EP4630028A1 - Pigment epithelium-derived factor peptides and use for treating retinal degeneration - Google Patents

Abstract

Provided herein are compositions and methods for treating retinal degeneration, such as an inherited retinal degeneration. In some examples, the compositions include a pigment epithelium-derived factor (PEDF) protein or a peptide thereof. Compositions including a PEDF 17mer[H105A] peptide in an eye drop formulation or an adeno-associated virus vector including a nucleic acid encoding a PEDF protein or PEDF 17mer[H105A] peptide are provided. Methods for treating retinal degeneration include administering a provided eyedrop formulation or adeno-associated virus vector to an eye of a subject with retinal degeneration.

Description

PIGMENT EPITHELIUM-DERIVED FACTOR PEPTIDES AND USE FOR TREATING

RETINAL DEGENERATION

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to US Provisional Application No. 63/430,251 filed December 5, 2022, herein incorporated by reference in its entirety.

FIELD

This disclosure relates to pigment epithelium-derived factor and pigment epithelium-derived factor peptides and methods of their use in treating retinal degeneration.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with Government support under project number Z0120892 by the National Institutes of Health, National Eye Institute. The Government has certain rights in the invention.

INCORPORATION OF SEQUENCE LISTING

The Sequence Listing is submitted as an XML file in the form of the file named 4239- 109378-02_sequence_listing.xml, which was created on March 22, 2023, and is 13,627 bytes, which is incorporated by reference herein.

BACKGROUND

Inherited Retinal Degenerations (IRD), like Retinitis Pigmentosa (RP), are characterized by a progressive loss of visual acuity caused by degeneration of photoreceptor cells. The main issue with IRDs is their heterogeneity in both symptoms and genetics. RP can arise from mutations in more than 90 genes and the phenotype of the disease is highly variable. Due to this high heterogeneity, therapeutic approaches targeting specific genes generally benefit few patients, while for most forms of RP few or no medical options are available. Thus, there remains a need to identify new and more effective treatments for RP and other inherited retinal degenerations.

SUMMARY

The common clinical feature among IRDs is progressive photoreceptor degeneration or photoreceptor cell death that leads to irreversible blindness in patients with the different types of RD. Provided herein are compositions and methods that utilize neurotrophic factors such as PEDF, to inhibit or delay photoreceptor cell loss and blindness. This delay may also provide opportunities for further interventions that target the specific cause of the degeneration, e.g. , gene replacement, gene correction, etc. Thus, provided herein are compositions and methods for treating retinal degeneration, such as an inherited retinal degeneration. In some examples, the compositions include a pigment epithelium-derived factor protein or a peptide thereof (for example, PEDF 17mer[H105A] peptide).

In some embodiments, provided herein is a composition including a PEDF peptide including the amino acid sequence of SEQ ID NO: 1 (also referred to herein as 17mer[H105A] or H105A peptide), wherein the composition is formulated as an eye drop. In some examples, the PEDF peptide is about 15-19 amino acids long, such as about 17 amino acids long. In some examples, the eye drop formulation includes a saline solution, such as a buffered saline solution (for example, Hank’s balanced salt solution). In one example, the composition includes about 1 mg/ml of PEDF peptide.

Also provided are methods of treating a retinal degeneration with the eye drop composition. In some embodiments, the methods include administering the composition including the PEDF peptide to an eye of a subject with retinal degeneration (such as a subject with retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), or age-related macular degeneration). In some examples, the composition is administered topically to the eye of the subject. In some examples, the composition is administered to the eye of the subject once daily.

In other embodiments, provided herein are adeno- associated virus (AAV) vectors including a nucleic acid encoding a pigment epithelium-derived factor protein. In further embodiments, provided herein are AAV vectors including a nucleic acid encoding a pigment epithelium-derived factor peptide comprising SEQ ID NO: 1, wherein the nucleic acid encoding the peptide is operably linked to a nucleic acid encoding a signal peptide. In some examples, the vector is an AAV2 vector (for example, an AAV2.1 vector or an AAV2/2 vims). In further examples, a composition including the AAV vector including a nucleic acid encoding the PEDF protein or PEDF peptide in a composition formulated for injection is provided.

In some examples, the signal peptide operably linked to the nucleic acid encoding the PEDF peptide is an interferon beta signal peptide. In one example, the interferon beta signal peptide includes the amino acid sequence of SEQ ID NO: 2, for example, is encoded by the nucleic acid sequence of SEQ ID NO: 3. In additional examples, the PEDF peptide is encoded by the nucleic acid sequence of SEQ ID NO: 4. In other examples, the nucleic acid encoding the PEDF peptide operably linked to a nucleic acid encoding the signal peptide includes the nucleic acid sequence of SEQ ID NO: 5. In further examples, the PEDF protein is encoded by the nucleic acid sequence of SEQ ID NO: 8. In other examples, the nucleic acid encoding the PEDF protein or peptide is operably linked to a promoter.

Also provided are methods of treating a retinal degeneration with the AAV vectors disclosed herein. In some embodiments, the methods include administering the vector or a composition including the vector to an eye of a subject with retinal degeneration (such as a subject with retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), or age-related macular degeneration). In some examples, the composition is administered to the eye of the subject by injection (for example, by intravitreal injection or subretinal injection). In some examples, the composition is administered to the eye of the subject one or more times.

The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and IB show detection of surface phosphatidylserine on dying photoreceptors (PRs) of retinas in culture ex vivo. FIG. 1A is a schematic showing the experimental design. FIG. IB shows representative fluorescent image projections of PRs in retinal flatmounts using confocal super-resolution microscopy (left) and quantification of TUNEL and PSVue® detection (right).

FIG. 2 is representative fluorescent images of rdl 0 retinas showing TUNEL and DAPI staining at postnatal days P15, P21, and P25.

FIGS. 3A-3C show in vivo detection of apoptotic PRs in rdlO mice. FIG. 3A is representative fluorescence images of fundoscopy 24 hours after PSVue® administration. FIG. 3B is a graph of mean fluorescent intensity in the control (HBSS) or PSVue® treated mice. FIG. 3C is a graph showing measurement of three regions of interest (ROI) for each postnatal day and averaged to generate the data and then normalized to HBSS.

FIG. 4 is representative fluorescent images of rdl O/Serpinfl null retinas showing TUNEL and DAPI staining at postnatal days P15, P21, and P25.

FIGS. 5A-5C show in vivo detection of apoptotic PRs in rdlO/Serpinfl null mice. FIG. 5 A is representative fluorescence images of fundoscopy 24 hours after PSVue® administration. FIG. 5B is a graph of mean fluorescent intensity in the control (HBSS) or PSVue® treated mice. FIG. 5C is a graph showing measurement of three regions of interest (ROI) for each postnatal day averaged to generate the data and then normalized to HBSS.

FIGS. 6A-6C show the effect of 17mer[H105A] peptide administration on PR cell death. FIG. 6A is a schematic illustrating the experimental design. Decreased cell death was observed following administration of 17mer[H105A] in both rdlO (FIG. 6B) and rdlO/Serpinfl null (FIG. 6C) mice at day P21.

FIGS. 7A and 7B show dose response of 17mer[H105A] peptide eyedrops in retinas of mice treated as shown in FIG. 6A. Dose response curves for rdlO (FIG. 7 A) and rdlO/Serpinfl null (FIG. 7B) mice are shown.

FIGS. 8 A and 8B show histological evaluation of retinas in mice treated as shown in FIG. 6A. Hematoxylin and eosin staining and measurement of outer nuclear layer (ONL) thickness are shown for rdlO (FIG. 8 A) and rdlO/Serpinfl null (FIG. 8B) mice.

FIGS. 9 A and 9B show assessment of BAX2 (a cell death marker) and BCL2 (a cell survival marker) in rdlO (FIG. 9 A) and rdlO/Serpinfl null (FIG. 9B) mice.

FIGS. 10A-10C show ERG assessment of mice treated as shown in FIG. 6A. In both rdlO (FIG. 10A) and rdlO/Serpinfl null (FIG. 10B) mice, an improvement of the a wave was observed following treatment with 17mer[H105A]. FIG. IOC shows scotopic threshold response (STR) for rdlO (left) and rdlO/Serpinfl null (right) mice at P21. Each data point corresponds to the average ± SEM of each genotype by unpaired t-test. *p < 0.05, **p < 0.001, ****p < 0.00001.

FIG. 11 shows an alignment of WT 17mer (SEQ ID NO: 10), H105A 17mer (SEQ ID NO: 1), WT 29mer (SEQ ID NO: 11) and H105A 29mer (SEQ ID NO: 12) peptides.

FIGS 12A and 12B show a comparison of cell death following administration of 1 mg/ml 17mer[H105A] or wild type 17mer to rdlO (FIG. 12A) or rdlO/Serpinfl null (FIG. 12B) mice treated as shown in FIG. 6A. Each data point corresponds to the average ± SEM of fluorescence relative to HBSS by unpaired t-test. **p < 0.001, ****p < 0.00001.

FIGS 13A and 13B show a comparison of cell death following administration of 1 mg/ml 29mer[H105A] or wild type 29mer to rdlO (FIG. 13A) or rdlO/Serpinfl null (FIG. 13B) mice treated as shown in FIG. 6A. Each data point corresponds to the average ± SEM of fluorescence relative to HBSS by unpaired t-test. **p < 0.001, ***p < 0.0001, ****p < 0.00001.

FIGS. 14A and 14B are graphical representations of efficacy of the efficacy of the peptides tested in FIGS. 12A-12B (rdlO) and 13A-13B (rdlO/Serpinfl null), respectively, in protecting against photoreceptor cell death. 17mer[H105A] was the most efficacious of the tested peptides (arrows).

FIGS. 15A-15C show effect of 17mer[H105A] peptide administration on PR cell death in mice treated every other day. FIG. 15A is a schematic illustrating the experimental design. Decreased cell death was observed following administration of 17mer[H105A] in both rdlO (FIG. 15B) and rdlO/Serpinfl null (FIG. 15C) mice at day P25. FIGS. 16A and 16B show histological evaluation of retinas in mice treated as shown in FIG. 15A. Tissue collection was performed at P25. Hematoxylin and eosin staining (FIG. 16A) and measurement of outer nuclear layer (ONL) thickness (FIG. 16B) are shown for rdlO mice.

FIGS. 17A and 17B show histological evaluation of retinas in mice treated as shown in FIG. 15A. ERG was performed at P25. ERG a wave (FIG. 17A) and ERG b wave (FIG. 17B) are shown for rdl 0 mice.

FIGS. 18A and 18B show in vitro detection of AlexaFluor488-labeled 17mer|H105AJ peptide. Labeled peptide was diluted in HBSS (FIG. 18A) or retinal extract (FIG. 18B) and fluorescence was determined.

FIG. 19 shows penetration or bioavailability of AlexFluor488-labeled 17mer[H105A] peptide administered as eyedrops in P21 C57/B16J mice. Fluorescence was measured at the indicated times after administration.

FIG. 20 shows the nucleic acid sequence (SEQ ID NO: 5), complement nucleic acid sequence (SEQ ID NO: 7), and amino acid sequence (SEQ ID NO: 6) of a IFNP signal peptide/PEDF 17mer[H105A] construct (SP-17mer[H105A]).

FIG. 21 is a map of pAAV2.1_PEDF vector. PEDF was replaced with the SP- 17mer[H105A] construct or GFP encoding nucleic acid to create pAAV2.1_SP-17mer[H105A] or pAAV2.1_GFP vectors, respectively.

FIG. 22 is a schematic showing the experimental design for testing recombinant AAV2/2 viral infection in Rho^p23H/+ murine retina. The plasmid used for cloning was AAV2.1 (e.g., as shown in FIG. 21), and the virus was produced as serotype 2. Thus, the virus injected was AAV2/2, that is, an AAV2 genome in an AAV2 capsid.

FIG. 23 shows protein expression at postnatal day (PN)19 in mice infected with the indicated recombinant viruses at PN5. Transduced cells can be identified as Muller cells.

FIG. 24 shows sections from AAV2/2_GFP infected retinas co-labeled with anti-GFP and anti-GS (expressed in Muller cells). Muller glia (arrows), ganglion cells (#), and bipolar cells (*) are indicated in the right panel.

FIGS. 25A-25E are digital images and bar graphs showing (A) ganglion cells and Muller glia cells are transduced by AAV2 infection at PN5 (arrows); (B) PEDF or 17mer[H105A] expression reduced inflammation in the retina, based on number of Ibal⁺ cells (immunofluorescence micrographs with Ibal, rhodopsin, and nuclei stained); (C) photoreceptor cell death, based on TUNEL assay (TUNNEL micrographs with TUNEL, rhodopsin, and nuclei stained) when compared to retinas transduced with control AAV2/2_EGFP. FIGS. 25D and 25E show graphical representation of the data for Ibal positive cells (FIG. 25D) and TUNEL analysis (FIG. 25E).

FIGS. 26A-26B are digital images showing qPCR and western blotting analyses at PN90 following intravitreal injection at PN5 of AAV2_PEDF, AAV2_SP-17mer[H105A], or AAV2_EGFP. (A) mRNA level based on qRT-PCR and (B) protein expression by western blotting for EGFP and PEDF were analyzed. Three months after viral injection expression of the therapeutic agents were detectable. Gene expression at mRNA level was normalized on S26 and quantified (Fold change shown below), proteins were normalized on tubulin. PEDF could be detected only in AAV2_PEDF transduced retinas and EGFP could be detected only in AAV2_EGFP transduced retinas.

FIGS. 27A-27C are digital images and graphs showing preservation of the photoreceptor layer. (A) AAV2_PEDF or (B) AAV2_SP-17mer[H105A] or AAV2_EGFP were intravitreally injected at PN5 and retinas were histologically analyzed 6 months after delivery. (A and B) Retinas were stained with hematoxylin/eosin and a significant preservation of the thickness of ONL, containing photoreceptor nuclei, could be detected in retinas transduced with AAV2_PEDF (A) or AAV2_ SP-17mer[H105A] (B). Spider graphs with counts of photoreceptor rows are presented on the right-hand side. Statistical analysis was based on multiple unpaired t-test with no correction for multiple comparison. (C) Rod photoreceptors were analyzed by immunofluorescence by labelling with an antibody anti rhodopsin and cone photoreceptors were analyzed by labelling with FITC- peanut agglutinin. White bars highlight the preservation of the photoreceptor cell layer upon delivery of PEDF or 17mer[H105A].

FIGS. 28A-28B are graphs showing the functionality of injected retinas. AAV2_PEDF or AAV2_SP-17mer[H105A] or AAV2_EGFP were intravitreally injected at PN5 and functionality of photoreceptors was assessed by ERG at the age of 6 months. (A) Both PEDF and 17mer[H105A] could preserve rod functionality, but only (B) PEDF could preserve cone functionality.

SEQUENCES

The nucleic and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases and amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

SEQ ID NO: 1 is the amino acid sequence of PEDF 17mer H105A peptide:

QRTESIIARALYYDLIS SEQ ID NO: 2 is the amino acid sequence of a modified interferon beta signal peptide in which a glycine was added after the first methionine:

MGTNKCLLQIALLLCFSTTALS

SEQ ID NO: 3 is a nucleic acid sequence encoding the modified interferon beta signal peptide:

ATGGGCACCAACAAGTGTCTCCTCCAAATTGCTCTCCTGCTTTGCTTCTCCACTA

CAGCTCTTTCC

SEQ ID NO: 4 is a nucleic acid sequence encoding the PEDF 17mer[H105A] peptide:

CAGCGAACAGAATCCATCATTGCACGGGCTCTCTACTATGACTTGATCAGC

SEQ ID NO: 5 is a nucleic acid sequence encoding an operably linked interferon beta signal peptide and PEDF 17mer[H105A] peptide (SP-17mer[H105A]):

ATGGGCACCAACAAGTGTCTCCTCCAAATTGCTCTCCTGCTTTGCTTCTCCACTA

CAGCTCTTTCCGGGATCCAGCGAACAGAATCCATCATTGCACGGGCTCTCTACT

ATGACTTGATCAGC

SEQ ID NO: 6 is the amino acid sequence of an operably linked interferon beta signal peptide and PEDF 17mer[H105A] peptide (SP-17mer[H105A]). GI linker was added between the signal peptide and PEDF 17mer[H105A]:

MGTNKCLLQIALLLCFSTTALSGIQRTESIIARALYYDLIS

SEQ ID NO: 7 is the complement of a nucleic acid sequence encoding an operably linked interferon beta signal peptide and PEDF 17mer[H105A] peptide:

TACCCGTGGTTGTTCACAGAGGAGGTTTAACGAGAGGACGAAACGAAGAGGTG

ATGTCGAGAAAGGCCCTAGGTCGCTTGTCTTAGGTAGTAACGTGCCCGAGAGAT GATACTGAACTAGTCG

SEQ ID NO: 8 is an exemplary human PEDF nucleic acid sequence:

ATGCAGGCCCTGGTGCTACTCCTCTGCATTGGAGCCCTCCTCGGGCACAGCAGC

TGCCAGAACCCTGCCAGCCCCCCGGAGGAGGGCTCCCCAGACCCCGACAGCAC

AGGGGCGCTGGTGGAGGAGGAGGATCCTTTCTTCAAAGTCCCCGTGAACAAGC TGGCAGCGGCTGTCTCCAACTTCGGCTATGACCTGTACCGGGTGCGATCCAGCA TGAGCCCCACGACCAACGTGCTCCTGTCTCCTCTCAGTGTGGCCACGGCCCTCT CGGCCCTCTCGCTGGGAGCGGAGCAGCGAACAGAATCCATCATTCACCGGGCT CTCTACTATGACTTGATCAGCAGCCCAGACATCCATGGTACCTATAAGGAGCTC CTTGACACGGTCACTGCCCCCCAGAAGAACCTCAAGAGTGCCTCCCGGATCGTC TTTGAGAAGAAGCTGCGCATAAAATCCAGCTTTGTGGCACCTCTGGAAAAGTCA TATGGGACCAGGCCCAGAGTCCTGACGGGCAACCCTCGCTTGGACCTGCAAGA GATCAACAACTGGGTGCAGGCGCAGATGAAAGGGAAGCTCGCCAGGTCCACAA AGGAAATTCCCGATGAGATCAGCATTCTCCTTCTCGGTGTGGCGCACTTCAAGG GGCAGTGGGTAACAAAGTTTGACTCCAGAAAGACTTCCCTCGAGGATTTCTACT TGGATGAAGAGAGGACCGTGAGGGTCCCCATGATGTCGGACCCTAAGGCTGTT TTACGCTATGGCTTGGATTCAGATCTCAGCTGCAAGATTGCCCAGCTGCCCTTG ACCGGAAGCATGAGTATCATCTTCTTCCTGCCCCTGAAAGTGACCCAGAATTTG ACCTTGATAGAGGAGAGCCTCACCTCCGAGTTCATTCATGACATAGACCGAGAA CTGAAGACCGTGCAGGCGGTCCTCACTGTCCCCAAGCTGAAGCTGAGTTACGAA GGCGAAGTCACCAAGTCCCTGCAGGAGATGAAGCTGCAATCCTTGTTTGATTCA CCAGACTTTAGCAAGATCACAGGCAAACCCATCAAGCTGACTCAGGTGGAACA CCGGGCTGGCTTTGAGTGGAACGAGGATGGGGCGGGAACCACCCCCAGCCCAG GGCTGCAGCCTGCCCACCTCACCTTCCCGCTGGACTATCACCTTAACCAGCCTTT CATCTTCGTACTGAGGGACACAGACACAGGGGCCCTTCTCTTCATTGGCAAGAT TCTGGACCCCAGGGGCCCCTAA

SEQ ID NO: 9 is the amino acid sequence of an exemplary human PEDF protein:

MQALVLLLCIGALLGHSSCQNPASPPEEGSPDPDSTGALVEEEDPFFKVPVNKLAAA VSNFGYDLYRVRS SMSPTTNVLLSPLS VATALS ALSLGAEQRTESIIHRALYYDLIS S PDIHGTYKELLDTVTAPQKNLKSASRIVFEKKLRIKSSFVAPLEKSYGTRPRVLTGNP RLDLQEINNWVQAQMKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLE DFYLDEERTVRVPMMSDPKAVLRYGLDSDLSCKIAQLPLTGSMSIIFFLPLKVTQNL TLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQEMKLQSLFDSPDFS KITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDT DTGALLFIGKILDPRGP

SEQ ID NO: 10 is the amino acid sequence of wild type PEDF 17mer peptide: QRTESIIHRALYYDLIS

SEQ ID NO: 11 is the amino acid sequence of wild type PEDF 29mer peptide:

SLGAEQRTESIIHRALYYDLISSPDIHGT

SEQ ID NO: 12 is the amino acid sequence of PEDF 29mer[H105A] peptide: SLGAEQRTESIIARALYYDLISSPDIHGT

DETAILED DESCRIPTION

Provided herein are compositions and methods for treating retinal degeneration, such as an inherited retinal degeneration. In some examples, the compositions include a pigment epithelium- derived factor protein or a peptide thereof (for example, PEDF 17mer[H105A] peptide), or a nucleic acid encoding the PEDF protein or peptide.

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a peptide” includes singular or plural peptides and can be considered equivalent to the phrase “at least one peptide.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated.

Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

Adeno-associated virus (AAV): A small, non-enveloped virus that infects humans and some other primate species. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV an attractive viral vector for gene therapy. In some examples, the AAV is a recombinant AAV and is replication-deficient.

As used herein, “recombinant AAV” (rAAV) refers to an AAV particle in which a heterologous nucleic acid molecule has been packaged. The heterologous nucleic acid molecule of the recombinant AAV includes one or more nucleic acid sequences that do and do not occur within the AAV genome; for example, a therapeutic nucleic acid sequence flanked by the inverted terminal repeat (ITR) nucleic acid sequences of AAV.

Administering: To provide or give a subject an agent, such as a therapeutic agent (e.g. a nucleic acid molecule or peptide), by any effective route. Exemplary routes of administration include, but are not limited to, topical administration (for example, eye drops) or injection (such as intravitreal or subretinal injection). Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein, or virus) has been substantially separated or purified away from other biological components (e.g., other nucleic acids, proteins, and/or organelles). Nucleic acids, proteins, and/or viruses that have been “isolated” include nucleic acids, proteins, and viruses purified by standard purification methods. The term also embraces nucleic acids, proteins, and viruses prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins.

The term “isolated” (or purified) does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated or purified nucleic acid, protein, virus, or other active compound is one that is isolated in whole or in part from associated nucleic acids, proteins, and other contaminants. In certain examples, the term “substantially purified” refers to a nucleic acid, protein, virus, or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation.

Operably linked: A first nucleic acid is operably linked to a second nucleic acid when the first nucleic acid is placed in a functional relationship with the second nucleic acid. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked nucleic acid sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carrier: Pharmaceutically acceptable carriers (vehicles) useful in this disclosure are known. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21^st Edition (2005), describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compounds, molecules, or agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, injectable formulations usually include fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Pigment epithelium-derived factor (PEDF): Also referred to as SERPINF1 (serpin family F member 1). A member of the serpin family that does not exhibit demonstrable serpin inhibitory activity against proteases. PEDF is a secreted protein with anti- angiogenic and neurotrophic properties. PEDF nucleic acid and protein sequences are publicly available. Human PEDF nucleic acid sequences include GenBank Accession Nos. NM_002615.7, NM_001329904.2, and NM_003129903.2 (all of which are incorporated by reference as present in GenBank on December 2, 2022) and SEQ ID NO: 8. Human PEDF amino acid sequences include GenBank Accession Nos. NP_002606.3, NP_001316833.1, and NP_001316832.1 (all of which are incorporated by reference as present in GenBank on December 2, 2022) and SEQ ID NO: 9.

Retinal Degeneration: Deterioration of the retina, including progressive death of the photoreceptor cells of the retina or associated structures (such as retinal pigment epithelium). Retinal degeneration includes diseases or conditions such as retinitis pigmentosa, cone-rod dystrophy, macular degeneration (such as age-related macular degeneration and Stargardt-like macular degeneration), Leber congenital amaurosis (LCS), and maculopathies.

Retinitis pigmentosa (RP): An inherited, degenerative eye disease that causes severe vision impairment due to the progressive degeneration of the rod photoreceptor cells in the retina. This form of retinal dystrophy manifests initial symptoms independent of age. The initial retinal degenerative symptoms of RP are characterized by decreased night vision (nyctalopia) and the loss of the mid-peripheral visual field. The rod photoreceptor cells, which are responsible for low-light vision and are orientated in the retinal periphery, are the retinal processes affected first during non- syndromic forms of this disease. Visual decline progresses relatively quickly to the far peripheral field, eventually extending into the central visual field as tunnel vision increases. Visual acuity and color vision can become compromised due to accompanying abnormalities in the cone photoreceptor cells, which are responsible for color vision, visual acuity, and sight in the central visual field. The progression of disease symptoms occurs in a symmetrical manner, with both the left and right eyes experiencing symptoms at a similar rate. There are multiple genes that, when mutated, can cause the retinitis pigmentosa phenotype. Inheritance patterns of RP have been identified as autosomal dominant, autosomal recessive, X-linked, and maternally (mitochondrially) acquired, and are dependent on the specific RP gene mutations present in the parental generation.

Subject: A living multi-cellular vertebrate organism, a category that includes human, laboratory, and veterinary subjects, including human and non-human mammals.

Therapeutically effective amount: An amount of a compound sufficient to treat a specified disorder or disease, or to ameliorate or eradicate one or more of its symptoms and/or to prevent the occurrence of the disease or disorder, such as retinal degeneration. The amount of a compound which constitutes a “therapeutically effective amount” will vary depending on the compound, the route of administration, the disease state and its severity, the age of the subject to be treated, and the like. The therapeutically effective amount can be determined by a person of ordinary skill in the art, for example, through various in vitro, in vivo, or ex vivo assays. Treating, Treatment, and Therapy: Any success or indicia of success in the attenuation or amelioration of an injury, pathology, or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the subject, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, or improving vision. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination, neurological examination, or psychiatric evaluations. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters known in the art that are specific to the particular disease, such as improved vision. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

Vector: A vector is a nucleic acid molecule allowing insertion of foreign nucleic acid without disrupting the ability of the vector to replicate and/or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. In some embodiments herein, the vector is a plasmid vector. In other embodiments, the vector is a viral vector, such as an AAV vector or virus.

II. PEDF Peptide Eye Drop Compositions and Methods of Use

In some embodiments, provided herein is a composition including a PEDF peptide including the amino acid sequence of SEQ ID NO: 1 (also referred to herein as 17mer[H105A] or H105A peptide). The PEDF peptide includes amino acids 98 to 114 of human PEDF protein (e.g., SEQ ID NO: 9), with an alanine substitution at the position corresponding to amino acid position H105 of SEQ ID NO: 9. The disclosed compositions are formulated as an eye drop, for example for topical administration to the eye. In some examples, the PEDF peptide is about 15-19 amino acids long, such as about 17 amino acids long. In some examples, the PEDF peptide includes or consists of the amino acid sequence of SEQ ID NO: 1. In some examples, the eye drop formulation includes a saline solution, such as a buffered saline solution. In some examples, the composition is formulated in Hank’s buffered saline solution (HBSS), balanced salt solution (BSS), or phosphate buffered saline (PBS). In other examples, the composition is formulated in a nanoparticle, for example utilizing cyclodextrins. One of ordinary skill in the art can select appropriate alternative vehicles or one or more additives for eye drop formulations.

In some embodiments, the disclosed compositions include a therapeutically effective amount of the PEDF peptide. In some examples, the composition includes about 0.1 mg/ml to about 2 mg/ml of the peptide (for example, about 0.1 mg/ml to about 0.5 mg/ml, about 0.25 mg/ml to about 1 mg/ml, about 0.75 mg/ml to about 1.5 mg/ml, or about 1.25 mg/ml to about 2 mg/ml peptide). In one example, the composition includes about 1 mg/ml of PEDF peptide.

Also provided are methods of treating a retinal degeneration with the eye drop compositions provided herein. In some embodiments, the methods include administering the composition including the PEDF peptide to one or both eyes of a subject with retinal degeneration. In some examples, the subject has an inherited retinal degeneration, including, but not limited to retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), or age-related macular degeneration.

In some examples, the composition is administered topically to one or both eyes of the subject. In some examples, the amount of the composition administered is about 1 pl to about 10 pl (such as about 1-3 pl, about 2-4 pl, about 3-5 pl, about 4-6 pl, about 5-7 pl, about 6-8 pl, about 7-9 pl, or about 8-10 pl), for example, about 5 pl. The composition may be administered one or more times, such as twice daily, once daily, every other day, once weekly, or every other week. In some examples, the composition is administered to one or both eyes of the subject once daily or every other day. The subject may be treated for a set period of time (such as once daily or every other day for 5-10 days) or on a continuing basis. In some examples, the subject receives the treatment unless or until a therapeutic effect is no longer observed. Efficacy of treatment can be evaluated by methods known to one of ordinary skill in the art, including, but not limited to use of fluorescent dye (such as PSVue® 550) for evaluating retinal cell death, optical coherence tomography (OCT) for evaluating retinal morphology, and/or electroretinogram (ERG) for evaluating retinal function. Other methods such as histology and immunofluorescent labeling of cell death markers (such as BAX and BCL2) can also be used.

III. Nucleic Acid Compositions and Methods of Use

In other embodiments, provided herein are compositions including a nucleic acid encoding a pigment epithelium-derived factor protein or a nucleic acid encoding a pigment epithelium-derived factor peptide. In some examples, the nucleic acid (for example, a DNA or mRNA nucleic acid) is a naked nucleic acid or is incorporated in a liposome or a nanoparticle. In other examples, the nucleic acid is incorporated in a viral vector, such as an AAV vector.

In some embodiments, adeno-associated virus (AAV) vectors including a nucleic acid encoding a pigment epithelium-derived factor protein are provided. In further embodiments, provided herein are AAV vectors including a nucleic acid encoding a pigment epithelium-derived factor peptide comprising SEQ ID NO: 1, wherein the nucleic acid encoding the peptide is operably linked to a nucleic acid encoding a signal peptide. In some examples, the vector is an AAV2 vector (for example, an AAV2.1 vector). In other examples, the vector is a recombinant AAV2 virus, for example, an AAV2/2 virus (see, e.g., Allocca et al. Journal of Virology 81:11372-11380, 2007). However, one of ordinary skill in the art can select alternative AAV vectors, including alternative AAV serotypes, such as AAV5, AAV8, AAV2.7m8, and AAV9, that can be used for the compositions and methods described herein.

In some examples, the signal peptide operably linked to the nucleic acid encoding the PEDF peptide is an interferon beta signal peptide (see, e.g., Jouanneau et al., Proc. Natl. Acad. Sci. USA 88:2893-2987, 1991) or a modified interferon beta signal peptide. In one example, a modified interferon beta signal peptide includes or consists of the amino acid sequence of SEQ ID NO: 2. In some examples, the modified interferon beta signal peptide is encoded by the nucleic acid sequence of SEQ ID NO: 3. In additional examples, the PEDF peptide includes or consists of the amino acid sequence of SEQ ID NO: 1, or is encoded by the nucleic acid sequence of SEQ ID NO: 4. In other examples, the nucleic acid encoding the PEDF peptide operably linked to a nucleic acid encoding the signal peptide includes or consists of the nucleic acid sequence of SEQ ID NO: 5. In some examples, the nucleic acid encodes the amino acid sequence of SEQ ID NO: 6. In further examples, the PEDF protein is encoded by the nucleic acid sequence of SEQ ID NO: 8 and/or includes the amino acid sequence of SEQ ID NO: 9. In other examples, the nucleic acid encodes a PEDF protein with an alanine at amino acid position 105 (H105A), for example, an alanine at amino acid corresponding to position 105 of SEQ ID NO: 9. In other examples, the signal peptide is a PEDF signal peptide, such as amino acids 1-20 of SEQ ID NO: 9.

Also contemplated are polynucleotides encoding a disclosed PEDF protein or peptide that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included as long as the amino acid sequence of the PEDF protein or peptide is unchanged. In some embodiments the nucleic acid encoding the PEDF protein or peptide is operably linked to a promoter. In some examples, the promoter is a constitutive promoter, such as a cytomegalovirus (CMV) promoter. In other examples, the promoter is a retina-specific promoter, for example, a promoter targeting retinal ganglion cells (such as SYN or NEFH promoters), a promoter targeting Muller glia cells (such as RLBP1 or GFAP promoters), or a promoter targeting retina pigment epithelial cells (such as RPE65 or Bestl promoters). In further examples, the promoter is a 0-actin promoter (for example, a chicken P-actin (CBA) promoter) or is a PEDF promoter.

In further examples, a composition including the AAV vector or virus including a nucleic acid encoding the PEDF protein or PEDF peptide in a composition formulated for injection is provided. Formulations for delivery of AAV vectors to the eye include injectable fluids including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.

Also provided are methods of treating a retinal degeneration with the AAV vectors disclosed herein. In some embodiments, the methods include administering the vector or a composition including the vector to one or both eyes of a subject with retinal degeneration. In some examples, the subject has an inherited retinal degeneration, including, but not limited to retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), or age-related macular degeneration.

In some embodiments, the composition is administered to one or both eyes of the subject by injection (for example, by intravitreal injection or subretinal injection). In some examples, the subject is administered about 1 x 10^s to about 1 x 10¹² viral particles (e.g. , genome copies) in one or both eyes, such as about 1 x 10⁸ to about 1 x 10⁸³, about 1 x IO⁸⁵ to about 1 x 10⁹, about 1 x 10⁹ to about 1 x 10⁹ \ about 1 x 10⁹³ to about 1 x IO¹⁰ viral particles, about 1 x IO¹⁰ to about 1 x IO¹⁰⁵, about 1 x IO¹⁰'⁵ to about 1 x 10¹¹, about 1 x 10¹¹ to about 1 x 10^{11 5}, or about 1 x IO^{11 5} to about 1 x 10¹² viral particles). In other examples, the subject is administered about 2 x 10⁹ genome copies in one or both eyes. In some examples, the composition is administered to one or both eyes of the subject one or more times (for example, 1, 2, 3, 4, or more times). Efficacy of treatment can be evaluated by methods known to one of ordinary skill in the art, including, but not limited to use of fluorescent dye (such as PSVue® 550) for evaluating retinal cell death, optical coherence tomography (OCT) for evaluating retinal morphology, and/or electroretinogram (ERG) for evaluating retinal function. Other methods such as histology and immunofluorescent labeling of cell death markers (such as BAX and BCL2) can also be used. EXAMPLES

The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

Example 1 Methods of Detecting Photoreceptor Cell Death In Vivo and Ex Vivo in Retinal Degeneration Models

Animals were used following the Association for Research in Vision and Ophthalmology statement for the Use of Animals in Ophthalmic and Vision Research, USA ARVO guidelines. The mice were wild type C57BL/6 J mice, rdlO (a mouse model for a human inherited autosomal recessive RP), and rdlO/Serpinfl null, a mouse without the Serpinfl gene in rd 10 background. Retinal degeneration in the rdl 0 mice is caused by a spontaneous mutation in the gene for the P- subunit of phosphodiesterase (Dixit et al., Exp. Eye Res. 198:108121, 2020).

Whole eyes were enucleated, and retinas dissected. Cultured retinal explants from wild type mice were prepared and cell death was induced by Zaprinast, an inhibitor of phosphodiesterase, to mimic the rdlO photoreceptor cell death (Hernandez-Pinto et al., Exp. Eye Res. 184:23-29, 2019). Cell death was detected using either TUNEL (Roche) or PSVue® (M.T.T.I.). Frozen retinal sections or retinal flat mounts from enucleated whole eyes were used for TUNEL, followed by confocal microscopy. PSVue® was added to the retinal explant cultures ex vivo and detected by confocal microscopy or administered as drops to the left eye of live mice and detected by fundoscopy using MICRON III in vivo. Control reference was eyedrops of HBBS in the right eye.

Phosphatidylserine-binding conjugate of Bis(zinc(II)-dipicolylamine(Zn-DPA) with Texas- red (PSVue®) is a robust probe for detecting phosphatidylserine on the surface of cells undergoing death. PSVue® was used to optimize the cell death read-out in cultured retinal explants (FIG. 1 A). This method yielded higher fluorescence detection with lower background compared to TUNEL assay (FIG. IB).

Both TUNEL and PSVue® were used to detect PR cell death in rdlO mice from postnatal days P15 to P25. As shown in FIG. 2, TUNEL assay did not detect cell death at days P15 to P25; however the PSVue® assay detected cell death peaking around days P17 to P21 (FIGS. 3A-3C). Similar experiments were performed in rdlO/Serpinfl null mice (FIGS. 4, 5A-5C), with similar results. These data illustrate the increased sensitivity of the PSVue® assay for detecting retinal cell death. This methodology is simple, fast, and can be used as a screening assay for potential therapeutics for treating retinal degenerations. Example 2

Effect of PEDF peptide H105A Eyedrops on Photoreceptor Cells

Eyedrops (5 |11) of a solution of 1 mg/ml of 17mer[H105A] peptide in HBSS were administered to the right eye (OD) and of HBSS, without the peptide, to the left eye (OS) as control to rdlO and rdlO x Serpinfl null mice. Administration was daily to mice at ages between Pl 5 and P20. PSVue® was administered at age P20 to detect PR cell death (FIG. 6A). The PSVue® assay detected cell death in eyes without the peptide but decreased with 17mer[H105A] in rdlO mice (FIG. 6B). Similar experiments were performed in rdlO/Serpinfl null mice (FIGS. 6C) with similar results. These data illustrate the efficacy of eyedrops of the 17mer[H105A] peptide for decreasing retinal cell death in both models.

Dose response curves were also performed using the same mouse models. Eyedrops of a solution of 17mer[H105A] in HBSS (5 pL) at concentrations as indicated were administered daily between P15 - P20. At P20, PSVue (5 pl at 1 mM) was administered. At P21, fundoscopy was performed, fluorescence was determined and pictures were acquired. Data were analyzed using GraphPad. Dose curve response for rdlO (FIG. 7 A) and rdlO/Serpinfl null (FIG. 7B) mice are shown. The plots were analyzed using ‘Interpolate standard curve’ and an IC50 was determined, as shown.

At end point, eyes were enucleated and cross section of retinas from the treated animals were obtained. First, histological evaluation of retina cross section stained with hematoxylin and eosin demonstrated that the outer nuclear layer (ONL) was thicker for eyes treated with eyedrops of 17mer[H105A] than without the peptide for both rdlO (FIG. 8A) and rdlO/Serpinfl null (FIG. 8B) mouse models. Spider plots showed differences in ONL thickness in retinal areas spanning away from the optic nerve (ON) (FIGS. 8 A and 8B). Second, immunofluorescence of the retinal cross sections to detect pro-apoptotic BAX2 and anti-apoptotic BCL2 proteins showed a decrease of the pro-death marker and increase in the pro-survival marker in retinas from mouse eyes treated with the 17mer[H105A] peptide relative to those without the peptide for both rdlO (FIG. 9A) and rdlO/Serpinfl null (FIG. 9B) mouse models. Third, electroretinography was performed in untreated and eyes treated with the 17mer[H105A] peptide eyedrops. Following an intense brief flash of light from darkness, the negative-going a- wave is generated by rod photocurrents and the positive-going b-wave by depolarizing bipolar-cell currents in combination with bipolar celldependent K⁺ currents affecting Muller cells. The data show an improvement of the amplitude of the a-wave in eyes of rdlO (FIG. 10A) and rdlO x SerpinFl (FIG. 10B) mice at P21 after treatment with eyedrops of 17mer[H105A] peptide. Scotopic b wave was also measured for both rdlO and rdlO/SerpinFl null mice (FIG. IOC). The data from these different assays corroborate the PSVue® data and illustrate the efficacy of eyedrops of the 17mer[H105A] peptide for protecting the photoreceptors against malformation, death, and misfunction in vivo.

Experiments comparing wild type and H105A 17mer and 29mer peptides (FIG. 11) were also performed. Eyedrops containing 1 mg/ml of the indicated peptide were administered to rdl 0 and rdlO/Serpinfl null mice daily from P15 to P20. At P20, eyedrops of PSVue (5 pl at 1 mM) were added. Fluorescence fundoscopy was performed at P21. Quantification of fluorescence intensity was performed using ImageJ by subtracting the background of HBSS. The lineal trendline was determine using GraphPad. For 17mer experiments, 3-5 retinas per group were evaluated. For 29mer experiments, 3 retinas per group were evaluated. The 17mer peptides protected against PR cell death, with 17mer[H105A] being more effective than wild type 17mer (FIGS. 12A-12B). The wild type and H105A 29mer peptides were not efficacious in protecting against PR cell death (FIGS. 13A-13B). FIGS. 14A-14B show a summary of the effects of 17mer, 17mer[H105A], 29mer and 29mer[H105A] peptides for rdlO and rdlO/Serpinfl null mice, respectively.

Administration of eyedrops of 17mer[H105A] peptide every other day between P15 and P24 (FIG. 15A) showed that at P25 the PSVue® assay detected cell death in eyes without the peptide but decreased with 17mer[H105A] in rdlO mice (FIG. 15B). Similar experiments were performed in rdlO/Serpinfl null mice (FIGS. 15C) with similar results. These data illustrate the efficacy of eyedrops of the 17mer[H105A] peptide for decreasing retinal cell death in both models when administered every other day.

At end point, eyes were enucleated and cross section of retinas from the treated animals were obtained. Histological evaluation of retina cross section stained with hematoxylin and eosin demonstrated that the outer nuclear layer (ONE) was thicker for eyes treated with eyedrops of 17mer[H105A] than without the peptide for rdlO mouse model (FIG. 16A). Spider plots showed differences in ONE thickness in retinal areas spanning away from the optic nerve (ON) (FIG. 16B). Electroretinography was also performed in untreated and eyes of rdl 0 mice treated with the 17mer[H105] peptide eyedrops. The data show an improvement of the amplitude of the a-wave (FIG. 17A) and b-wave (FIG. 17B) at P25 after treatment with eyedrops of 17mer[H105A] peptide. There is a trend, but there was no statistically significant difference in b-wave between treated and untreated. The work was done with 3 animals, which may not give statistical significance.

To detect labeled peptide in vitro, the labeled Alexa488-17-mer[H105A] peptide was diluted in HBSS at the indicated concentrations (X-axis). A total of 30 pl of each was added to a well in 96-well plate in triplicates. RIPA buffer was added to one dissected mouse retina at 80 pl/retina. The suspension was sonicated for 30 sec at 4°C followed by centrifugation at 4°C 10 min at 14,00 rpm (Eppendorf centrifuge) to separate the soluble retinal extract from the particulate material. The labeled peptide was diluted in retinal extract. A total of 30 pl of each was added to a well in 96-well plate. HBSS (FIG. 18 A) or retinal extract (FIG. 18B) were used as a control at zero and different concentrations of 17mer[H105A]-488 as indicated in the x-axis. Fluorescence was determined using a fluorometer [BMG LabTech POLARstar OPTIMA] using wavelength at [Excitation 485-P, Emission 520-P]. Plots were obtained using GraphPad and non-linear fit were analyzed.

To evaluate penetration or bioavailability of 17mer[H105AJ peptide administered as eyedrops, 5 pl of labeled Alexa488-17-mer[H105A] peptide was administered to C57/B16J mice at P21 days old. At 1, 3, 6 and 24 hours after administration, retinas were dissected, their proteins were extracted with RIPA (80 pl/retina) and clarified by sonication and centrifugation. A total of 30 pl of extracts were used to determine the fluorescence of the peptide with a fluorometer (FIG. 19).

17-mer[H105A]-488 was detected in the dissected retina after 1 h and even after 24 h post administration. These results indicate that the 17mer[H105A] administered as eyedrops reached the retina after 1-24 hours, penetrating through the layers of the eye from the anterior to the posterior regions of the eye and becoming available for retina cells. At 24 hours there was one third of the amount at one hour. After 24 hours, an additional daily eyedrop was administered to the animal.

Example 3

Effect of PEDF peptide H105A AAV Delivery on Photoreceptor Cell Death

A recombinant AAV2/2-based delivery system was developed for sustained production and secretion of PEDF and SP-17mer[H105A] (FIGS. 20 and 21). The AAV2/2 recombinant virus for delivery of 17mer[H105A] included the nucleic acid sequence of SEQ ID NO: 5. The photoreceptor-protective efficacies were assessed using the Rho^p23H/+ mouse model of RP (FIG. 22). The AAV2/2 viruses (produced from the recombinant AAV2.1 vectors) were intravitreally injected (0.5 pL undiluted viral batch) for expression and secretion of full-length PEDF (n=8 animal) or of SP-17mer[H105A] (n=8 animals) at post-natal day 5. The contralateral eye of each animal was injected with AAV2/2_GFP. The viral batch concentrations and amounts injected are shown in Table 1. In these experiments, the amount of AAV2/2_SP-17mer[H105A] injected was less than half the dose of viral particles for AAV2/2_PEDF or AAV2/2_GFP. Table 1. AAV2/2 viral batches

GC/mL = genome copies per milliliter

Infected cells with AAV2/2_PEDF and AAV2/2_GFP (reporter) were tracked in retinal ganglion and Muller glial cells (FIGS. 23 and 24). In additional experiments, the AAV2_SP- 17mer[H105A] (10⁹ gc or genome copies) and the AAV2_PEDF (2xl0⁹ gc) viruses, or AAV2_EGFP (2xl0⁹ gc), as control, were intravitreally injected in murine Rho^p23H/+ mutant pups at the age of 5 days (postnatal day 5, PN5). At 14 days after viral transduction, mice were sacrificed and eyes analyzed by immunofluorescence to detect EGFP or PEDF. Proteins from viral transduction were detected mostly in ganglion cells and Muller glia cells (FIGS. 23, 24, and 25 A, arrows). In PEDF and 17mer[H105A] expressing retinas reduced inflammation was observed, based on the number of Ibal⁺ cells (FIGS. 25B and 25D). The number of photoreceptors undergoing degeneration, evaluated by TUNEL assay at PN19, the peak of cell death in this model of retinitis pigmentosa, was significantly reduced in retinas expressing either PEDF or 17mer[HI05A] (FIGS. 25C and 25E).

Expression of the viruses was confirmed 3 months after transduction by RT-PCR (FIG. 26A) and western blotting (FIG. 26B).

Preservation of photoreceptor cells was analyzed 6 months after viral delivery. Significant preservation of the photoreceptor layer was observed using histological analysis (FIGS. 27 A, 27B). Immunostaining of rod photoreceptors with anti-rhodopsin, and labelling of cone photoreceptors by FITC-Peanut Agglutinin confirmed preservation of photoreceptor cells 6 months after AAV2_SP- 17mer|HI05AJ and AAV2 PEDF viral transduction of the retina (FIG. 27C).

Functionality of the injected retinas was evaluated by ERG. Rod photoreceptor functionality was preserved by sustained expression of PEDF and I7mer[H105A] (FIG. 28A). Cones were better preserved by sustained expression of PEDF (FIG. 28B).

It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

We claim:

1. A composition comprising a pigment epithelium-derived factor peptide comprising SEQ ID NO: 1, wherein the composition is formulated as an eye drop.

2. The composition of claim 1, wherein the peptide is about 15-19 amino acids long.

3. The composition of claim 2, wherein the peptide consists of 17 amino acids.

4. The composition of any one of claims 1 to 3, wherein the eye drop formulation comprises a saline solution.

5. The composition of claim 4, wherein the saline solution is Hank’s balanced salt solution.

6. The composition of any one of claims 1 to 5, wherein the composition comprises about 1 mg/ml of the peptide.

7. A method of treating retinal degeneration, comprising administering the composition of any one of claims 1 to 6 to an eye of a subject with retinal degeneration.

8. The method of claim 7, wherein the composition is administered topically to the eye of the subject.

9. The method of claim 7 or claim 8, wherein the composition is administered to the eye of the subject once daily, every other day, or every three days.

10. An adeno-associated virus (AAV) vector comprising: a nucleic acid encoding pigment epithelium-derived factor (PEDF) protein; or a nucleic acid encoding a PEDF peptide comprising SEQ ID NO: 1, wherein the nucleic acid encoding the PEDF peptide is operably linked to a nucleic acid encoding a signal peptide.

11. The composition of AAV vector of claim 10, wherein the PEDF peptide is about 15-19 amino acids long.

12. The AAV vector of claim 11, wherein the PEDF peptide consists of 17 amino acids.

13. The AAV vector of any one of claims 10 to 12, wherein the vector is an AAV2 vector or an AAV2 recombinant virus.

14. The AAV vector of claim 13, wherein the vector is an AAV2.1 vector or an AAV2/2 recombinant virus.

15. The AAV vector of any one of claims 10 to 14, wherein the signal peptide is an interferon beta signal peptide.

16. The AAV vector of claim 15, wherein the interferon beta signal peptide comprises the amino acid sequence of SEQ ID NO: 2.

17. The AAV vector of claim 16, wherein the interferon beta signal peptide is encoded by the nucleic acid sequence of SEQ ID NO: 3.

18. The AAV vector of any one of claims 10 to 17, wherein the PEDF peptide is encoded by the nucleic acid sequence of SEQ ID NO: 4.

19. The AAV vector of any one of claims 10 to 18, wherein the nucleic acid encoding a pigment epithelium-derived factor peptide comprising SEQ ID NO: 1 operably linked to a nucleic acid encoding a signal peptide comprises the nucleic acid sequence of SEQ ID NO: 5.

20. The AAV vector of any one of claims 10 to 14, wherein the PEDF protein is encoded by the nucleic acid sequence of SEQ ID NO: 8.

21. The AAV vector of any one of claims 10 to 20, wherein the nucleic acid encoding the pigment epithelium-derived factor protein or peptide is operably linked to a promoter.

22. A composition comprising the AAV vector of any one of claims 10 to 21, formulated for injection.

23. A method of treating retinal degeneration, comprising administering the vector of claim any one of claims 10 to 21 or the composition of claim 22 to an eye of a subject with retinal degeneration.

24. The method of claim 23, wherein the vector or composition is administered to the eye of the subject by intravitreal injection or subretinal injection.

25. The method of claim 24, wherein the vector or composition is administered one or more times.

26. The method of any one of claims 7 to 9 or claims 23 to 25, wherein the retinal degeneration is retinitis pigmentosa, Leber congenital amaurosis, cone-rod dystrophy, age-related macular degeneration, Stargardt-like macular degeneration, or a maculopathy.