WO2025165877A1

WO2025165877A1 - High acuity vision restoration using soma targeted optogenetics

Info

Publication number: WO2025165877A1
Application number: PCT/US2025/013593
Authority: WO
Inventors: Or A. SHEMESH; Pururav RAMAKRISHNA
Original assignee: University of Pittsburgh
Current assignee: University of Pittsburgh
Priority date: 2024-01-29
Filing date: 2025-01-29
Publication date: 2025-08-07
Anticipated expiration: 2026-07-29

Abstract

The presently disclosed subject matter relates to polypeptides and their encoding nucleic acid molecules for targeting fusion proteins to the soma of cells in which they are expressed. The presently disclosed subject matter further relates to methods for treating a retinal disease, e.g., retinitis pigmentosa, macular degeneration, and diabetic retinopathy.

Description

HIGH ACUITY VISION RESTORATION USING SOMA TARGETED OPTOGENETICS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/626,235 filed January 29, 2024, the content of which is incorporated by reference in its entirety.

SEQUENCE LISTING

A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on January 22, 2025, is entitled “072396.1052_ST26.xml”, and is 234,067 bytes in size.

FIELD

The presently disclosed subject matter relates to methods of treating retinal diseases by expressing soma-targeted opsins in the retina The presently disclosed subject matter further relates to methods of treating retinal diseases by administering nucleic acid constructs encoding soma-targeted opsins.

BACKGROUND

Optogenetics using conventional opsins have been used for vision restoration previously in mice, non-human primates, and human clinical trials, but have had limited success in achieving high acuity. This is due to the expression of opsins throughout the entirety of the processes of retinal ganglion cells (RGCs) and bipolar cells (BCs) in the retina. The neuronal process extensively overlap surrounding cells leading to significant crossactivation. Due to this, single-cell activation from a photostimulation has not been achieved resulting in low-acuity vision restoration. There is a need for soma-targeted opsins whose expression is restricted to the soma of RGCs, which are capable of producing single-cell- resolution activation from a photostimulation, resulting in high-acuity vision restoration.

SUMMARY OF THE INVENTION

The presently disclosed subject matter provides a method of treating a subject having retinal disease, comprising administering a therapeutically effective amount of a fusion protein to one or more cells of the retina, wherein the fusion protein comprises one or more soma-targeting polypeptides and wherein the one or more soma-targeting polypeptides have amino acid sequences that are at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof. In certain embodiments, the one or more soma-targeting polypeptides have amino acid sequences that are at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof. In certain embodiments, the one or more soma-targeting polypeptides have amino acid sequences that are at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof. In certain embodiments, the one or more soma-targeting polypeptides have amino acid sequences that are identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptide, wherein each soma-targeting polypeptides comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

In certain embodiments, the retinal disease is retinitis pigmentosa, macular degeneration, or diabetic retinopathy. In certain embodiments, the one or more cells of the retina comprise retinal ganglion cells (RGCs), bipolar cells (BCs), or a mixture thereof. In certain embodiments, the fusion protein is administered by contacting the one or more cells of the retina with virus particles comprising a nucleic acid that encodes the fusion protein. In certain embodiments, the fusion protein is administered by introducing the one or more cells of the retina with a nucleic acid that encodes the fusion protein by fusion, electroporation, biolistics, transfection, or lipofection.

In certain embodiments, the one or more soma-targeting polypeptides are positioned at the N-terminus of the fusion protein. In certain embodiments, the one or more somatargeting polypeptides are positioned at the C-terminus of the fusion protein. In certain embodiments, one or more soma-targeting polypeptides are positioned at the N-terminus and one or more soma-targeting polypeptides are positioned at the C-terminus. In certain embodiments, the fusion protein further comprises an opsin. In certain embodiments, the opsin is selected from the group consisting of CoChR, ChrimsonR, CsChrimson, ChR2, CsChrimsonR, MW-opsin (0PN1MW), LW-opsin (0PN1LW), SW-opsin (0PN1SW), Chronos, Rhodopsin (0PN2/RH0), mGluR6, Melanopsin, ChroME, CatCh, ReaChR, CsChR, ChETA, CheRiff, PsChR2, C1C1, C1V1, GtACR, Halo, GtACR2, CoChR-3M, Encephalopsin, panopsin (0PN3), Melanopsin (0PN4), Neuropsin (0PN5), Peropsin (RRH), and Retinal G protein coupled receptor (RGR). In certain embodiments, the fusion protein further comprises a fluorescent protein. In certain embodiments, the fluorescent protein is selected from the group consisting of GFP, eGFP, YFP, eYFP, YFP3, miRFP680, miRFP670, miRFP670nano, miRFP670nano3, dTomato, tdTomato, mCherry, DsRed, iRFP, Electra2, Electra, mNeonGreen, mClover3, Clover, VFP, mVFP, mScarlet3, mScarlet, mGreenLantem, Citrane2, mGold, mRuby3, mRuby, mRuby2, mOrange, m0range2, mEmerald, smURFP, DsRed2, mCardinal, mRFPl, iRFP670, miRFP, BFP, miRFP670, FusionRed, EBFP2, mVenus, Venus, Emerald, TagRFP, mApple, and mKate. In certain embodiments, the fusion protein further comprises one or more linkers having amino acid sequences that are identical to the amino acid sequences set forth in SEQ ID NO.: 116-140, or mixtures thereof.

In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 80% identical to the amino acid sequences set forth in SEQ ID NO.: 1- 112 or fragments thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 80% identical to the amino acid sequences set forth in SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or fragments thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequences set forth in SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or fragments thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequences set forth in SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or fragments thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is identical to the amino acid sequences set forth in SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or fragments thereof.

In certain embodiments, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof, at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof, or mixtures thereof. In certain embodiments, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof, at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof, or mixtures thereof. In certain embodiments, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof, at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof, or mixtures thereof. In certain embodiments, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof, identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof, or mixtures thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a a first region comprising a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each somatargeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are included to illustrate certain aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations, and equivalents in form and function, without departing from the scope of this disclosure.

Figure 1 shows soma-targeted CoChR opsins in primary cortical neuron. Images were collected from mice primary neurons transfected with CoChR-GFP, fused with or without soma targeting protein fragments. Cells were simultaneously transfected with Electra2 fluorescent protein as a neuronal tracer. Top row, untargeted CoChR; bottom rows, targeted CoChR rSKl, MBD and MVIBD are completely novel soma targeting protein fragments. Scalebar for all images - 100pm.

Figure 2 shows soma-targeted ChrimsonR opsins in primary cortical neuron. Images were collected from mice primary neurons transfected with ChrimsonR-miRFP680, fused with or without soma targeting protein fragments. Cells were simultaneously transfected with Electra2 fluorescent protein as a neuronal tracer. Top row, untargeted ChrimsonR; bottom rows, targeted ChrimsonR rSKl, MBD and MVIBD are completely novel soma targeting protein fragments. Scalebar for all images - 100pm.

Figures 3A-3C show soma-targeted CoChR opsins. Figure 3A shows neurons expressing CoChR opsins AAVs. Figure 3B shows whole-cell recordings in mouse brain slices. Figure 3C shows cell-body restricted photoactivation using novel CoChR opsin in mouse brain slices. Figure 4 shows soma-targeted CsChrimson-DsRed in a retinal explant. Eyes were enucleated from P23H homozygous mice. Explants were prepared enucleated and mounted on polycarbonate membranes at days-in-vitro 0 (DIV 0), transduced on DIV 1 and imaged on DIV 11 using a confocal microscope. Red - CsChrimson-DsRed (scalebar - lOOum in all images).

Figures 5A-5B show high-acuity vision restoration using soma-targeted opsins. Figure 5A shows retinal cell layers in healthy human eyes vs the pathological loss of rods and cones in retinitis pigmentosa. Figure 5B shows conventional optogenetic strategies for vision restoration (top, i) and the novel strategy using soma-targeting (bottom, ii). (i) RGCs in human retina expressing conventional opsins throughout the soma and the processes for restoring light sensitivity. Neighboring cells whose processes come in contact with the photostimulated cell are also stimulated due to opsin expression. This non-specific photostimulation translates to low acuity vision restoration, (ii) RGCs in human retina expressing soma-targeted opsins for restoring light sensitivity, leading to high spatial resolution of activation. This translates to high acuity vision restoration.

Figure 6 shows amino acid and nucleotide sequences encoding soma-targeting polypeptides.

DETAILED DESCRIPTION

The presently disclosed subject matter relates to soma-targeted polypeptides, and methods of treating retinal diseases, e.g., retinitis pigmentosa, macular degeneration, and diabetic retinopathy using soma-targeted polypeptides.

For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

1. Definitions;

2. Soma-targeting Polypeptides; and

3. Methods of Treatment.

1. Definitions

The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them. As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Still further, the terms “having,” “including,” “containing” and “comprising” are interchangeable and one of skill in the art is cognizant that these terms are open ended terms.

As used herein, the terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The present disclosure also contemplates other embodiments “comprising,” “consisting of’, and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

As used herein, the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

As used herein, the term “increase” is meant to alter positively by at least about 5%. An alteration can be an increase of about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100% or more.

As used herein, the term “reduce” is meant to alter negatively by at least about 5%. An alteration can be a decrease of about 5%, about 10%, about 25%, about 30%, about 50%, about 75% or more, even by about 100%.

As used herein, the term “cell” refers to any suitable cell for use in the present disclosure, e.g., eukaryotic cells. For example, but not by way of limitation, suitable eukaryotic cells include animal cells, e.g., mammalian cells. In certain embodiments, suitable cells are cultured cells. In certain embodiments, suitable cells are host cells, recombinant cells, and recombinant host cells. In certain embodiments, suitable cells are cell lines obtained or derived from mammalian tissues which are able to grow and survive when placed in media containing appropriate nutrients and/or growth factors. As used herein, the terms “host cell,” “host cell line” and “host cell culture” are used interchangeably and refer to cells and their progeny into which exogenous nucleic acid can be subsequently introduced to create recombinant cells. In certain embodiments, these host cells can also be modified (i.e., engineered) to alter or delete the expression of certain endogenous host cell proteins. Host cells can include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny does not need to be completely identical in nucleic acid content to a parent cell, but can contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The introduction of exogenous nucleic acid (e.g., by transfection) to these host cells would create recombinant cells that are derived from the original “host cell,” “host cell line” or “host cell line”. The terms “host cell,” “host cell line” and “host cell culture” can also refer to such recombinant cells and their progeny.

As used herein, the terms “expression” or “expresses,” refer to transcription and translation occurring within a cell, e.g., mammalian cell. In certain embodiments, the level of expression of a gene and/or nucleic acid in a cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the gene and/or nucleic acid that is produced by the cell. For example, mRNA transcribed from a gene and/or nucleic acid is desirably quantitated by northern hybridization. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a gene and/or nucleic acid can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. For example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, overexpressed or not expressed at all.

As used herein, the terms “vector” or “plasmid”, which can be used interchangeably, refer to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

As used herein, the terms “nucleic acid molecule” and “nucleotide sequence,” refer to a single or double-stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodi ester bonds. The nucleic acid molecule can include deoxyribonucleotide bases or ribonucleotide bases, and can be manufactured synthetically in vitro or isolated from natural sources.

As used herein, the terms “polypeptide,” “peptide,” “amino acid sequence” and “protein,” used interchangeably herein, refer to a molecule formed from the linking of at least two amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. A polypeptide can be obtained by a suitable method known in the art, including isolation from natural sources, expression in a recombinant expression system, chemical synthesis or enzymatic synthesis. The terms can apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.

As used herein, the term “protein” is meant to refer to a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from “peptides” or other small molecular weight polypeptides that do not have such structure. In certain embodiments, the protein herein will have a molecular weight of at least about 15-20 kDa, e.g., about 20 kDa or greater. Examples of proteins encompassed within the definition herein include host cell proteins as well as all mammalian proteins, in particular, therapeutic and diagnostic proteins, such as therapeutic and diagnostic antibodies, and, in general proteins that contain one or more disulfide bonds, including multi-chain polypeptides comprising one or more inter- and/or intrachain disulfide bonds.

As used herein, “a functional fragment” or “functional variant” of a molecule or polypeptide includes nucleic acids or polypeptides that comprise modifications and/or truncations compared to a parent or wild type nucleic acid or a sequence and retain at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% of the primary function of the molecule or polypeptide. In certain embodiments, a functional variant can differ from the parent or wild type sequence by at least one modification, e.g., from about one to about ten modifications. In certain embodiments, the sequence of a functional variant sequence has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity to a parent or wild type sequence. In certain embodiments, a functional variant can differ from another variant of the parent by at least one modification, e.g., from about one to about ten modifications. In certain embodiments, the sequence of a functional variant has at least about 80%, at least about 90%, at least about 95% or at least about at least about 99% identity to a different variant of the parent.

As used herein, the term “substantially identical” or “substantially homologous” refers to a polypeptide or a nucleic acid molecule exhibiting at least about 50% identical or homologous to a reference amino acid sequence (for example, any of the amino acid sequences described herein) or a reference nucleic acid sequence (for example, any of the nucleic acid sequences described herein). In certain embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identical or homologous to the amino acid sequence or the nucleic acid sequence used for comparison.

As used herein, the term “linker” or “spacer” refers to a polypeptide which is used to connect adjacent polypeptides. Linker sequences known in the art are suitable for use in the present disclosure.

As used herein, an “individual” or “subject” is a vertebrate, such as a human or nonhuman animal, for example, a mammal. Mammals include, but are not limited to, humans, non-human primates, farm animals, sport animals, rodents and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters, and guinea pigs; rabbits; dogs; cats; sheep; pigs; goats; cattle; horses; and non-human primates such as apes and monkeys.

As used herein, the term “disease” refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of a composition that is sufficient for obtaining beneficial or desired results, including clinical results. The amount of a virus composition or nucleic acid composition that is therapeutically effective or effective can vary depending on the context. An effective amount will vary with the particular intended use and means of detection, for example, the location and condition of the cells in which the fusion protein is to be expressed, the specific route of administration. An effective amount can be administered in one or more administrations.

As used herein, the terms “administer”, “administering”, or “administration” include the delivery of one or more fusion protein disclosed herein to target cells such that the one or more fusion protein is expressed in the target cells. These terms additionally include the delivery of one or more nucleic acids encoding one or more fusion protein disclosed herein to target cells, such that the one or more fusion protein is expressed in the target cells. These terms additionally include the delivery of a pharmaceutical composition comprising one or more fusion protein or one or more nucleic acids disclosed herein to target cells, such that the one or more fusion protein or one or more nucleic acid is expressed in the target cells. In a non-limiting example, a nucleic acid encoding a fusion protein disclosed herein can be administered to target cells by techniques known in the art, such as fusion, electroporation, biolistics, transfection, or lipofection. In another non-limiting example, virus particles can be administered to the target cells, wherein the virus particles comprise a nucleic acid encoding a fusion protein disclosed herein. As used herein, and as well-understood in the art, “treatment” is an approach for obtaining beneficial or desired results, including clinical results. For purposes of this subject matter, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more sign or symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, prevention of disease, delay or slowing of disease progression, and/or amelioration or palliation of the disease state. The decrease can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99% decrease in severity of complications or symptoms. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.

2. Soma-targeting Polypeptides

The present disclosure relates to fusion proteins and their encoding nucleic acids which comprise soma-targeting sequences. Soma-targeting sequences of the present disclosure include the nucleic acid sequences SEQ ID NO.: 113, SEQ ID NO.: 114, and SEQ ID NO.: 115. Soma-targeting polypeptides of the present disclosure further include the amino acid sequences SEQ ID NO.: 1-112.

Fusion proteins and their encoding nucleic acids can comprise one or more somatargeting sequence of the present disclosure or functional variant thereof. Functional variants have one or more substitutions or modifications while retaining at least a portion of the function of the parent molecule of which they are a variant. For example, the soma-targeting nucleic acid or amino acid sequence disclosed herein can be modified with one or more substitutions, deletions, insertions, or combinations thereof. Functional variants can be tested using methods disclosed herein for characteristics including, but not limited to, expression, cell localization, and targeting of one or more polypeptides of interest to the soma of a cell in which they are expressed. Fusion proteins can be produced using methods known in the art, for example, as described in WO Patent Publication No. 2020/123688, U.S. Patent 10,545,145, and U.S. Patent 11,324,824, which are herein incorporated by reference which are herein incorporated by reference.

In certain embodiments, a functional variant of a soma-targeting polypeptide- encoding nucleic acid has a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof.

In certain embodiments, a functional variant of a soma-targeting polypeptide- encoding nucleic acid has a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

In certain embodiments, a functional variant of a soma-targeting polypeptide- encoding nucleic acid has a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof.

In certain embodiments, a functional variant of a soma-targeting polypeptide has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequences set forth in SEQ ID NO.: 1-112 or fragments thereof. In certain embodiments, a functional variant of a soma-targeting polypeptide has an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homologous or identical to the amino acid sequences set forth in SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, or fragments thereof.

Fusion proteins and their encoding nucleic acids can comprise one soma-targeting polypeptide or a plurality of soma-targeting polypeptides. Fusion proteins can comprise one or more, two or more, three or more, four or more, five or more, or six or more somatargeting polypeptides. When fusion proteins comprise a plurality of soma-targeting polypeptides, the plurality of soma-targeting polypeptides can have identical or different sequences. For example, the nucleic acid sequence encoding the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 113 or fragments thereof, wherein the amino acid sequences of the functional variants are the same or different. The nucleic acid sequence encoding the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 114 or fragments thereof, wherein the amino acid sequences of the functional variants are the same or different. The nucleic acid sequence encoding the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 115 or fragments thereof, wherein the amino acid sequences of the functional variants are the same or different. The amino acid sequence of the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 1 or fragments thereof, wherein the amino acid sequences of the functional variants are the same or different. The amino acid sequence of the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 2 or fragments thereof, wherein the amino acid sequences of the functional variants are the same or different. The amino acid sequence of the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 3 or fragments thereof, wherein the amino acid sequences of the functional variants are the same or different.

In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof.

Fusion proteins and their encoding nucleic acids can comprise a combination of functional variants of SEQ ID NO.: 1 and SEQ ID NO.: 2, wherein the amino acid sequences of the functional variants of SEQ ID NO.: 1 are the same or different and wherein the amino acid sequences of the functional variants of SEQ ID NO.: 2 are the same or different. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises a first region comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and a second region comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

Alternatively, the nucleic acid sequence encoding the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 113, wherein the nucleic acid sequences of the functional variants are the same or different. The nucleic acid sequence encoding the fusion protein can comprise a plurality of functional variants of SEQ ID NO.: 114, wherein the nucleic acid sequences of the functional variants are the same or different. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof.

Fusion proteins and their encoding nucleic acids can comprise a combination of functional variants of SEQ ID NO.: 113 and SEQ ID NO.: 114, wherein the nucleic acid sequences of the functional variants of SEQ ID NO.: 113 are the same or different and wherein the nucleic acid sequences of the functional variants of SEQ ID NO.: 114 are the same or different. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises a first region comprising a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and a second region comprising a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% homologous or identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 somatargeting polypeptides encoded by a nucleic acid sequence at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% homologous or identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

The soma-targeting polypeptides can be positioned at the N-terminus and/or C- terminus of the fusion protein. In certain embodiments, the soma-targeting polypeptide is positioned at the N-terminus of the fusion protein. In certain embodiments, the somatargeting polypeptide is positioned at the C-terminus of the fusion protein. In certain embodiments, one or more soma-targeting polypeptides are positioned at the N-terminus and one or more soma-targeting polypeptides are positioned at the C-terminus.

Fusion proteins and their encoding nucleic acids can comprise opsins. Non-limiting examples of opsins include CoChR, ChrimsonR, CsChrimson, ChR2, CsChrimsonR, MW- opsin (0PN1MW), LW-opsin (OPN1LW), SW-opsin (OPN1SW), Chronos, Rhodopsin (OPN2/RHO), mGluR6, Melanopsin, ChroME, CatCh, ReaChR, CsChR, ChETA, CheRiff, PsChR2, C1C1, C1V1, GtACR, Halo, GtACR2, CoChR-3M, Encephal opsin, panopsin (OPN3), Melanopsin (OPN4), Neuropsin (OPN5), Peropsin (RRH), and Retinal G protein coupled receptor (RGR).

Fusion proteins can comprise linkers. Non-limiting examples of linkers are listed in Table 1. In certain embodiments, the fusion protein comprises two or more soma-targeting polypeptides and one or more linkers between the two or more soma-targeting polypeptides. In certain embodiments, the fusion protein comprises one or more soma-targeting polypeptide an opsin, and a linker between the one or more soma-targeting polypeptide and the opsin.

Table 1. Linker sequences.

Fusion proteins and their encoding nucleic acids can comprise fluorescent proteins. Non-limiting examples of fluorescent proteins include GFP, eGFP, YFP, eYFP, YFP3, miRFP680, miRFP670, miRFP670nano, miRFP670nano3, dTomato, tdTomato, mCherry, DsRed, iRFP, Electra2, Electra, mNeonGreen, mClover3, Clover, VFP, mVFP, mScarlet3, mScarlet, mGreenLantern, Citrane2, mGold, mRuby3, mRuby, mRuby2, mOrange, mOrange2, mEmerald, smURFP, DsRed2, mCardinal, mRFPl, iRFP670, miRFP, BFP, miRFP670, FusionRed, EBFP2, mVenus, Venus, Emerald, TagRFP, mApple, and mKate.

In certain embodiments, the nucleic acid molecule encoding the fusion protein is integrated into the genome of the virus, where the expression of the nucleic acid molecule is operably linked to a promoter that is active or activatable in the virus infected cell. As used herein, “operably linked” means that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid locus to control transcriptional initiation and/or expression of that locus. In certain embodiments, the nucleic acid encoding the fusion protein is operably linked to a promoter which induces expression in retinal ganglion cells and bipolar cells. Non-limiting examples of promoters include chicken P-actin (CBA), cytomegalovirus (CMV), short CMV early enhancer/chicken P-actin/short P-globulin intron (sCAG), mouse phosphoglycerate kinase (PGK), and human synapsin (SYN), Neurofilament heavy (Nefh), mouse y-synuclein (mSncg), human y-synuclein (hSncg), Piel 55, Ple265 and Ple341 (PCP2), mGluR6. In certain embodiments, this can also be used in conjunction with gene expression enhancers for specific cells such as, but not limited to, AT0H7, Grm6, Cabp5, ChxlO, 200En, 407En, 770En, introns In4, In3 of the mGluR6 gene.

Fusion proteins with soma-targeting polypeptides, and their encoding nucleic acid molecules, can be produced using methods know in the art. Constructs encoding soma- targeted opsins, or constructs encoding related functional variants, as described herein, can be introduced into cells as one or more DNA molecules or constructs, in many cases in association with one or more markers to allow for selection of host cells which contain the construct s). The constructs can be prepared in conventional ways, where the coding sequences and regulatory regions can be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means.

In certain embodiments, the expression construct encoding the polypeptide or protein of interest is integrated into one or more expression vectors. In certain embodiments, the expression vector is a nucleic acid and provides all required elements for the amplification of said vector in a mammalian cell. In certain embodiments, an expression vector is a vehicle for the introduction of an expression construct into a modified mammalian cell according to the subject matter of the present disclosure. In certain embodiments, a construct can be introduced as a single DNA molecule encoding multiple genes, or different DNA molecules having one or more genes. In certain embodiments, multiple constructs can be introduced simultaneously or consecutively, each with the same or different DNA molecule.

3. Methods of Treatment

The present disclosure provides methods of treating a subject having a retinal disease, e.g., retinitis pigmentosa, macular degeneration, and diabetic retinopathy. The method comprises expressing soma-targeted opsins, as disclosed herein, in cells of the retina. In certain embodiments, methods comprise administering a therapeutically effective amount of a fusion protein comprising one or more soma-targeting polypeptides as disclosed herein. In certain embodiments, the soma-targeted opsins are expressed in the retinal ganglion cells (RGC), bipolar cells (BCs), or mixtures thereof.

Soma-targeted opsins are capable of producing single-cell-resolution activation from a photostimulation, resulting in high-acuity vision restoration. Soma-targeted opsins are additionally capable of producing single-cell resolution activation of neurons of the brain, e.g., for precise manipulation of cellular biology or cell signaling pathways, precise deep brain stimulation, high resolution hearing restoration, drug screening, and/or sensory restoration and basic research. Retinal diseases, e.g., retinitis pigmentosa, macular degeneration, and diabetic retinopathy, result in the loss of photoreceptors in the retina. As a result, subjects with retinal diseases experience reduced ability to detect or transmit light- triggered signals to the brain. These phenotypes can be studied using mouse and rat models of retinal disease which include, but are not limited to, Rpe65-/-, Gnatl-/-, Lrat-/-, Rdhl2-/-, Gel-/-, Cep290-/-, Crx-/-, Aipll— /— , Rgr-/-, Cnga3 Opn4 , , , , , ave phenotypes resembling retinitis pigmentosa, e.g., loss of rods and cones or retinal pigment epithelium, neovascularization, or drusen deposits in the retina, etc.

Methods for increasing the light sensitivity of cells in the retina have been proposed for treating retinal diseases. The purpose of the increased sensitivity is to mitigate the loss of light-triggered signal transduction caused by retinal disease. The present disclosure provides methods for treating retinal disease comprising expressing a soma-targeted opsin in the cells of the retina (e.g., retinal ganglion cells (RGCs) and bipolar cells (BCs)). The expression of opsins increases the light sensitivity of the cells. Targeting opsins to the soma of the cells reduces the presence of opsin at the processes of the cells thereby reducing cross-activation between nearby cells following photostimulation. As a result, the method yields single-cell- resolution activation from a photostimulation and results in high-acuity vision restoration.

The present disclosure further provides methods of treating a subject having a neurodegeneration disorder or psychiatric condition, comprising expressing a therapeutically effective amount of a fusion protein comprising one or more soma-targeting polypeptides as disclosed herein. Soma-targeted polypeptides can be used for precise in vitro and in vivo cortical and peripheral neuron manipulation, which facilitates study and/or treatment of healthy neurons, animal behavior, neurodegeneration, and psychiatric conditions. Nonlimiting examples of neurodegeneration include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, epilepsy, stroke, and chronic pain. Non-limiting examples of psychiatric conditions include obsessive-compulsive disorder, drug addiction, depression, social dysfunction, and anxiety.

Expression and activity of soma-targeted opsins can be evaluated, for example, using optogenetic stimulations. Cells expressing soma-targeted opsins can be contacted with a light under suitable conditions to activate the opsin polypeptide and modulate electrical activity and/or physiological activity. The present disclosure provides a method of treating a subject having retinal disease, comprising administering a therapeutically effective amount of a fusion protein to one or more cells of the retina, wherein the fusion protein comprises one or more soma-targeting polypeptides and wherein the one or more soma-targeting polypeptides have amino acid sequences that are at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof. Alternatively, the one or more soma-targeting polypeptides have amino acid sequences that are at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3, or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3 or fragments thereof. Alternatively, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. Alternatively, the fusion protein comprises up to 3 somatargeting polypeptides comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the retinal disease is retinitis pigmentosa, macular degeneration, or diabetic retinopathy. In certain embodiments, the one or more cells of the retina comprise retinal ganglion cells (RGCs), bipolar cells (BCs), or a mixture thereof. In certain embodiments, the fusion protein is administered by contacting the one or more cells of the retina with virus particles comprising a nucleic acid that encodes the fusion protein. In certain embodiments, the fusion protein is administered by introducing the one or more cells of the retina with a nucleic acid that encodes the fusion protein by fusion, electroporation, biolistics, transfection, or lipofection. In certain embodiments, the one or more soma-targeting polypeptides are positioned at the N- terminus and/or the C-terminus of the fusion protein. In certain embodiments, the fusion protein further comprises an opsin. In certain embodiments, the opsin is selected from the group consisting of CoChR, ChrimsonR, CsChrimson, ChR2, CsChrimsonR, MW-opsin (0PN1MW), LW-opsin (OPN1LW), SW-opsin (OPN1SW), Chronos, Rhodopsin (OPN2/RHO), mGluR6, Melanopsin, ChroME, CatCh, ReaChR, CsChR, ChETA, CheRiff, PsChR2, C1C1, C1V1, GtACR, Halo, GtACR2, CoChR-3M, Encephal opsin, panopsin (OPN3), Melanopsin (OPN4), Neuropsin (OPN5), Peropsin (RRH), and Retinal G protein coupled receptor (RGR). In certain embodiments, the fusion protein further comprises a fluorescent protein. In certain embodiments, the fusion protein further comprises one or more linkers having amino acid sequences that are identical to the amino acid sequences set forth in SEQ ID NO.: 116-140, or mixtures thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 80% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112 or fragments thereof. Alternatively, the somatargeting polypeptide has an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof. In certain embodiments, the fusion protein comprises one or more somatargeting polypeptides encoded by nucleic acid sequences that are at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. Alternatively, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a a first region comprising a nucleic acid sequence that is at least about 90%, at least about 95% or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. Alternatively, the fusion protein comprises up to 3 somatargeting polypeptides encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

The present disclosure further provides methods of increasing the light sensitivity of one or more cells of the retina, comprising administering a therapeutically effective amount of a fusion protein to one or more cells of the retina, wherein the fusion protein comprises one or more soma-targeting polypeptides and wherein the one or more soma-targeting polypeptides have amino acid sequences that are at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof. Alternatively, the one or more soma-targeting polypeptides have amino acid sequences that are at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3, or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3 or fragments thereof. Alternatively, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the retinal disease is retinitis pigmentosa, macular degeneration, or diabetic retinopathy. In certain embodiments, the one or more cells of the retina comprise retinal ganglion cells (RGCs), bipolar cells (BCs), or a mixture thereof. In certain embodiments, the fusion protein is administered by contacting the one or more cells of the retina with virus particles comprising a nucleic acid that encodes the fusion protein. In certain embodiments, the fusion protein is administered by introducing the one or more cells of the retina with a nucleic acid that encodes the fusion protein by fusion, electroporation, biolistics, transfection, or lipofection. In certain embodiments, the one or more soma-targeting polypeptides are positioned at the N-terminus and/or the C-terminus of the fusion protein. In certain embodiments, the fusion protein further comprises an opsin. In certain embodiments, the opsin is selected from the group consisting of CoChR, ChrimsonR, CsChrimson, ChR2, CsChrimsonR, MW-opsin (0PN1MW), LW-opsin (0PN1LW), SW- opsin (0PN1SW), Chronos, Rhodopsin (0PN2/RH0), mGluR6, Melanopsin, ChroME, CatCh, ReaChR, CsChR, ChETA, CheRiff, PsChR2, C1C1, C1V1, GtACR, Halo, GtACR2, CoChR-3M, Encephalopsin, panopsin (0PN3), Melanopsin (0PN4), Neuropsin (0PN5), Peropsin (RRH), and Retinal G protein coupled receptor (RGR). In certain embodiments, the fusion protein further comprises a fluorescent protein. In certain embodiments, the fusion protein further comprises one or more linkers having amino acid sequences that are identical to the amino acid sequences set forth in SEQ ID NO.: 116-140, or mixtures thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 80% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112 or fragments thereof. Alternatively, the soma-targeting polypeptide has an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof. In certain embodiments, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. Alternatively, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a a first region comprising a nucleic acid sequence that is at least about 90%, at least about 95% or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 somatargeting polypeptides encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

The present disclosure further provides methods of increasing single-cell-resolution activation from a photostimulation, comprising administering a therapeutically effective amount of a fusion protein to one or more cells of the retina, wherein the fusion protein comprises one or more soma-targeting polypeptides and wherein the one or more somatargeting polypeptides have amino acid sequences that are at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof. Alternatively, the one or more soma-targeting polypeptides have amino acid sequences that are at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3, or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3 or fragments thereof. Alternatively, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1-3 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 90%, 95%, or 100% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof. In certain embodiments, the retinal disease is retinitis pigmentosa, macular degeneration, or diabetic retinopathy. In certain embodiments, the one or more cells of the retina comprise retinal ganglion cells (RGCs), bipolar cells (BCs), or a mixture thereof. In certain embodiments, the fusion protein is administered by contacting the one or more cells of the retina with virus particles comprising a nucleic acid that encodes the fusion protein. In certain embodiments, the fusion protein is administered by introducing the one or more cells of the retina with a nucleic acid that encodes the fusion protein by fusion, electroporation, biolistics, transfection, or lipofection. In certain embodiments, the one or more soma-targeting polypeptides are positioned at the N-terminus and/or the C-terminus of the fusion protein. In certain embodiments, the fusion protein further comprises an opsin. In certain embodiments, the opsin is selected from the group consisting of CoChR, ChrimsonR, CsChrimson, ChR2, CsChrimsonR, MW-opsin (0PN1MW), LW-opsin (0PN1LW), SW- opsin (0PN1SW), Chronos, Rhodopsin (0PN2/RH0), mGluR6, Melanopsin, ChroME, CatCh, ReaChR, CsChR, ChETA, CheRiff, PsChR2, C1C1, C1V1, GtACR, Halo, GtACR2, CoChR-3M, Encephalopsin, panopsin (0PN3), Melanopsin (0PN4), Neuropsin (0PN5), Peropsin (RRH), and Retinal G protein coupled receptor (RGR). In certain embodiments, the fusion protein further comprises a fluorescent protein. In certain embodiments, the fusion protein further comprises one or more linkers having amino acid sequences that are identical to the amino acid sequences set forth in SEQ ID NO.: 116-140, or mixtures thereof. In certain embodiments, the soma-targeting polypeptide has an amino acid sequence that is at least about 80% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112 or fragments thereof. Alternatively, the soma-targeting polypeptide has an amino acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof. In certain embodiments, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. Alternatively, the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113-115 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a a first region comprising a nucleic acid sequence that is at least about 90%, at least about 95% or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. In certain embodiments, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof. Alternatively, the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 somatargeting polypeptides encoded by a nucleic acid sequence that is at least about 90%, at least about 95%, or about 100% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

3.1 Pharmaceutical compositions

Nucleic acid constructs encoding soma-targeting polypeptides can be introduced into a host cell by any convenient means. The constructs can be incorporated into a pharmaceutical composition and introduced into the one or more cells by any technique known in the art such as by transduction, electroporation, and transfection.

In certain embodiments, the pharmaceutical composition described herein further includes a pharmaceutically acceptable carrier, e.g., an excipient. In certain embodiments, the pharmaceutically acceptable carrier includes any carrier which does not interfere with the effectiveness of the biological activity of the active ingredients and/or that is not toxic to the patient to whom it is administered. Non-limiting examples of suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents and sterile solutions. Additional non-limiting examples of pharmaceutically acceptable carriers include gels, bioadsorbable matrix materials, implantation elements containing the virus, and any other suitable vehicle, delivery, or dispensing means or material.

In certain embodiments, the pharmaceutically acceptable carrier can be a buffering agent. Non-limiting examples of suitable buffering agents can include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As a buffering agent, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium glucomate, aluminium hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide other calcium salts, and combinations thereof.

In certain embodiments, the virus disclosed herein can be propagated in suitable host cells, isolated from host cells, and stored in conditions that promotes stability and integrity of the virus, such that loss of infectivity over time is minimized. In certain embodiments, the virus disclosed herein can be stored by freezing or drying, such as by lyophilization. In certain embodiments, prior to administration, the stored virus can be reconstituted (if dried for storage) and diluted in a pharmaceutically acceptable carrier for administration.

The manner and dosage administered can be adjusted by the individual practitioner. The absolute amount administered can depend upon a variety of factors, including the material selected for administration, whether the administration is in single or multiple doses, and individual subject parameters including age, physical condition, size, weight, etc. In certain embodiments, the pharmaceutical composition is administered from about 1 week to about 4 weeks, about 1 week to about 2 weeks, about 2 weeks to about 4 weeks, about 1 week, about 2 weeks, about 3 weeks, or about 4 weeks. In some embodiments, the composition is administered daily. In some embodiments, the composition is administered once a week, twice a week, three times a week, or four times a week.

The presently disclosed viruses or pharmaceutical compositions can be directly injected into an organ of interest. For example, the presently disclosed viruses or pharmaceutical compositions can be administered to the retina via intraocular injection. Alternatively, the presently disclosed viruses or pharmaceutical compositions are provided indirectly to the organ of interest, for example, by administration into the circulatory system. Alternatively, the presently disclosed viruses or pharmaceutical compositions can be administered via stereotactic techniques, e.g., for intracranial injections.

When administering a therapeutic composition of the presently disclosed subject matter (e.g., a pharmaceutical composition comprising a presently disclosed virus), it can be formulated in a unit dosage injectable form (solution, suspension, emulsion).

3.1.1. Pharmaceutical compositions for transduction

For example, but not by way of limitation, the methods can include administering to the subject a virus that comprises a nucleic acid that encodes a fusion protein disclosed herein. In certain embodiments, the fusion protein comprises a soma-targeted opsin or a functional fragment thereof. In certain embodiments, the pharmaceutical composition includes a therapeutically effective amount of the virus. In certain embodiments, the fusion protein is administered by contacting the one or more cells of the retina with virus particles comprising a nucleic acid that encodes the fusion protein.

The constructs can be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors, for infection or transduction into cells. The fusion protein comprising the soma-targeted polypeptide can be encoded in an adenoviral-associated viral (AAV) vector, a standard, non-integrating virus that is routinely used for gene therapy in humans. In certain embodiments, the virus isotypes can include, but are not limited to, AAV1, AAV2, AAV3, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrhlO, AAV/DJ, AAV/DJ8, AAV2.7m8, AAV2.GL, AAV2.NN, AAV2.GA, AAV2.NS, AAV2.SS, AAV 2/2- 4YF, or Y444F mutants. In certain embodiments, the constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like. The host cells will in some cases be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct s) and integration of the construct s). The cells will then be expanded and screened by virtue of a marker present in the construct.

In certain embodiments, the virus is administered in an amount between about 10⁵ to about 10^2u genome copies per kg, between about 10⁵ to about IO¹⁰ genome copies per kg, between about 10^w to about I0^{! 5} genome copies per kg, between about 10^l3 to about IO²⁰ genome copies per kg, about 10⁵ genome copies per kg, about 10⁶ genome copies per kg, about 10 ' genome copies per kg, about 10⁸ genome copies per kg, about IO⁹ genome copies per kg, about IO¹⁰ genome copies per kg, about 10¹¹ genome copies per kg, about 10¹² genome copies per kg, about 10¹³ genome copies per kg, about 10¹⁴ genome copies per kg, about 10¹⁵ genome copies per kg, about 10¹⁶ genome copies per kg, about 10¹ ' genome copies per kg, about IO¹⁸ genome copies per kg, about IO¹⁹ genome copies per kg, or about IO²⁰ genome copies per kg. In certain embodiments, the virus is administered in an amount that is at least about 10⁵ genome copies per kg, at least about 10⁶ genome copies per kg, at least about 10'' genome copies per kg, at least about 10^s genome copies per kg, at least about 10⁹ genome copies per kg, at least about IO¹⁰ genome copies per kg, at least about 10¹¹ genome copies per kg, at least about 10¹² genome copies per kg, at least about 10^!J genome copies per kg, at least about 10¹⁴ genome copies per kg, at least about 10¹⁵ genome copies per kg, at least about 10¹⁶ genome copies per kg, at least about 10¹⁷ genome copies per kg, at least about 10¹⁸ genome copies per kg, at least about 10¹⁹ genome copies per kg, or at least about IO²⁰ genome copies per kg.

3, 1.2. Pharmaceutical compositions for non-viral delivery

The fusion protein can be administered by introducing the one or more cells of the retina with a nucleic acid that encodes the fusion protein by fusion, electroporation, biolistics, transfection, or lipofection, such that the one or more cells express the fusion protein.

In certain embodiments, expressing one or more recombinant proteins of interest in a host cell includes culturing a cell comprising one or more nucleic acid(s) encoding the polypeptide or protein of interest, under conditions suitable for expression of the polypeptide or protein. Non-limiting examples of such cells are disclosed herein, e.g., mammalian cells can be used to express the polypeptide or protein. In certain embodiments, a host cell is transfected with a vector containing the nucleic acid sequence suitable for expression of said polypeptide or protein of interest.

The expression construct encoding the polypeptide or protein of interest can be integrated into a host cell using genome editing techniques. Genome editing is a technique in which endogenous chromosomal sequences present in one or more cells within a subject, can be edited, e.g., modified, using targeted endonucleases and single-stranded nucleic acids. The genome editing method can result in the insertion of a nucleic acid sequence at a specific region within the genome, the excision of a specific sequence from the genome and/or the replacement of a specific genomic sequence with a new nucleic acid sequence. Non-limiting examples of genome editing techniques for use in the disclosed methods is the CRISPR system, e.g., CRISPR/Cas 9 system, and RNA-based gene delivery methods. Non-limiting examples of such genome editing techniques are disclosed in PCT Application Nos. WO 2014/093701 and WO 2014/165825, the contents of which are hereby incorporated by reference in their entireties.

In certain embodiments, the genome editing technique can include the use of one or more guide RNAs (gRNAs), complementary to a specific sequence within a genome, e.g., a ga mutation site or a chromosomal breakpoint associated with a fusion gene, including protospacer adjacent motifs (PAMs), to guide a nuclease, e.g., an endonuclease, to the specific genomic sequence. In certain embodiments, the genome editing technique can include the use of one or more guide RNAs (gRNAs), complementary to the sequences that are adjacent to and/or overlap the mutation site or chromosomal breakpoint, to guide one or more nucleases.

In certain embodiments, the one or more gRNAs can include a targeting sequence that is complementary to a sequence present within the gene. In certain embodiments, the targeting sequences are about 10 to about 50 nucleotides in length, e.g., from about 10 to about 45 nucleotides, from about 10 to about 40 nucleotides, from about 10 to about 35 nucleotides, from about 10 to about 30 nucleotides, from about 10 to about 25 nucleotides, from about 10 to about 20 nucleotides, from about 10 to about 15 nucleotides, from about 15 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 25 to about 50 nucleotides, from about 30 to about 50 nucleotides, from about 35 to about 50 nucleotides, from about 40 to about 50 nucleotides or from about 45 to about 50 nucleotides in length. In certain embodiments, the targeting sequence is greater than about 50, 51, 52, 53, 54, 55, 56,

57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more nucleotides in length.

In certain embodiments, the one or more gRNAs comprise a pair of offset gRNAs complementary to opposite strands of the target site. In certain embodiments, the one or more gRNAs comprises a pair of offset gRNAs complementary to opposite strands of the target site to generate offset nicks by an endonuclease. In certain embodiments, the offset nicks are induced using a pair of offset gRNAs with a nickase, e.g., a Cas9 nickase such as Cas9^D10A. In certain embodiments, the pair of offset gRNAs are offset by at least about 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,

33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,

58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 88, 89, 90, 91, 92,93, 94, 95 ,96, 97, 98, 99, or at least 100 nucleotides. In certain embodiments, the pair of offset sgRNAs are offset by about 5 to about 100 nucleotides, about 10 to about 50 nucleotides, about 10 to about 40 nucleotides, about 10 to about 30 nucleotides, about 10 to about 20 nucleotides or about 15 to 30 nucleotides.

In certain non-limiting embodiments, a PAM can be recognized by a CRISPR endonuclease such as a Cas protein. Non-limiting examples of Cas proteins include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl , Csy2, Csy3, Cse 1, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Csf2, CsO, Csf4, Cpfl, c2cl, c2c3, Cas9HiFi, homologues thereof or modified versions thereof.

In certain embodiments, the endonuclease can be the clustered, regularly interspaced short palindromic repeat (CRISPR) associated protein 9 (Cas9) endonuclease. In certain embodiments, the Cas9 endonuclease is obtained from Streptococcus pyogenes. In certain embodiments, the Cas9 endonuclease is obtained from Staphylococcus aureus. In certain embodiments, the endonuclease can result in the cleavage of the targeted genome sequence and allow modification of the genome at the cleavage site through nonhomologous end joining (NHEJ) or homologous recombination. In certain embodiments, the Cas9 endonuclease can be a mutated form of Cas9, e.g., that generates a single-strand break or “nick.” For example, and not by way of limitation, the Cas9 protein can include the D10A mutation, i.e., Cas9^D10A (Cong et al. Science. 339:819-823 (2013); Gasiunas et al. PNAS 109:E2579-2586 (2012); and Jinek et al. Science. 337:816-821 (2012), the contents of which are incorporated by reference herein).

In certain embodiments, the genome editing method and/or technique can be used to promote homologous recombination to insert a nucleic acid into the genome of the cell, e.g., to insert any of the nucleic acids encoding fusion proteins comprising soma-targeting polypeptides disclosed herein.

EXAMPLES

The present disclosure will be better understood by reference to the following Examples, which are provided as exemplary of the present disclosed subject matter, and not by way of limitation. EXAMPLE 1: In vitro analysis of soma-targeted opsins

The present example demonstrates the use of novel soma-targeting polypeptides for localizing opsins to the soma of neurons.

Methods

DNA design. A pAAV vector backbone containing a human synapsin promoter, WPRE, bGH poly(A) tail, and flanked by ITRs, was used for all constructs. CoChR opsin and a C-terminal EGFP was subcloned into the aforementioned backbone. This construct served as our control. Subsequent soma-targeted constructs containing the Kv2.1, rSKl (SEQ ID NO.: 3), and KA2(l-150) sequences were prepared by inserting the appropriate sequence at the 3’ terminal of the EGFP. Alternative variants were prepared by inserting it at the 5’ terminal of the opsin. Variants were prepared using the MVIBD and MBD sequences by inserting them at the 3’ terminal of the EGFP in consecutive and sequential repeats, e.g., (“(MVIBD-MBD)x3” (SEQ ID NO.: 14), “(MVIBD)x3-(MBD)x3” (SEQ ID NO.: 30), and “(MVIBD)x2-(MBD)x2” (SEQ ID NO.: 26)). The soma-targeting sequences were separated from CoChR opsin or the EGFP using a GGSGGTGGSGGT (SEQ ID NO.: 140) linker. The ChrimsonR constructs were prepared similarly by replacing CoChR with the red-shifted opsin known as ChrimsonR, and EFGP with the NIR-FP called miRFP680. A neuron tracer construct was prepared using the aforementioned backbone by inserting the blue FP, Electra2.

DNA storage and handling. The plasmid constructs were transformed into OneShot Top 10 competent cells according to manufacturer’s protocol. Lyophilized DNA were resuspended using Ultrapure water at a concentration of 100 ng/pL and used 5 pL for each transformation reaction. 50 pL of the resulting bacteria solution were used following the shaking incubator step of the aforementioned protocol to grow bacterial colonies on LB agar plates. LB Agar plates were prepared in 100 mm dishes with ampicillin antibiotic at a concentration of 100 pg/mL. LB Broth was prepared according to Addgene’s plasmid protocols to isolate and inoculate bacterial colonies, and bacterial glycerol stocks were created. Plasmid DNA was amplified for transfection and virus preparation using GeneJET Miniprep and Maxiprep Kits according to manufacturer’s protocols.

Virus packaging. Plasmids were amplified for virus packaging as described above. All plasmid constructs were packaged into AAVs (serotype 2). Virus titers ranged from 7.5xlOⁿ - 3.2xl0¹².

Culture preparation. Mice primary cortical cultures were prepared using a protocol previously described (Price, P.J., Brewer, G.J. (2001). Serum-Free Media for Neural Cell Cultures. In: Fedoroff, S., Richardson, A. (eds) Protocols for Neural Cell Culture. Springer Protocols Handbooks. Humana Press. https://doi.Org/10.1385/l-59259-207-4:2550). Mice cortical cultures were prepared in 12 well plates and 24 well plates, and cultured in 1000 pL and 500 pL of media, respectively. Mice primary cortical cultures were maintained in Neurobasal Plus medium containing 2% B27 Plus supplement and 1% Penicillin- Streptomycin. One half of the existing culture media was replaced every 2-3 days. The cultures were maintained for approximately 2-3 weeks.

Transfection. Opsin constructs were expressed in mice primary cortical cultures through transfection using commercially available Lipofectamine 2000 reagent according to manufacturer’s protocol. The transfection was performed between the 6-7th day in vitro (DIV) and imaging was performed 24-76 hours later.

Transduction. Opsin constructs were expressed in mice primary cortical cultures through transduction by first diluting virus in media. CoChR opsins were transduced at titers of IxlO⁸ and 2xl0⁸ per well and the Electra2 tracer at 1.4xl0⁹ and 0.7xl0⁹. Existing media was replaced with fresh media containing viruses. The viruses were incubated for 7 days before returning to the regular media replenishment. The transduction was performed between the 4-5th day in vitro (DIV) and imaging was performed 10-14 days (DIV 14-19) later.

Animal handling. All procedures involving animals were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Pittsburgh Institute of Technology Animal Care and Use Committee. Female and Female mice of ~40 days old were used for electrophysiology experiments. Wild-type mice (C57BL/6J) were used as controls for electrophysiology experiments. Transgenic P23H, 129Sv mice were used as RP models for retinal explants.

Retinal explants. Retinal explants were prepared using a protocol previously described (Vats, A., Xi, Y., Feng, B., Clinger, O. D., St Leger, A. J., Liu, X., Ghosh, A., Dermond, C. D., Lathrop, K. L., Tochtrop, G. P., Picaud, S., & Chen, Y. (2022). Nonretinoid chaperones improve rhodopsin homeostasis in a mouse model of retinitis pigmentosa. JCI insight, 7(10), el53717. https://doi.org/10.1172/jci.insight.153717). The retinal explants were maintained in Neurobasal-Plus medium (Gibco) containing 2% B27 Plus supplement (Gibco) and 1% Penicillin-Streptomycin (Gibco, Catalog Number: 15140122). The culture media was replaced the day after explant preparation, and every 2-3 days thereafter. The explant cultures were maintained for 14 days in total. Confocal Imaging. Imaging was performed using Nikon Eclipse Ti-2 Confocal microscope equipped with a 10X objective, 40X objective, high resolution galvano scanner, and NIS-Elements AR 5.40.01 (Build 1657) 64-bit software to image all primary cultures and retinal explants.

Stereotaxic surgeries and injections. Mice were anesthetized with isoflurane. Their head was fixed in a stereotaxic frame while isoflurane was administrated via inhalation. Their body temperature was maintained at ~37 °C with a heating pad. A small craniotomy was drilled to perform intracranial injections on the auditory cortex (ACx) (Lambda (mm); a/p: 0.0, m/1: 3.8, d/v: -1.0, w/ needle angled 30°. To express CoChR variants, a 32G needle attached to a lOpe-tubing and connected to a 10-pL glass syringe was used. Then, a syringe pump was used to inject -200 nl of AAV (titer: -2.9 X 10¹² GC/mL) diluted [1 :25] in PBS and delivered at 200 nl/min. After completing the injection, the needle capillary remained in position for 5 min to allow the virus to diffuse at the injection site, at the end the needle was slowly withdrawn. Virus was allowed to express themselves for 15 or 30 days.

Slice preparation. Mice were anesthetized with isoflurane and decapitated. The brain was removed from the skull and immersed in ice-cold oxygenated (95% 02-5% CO2) low Ca²⁺ solution (in mM: 125 NaCl, 2.5 KC1, 26 NaHCO₃, 1.25 NaH₂PO₄, 2 MgCh, 10 glucose, 205 sucrose, 0.5 CaCh). Coronal slices (300 pm) were made using a vibratome in ice-cold low Ca²⁺ solution. Then, the slices were transferred in a chamber at room temperature for 60 min in recording ACSF solution (in mM: 125 NaCl, 2.5 KC1, 26 NaHCOs, 2 CaCh,l MgCh, and 20 glucose, pH 7.4, ~305 mOsm, oxygenated w/ 95% 02-5% CO2). After incubation, slices were maintained at room temperature to perform patch clamp recordings in the same conditions (room temp, normal ACSF).

Electrophysiology. Whole cell patch clamps recordings were performed in voltage or current clamp modalities by using a MultiClamp 700B amplifier. Data were low pass filtered (5 kHz) and sampled at 10 kHz using a Digidata 1440-A controlled by p-clamp software. Recording pipettes (-5 MQ) were pulled from borosilicate glass (1.5 mm, outer diameter) on a Flaming/Brown micropipette puller. Pipette capacitance and series (Rs) resistance were compensated priory data acquisition from each cell. Series (Rs) and input (Ri) resistance were determined by using a 50 ms -10 mV hyperpolarizing voltage step from - 70 mV (holding potential) and monitored throughout the experiment. R_s and Neurons with Rs > 20 MW were discarded. We also discarded cells that showed more than 20% change in either Rs or Ri changed during experiment. We employed an intracellular solution consisting of (in mM) 113 K-gluconate, 4.5 MgCh, 9 HEPES, 4 Na2ATP, 0.3 TrisGTP, 14 Tris-phosphocreatine, 0.1 EGTA, and 10 Sucrose (pH=7.3, 295 mOsm). Neurons were visualized digitally using a Retiga 2000 camera attached to an Olympus microscope. Experiments were performed in presence of a cocktail of Gabazine, NBQX, and Strychnine (1-5 pM) or TTX (1.0 pM) in the bath solution.

Ontogenetic Stimulation. CoChR variants were activated using a white LED light source mounted on an Olympus microscope with FITC-filters set (ET-EGFP/FITC/CY2 Filter Set 470/40X, BS495, 525/50M) that delivered a blue light (470 nm) through the epifluorescence pathway of the microscope (Olympus) using a water-immersion objective (40x). The duration of the light pulses was 3-5 ms (light intensity of 3-4 mW) adjusted to the maximum in all the experiments.

Results

Soma-targeted opsins in primary cortical neurons. Primary cortical neurons were isolated and transfected with channelrhodopsin-GFP (CoChR-GFP) fused with or without soma-targeting polypeptide. The soma-targeting protein fragments used in this study included a 65-amino-acid C-terminal fragment of potassium channel Kv2.1 (herein “Kv2.1”), a 108- amino-acid fragment from a rat small conductance potassium channel (herein “rSKl”; (SEQ ID NO.: 3), a 150-amino-acid N-terminal fragment of kainite receptor subunit 2 (herein “KA2(l-150)”), a 178-amino-acid fragment of a myosin VI binding domain (herein “MVIBD”; SEQ ID NO.: 1), and a 26-amino-acid of a myosin binding domain (herein “MBD”; SEQ ID NO.: 2). MVIBD and MBD were combined as 1) one, two, or three repeats of MVIBD-MBD in tandem or 2) one, two, or three repeats of MVIBD followed by one, two, or three repeats of MBD. Soma-targeting polypeptides were tested at either the N-terminus or C -terminus. Cortical neurons were additionally transfected with Electra2 fluorescent protein as a neuronal tracer.

For neurons transfected with CoChR-GFP without soma-targeting polypeptide, diffuse green fluorescence was observed throughout the soma and neurites in an overlapping pattern with Electra2 (Figure 1). For neurons transfected with CoChR-GFP comprising somatargeting polypeptides, green fluorescence remained in the cell body but was decreased in neurites. Green fluorescence was absent in neurites of neurons transfected with CoChR-GFP comprising rSKl, (MVIBD-MBD)x3, or (MVIBD)x3-(MBD)x3 soma-targeting polypeptides.

The above experiment was repeated using a different opsin-reporter combination, specifically, ChrimsonR fused to miRFP680 (ChrimsonR-miRFP680). The results were consistent with the above experiment: for neurons transfected with ChrimsonR-miRFP680 comprising soma-targeting polypeptides, red fluorescence remained in the cell body but was decreased in neurites (Figure 2). Red fluorescence was absent in neurites of neurons transfected with ChrimsonR-miRFP680 comprising rSKl, (MVIBD-MBD)x3, or (MVIBD)x3-(MBD)x3 soma-targeting polypeptides.

This disclosed subject matter is applicable to all optogenetic molecules of microbial and mammalian origin, including channel opsins and GPCR proteins.

Photostimulation of soma-targeted opsins in brain slices. Stereotactic injections of virus encoding CoChR-GFP, with or without (MVIBD-MBD)x3 soma-targeting polypeptide, were performed in mice. Brain slices were prepared under a fluorescence microscope, and neuron voltage clamp photostimulation experiments were performed.

For neurons expressing CoChR-GFP without soma-targeting polypeptide, diffuse green fluorescence was observed throughout the soma and neurites (Figure 3 A). However, for neurons expressing CoChR-GFP with (MVIBD-MBD)x3 soma-targeting polypeptide, green fluorescence was observed only in the cell body of neurons.

CoChR-GFP mediates action potentials in neurons in response to blue light pulses. When blue light pulses were directed to neuron cell bodies, no significant differences were observed in current amplitude, current rise time, or current decay between CoChR-GFP with or without (MVIBD-MBD)x3 soma-targeting polypeptide (Figure 3B). In a separate experiment, blue light pulses were directed at different layers of the cortex comprising neurites of a patched neuron (Figure 3C). In neurons expressing CoChR-GFP without somatargeting polypeptide, action potentials and spikes in current were observed when blue light was directed to layers L4, L5 or the layer comprising the cell body (L6). In neurons expressing CoChR-GFP with (MVIBD-MBD)x3 soma-targeting polypeptide, action potentials and spikes were observed only when blue light was directed to layer L6.

Soma-targeted opsins in retinal explants. Eyes were harvested from P23H mice, a murine model of retinitis pigmentosa. Retinal explants were mounted on polycarbonate membranes. The day of mounting was considered “days-zri-vztro 0” (DIV 0). The explants were transduced with CsChrimson-DsRed on DIV 1 and imaged on DIV 11. CsChrimson- DsRed was fused with soma-targeting polypeptides comprising two repeats of MVIBD followed by two repeats of MBD (herein “(MVIBD)x2-(MBD)x2”). On DIV 11, images showed red fluorescence in the cell body of neurons of the retina (Figure 4).

Discussion

Soma-targeted optogenetic molecules are instrumental in the development of achieve high-acuity vision restoration therapeutics. This can lead to cures for retinal diseases, e.g., retinitis pigmentosa, macular degeneration, and diabetic retinopathy, causing blindness that are a result of a loss/degeneration of rods, cones, and retinal pigment epithelium (Figure 5A). While optogenetics have been used for vision restoration for retinal diseases causing blindness in mice, non-human primates, and humans, treatments using conventional opsins have had limited success in achieving a high-acuity vision restoration. This is due to the expression of opsins throughout the soma and processes of retinal ganglion cells (RGCs) within the retina. The neuronal process extensively overlap surrounding cells leading to significant cross-activation. This multicellular activation translates to a low acuity vision restoration.

This study demonstrates the restriction of soma-targeted opsins to the soma of cells in the retina in a model of retinal disease. The restriction of opsins to the soma, in contrast with expression in both soma and processes, permits single-cell-resolution activation from a photostimulation which promotes high-acuity vision (Figures 4 and 5B). As such, these data support that application of the soma-targeted opsins disclosed herein can restore high-acuity vision in models of retinal disease. Soma-targeted opsins can also be restricted to specific cells of the retina, e.g., RGCs and/or bipolar cells (BCs), as necessary for treating retinal disease.

References

1. Baker, C. A., Elyada, Y. M., Parra, A., & Bolton, M. M. (2016). Cellular resolution circuit mapping with temporal-focused excitation of soma-targeted channelrhodopsin. eLife, 5, el4193. https://doi.org/10.7554/eLife.14193.

2. Shemesh, O. A., Tanese, D., Zampini, V., Linghu, C., Piatkevich, K., Ronzitti, E., Papagiakoumou, E., Boyden, E. S., & Emiliani, V. (2017). Temporally precise single-cell- resolution optogenetics. Nature neuroscience, 20(12), 1796-1806. https://doi.org/10.1038/s41593-017-0018-8.

3. Mardinly, A. R., Oldenburg, I. A., Pegard, N. C., Sridharan, S., Lyall, E. EL, Chesnov,

K., Brohawn, S. G., Waller, L., & Adesnik, H. (2018). Precise multimodal optical control of neural ensemble activity. Nature neuroscience, 21(6), 881-893. https://doi.org/10.1038/s41593-018-0139-8.

4. Collin, G. B., Gogna, N., Chang, B., Damkham, N., Pinkney, J., Hyde, L. F., Stone,

L., Naggert, J. K., Nishina, P. M., & Krebs, M. P. (2020). Mouse models of inherited retinal degeneration with photoreceptor loss. Cells, 9(4):931. doi: 10.3390/cells9040931. Veleri, S., Lazar, C. H., Chang, B., Sieving, P. A., Banin, E., & Swaroop, A. (2015). Biology and therapy of inherited retinal degenerative disease: insights from mouse models. Dis Model Meeh. 8(2):109-129. doi: 10.1242/dmm.017913.

* * *

Although the presently disclosed subject matter and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the present disclosure. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, and compositions of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the present disclosure of the presently disclosed subject matter, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the presently disclosed subject matter. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps. Various patents, patent applications, publications, product descriptions, protocols, and sequence accession numbers are cited throughout this application, this present disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a subject having retinal disease, comprising administering a therapeutically effective amount of a fusion protein to one or more cells of the retina, wherein the fusion protein comprises one or more soma-targeting polypeptides and wherein the one or more soma-targeting polypeptides have amino acid sequences that are at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof.

2. The method of claim 1, wherein the one or more soma-targeting polypeptides have amino acid sequences that are at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof.

3. The method of claim 1 or 2, wherein the one or more soma-targeting polypeptides have amino acid sequences that are at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof.

4. The method of any one of claims 1-3, wherein the one or more soma-targeting polypeptides have amino acid sequences that are identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof, identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof, or mixtures thereof.

5. The method of claim 1, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof.

6. The method of claim 1 or 5, wherein the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof.

7. The method of any one of claims 1 and 5-6, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof.

8. The method of any one of claims 1 and 5-7, wherein the fusion protein comprises up to 6 soma-targeting polypeptide, wherein each soma-targeting polypeptides comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof.

9. The method of claim 1, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

10. The method of claim 1 or 9, wherein the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

11. The method of any one of claims 1 and 9-10, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

12. The method of any one of claims 1 and 9-11, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

13. The method of claim 1, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof.

14. The method of claim 1 or 13, wherein the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof.

15. The method of any one of claims 1 and 13-14, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof.

16. The method of any one of claims 1 and 13-15, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 3 or fragments thereof.

17. The method of claim 1, wherein the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

18. The method of claim 1 or 17, wherein the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

19. The method of any one of claims 1 and 17-18, wherein the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

20. The method of any one of claims 1 and 17-19, wherein the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide comprises: a. a first region comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and b. a second region comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

21. The method of claim 1, wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

22. The method of claim 1 or 21, wherein the fusion protein comprises up to 3 somatargeting polypeptides comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

23. The method of any one of claims 1 and 21-22, wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

24. The method of any one of claims 1 and 22-23, wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 1 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides comprising an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 2 or fragments thereof.

25. The method of any one of claims 1-24, wherein the one or more soma-targeting polypeptides are positioned at the N-terminus of the fusion protein.

26. The method of any one of claims 1-24, wherein the one or more soma-targeting polypeptides are positioned at the C-terminus of the fusion protein.

27. The method of any one of claims 1-24, wherein one or more soma-targeting polypeptides are positioned at the N-terminus and one or more soma-targeting polypeptides are positioned at the C-terminus.

28. The method of any one of claims 1-27, wherein the fusion protein further comprises an opsin.

29. The method of claim 28, wherein the opsin is selected from the group consisting of CoChR, ChrimsonR, CsChrimson, ChR2, CsChrimsonR, MW-opsin (0PN1MW), LW-opsin (0PN1LW), SW-opsin (0PN1SW), Chronos, Rhodopsin (0PN2/RH0), mGluR6, Melanopsin, ChroME, CatCh, ReaChR, CsChR, ChETA, CheRiff, PsChR2, C1C1, C1V1, GtACR, Halo, GtACR2, CoChR-3M, Encephalopsin, panopsin (0PN3), Melanopsin (0PN4), Neuropsin (0PN5), Peropsin (RRH), and Retinal G protein coupled receptor (RGR).

30. The method of any one of claims 1-29, wherein the fusion protein further comprises a fluorescent protein.

31. The method of claim 30, wherein the fluorescent protein is selected from the group consisting of GFP, eGFP, YFP, eYFP, YFP3, miRFP680, miRFP670, miRFP670nano, miRFP670nano3, dTomato, tdTomato, mCherry, DsRed, iRFP, Electra2, Electra, mNeonGreen, mClover3, Clover, VFP, mVFP, mScarlet3, mScarlet, mGreenLantem, Citrane2, mGold, mRuby3, mRuby, mRuby2, mOrange, mOrange2, mEmerald, smURFP, DsRed2, mCardinal, mRFPl, iRFP670, miRFP, BFP, miRFP670, FusionRed, EBFP2, mVenus, Venus, Emerald, TagRFP, mApple, and mKate.

32. The method of any one of claims 1-31, wherein the fusion protein further comprises one or more linkers having amino acid sequences that are identical to the amino acid sequences set forth in SEQ ID NO.: 116-140, or mixtures thereof.

33. The method of any one of claims 1-32, wherein the soma-targeting polypeptide has an amino acid sequence that is at least about 80% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112 or fragments thereof.

34. The method of claim 33, wherein the soma-targeting polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof.

35. The method of claim 33 or 34, wherein the soma-targeting polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof.

36. The method of any one of claims 33-35, wherein the soma-targeting polypeptide has an amino acid sequence that is identical to the amino acid sequences set forth in SEQ ID NO.: 1-112, or fragments thereof.

37. The method of claim 33, wherein the soma-targeting polypeptide has an amino acid sequence that is at least about 80% identical to the amino acid sequences set forth in SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or fragments thereof.

38. The method of claim 33 or 37, wherein the soma-targeting polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequences set forth in SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or fragments thereof.

39. The method of any one of claims 33 and 37-38, wherein the soma-targeting polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequences set forth in SEQ ID NO.: 14, SEQ ID NO.: 26, SEQ ID NO.: 30, SEQ ID NO.: 1, SEQ ID NO.: 2, SEQ ID NO.: 3, or fragments thereof.

40. The method of any one of claims 33 and 37-39, wherein the soma-targeting polypeptide has an amino acid sequence that is identical to the amino acid sequences set forth in SEQ ID NO. : 14, SEQ ID NO. : 26, SEQ ID NO. : 30, SEQ ID NO. : 1, SEQ ID NO. : 2, SEQ ID NO.: 3, or fragments thereof.

41. The method of claim 1, wherein the fusion protein comprises one or more somatargeting polypeptides encoded by nucleic acid sequences that are at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof, at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof, or mixtures thereof.

42. The method of claim 41, wherein the fusion protein comprises one or more somatargeting polypeptides encoded by nucleic acid sequences that are at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof, at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof, or mixtures thereof.

43. The method of claim 41 or 42, wherein the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof, at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof, or mixtures thereof.

44. The method of any one of claims 41-43, wherein the fusion protein comprises one or more soma-targeting polypeptides encoded by nucleic acid sequences that are identical to the nucleic acid sequence set forth in SEQ ID NO 113 or fragments thereof, identical to the nucleic acid sequence set forth in SEQ ID NO 114 or fragments thereof, identical to the nucleic acid sequence set forth in SEQ ID NO 115 or fragments thereof, or mixtures thereof.

45. The method of claim 41, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof.

46. The method of claim 41 or 45, wherein the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof.

47. The method of any one of claims 41 and 45-46, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof.

48. The method of any one of claims 41 and 45-47, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.:

113 or fragments thereof.

49. The method of claim 41, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

50. The method of claim 41 or 49, wherein the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

51. The method of any one of claims 41 and 49-50, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

52. The method of any one of claims 41 and 49-51, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.:

114 or fragments thereof.

53. The method of claim 41, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof.

54. The method of claim 41 or 53, wherein the fusion protein comprises up to 6 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof.

55. The method of any one of claims 41 and 53-54, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof.

56. The method of any one of claims 41 and 53-55, wherein the fusion protein comprises up to 6 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 115 or fragments thereof.

57. The method of claim 41, wherein the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

58. The method of claim 41 or 57, wherein the fusion protein comprises up to 3 somatargeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

59. The method of any one of claims 41 and 57-58, wherein the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

60. The method of any one of claims 41 and 57-59, wherein the fusion protein comprises up to 3 soma-targeting polypeptides, wherein each soma-targeting polypeptide is encoded by a nucleic acid sequence that comprises: a. a first region comprising a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and b. a second region comprising a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

61. The method of claim 41, wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 80% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

62. The method of claim 41 or claim 61, wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 90% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 90% identical to the amino acid sequence set forth in SEQ ID NO. : 114 or fragments thereof.

63. The method of any one of claims 41 and 61-62, wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 95% identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is at least about 95% identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

64. The method of any one of claims 41 and 61-63, wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is identical to the nucleic acid sequence set forth in SEQ ID NO.: 113 or fragments thereof, and wherein the fusion protein comprises up to 3 soma-targeting polypeptides encoded by a nucleic acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO.: 114 or fragments thereof.

65. The method of any one of claims 1-64, wherein the retinal disease is retinitis pigmentosa, macular degeneration, or diabetic retinopathy.

66. The method of any one of claims 1-65, wherein the one or more cells of the retina comprise retinal ganglion cells (RGCs), bipolar cells (BCs), or a mixture thereof.

67. The method of any one of claims 1-66, wherein the fusion protein is administered by contacting the one or more cells of the retina with virus particles comprising a nucleic acid that encodes the fusion protein.

68. The method of any one of claims 1-66, wherein the fusion protein is administered by introducing the one or more cells of the retina with a nucleic acid that encodes the fusion protein by fusion, electroporation, biolistics, transfection, lipofection.