology (see, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, N.J.; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, N.J.; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, N.J.).

[0138] Production of Recombinant AAVs

[0139] The present disclosure provides for virus particles comprising the present viral vectors. 01001 / 012745-WQ0

[0140] Also provided are methods of producing recombinant adeno-associated virus (AAV) particles comprising any of the AAV vectors provided herein. The methods comprise culturing a cell that has been transfected with any of the AAV vectors provided herein (in association with various AAV cap and rep genes) and recovering recombinant therapeutic AAV virus particles from the supernatant of the transfected cell. Generation of the viral vector can be accomplished using any suitable genetic engineering techniques known in the art, including, without limitation, the standard techniques of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, N.Y. (1989)).

[0141] The cells useful for recombinant AAV production may be any cell type susceptible to baculovirus infection, including insect cells such as High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf9, Sf900+, Sf21, BTI-TN-5B1-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5, and Ao38. In another embodiment, mammalian cells such as HEK293, HeLa, CHO, NSO, SP2 / 0, PER.C6, Vero, RD, BHK, HT 1080, A549, Cos-7, ARPE-19, and MRC-5 can be used.

[0142] Methods for obtaining recombinant AAVs having a desired capsid protein have been described (See, for example, US 2003 / 0138772, the contents of which are incorporated herein by reference in their entirety). A number of different AAV capsid proteins have been described, for example, those disclosed in Gao et al. (2004) J. Virol, 78(12):6381-6388; Gao et al. (2004) Proc Natl Acad Sci USA, 100(10):6081-6086. In one embodiment, the AAV9 vector and capsid are used. Other suitable AAVs, such as rAAVrh.8 and rAAVrh.10, or other similar vectors may be adapted for use. Typically, the methods involve culturing a host cell which contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and a transgene; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins.

[0143] The components to be cultured in the host cell to package an AAV vector in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. For example, such a stable host cell may contain the required component(s) under the control of an inducible promoter. 01001 / 012745-WQ0

[0144] However, the required component(s) may be under the control of a constitutive promoter. In still another alternative, a selected stable host cell may contain selected componcnt(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and / or cap proteins under the control of inducible promoters.

[0145] The recombinant AAV vector, rep sequences, cap sequences, and helper functions for producing the AAV may be delivered to the packaging host cell using any appropriate genetic element (vector). The selected genetic element may be delivered by any suitable method, including those described herein. See, e.g., Fisher et al. (1993) J. Virol., 10'.520-532 and U.S. Patent No. 5,478,745.

[0146] In some embodiments, recombinant AAVs may be produced using the triple transfection method (e.g., as describedin detail in U.S. Patent No. 6,001,650). Typically, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising a transgene) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the “AAV helper function” sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions i.e., AAV virions containing functional rep and cap genes). Nonlimiting examples of vectors suitable for use with the present disclosure include pHLP19 (described in U.S. Patent No. 6,001,650) and pRep6cap6 vector (described in U.S. Patent No. 6,156,303). The accessory function vector encodes nucleotide sequences for non-AAV derived viral and / or cellular functions upon which AAV is dependent for replication i.e., "accessory functions"). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.

[0147] A nucleic acid molecule provided herein may be produced, for example, using chemical synthesis of a given sequence. Further, suitable methods would be apparent to those skilled in the 01001 / 012745-WQ0 art for determining whether a nucleic acid described herein expresses a functional protein. For example, one suitable in vitro method involves inserting the nucleic acid into a vector, such as an AAV vector, transducing host cells, such as 293T or HeLa cells, with the vector, and assaying for PEDF activity. Alternatively, a suitable in vivo method involves transducing a vector containing the nucleic acid into a mouse and assaying for functional PEDF in the plasma of the mouse.

[0148] In one embodiment, AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

[0149] The term “inverted terminal repeat (ITR)” as used herein may refer to the art-recognized regions found at the 5' and 3' termini of the AAV genome which function in cis as origins of DNA replication and as packaging signals for the viral genome. AAV ITRs, together with the AAV rep coding region, provide for efficient excision and rescue from, and integration of a nucleotide sequence interposed between two flanking ITRs into a host cell genome. Sequences of certain AAV- associated ITRs are disclosed by Yan et al., J. Virol. (2005) vol. 79, pp. 364-379 which is herein incorporated by reference in its entirety. ITR sequences may be full length, wildtype AAV ITRs, or fragments thereof that retain functional capability, or may be sequence variants of full-length, wild-type AAV ITRs that are capable of functioning in cis as origins of replication. AAV ITRs useful in the present recombinant AAV vectors may be derived from any known AAV serotype. AAV ITRs may be derived from the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype. In certain embodiments, AAV ITRs may be derived from the AAV2 or AAV5 serotype. The ITRs employed in the vectors of the present embodiment may correspond to the same serotype as the associated cap genes, or may differ. In one embodiment, the ITRs employed herein correspond to an AAV2 serotype and the cap genes correspond to an AAV5 serotype.

[0150] A “transcription regulatory element” refers to nucleotide sequences of a gene involved in regulation of genetic transcription including a promoter, plus response elements, activator and enhancer sequences for binding of transcription factors to aid RNA polymerase binding and promote expression, and operator or silencer sequences to which repressor proteins bind to block RNA polymerase attachment and prevent expression.

[0151] The term “control sequences” refers to DNA sequences necessary for the expression of an 01001 / 012745-WQ0 operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0152] As used herein, the term “variant” refers to a polynucleotide (or polypeptide) having a sequence substantially similar- to a reference polynucleotide (or polypeptide). Procedures for the introduction of nucleotide and amino acid changes in a polynucleotide, protein or polypeptide are known to the skilled artisan (see, e.g., Sambrook et al. (1989)). In the case of a polynucleotide, a variant can have deletions, substitutions, additions of one or more nucleotides at the 5' end, 3' end, and / or one or more internal sites in comparison to the reference polynucleotide. Similarities and / or differences in sequences between a variant and the reference polynucleotide can be detected using conventional techniques known in the art, for example polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis. Generally, a variant of a polynucleotide, including, but not limited to, a DNA, can have at least or about 50%, at least or about 55%, at least or about 60%, at least or about 65%, at least or about 70%, at least or about 75%, at least or about 80%, at least or about 85%, at least or about 90%, at least or about 91%, at least or about 92%, at least or about 93%, at least or about 94%, at least or about 95%, at least or about 96%, at least or about 97%, at least or about 98%, at least or about 99% or more sequence identity to the reference polynucleotide (e.g., a polynucleotide encoding wildtype PEDF) as determined by sequence alignment programs known by skilled artisans. In the case of a polypeptide, a variant can have deletions, substitutions, additions of one or more amino acids in comparison to the reference polypeptide. Similarities and / or differences in sequences between a variant and the reference polypeptide can be detected using conventional techniques known in the art, for example Western blot. Generally, a variant of a polypeptide, can have at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to the reference polypeptide as determined by sequence alignment programs known by skilled artisans.

[0153] As used herein, an “intron” is broadly defined as a sequence of nucleotides that is removable by RNA splicing. RNA splicing means the excision of introns from a pre-mRNA to 01001 / 012745-WQ0 form a mature mRNA. Insertion of an intron into an expressed sequence can be accomplished by any method known in the ail.

[0154] Host Organism and / or Cells

[0155] The present disclosure provides for a cell or host comprising the present nucleic acid molecule or vector.

[0156] As used herein, the term “host” refers to organisms and / or cells which harbor / comprise / express a nucleic acid molecule or a vector of the present disclosure, as well as organisms and / or cells that are suitable for use in expressing a protein. A host cell may be in the form of a single cell, a population of similar or different cells, for example in the form of a culture (such as a liquid culture or a culture on a solid substrate), an organism or part thereof. In one embodiment, a host cell may permit the expression of a nucleic acid molecule provided herein. Thus, the host cell may be, for example, a bacterial, a yeast, an insect or a mammalian cell.

[0157] In another embodiment, provided is a means for delivering a nucleic acid provided herein into a broad range of cells, including dividing and non-dividing cells. The present disclosure may be employed to deliver a nucleic acid provided herein to a cell in vitro, e.g., to produce a polypeptide encoded by such a nucleic acid molecule in vitro or for ex vivo gene therapy. The nucleic acid molecule, vector, cells and methods / use of the present disclosure are additionally useful in a method of delivering a nucleic acid provided herein to a subject, typically a host suffering from a disorder.

[0158] The present disclosure finds use in both veterinary and medical applications. Suitable subjects for gene delivery methods as described herein include both avians and mammals, with mammals being preferred.

[0159] As used herein, a “subject” refers to an animal that is the object of treatment, observation or experiment. Mammals include, but are not limited to, humans, domestic and farm animals, zoo animals, sports and pet animals. Non-limiting examples of mammals include mice; rats; rabbits; guinea pigs; dogs; cats; sheep; goats; cows; horses; primates, such as monkeys, chimpanzees and apes, and, in particular, humans. In some embodiments, the mammal is a human. Human subjects include neonates, infants, juveniles, and adults. 01001 / 012745-WQ0

[0160] Conditions to be treated

[0161] The present nucleic acid molecule, vector, cell, or pharmaceutical composition may be used to treat or prevent a disorder or condition in a subject. The disorder or condition may be an age-related disease, a neurodegenerative disease, an ocular degenerative disease, a retinal degenerative disease, a cancer, or a cardiovascular disease.

[0162] The present nucleic acid molecule, vector, cell, or pharmaceutical composition may be used to treat retinal disorders, age-related diseases, or neoplastic diseases, including, but not limited to, glaucoma, diabetic macular degeneration, retinitis pigmentosa, Stargardt disease, neurodegenerative disease (Alzheimer’s, Frontotemporal dementia, Parkinson’s), cardiovascular disease, arthritis, osteoporosis, chronic kidney disease, chronic obstructive pulmonary disease, cancers, etc.

[0163] The present nucleic acid molecule, vector, cell, or pharmaceutical composition may be used for prophylaxis as well as treating a disorder, for example, a neurodegenerative disease such as an ocular (or retinal) neurodegenerative disease (e.g., amelioration of signs and / or symptoms of the retinal degenerative disease or other neurodegenerative diseases).

[0164] Retinal degenerative diseases include, but are not limited to, retinitis pigmentosa (RP), age-related macular degeneration (AMD), and glaucoma. Neurodegenerative diseases include, but are not limited to, Alzheimer’s, Parkinson’s, Huntington’s, Amyotrophic lateral sclerosis (ALS), or Lewy body dementia.

[0165] A neurodegenerative disease may be caused by the progressive loss of structure or function of neurons, in the process known as neurodegeneration. Such neuronal damage may ultimately involve cell death. Neurodegeneration can be found in the brain at many different levels of neuronal circuitry, ranging from molecular to systemic. Neurodegenerative diseases may include abnormalities in signaling pathways, for example aberrant phosphorylation due to dysregulated kinase activity, mutant proteins (mutant tau, mutant APP) and chaperone unbalance leading to misfolding. Neurodegenerative diseases may be characterized by a slow progressive loss of neurons in the central nervous system (CNS), which often leads to deficits in specific brain functions (e.g., memory, movement, cognition, etc.) performed by the affected CNS region.

[0166] Neurodegenerative diseases may include amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, Batten disease, and prion diseases such as Creutzfeldt-Jakob disease. 01001 / 012745-WQ0

[0167] Neurodegenerative diseases include, but are not limited to, Alzheimer's disease (sporadic or familial), amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), multiple sclerosis, Huntington's disease, multiple system atrophy, argyrophilic grain dementia, dementia pugilistica, chronic traumatic encephalopathy, diffuse neurofibrillary tangles with calcification, Down syndrome, Gerstmann-Straussler-Scheinker disease, Hallervorden-Spatz disease, hereditary frontotemporal dementia, parkinsonism linked to chromosome 17 (FTDP-17), inclusion body myositis, Creutsfeld- Jakob disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, prion protein cerebral amyloid angiopathy, sporadic corticobasal degeneration, progressive supranuclear palsy, subacute sclerosing panencephalitis, myotonic dystrophy, motor neuron disease with neurofibrillary tangles, tangle only dementia, and progressive subcortical gliosis. Neurodegenerative diseases also include alcohol-induced neurodegeneration; brain ischemia; cocaine addiction; diffuse Lewy body disease; electroconvulsive seizures; fetal alcohol syndrome; focal cortical dysplasia; hereditary canine spinal muscular atrophy; inclusion body myositis; multiple system atrophy; Niemann-Pick type C; Parkinson's disease; and peripheral nerve injury.

[0168] Neurodegenerative diseases or conditions may include Parkinson's disease, Alzheimer's disease, prion disease, a motor neuron disease (MND) such as amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's ataxia, Lewy body disease, epilepsy, encephalitis, hydrocephalus, stroke, chronic traumatic encephalopathy (CTE); a synucleinopathy; a tauopathy, a spongiform encephalopathy; familial amyloidotic polyneuropathy; Dutch hereditary cerebral hemorrhage with amyloidosis; congophilic angiopathy; corticobasal degeneration; Pick's disease; progressive supranuclear palsy; Creutzfeldt-Jacob disease; Gerstmann-Straussler-Schneiker syndrome; fatal familial insomnia; kuru; bovine spongiform encephalopathy; scrapie; chronic wasting disease; Lewy body variant of Alzheimer's disease; diffuse Lewy body disease; dementia with Lewy bodies; multiple system atrophy; neurodegeneration with brain iron accumulation type I; diffuse Lewy body disease; frontotemporal lobar degeneration; hereditary dentatorubraLpallidoluysian atrophy; Kennedy's disease; Alexander's disease; Cockayne syndrome; Icelandic hereditary cerebral hemorrhage with amyloidosis; and neuroinflammation.

[0169] As used herein, the terms “neurodegenerative disease”, “neurodegenerative disorder”, and “neurodegenerative condition” generally refer to any disease, disorder, and / or condition that 01001 / 012745-WQ0 affects the neurons (sometimes referred to as “nerve cells”), such as neurons of a brain and / or neurons of a nervous system which is associated with the degeneration or loss of neural cells. Neurodegenerative diseases may result in progressive degeneration and / or death of nerve cells. In general, neurodegeneration is the progressive loss of structure and / or function of neurons, including the death of neurons. Neurodegenerative diseases may cause problems with movement (e.g., ataxias), or mental or cognitive functioning (e.g., dementias). Frequently neurodegeneration is associated with neuroinflammation. Therefore, it is to be understood that neurodegenerative diseases or disorders encompass neural diseases which are characterized by neuroinflammation. Some neurodegenerative diseases and / or conditions are associated with microglia cell over-activation, increased numbers of microglia cells, production of inflammatory proteins and / or inflammatory activities, and / or neuronal death.

[0170] Non-limiting examples of neurodegenerative diseases include Alzheimer's disease and other dementias, Parkinson's disease and other Parkinson's disease related disorders, prion disease, motor neuron diseases other than ALS, Huntington's disease, Spinocerebellar ataxia (SCA), Spinal muscular atrophy (SMA), Friedreich's ataxia, Lewy body disease, epilepsy, multiple sclerosis, encephalitis, hydrocephalus, stroke, chronic traumatic encephalopathy (CTE); synucleinopathies; tauopathies; spongiform encephalopathies; familial amyloidotic polyneuropathy; Dutch hereditary cerebral hemorrhage with amyloidosis; congophilic angiopathy; corticobasal degeneration; Pick's disease; progressive supranuclear palsy; Creutzfeld- Jacob disease; Gerstmann-Straussler-Schneiker syndrome; fatal familial insomnia; kuru; bovine spongiform encephalopathy; scrapie; chronic wasting disease; Lewy body variant of Alzheimer's disease; diffuse Lewy body disease; dementia with Lewy bodies; multiple system atrophy; neurodegeneration with brain iron accumulation type I; diffuse Lewy body disease; frontotemporal lobar degeneration; hereditary dentatorubral-pallidoluysian atrophy; Kennedy's disease; Alexander's disease; Cockayne syndrome; Icelandic hereditary cerebral hemorrhage with amyloidosis.

[0171] In some embodiments, the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may decrease or prevent at least one symptom associated with a disorder (e.g., a neurodegenerative disease).

[0172] Conditions to be treated by the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition also include, but are not limited to, spinal muscular atrophy (SMA), 01001 / 012745-WQ0 amytrophic lateral sclerosis (ALS), spinal bulbar muscular atrophy (SBMA), spinal cerebellar ataxia, primary lateral sclerosis (PLS), or traumatic spinal cord injury, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), hereditary spastic paraparesis (HSP), X-linked spinobulbar muscular atrophy (SBMA; Kenney disease), progressive bulbar palsy, pseudo-bulbar palsy, post-polio syndrome (PPS), Huntington's disease, Essential tremor (ET), paralysis, and Parkinson's disease.

[0173] Pharmaceutical Compositions and Administration

[0174] The present disclosure provides for a pharmaceutical composition comprising the present nucleic acid molecule, the present vector, or the present cell comprising the nucleic acid molecule or vector.

[0175] The present compositions may be used in vitro or administered to a subject. The administration may be topical, intravenous, intranasal, or any other suitable route as described herein. The present compositions may be administered by intravitreal injection or subretinal injection.

[0176] In certain embodiments, the concentration or titer of the vector in the composition is at least: (a) 5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 (xlO12genomic particles (gp) / ml); or (b) 5, 6, 7, 8, 9, 10, 15, 20, 25, or 50 (x 1010infectious units (IU) / ml).

[0177] The pharmaceutical compositions can further comprise one or more pharmaceutically acceptable excipient, ligand, a conjugate, a lipid, a nanoparticle, a liposome, a carrier, an adjuvant or a diluent.

[0178] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like can be used to deliver the nucleic acid molecules or vectors described herein.

[0179] The formation and use of liposomes is generally known to those of skill in the art. Recently, liposomes were developed with improved serum stability and circulation half-times (U.S. Patent No. 5,741,516). Further, various methods of liposome and liposome like preparations as potential drug carriers have been described (U.S. Patent Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868; and 5,795,587).

[0180] Liposomes have been used successfully with a number of cell types that are normally resistant to transfection by other procedures. Liposomes have been used effectively to introduce 01001 / 012745-WQ0 genes, drugs, radiotherapeutic agents, viruses, transcription factors and allosteric effectors into a variety of cultured cell lines and animals.

[0181] Liposomes may be formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs). MLVs generally have diameters of from 25 nm to 4 pm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500A, containing an aqueous solution in the core.

[0182] Alternatively, nanocapsule or nanoparticle formulations may be used. Nanocapsules can generally entrap substances in a stable and reproducible way. Nanoparticles can be used to transport drugs through the blood-brain barrier (BBB) when administered intravenously as well as the factors that influence its transportation.

[0183] Nanoparticles (NPs) are colloidal carriers that can have a natural or synthetic origin and can vary from 1 to 1000 nm in size. Synthetic NPs may be prepared from polymeric materials such as poly(ethylenimine) (PEI), poly(alkylcyanoacrylates), poly(amidoamine) dendrimers (PAMAM), poly(s-caprolactone) (PCL), poly(lactic-co-glycolic acid) (PLGA), polyesters (poly(lactic acid) (PLA), or from inorganic materials such as gold, silicon dioxide (silica), among others. These carriers can transport drugs by adsorbing, entrapping or bounding covalently to them. Natural NPs are produced from natural polymers, such as polysaccharides (chitosan and alginate), amino acids (poly(lysine), poly(aspartic acid) (PASA)), or proteins (gelatin and albumin). Natural NPs have the advantage of providing biological signals to interact with specific receptors / transporters expressed by endothelial cells.

[0184] A number of ligands have been conjugated to NPs to facilitate BBB penetration. Such molecules can be grouped into four different types: (i) ligands that mediate the adsorption of proteins from the bloodstream that interact directly with BBB receptors or transporters; (ii) ligands that have direct interaction per se with BBB receptors or transporters; (iii) ligands that increase charge and hydrophobicity; and (iv) ligands that improve blood circulation time (e.g. PEG).

[0185] Other methods for assisting the NPs to cross the blood-brain barrier would include but are not limited to receptor mediated transport, transporter mediated transport, absorptive mediated transport, and cell penetrating transport.

[0186] Mammalian vims vectors that can be used to deliver RNA include oncoretroviral vectors, adenovirus vectors, herpes simplex virus (HSV) vectors, and lentiviruses. 01001 / 012745-WQ0

[0187] In particular, HSV vectors are tropic for the central nervous system (CNS) and can establish lifelong latent infections in neurons.

[0188] The AAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. The AAV, preferably suspended in a physiologically compatible carrier (e.g., in a pharmaceutical composition), may be administered to a subject, e.g., a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, or a nonhuman primate. In certain embodiments, compositions may comprise an AAV alone, or in combination with one or more other viruses (e.g., a second AAV encoding having one or more different transgenes).

[0189] Suitable carriers may be readily selected by one of skill in the ail in view of the indication for which the AAV is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, and water. The selection of the carrier is not a limitation of the present invention.

[0190] Optionally, the present compositions may contain, in addition to the AAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers. Suitable exemplary preservatives include chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0191] The dose of AAV virions required to achieve a desired effect or "therapeutic effect," e.g., the units of dose in vector genomes / per kilogram of body weight (vg / kg), will vary based on several factors including, but not limited to: the route of AAV administration, the level of gene or RNA expression required to achieve a therapeutic effect, the specific disease or disorder being treated, and the stability of the gene or RNA product. One of skill in the art can readily determine an AAV virion dose range to treat a subject having a particular disease or disorder based on the aforementioned factors, as well as other factors that are well known in the art. An effective amount of the AAV is generally in the range of from about 10 pl to about 100 ml of solution containing from about 109to 1016genome copies per subject. Other volumes of solution may be used. The volume used will typically depend, among other things, on the size of the subject, the dose of the AAV, and the route of administration. For example, for intrathecal or intracerebral administration 01001 / 012745-WQ0 a volume in range of 1 l to 10 pl or 10 pl to 100 pl may be used. For intravenous administration a volume in range of 10 pl to 100 pl, 100 pl to 1 ml, 1 ml to 10 ml, or more may be used. In some cases, a dosage between about 1010to 1012AAV genome copies per subject may be used. In certain embodiments, 1012AAV genome copies per subject is effective to target CNS tissues. In some embodiments the AAV is administered at a dose of 1010, 1011, 1012, 1013, 1014, or 1015genome copies per subject. In some embodiments the AAV is administered at a dose of 1010, 1011, 1012, 1013, or 1014genome copies per kg.

[0192] In some embodiments, AAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high AAV concentrations are present (e.g., about 1013GC / ml or more). Methods for reducing aggregation of AAVs are well known in the ail and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright et al. (2005) Molecular Therapy 12:171-178.)

[0193] Formulation of pharmaceutically acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. Typically, these formulations may contain at least about 0.1% of the active agent (e.g., the present nucleic acid molecule, vector, cell, or host) or more, although the percentage of the active agent(s) may, of course, be varied and may conveniently be between about 1 or 2% and about 70% or 80% or more of the weight or volume of the total formulation. Naturally, the amount of active agent (e.g., the present nucleic acid molecule, vector, cell, or host) in each therapeutically useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the agent. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.

[0194] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. In many cases the form is sterile and fluid to the extent that easy syringability exists. It must be stable under the conditions of 01001 / 012745-WQ0 manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0195] For administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, a sterile aqueous medium that can be employed will be known to those of skill in the art. For example, one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition). Some variation in dosage will necessarily occur depending on the condition of the host. The person responsible for administration will, in any event, determine the appropriate dose for the individual host.

[0196] Sterile injectable solutions are prepared by incorporating the active AAV in the required amount in the appropriate solvent with various of the other ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of prepar ation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0197] As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, 01001 / 012745-WQ0 buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically-acceptable" refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a host.

[0198] Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition into suitable host cells. In particular, present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

[0199] In addition to the methods of delivery described above, the following techniques are also contemplated as alternative methods of delivering the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition to a host. Sonophoresis (i.e., ultrasound) has been used and described in U.S. Patent No. 5,656,016 as a device for enhancing the rate and efficacy of drug permeation into and through the circulatory system. Other drug delivery alternatives contemplated are intraosseous injection (U.S. Patent. No. 5,779,708), microchip devices (U.S. Pat. No. 5,797,898), ophthalmic formulations, transdermal matrices (U.S. Patent Nos. 5,770,219 and 5,783,208) and feedback-controlled delivery (U.S. Patent No. 5,697,899).

[0200] To prepare the present pharmaceutical compositions, a conjugate, a lipid, a nanoparticle, a liposome, an adjuvant or a diluent may be further admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0201] Formulations of present nucleic acid molecule, vector, cell, host, or pharmaceutical composition may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical 01001 / 012745-WQ0

[0202] Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0203] Toxicity and therapeutic efficacy of the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD50 / ED50). In particular aspects, compositions exhibiting high therapeutic indices are desirable. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such nucleic acid molecule, vector, cell, host, or pharmaceutical composition lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

[0204] The mode of administration can vary. Suitable routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra-arterial.

[0205] In particular embodiments, the composition or therapeutic can be administered by an invasive route such as by injection. In further embodiments, the present nucleic acid molecule, vector, cell, host, or pharmaceutical composition, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra-articularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery. Administration by non-invasive routes (e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present disclosure.

[0206] In order to overcome any issue of the pharmacological agents crossing the blood / brain barrier, intrathecal administration may be used. Intrathecal administration involves injection of the drug into the spinal canal, more specifically the subarachnoid space such that it reaches the cerebrospinal fluid. Intrathecal administration can be performed by lumbar puncture (bolus injection) or by a port-catheter system (bolus or infusion). The catheter is most commonly inserted between the laminae of the lumbar vertebrae and the tip is threaded up the thecal space to the desired level (generally L3-L4). Intrathecal formulations most commonly use water, and saline as excipients but EDTA and lipids have been used as well. 01001 / 012745-WQ0

[0207] Compositions can be administered with medical devices known in the art. For example, a present pharmaceutical composition can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.

[0208] The present pharmaceutical compositions may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

[0209] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injecting directly into the desired target site, often in a depot or sustained release formulation. Furthermore, one may administer the composition in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody, targeting, for example, the brain. The liposomes will be targeted to and taken up selectively by the desired tissue.

[0210] The administration regimen depends on several factors, including the serum or tissue turnover rate of the therapeutic composition, the level of symptoms, and the accessibility of the target cells in the biological matrix. Preferably, the administration regimen delivers sufficient therapeutic composition to effect improvement in the target disease state, while simultaneously minimizing undesired side effects. Accordingly, the amount of biologic delivered depends in part on the particular therapeutic composition and the severity of the condition being treated.

[0211] Determination of the appropriate dose is made by the clinician, e.g., using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects.

[0212] Kits

[0213] The present disclosure also provides kits comprising the present composition / agent (nucleic acid molecules, vectors, and / or cells) in kit form. A kit of the present disclosure includes one or more components described herein, in association with one or more additional components including, but not limited to a pharmaceutically acceptable ligand, a conjugate, a lipid, a nanoparticle, a liposome, an adjuvant, a diluent, carrier or excipient.

[0214] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example, the kit can include 01001 / 012745-WQ0 one or more hypodermic needles or other injection devices as discussed above.

[0215] The kit can include a package insert including information concerning the pharmaceutical compositions and dosage forms in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and patent information.

[0216] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 5%, 10% or 20%.

[0217] The present invention may be better understood by reference to the following non-limiting examples, which are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed to limit the broad scope of the invention.

[0218] Example 1

[0219] Pigment epithelium-derived factor (PEDF) is a conserved multifunctional secreted protein encoded by the SERPINF1 gene that is widely expressed across tissues in vertebrates (The GTEx Consortium atlas of genetic regulatory effects across human tissues, Science 369, 1318-1330 (2020)). Studies have shown that PEDF expression decreases with cellular senescence in vitro (DiPaolo et al., Identification of proteins differentially expressed in quiescent and proliferatively senescent fibroblast cultures, Exp Cell Res 220, 178-85 (1995)), and is lost during human skin aging in vivo (Francis et al., Loss of EPC-l / PEDF expression during skin aging in vivo, J Invest Dermatol 488 122, 1096-105 (2004)). Studies of animal models of injury and disease have implicated PEDF in the maintenance of physiological homeostasis through its demonstrated neurotrophic, anti- angiogenic, anti-fibrotic, immunomodulatory, antiinflammatory, tumoricidal, and stem cell-supporting functions. Additionally, PEDF has also been identified as significantly increasing in abundance in plasma following exercise in mice (De 01001 / 012745-WQ0

[0220] Miguel et al. Exercise plasma boosts memory and dampens brain inflammation via clusterin, Nature 600, 494-499 (2021)), suggesting that PEDF may play a role in mediating the beneficial effects of exercise on organismal health.

[0221] We identified and characterized PEDF as a conserved circulating geroprotective factor, both in vitro and in vivo, demonstrating that PEDF mediates, in part, the protective effect of young serum on replicative lifespan (RLS) of human primary fibroblasts, and that systemic PEDF administration to aged mice has rejuvenating effects on multiple tissues (Wang et al., In vitro heterochronic parabiosis identifies pigment epithelium-derived factor as a systemic mediator of rejuvenation by young blood, bioRxiv, 2024 May 5:2024.05.02.592258). Our results demonstrate that PEDF supplementation induces protective transcriptional signatures in the hippocampus, liver, and kidney of aged mice, improving cognitive function and hepatic and renal integrity, indicating that maintaining or supplementing PEDF levels in aged humans can have therapeutic effects for age-related conditions and chronic diseases of these tissues.

[0222] For therapeutics applications, this study focused on age-related macular degeneration (AMD). We used a recombinant adeno-associated virus (rAAV) vector comprising an expression construct encoding a transgene insert of a codon-optimized version of the nucleotide sequence of SERPINF1 (PEDF), wherein the codon-optimized sequence preserves a conserved stem-loop hairpin sequence encoded by nucleotides 31-42 and 79-90 in the wild-type SERPINF1 nucleotide sequence.

[0223] The rAAV vector also contains a hybrid chicken beta (CBh) actin promoter element, a woodchuck hepatitis vims post-transcriptional regulatory element (WPRE), and a bovine growth hormone polyA signal tail. The rAAV vector includes wild-type AAV2 5’ and 3’ inverted terminal repeat (ITR) sequences.

[0224] The rAAV capsid protein is AAV9 capsid protein. Summary

[0225] This technology is a system of one-time gene therapy for dry AMD that uses adeno- associated virus serotype 9 (AAV9) for intravitreal delivery of PEDF. This is a codon-optimized vector system that preserves sequences associated with a key stem loop hairpin element that can facilitate substantially elevated expression of PEDF. The AAV9-PEDF system has been demonstrated to augment PEDF levels in human ARPE-19 retinal pigment epithelial cells and the mouse retina and confers protection against retinal thinning in a sodium iodate (SI) model of 01001 / 012745-WQ0 retinal degeneration. As such, this technology can provide needed therapeutic avenues for individuals with dry AMD and treat other retinal conditions or age-related diseases.

[0226] The preservation of the stem-loop hairpin sequence in the SERPINF1 mRNA transcript transcribed from the codon-optimized PEDF sequence is important for efficient and high expression of PEDF, compared to constructs lacking the stem-loop.

[0227] Results

[0228] The SERPINF1 open reading frame (ORF) sequences of Figure 1 were synthesized and cloned into an AAV vector backbone. The vector backbones contained AAV2 5’ and 3’ inverted terminal repeats (ITRs), flanking a promoter element, either wild-type or codon-optimized SERPINF1 ORFs, a regulatory element, and a polyA signal sequence. The promoter element is a CBh promoter, comprising a CMV early promoter sequence, a CB promoter sequence, and truncated chicken (Lactin intron and minute virus of mouse (MVM) intron sequences. Codonoptimization was performed using two different online tools: Integrated DNA Technologies codon optimization tool (Figure 1, CO1 and CO2), and Genscript GenSmart codon optimization tool (Figure 1, CO3 and CO4).

[0229] ARPE-19 human RPE cell line (CRL-2302, ATCC) was maintained in DMEM / F12 media (Gibco, 11320033), supplemented with 10% FBS (Gemini, 100-500), and 1% Pen / Strep (Gibco, 15070063). Cell cultures were maintained at 37°C and 5% CO2. Plasmid transfection of ARPE- 19 cells was performed using Lipofectamine LTX with Plus reagent (Thermo Fisher Scientific, A12621). ARPE-19 cells were seeded at 8 x 105cells per well of a 24- well plate and incubated overnight in cell culture medium prior to transfection. Transfection was performed with 1.5 pl / ml Lipofectamine LTX, 0.375 pl / ml Plus reagent and 400 ng / ml plasmid DNA, and cells were incubated for 48 hours at 37°C prior to collection of the supernatant. PEDF concentration in the supernatant of ARPE-19 cells was determined by Human PEDF ELISA Kit (Abeam, ab213815) according to the manufacturer's instructions.

[0230] Figure 2 shows the ELISA analyses of PEDF concentrations from the supernatants. Our data illustrate that preservation of the stem-loop hairpin sequence in codon-optimized PEDF ORF sequence leads to much higher expression as compared to codon-optimized sequences that do not preserve the stem-loop sequence.

[0231] For intravitreal injections, 3-month-old C57BL6 / I mice were first anaesthetized by 01001 / 012745-WQ0 ketamine and xylazine based on their body weight (0.08 mg ketamine / g and 0.01 mg xylazine / g) according to the Administrative Panel of Laboratory Animal Care (APLAC) and the Institutional Animal Care and Use Committee (lACUC)-approved protocols at Columbia University. A slight proptosis of the eye was induced by applying gentle pressure to expose the sclera approximately 1 mm behind the limbus. The eye was thoroughly rinsed with a sterile ophthalmic iodine solution to disinfect the surgical site. A sharp-pointed scalpel was used to create a small hole in the sclera at the injection site. A new micropipette containing either balanced salt solution alone, or balanced salt solution with diluted AAV was prepared for each injection and carefully guided through the scleral incision in a tangential direction to reach the vitreous cavity. The AAV solution (IxlO9vector genomes (vgs) / eye) was slowly injected into the vitreous cavity, taking care to avoid rapid changes in intraocular- pressure. Following the injection, the mice were placed on an adjustable temperature-controlled heating pad and monitored until full recovery from anesthesia. One-week post-injection, the mice were euthanized, and retinal tissue was harvested for analysis. Protein extraction was performed on fresh mouse retina samples using RIPA buffer supplemented with protease and phosphatase inhibitors. The tissues were homogenized, lysed, and centrifuged to obtain soluble protein extracts. PEDF levels in the extracted proteins were quantified using the Human PEDF ELISA Kit (Abeam, ab213815) according to the manufacturer's instructions.

[0232] Figure 3 shows the ELISA analyses of PEDF concentrations from the whole mouse retinas 1 week after intravitreal injection of the constructs. Our data illustrates that AAV9-CO4 shows superior expression in comparison to AAV9-PEDF(WT) and AAV9-CO2.

[0233] For the sodium iodate (Sl)-induced mouse model of dry AMD, NalOa (Sigma-Aldrich, St. Louis, MO) was dissolved in 0.9% saline at a concentration of 1% (w / v) solution. The solution was injected intraperitoneally to 3-month-old C57BL6 / J mice at 3.5 pl / g to yield a dose of 35 mg / kg. One week prior to SI treatment, mice that were treated with AAV underwent intravitreal injection of AAV9-CO4 (IxlO9vgs / eye) as described above.

[0234] Four weeks after SI treatment, the retinal structures of mice in the different experimental groups were assessed by optical coherence tomography (OCT). Mice were first anaesthetized by ketamine and xylazine based on their body weight (0.08 mg ketamine / g and 0.01 mg xylazine / g). Following anesthesia, the pupils were dilated using sterile tropicamide (0.5%) and phenylephrine (2.5%) eyedrops. A drop of each solution was applied to both eyes and allowed to take effect for 01001 / 012745-WQ0

[0235] 5-10 minutes. To prevent corneal desiccation and cataract formation during the imaging procedure, a layer of mcthylccllulosc lubricating gel was gently applied to the surface of the cornea. For image acquisition, the anesthetized mouse was placed on a platform that was adjusted to ensure proper alignment of the mouse's eye with the imaging lens and a heating pad was placed underneath the animal to maintain its body temperature within the normal physiological range. For imaging, a Heidelberg Spectralis SD-OCT (Heidelberg Engineering Inc., Dossenheim, Germany) system was utilized, and superior, inferior, nasal, and temporal optical sections of the retina were acquired to comprehensively assess the retinal structure. The acquired SD-OCT and fundoscopy images were processed and analyzed using the Bioptigen In Vivo Vue software provided by the imaging system manufacturer. Quantitative measurements of retinal layer thicknesses were performed at specific regions of interest, and comparisons were made between experimental groups to assess the effects of both the SI treatment and the AAV9- CO4 treatment on retinal structure.

[0236] Quantification of total retina thickness (Figure 4) shows that pre-treatment with AAV9- CO4 (IxlO9vgs / eye) prior to induction of retinal degeneration by SI treatment preserves retinal structure.

[0237] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the ail will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions and dimensions. Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety. Variations, modifications and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention. While certain embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the spirit and scope of the invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not as a limitation. 01001 / 012745-WQ0

[0238] SEO ID NO: 1: wildtype PEDF (RefSeq NM 001329903)

[0239] ATGCAGGCCCTGGTGCTACTCCTCTGCATTGGAGCCCTCCTCGGGCACAGCAGCTGC

[0240] CAGAACCCTGCCAGCCCCCCGGAGGAGGGCTCCCCAGACCCCGACAGCACAGGGGC

[0241] GCTGGTGGAGGAGGAGGATCCTTTCTTCAAAGTCCCCGTGAACAAGCTGGCAGCGG

[0242] CTGTCTCCAACTTCGGCTATGACCTGTACCGGGTGCGATCCAGCACGAGCCCCACGA

[0243] CCAACGTGCTCCTGTCTCCTCTCAGTGTGGCCACGGCCCTCTCGGCCCTCTCGCTGGG

[0244] AGCGGAGCAGCGAACAGAATCCATCATTCACCGGGCTCTCTACTATGACTTGATCAGC

[0245] AGCCCAGACATCCATGGTACCTATAAGGAGCTCCTTGACACGGTCACTGCCCCCCAG

[0246] AAGAACCTCAAGAGTGCCTCCCGGATCGTCTTTGAGAAGAAGCTGCGCATAAAATCC

[0247] AGCTTTGTGGCACCTCTGGAAAAGTCATATGGGACCAGGCCCAGAGTCCTGACGGGC

[0248] AACCCTCGCTTGGACCTGCAAGAGATCAACAACTGGGTGCAGGCGCAGATGAAAGG

[0249] GAAGCTCGCCAGGTCCACAAAGGAAATTCCCGATGAGATCAGCATTCTCCTTCTCGG

[0250] TGTGGCGCACTTCAAGGGGCAGTGGGTAACAAAGTTTGACTCCAGAAAGACTTCCCT

[0251] CGAGGATTTCTACTTGGATGAAGAGAGGACCGTGAGGGTCCCCATGATGTCGGACCC

[0252] TAAGGCTGTTTTACGCTATGGCTTGGATTCAGATCTCAGCTGCAAGATTGCCCAGCTG

[0253] CCCTTGACCGGAAGCATGAGTATCATCTTCTTCCTGCCCCTGAAAGTGACCCAGAATT

[0254] TGACCTTGATAGAGGAGAGCCTCACCTCCGAGTTCATTCATGACATAGACCGAGAAC

[0255] TGAAGACCGTGCAGGCGGTCCTCACTGTCCCCAAGCTGAAGCTGAGTTATGAAGGCG

[0256] AAGTCACCAAGTCCCTGCAGGAGATGAAGCTGCAATCCTTGTTTGATTCACCAGACT

[0257] TTAGCAAGATCACAGGCAAACCCATCAAGCTGACTCAGGTGGAACACCGGGCTGGCT

[0258] TTGAGTGGAACGAGGATGGGGCGGGAACCACCCCCAGCCCAGGGCTGCAGCCTGCC

[0259] CACCTCACCTTCCCGCTGGACTATCACCTTAACCAGCCTTTCATCTTCGTACTGAGGG

[0260] ACACAGACACAGGGGCCCTTCTCTTCATTGGCAAGATTCTGGACCCCAGGGGCCCCT

[0261] AA

[0262] SEO ID NO: 2: CO1 - Codon optimized variant 1

[0263] ATGCAAGCTCTTGTCTTGCTGCTGTGTATTGGTGCACTGCTGGGACACTCCTCTTGTC

[0264] AGAACCCGGCGAGTCCCCCCGAAGAAGGCTCACCCGATCCCGATTCTACGGGTGCAT

[0265] TGGTTGAAGAGGAGGATCCTTTCTTTAAGGTACCGGTAAATAAGCTCGCTGCCGCAGT 01001 / 012745-WQ0

[0266] CTCAAACTTTGGATATGACCTCTACCGGGTGCGGTCCTCTACTAGTCCGACAACCAAT

[0267] GTCTTGCTTTCACCTCTTTCCGTTGCTACAGCACTCTCCGCTTTGAGTTTGGGTGCCG

[0268] AGCAGAGGACCGAATCCATAATTCACAGGGCGCTCTACTACGATCTCATAAGTTCTCC

[0269] TGATATTCACGGTACGTATAAAGAGCTTCTTGATACAGTGACAGCGCCTCAAAAGAAC

[0270] CTCAAATCCGCCTCTCGGATAGTTTTCGAAAAGAAACTGAGAATTAAATCAAGTTTCG

[0271] TCGCGCCACTCGAAAAGTCTTACGGAACTAGGCCCAGGGTCTTGACCGGGAACCCTC

[0272] GACTCGATCTGCAGGAGATAAACAATTGGGTTCAAGCTCAAATGAAGGGAAAACTCG

[0273] CACGCAGTACCAAGGAAATACCAGATGAGATTAGTATACTCCTCCTTGGTGTCGCACA

[0274] CTTCAAAGGCCAGTGGGTTACGAAATTTGATTCCAGAAAAACATCTTTGGAGGACTT

[0275] CTACTTGGATGAGGAACGAACAGTGAGGGTACCAATGATGTCAGACCCCAAAGCAGT

[0276] ACTGCGATACGGTCTGGATAGCGACCTCTCCTGTAAAATAGCACAGTTGCCACTCACT

[0277] GGAAGCATGTCAATTATATTTTTCTTGCCACTGAAGGTTACCCAGAACTTGACCTTGAT

[0278] TGAGGAATCTCTGACGTCTGAGTTTATACATGACATAGATAGAGAGTTGAAGACCGTC

[0279] CAAGCCGTATTGACGGTCCCCAAGTTGAAGCTCTCTTACGAGGGTGAAGTTACTAAA

[0280] AGTCTGCAAGAGATGAAACTCCAGTCTCTCTTTGATAGTCCTGATTTTTCTAAGATCA

[0281] CTGGGAAACCCATAAAGTTGACCCAAGTTGAACATCGCGCCGGGTTCGAGTGGAACG

[0282] AAGATGGAGCGGGGACGACTCCATCTCCCGGTTTGCAGCCTGCTCATCTTACCTTCCC

[0283] GTTGGATTATCATTTGAATCAACCCTTTATCTTCGTCCTGCGGGATACTGACACGGGCG

[0284] CCCTGTTGTTCATCGGTAAGATTCTGGACCCTCGAGGCCCTTAA

[0285] SEO ID NO: 3 : CO2 - Codon optimized variant 2

[0286] ATGCAAGCTCTTGTCTTGCTGCTGTGTATTGGAGCCCTCCTCGGACACTCCTCTTGTC

[0287] AGAACCCGGCGAGTCCCCCCGAGGAGGGCTCCCCCGATCCCGATTCTACGGGTGCAT

[0288] TGGTTGAAGAGGAGGATCCTTTCTTTAAGGTACCGGTAAATAAGCTCGCTGCCGCAGT

[0289] CTCAAACTTTGGATATGACCTCTACCGGGTGCGGTCCTCTACTAGTCCGACAACCAAT

[0290] GTCTTGCTTTCACCTCTTTCCGTTGCTACAGCACTCTCCGCTTTGAGTTTGGGTGCCG

[0291] AGCAGAGGACCGAATCCATAATTCACAGGGCGCTCTACTACGATCTCATAAGTTCTCC

[0292] TGATATTCACGGTACGTATAAAGAGCTTCTTGATACAGTGACAGCGCCTCAAAAGAAC

[0293] CTCAAATCCGCCTCTCGGATAGTTTTCGAAAAGAAACTGAGAATTAAATCAAGTTTCG

[0294] TCGCGCCACTCGAAAAGTCTTACGGAACTAGGCCCAGGGTCTTGACCGGGAACCCTC

[0295] GACTCGATCTGCAGGAGATAAACAATTGGGTTCAAGCTCAAATGAAGGGAAAACTCG 01001 / 012745-WQ0

[0296] CACGCAGTACCAAGGAAATACCAGATGAGATTAGTATACTCCTCCTTGGTGTCGCACA

[0297] CTTCAAAGGCCAGTGGGTTACGAAATTTGATTCCAGAAAAACATCTTTGGAGGACTT

[0298] CTACTTGGATGAGGAACGAACAGTGAGGGTACCAATGATGTCAGACCCCAAAGCAGT

[0299] ACTGCGATACGGTCTGGATAGCGACCTCTCCTGTAAAATAGCACAGTTGCCACTCACT

[0300] GGAAGCATGTCAATTATATTTTTCTTGCCACTGAAGGTTACCCAGAACTTGACCTTGAT

[0301] TGAGGAATCTCTGACGTCTGAGTTTATACATGACATAGATAGAGAGTTGAAGACCGTC

[0302] CAAGCCGTATTGACGGTCCCCAAGTTGAAGCTCTCTTACGAGGGTGAAGTTACTAAA

[0303] AGTCTGCAAGAGATGAAACTCCAGTCTCTCTTTGATAGTCCTGATTTTTCTAAGATCA

[0304] CTGGGAAACCCATAAAGTTGACCCAAGTTGAACATCGCGCCGGGTTCGAGTGGAACG

[0305] AAGATGGAGCGGGGACGACTCCATCTCCCGGTTTGCAGCCTGCTCATCTTACCTTCCC

[0306] GTTGGATTATCATTTGAATCAACCCTTTATCTTCGTCCTGCGGGATACTGACACGGGCG

[0307] CCCTGTTGTTCATCGGTAAGATTCTGGACCCTCGAGGCCCTTAA

[0308] SEO ID NO: 4: CO3 - Codon optimized variant 3

[0309] ATGCAGGCACTGGTGCTGCTCCTCTGTATTGGTGCACTGCTGGGCCACAGCTCCTGCC

[0310] AAAACCCAGCCTCACCCCCAGAAGAAGGCTCACCTGACCCAGACTCGACTGGAGCC

[0311] CTGGTGGAGGAAGAAGACCCTTTCTTCAAGGTGCCTGTTAACAAACTGGCAGCTGCG

[0312] GTGAGCAACTTTGGCTACGATCTTTACAGAGTGCGGAGCAGCACGAGCCCCACCACC

[0313] AATGTTCTGCTGAGCCCCCTGAGTGTGGCCACTGCCCTGAGTGCCCTGAGCCTGGGC

[0314] GCTGAGCAGCGAACAGAAAGCATCATCCACAGGGCCCTCTACTATGACTTGATCAGC

[0315] AGCCCGGATATCCATGGCACCTACAAGGAACTGTTGGACACTGTAACAGCCCCCCAG

[0316] AAGAACCTCAAGAGTGCCTCCCGTATTGTGTTTGAAAAGAAGTTGAGAATCAAGAGC

[0317] AGTTTCGTGGCTCCTCTGGAGAAGAGCTACGGCACCAGACCCCGGGTGCTGACAGG

[0318] AAACCCCAGGCTGGACCTTCAGGAAATCAACAACTGGGTTCAGGCCCAGATGAAGG

[0319] GAAAGCTGGCCCGGTCTACCAAGGAGATCCCTGATGAAATCAGCATCCTACTGTTAG

[0320] GCGTGGCTCACTTCAAAGGACAGTGGGTGACCAAATTTGATTCAAGGAAGACCTCTC

[0321] TGGAGGACTTCTACCTAGATGAGGAAAGAACAGTGAGAGTTCCCATGATGAGTGACC

[0322] CGAAGGCAGTCCTGCGCTACGGTCTGGACTCTGACCTCAGCTGCAAAATCGCTCAAT

[0323] TGCCTTTAACAGGCAGTATGAGCATCATCTTCTTCCTCCCCTTGAAAGTAACCCAGAA

[0324] CCTGACCCTCATTGAAGAGTCCCTGACATCAGAGTTCATTCATGACATCGATAGAGAG

[0325] CTGAAGACGGTGCAGGCTGTGCTGACTGTGCCCAAATTGAAGCTGAGCTATGAGGGT 01001 / 012745-WQ0

[0326] GAGGTCACAAAGTCTCTGCAGGAGATGAAACTACAGTCCCTCTTCGACTCCCCTGAC

[0327] TTCAGCAAGATCACTGGGAAGCCAATTAAGCTGACGCAGGTGGAGCACCGCGCTGG

[0328] CTTTGAGTGGAATGAAGACGGAGCAGGCACCACACCTAGCCCTGGGCTGCAGCCAG

[0329] CCCATCTGACCTTCCCTCTGGACTACCACCTGAATCAACCCTTTATCTTTGTGCTAAGA

[0330] GACACAGACACCGGCGCCCTGCTCTTCATCGGCAAGATACTCGATCCTCGGGGGCCT TGA

[0331] SEO ID NO: 5: CO4 - Codon optimized variant 4

[0332] ATGCAGGCACTGGTGCTGCTCCTCTGTATTGGAGCCCTCCTCGGCCACAGCTCCTGCC

[0333] AAAACCCAGCCTCACCCCCAGAGGAGGGCTCCCCTGACCCAGACTCGACTGGAGCC

[0334] CTGGTGGAGGAAGAAGACCCTTTCTTCAAGGTGCCTGTTAACAAACTGGCAGCTGCG

[0335] GTGAGCAACTTTGGCTACGATCTTTACAGAGTGCGGAGCAGCACGAGCCCCACCACC

[0336] AATGTTCTGCTGAGCCCCCTGAGTGTGGCCACTGCCCTGAGTGCCCTGAGCCTGGGC

[0337] GCTGAGCAGCGAACAGAAAGCATCATCCACAGGGCCCTCTACTATGACTTGATCAGC

[0338] AGCCCGGATATCCATGGCACCTACAAGGAACTGTTGGACACTGTAACAGCCCCCCAG

[0339] AAGAACCTCAAGAGTGCCTCCCGTATTGTGTTTGAAAAGAAGTTGAGAATCAAGAGC

[0340] AGTTTCGTGGCTCCTCTGGAGAAGAGCTACGGCACCAGACCCCGGGTGCTGACAGG

[0341] AAACCCCAGGCTGGACCTTCAGGAAATCAACAACTGGGTTCAGGCCCAGATGAAGG

[0342] GAAAGCTGGCCCGGTCTACCAAGGAGATCCCTGATGAAATCAGCATCCTACTGTTAG

[0343] GCGTGGCTCACTTCAAAGGACAGTGGGTGACCAAATTTGATTCAAGGAAGACCTCTC

[0344] TGGAGGACTTCTACCTAGATGAGGAAAGAACAGTGAGAGTTCCCATGATGAGTGACC

[0345] CGAAGGCAGTCCTGCGCTACGGTCTGGACTCTGACCTCAGCTGCAAAATCGCTCAAT

[0346] TGCCTTTAACAGGCAGTATGAGCATCATCTTCTTCCTCCCCTTGAAAGTAACCCAGAA

[0347] CCTGACCCTCATTGAAGAGTCCCTGACATCAGAGTTCATTCATGACATCGATAGAGAG

[0348] CTGAAGACGGTGCAGGCTGTGCTGACTGTGCCCAAATTGAAGCTGAGCTATGAGGGT

[0349] GAGGTCACAAAGTCTCTGCAGGAGATGAAACTACAGTCCCTCTTCGACTCCCCTGAC

[0350] TTCAGCAAGATCACTGGGAAGCCAATTAAGCTGACGCAGGTGGAGCACCGCGCTGG

[0351] CTTTGAGTGGAATGAAGACGGAGCAGGCACCACACCTAGCCCTGGGCTGCAGCCAG

[0352] CCCATCTGACCTTCCCTCTGGACTACCACCTGAATCAACCCTTTATCTTTGTGCTAAGA

[0353] GACACAGACACCGGCGCCCTGCTCTTCATCGGCAAGATACTCGATCCTCGGGGGCCT TGA 01001 / 012745-WQ0

[0354] SEO ID NO: 6: CBh promoter

[0355] CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCC

[0356] ATTGACGTCAATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTAT

[0357] TTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCC

[0358] CTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTT

[0359] ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCG

[0360] AGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAAT

[0361] TTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGG

[0362] GGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGA

[0363] GAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGA

[0364] GGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCT

[0365] GCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGG

[0366] CTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG

[0367] GGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTA

[0368] TTAATGTTTAATTACCTGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGG

[0369] SEO ID NO: 7: EGFP

[0370] ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG

[0371] GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGC

[0372] CACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC

[0373] CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC

[0374] CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCA

[0375] GGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGA

[0376] AGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAG

[0377] GAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGT

[0378] CTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCA

[0379] CAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCA

[0380] TCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCC

[0381] TGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACC

[0382] GCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA 01001 / 012745-WQ0

[0383] SEO ID NO: 8: WPRE

[0384] CGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATG

[0385] TTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTT

[0386] CCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGG

[0387] AGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAA

[0388] CCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTT

[0389] CCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACA

[0390] GGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCC

[0391] TTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCT

[0392] ACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCT

[0393] GCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCC

[0394] GCCTCCCCGCATCGG

[0395] SEO ID NO: 9: Bovine Growth Hormone polyA

[0396] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACC

[0397] CTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATT

[0398] GTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGG

[0399] AGGATTGGGAAGAGAATAGCAGGCATGCTGGGGA

[0400] SEO ID NO: 10: AAV9-CBh-EGFP

[0401] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

[0402] CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT

[0403] CCATCACTAGGGGTTCCTTCTAGACAACTTTGTATAGAAAAGTTGCGTTACATAACTTA

[0404] CGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAG

[0405] TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC

[0406] CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATG

[0407] ACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTAC

[0408] TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGT

[0409] TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTT

[0410] TTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCG 01001 / 012745-WQ0

[0411] GGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAG

[0412] CCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGC

[0413] GGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGC

[0414] CCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCG

[0415] TTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTG

[0416] AGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACC

[0417] TGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCAAGTTTGTACAAAAAAGCA

[0418] GGCTGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCT

[0419] GGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCG

[0420] AGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGC

[0421] TGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCA

[0422] GCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAG

[0423] GCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCG

[0424] CCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATC

[0425] GACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAG

[0426] CCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAA

[0427] GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGA

[0428] ACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCC

[0429] AGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAG

[0430] TTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAACCCAG

[0431] CTTTCTTGTACAAAGTGGGAATTCCGATAATCAACCTCTGGATTACAAAATTTGTGAA

[0432] AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTA

[0433] ATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAAT

[0434] CCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGT

[0435] GTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA

[0436] GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCC

[0437] GCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTG

[0438] GTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCCTGTGTTGCCACCTGGA

[0439] TTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCC

[0440] TTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAG

[0441] ACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGGAATTCCTAGAGCTCGCTG 01001 / 012745-WQ0

[0442] ATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTG

[0443] CCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAA

[0444] TTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGA

[0445] CAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCTGGGGAGGGCCGCAGG

[0446] AACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG

[0447] CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGC

[0448] GAGCGAGCGCGCAGCTGCCTGCAGG

[0449] SEO ID NO: 11: AAV9-CBh-PEDF

[0450] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

[0451] CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT

[0452] CCATCACTAGGGGTTCCTTCTAGACAACTTTGTATAGAAAAGTTGCGTTACATAACTTA

[0453] CGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAG

[0454] TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC

[0455] CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATG

[0456] ACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTAC

[0457] TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGT

[0458] TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTT

[0459] TTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCG

[0460] GGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAG

[0461] CCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGC

[0462] GGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGC

[0463] CCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCG

[0464] TTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTG

[0465] AGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACC

[0466] TGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCAAGTTTGTACAAAAAAGCA

[0467] GGCTGCCACCATGCAGGCCCTGGTGCTACTCCTCTGCATTGGAGCCCTCCTCGGGCAC

[0468] AGCAGCTGCCAGAACCCTGCCAGCCCCCCGGAGGAGGGCTCCCCAGACCCCGACAG

[0469] CACAGGGGCGCTGGTGGAGGAGGAGGATCCTTTCTTCAAAGTCCCCGTGAACAAGC

[0470] TGGCAGCGGCTGTCTCCAACTTCGGCTATGACCTGTACCGGGTGCGATCCAGCACGA

[0471] GCCCCACGACCAACGTGCTCCTGTCTCCTCTCAGTGTGGCCACGGCCCTCTCGGCCC 01001 / 012745-WQ0

[0472] TCTCGCTGGGAGCGGAGCAGCGAACAGAATCCATCATTCACCGGGCTCTCTACTATG

[0473] ACTTGATCAGCAGCCCAGACATCCATGGTACCTATAAGGAGCTCCTTGACACGGTCAC

[0474] TGCCCCCCAGAAGAACCTCAAGAGTGCCTCCCGGATCGTCTTTGAGAAGAAGCTGCG

[0475] CATAAAATCCAGCTTTGTGGCACCTCTGGAAAAGTCATATGGGACCAGGCCCAGAGT

[0476] CCTGACGGGCAACCCTCGCTTGGACCTGCAAGAGATCAACAACTGGGTGCAGGCGC

[0477] AGATGAAAGGGAAGCTCGCCAGGTCCACAAAGGAAATTCCCGATGAGATCAGCATTC

[0478] TCCTTCTCGGTGTGGCGCACTTCAAGGGGCAGTGGGTAACAAAGTTTGACTCCAGAA

[0479] AGACTTCCCTCGAGGATTTCTACTTGGATGAAGAGAGGACCGTGAGGGTCCCCATGA

[0480] TGTCGGACCCTAAGGCTGTTTTACGCTATGGCTTGGATTCAGATCTCAGCTGCAAGAT

[0481] TGCCCAGCTGCCCTTGACCGGAAGCATGAGTATCATCTTCTTCCTGCCCCTGAAAGTG

[0482] ACCCAGAATTTGACCTTGATAGAGGAGAGCCTCACCTCCGAGTTCATTCATGACATAG

[0483] ACCGAGAACTGAAGACCGTGCAGGCGGTCCTCACTGTCCCCAAGCTGAAGCTGAGT

[0484] TATGAAGGCGAAGTCACCAAGTCCCTGCAGGAGATGAAGCTGCAATCCTTGTTTGAT

[0485] TCACCAGACTTTAGCAAGATCACAGGCAAACCCATCAAGCTGACTCAGGTGGAACAC

[0486] CGGGCTGGCTTTGAGTGGAACGAGGATGGGGCGGGAACCACCCCCAGCCCAGGGCT

[0487] GCAGCCTGCCCACCTCACCTTCCCGCTGGACTATCACCTTAACCAGCCTTTCATCTTC

[0488] GTACTGAGGGACACAGACACAGGGGCCCTTCTCTTCATTGGCAAGATTCTGGACCCC

[0489] AGGGGCCCCTAAACCCAGCTTTCTTGTACAAAGTGGGAATTCCGATAATCAACCTCTG

[0490] GATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCT

[0491] ATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCAT

[0492] TTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTG

[0493] TCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGG

[0494] GCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCC

[0495] ACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTG

[0496] GGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTC

[0497] GCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCC

[0498] TCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCG

[0499] TCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGG

[0500] GAATTCCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGT

[0501] TGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTT

[0502] TCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGG 01001 / 012745-WQ0

[0503] GGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCAT

[0504] GCTGGGGAGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCG

[0505] CTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCC

[0506] GGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0507] SEO ID NO: 12: AAV9-CBh-CO4

[0508] CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGAC

[0509] CTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACT

[0510] CCATCACTAGGGGTTCCTTCTAGACAACTTTGTATAGAAAAGTTGCGTTACATAACTTA

[0511] CGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAG

[0512] TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC

[0513] CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATG

[0514] ACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTTATGGGACTTTCCTAC

[0515] TTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGT

[0516] TCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTT

[0517] TTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCG

[0518] GGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAG

[0519] CCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGC

[0520] GGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGC

[0521] CCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCG

[0522] TTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTG

[0523] AGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACC

[0524] TGGAGCACCTGCCTGAAATCACTTTTTTTCAGGTTGGCAAGTTTGTACAAAAAAGCA

[0525] GGCTGCCACCATGCAGGCACTGGTGCTGCTCCTCTGTATTGGAGCCCTCCTCGGCCAC

[0526] AGCTCCTGCCAAAACCCAGCCTCACCCCCAGAGGAGGGCTCCCCTGACCCAGACTC

[0527] GACTGGAGCCCTGGTGGAGGAAGAAGACCCTTTCTTCAAGGTGCCTGTTAACAAACT

[0528] GGCAGCTGCGGTGAGCAACTTTGGCTACGATCTTTACAGAGTGCGGAGCAGCACGAG

[0529] CCCCACCACCAATGTTCTGCTGAGCCCCCTGAGTGTGGCCACTGCCCTGAGTGCCCT

[0530] GAGCCTGGGCGCTGAGCAGCGAACAGAAAGCATCATCCACAGGGCCCTCTACTATGA

[0531] CTTGATCAGCAGCCCGGATATCCATGGCACCTACAAGGAACTGTTGGACACTGTAACA

[0532] GCCCCCCAGAAGAACCTCAAGAGTGCCTCCCGTATTGTGTTTGAAAAGAAGTTGAGA 01001 / 012745-WQ0

[0533] ATCAAGAGCAGTTTCGTGGCTCCTCTGGAGAAGAGCTACGGCACCAGACCCCGGGTG

[0534] CTGACAGGAAACCCCAGGCTGGACCTTCAGGAAATCAACAACTGGGTTCAGGCCCA

[0535] GATGAAGGGAAAGCTGGCCCGGTCTACCAAGGAGATCCCTGATGAAATCAGCATCCT

[0536] ACTGTTAGGCGTGGCTCACTTCAAAGGACAGTGGGTGACCAAATTTGATTCAAGGAA

[0537] GACCTCTCTGGAGGACTTCTACCTAGATGAGGAAAGAACAGTGAGAGTTCCCATGAT

[0538] GAGTGACCCGAAGGCAGTCCTGCGCTACGGTCTGGACTCTGACCTCAGCTGCAAAAT

[0539] CGCTCAATTGCCTTTAACAGGCAGTATGAGCATCATCTTCTTCCTCCCCTTGAAAGTAA

[0540] CCCAGAACCTGACCCTCATTGAAGAGTCCCTGACATCAGAGTTCATTCATGACATCGA

[0541] TAGAGAGCTGAAGACGGTGCAGGCTGTGCTGACTGTGCCCAAATTGAAGCTGAGCTA

[0542] TGAGGGTGAGGTCACAAAGTCTCTGCAGGAGATGAAACTACAGTCCCTCTTCGACTC

[0543] CCCTGACTTCAGCAAGATCACTGGGAAGCCAATTAAGCTGACGCAGGTGGAGCACCG

[0544] CGCTGGCTTTGAGTGGAATGAAGACGGAGCAGGCACCACACCTAGCCCTGGGCTGC

[0545] AGCCAGCCCATCTGACCTTCCCTCTGGACTACCACCTGAATCAACCCTTTATCTTTGT

[0546] GCTAAGAGACACAGACACCGGCGCCCTGCTCTTCATCGGCAAGATACTCGATCCTCG

[0547] GGGGCCTTGAACCCAGCTTTCTTGTACAAAGTGGGAATTCCGATAATCAACCTCTGGA

[0548] TTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTAT

[0549] GTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTT

[0550] TCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTC

[0551] AGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGC

[0552] ATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCAC

[0553] GGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGG

[0554] CACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATGGCTGCTCGCC

[0555] TGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCA

[0556] ATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCT

[0557] TCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGGGA

[0558] ATTCCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTG

[0559] TTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTC

[0560] CTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGG

[0561] GTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAGAATAGCAGGCATGCT

[0562] GGGGAGGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTC GCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGG

[0563] GCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[0564] SEQ ID NO; 13: wildtype PEDF nucleotides 31-42 ggagccctcctc

[0565] SEQ ID NO: 14: wildtype PEDF nucleotides 79-90 gaggagggctcc SEQ ID NO: 15: wildtype PEDF amino acid sequence

[0566] MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAAVSN

[0567] FGYDLYRVRSSTSPTTNVLLSPLSVATALSALSLGAEQRTESIIHRALYYDLISSPDIHGTYK

[0568] ELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNPRLDLQEINNWV

[0569] QAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPM MSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELK

[0570] TVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNE

[0571] DGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTDTGALLFIGKILDPRGP

Claims

Claims1. A nucleic acid molecule encoding human pigment epithelium-derived factor (PEDF), comprising nucleotides 31-42 and 79-90 of the wildtype PEDF coding sequence which encode a stem- loop hairpin in a messenger RNA (mRNA) transcript of the nucleic acid molecule, wherein nucleotides 31-42 and 79-90 of the wildtype PEDF coding sequence have the nucleotide sequences set forth in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, wherein the nucleic acid molecule is human codon-optimized, and wherein the nucleic acid molecule is no greater than 95% identical to the wildtype PEDF coding sequence set forth in SEQ ID NO:1.

2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is no greater than 90% identical to the wildtype PEDF coding sequence.

3. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is no greater than 85% identical to the wildtype PEDF coding sequence.

4. The nucleic acid molecule of claim 1, comprising a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:5.

5. The nucleic acid molecule of claim 1, comprising a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

6. The nucleic acid molecule of any of the previous claims, wherein the nucleic acid molecule encodes a wildtype PEDF protein comprising the amino acid sequence set forth in SEQ ID NO: 15.

7. The nucleic acid molecule of any of the previous claims, further comprising AAV 5’ and 3’ inverted terminal repeat (ITR) sequences.

8. The nucleic acid molecule of any of the previous claims, further comprising AAV2 5’ and 3’ inverted terminal repeat (ITR) sequences.

9. The nucleic acid molecule of any of the previous claims, further comprising a promoter element.

10. The nucleic acid molecule of claim 9, wherein the promoter element comprises a chicken beta- actin (CB) promoter.

11. The nucleic acid molecule of claim 9, wherein the promoter element comprises a cytomegalovirus (CMV) early promoter sequence, a chicken beta-actin (CB) promoter sequence, a truncated chicken 0-actin intron sequence, and a minute vims of mouse (MVM) intron sequence.

12. The nucleic acid molecule of any of claims 9-11, wherein the promoter element comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:6.

13. The nucleic acid molecule of any of the previous claims, further comprising a post- transcriptional regulatory element.

14. The nucleic acid molecule of claim 13, wherein the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

15. The nucleic acid molecule of claim 13 or 14, wherein the post-transcriptional regulatory element comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:8.

16. The nucleic acid molecule of any of the previous claims, further comprising a polyadenylation signal sequence.

17. The nucleic acid molecule of claim 16, wherein the polyadenylation signal sequence is a bovine growth hormone polyadenylation signal sequence.

18. The nucleic acid molecule of claim 16 or 17, wherein the polyadcnylation signal sequence comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO:9.

19. The nucleic acid molecule of any of the previous claims, wherein the nucleic acid molecule comprises a recombinant adeno-associated virus (AAV) vector.

20. The nucleic acid molecule of claim 19, wherein the AAV vector is AAV9.

21. The nucleic acid molecule of any of the previous claims, wherein the nucleic acid molecule comprises a nucleotide sequence about 80% to about 100% identical to the nucleotide sequence set forth in SEQ ID NO: 12.

22. A vector comprising the nucleic acid molecule of any of the previous claims.

23. A cell comprising the nucleic acid molecule of any of claims 1-21.

24. A cell comprising the vector of claim 22.

25. A pharmaceutical composition comprising the nucleic acid molecule of any of claims 1- 21.

26. A pharmaceutical composition comprising the vector of claim 22.

27. A pharmaceutical composition comprising the cell of claim 23 or 24.

28. A method of treating a disorder in a subject, the method comprising administering to the subject the pharmaceutical composition of any of claims 25-27, the nucleic acid molecule of any of claims 1-21, the vector of claim 22, or the cell of claim 23 or 24.

29. The method of claim 28, wherein the disorder is an ocular degenerative disease or a retinal degenerative disease.

30. The method of claim 29, wherein the ocular degenerative disease is age-related macular degeneration (AMD), retinitis pigmentosa (RP), diabetic retinopathy (DR), or glaucoma.

31. The method of claim 28, wherein the disorder is a neurodegenerative disease.

32. The method of 31, wherein the neurodegenerative disease is amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, Batten disease, or a prion disease.

33. The method of claim 28, wherein the disorder is cancer.

34. The method of claim 28, wherein the disorder is a cardiovascular disease.

35. The method of any of claims 28-34, wherein the pharmaceutical composition is administered by intravitreal injection.

36. The method of any of claims 28-34, wherein the pharmaceutical composition is administered by subretinal injection.

37. The method of any of claims 28-34, wherein the pharmaceutical composition is administered to the central nervous system (CNS) of the subject.

38. The method of any of claims 28-34, wherein the pharmaceutical composition is administered to the spinal cord of the subject.

39. The method of any of claims 28-34, wherein the pharmaceutical composition is administered by intrathecal injection.

40. The method of any of claims 28-34, wherein the pharmaceutical composition isadministered orally, intravenously, intramuscularly, topically, arterially, or subcutaneously.

41. The method of any of claims 28-40, wherein the subject is a mammal.

42. The method of claim 41, wherein the mammal is a human, a rodent, or a simian.

43. The method of claim 41, wherein the mammal is a human.