Depolarizing ion channel therapeutics for treating neurodegenerative diseases

Abstract

Provided herein are depolarizing ion channel therapeutics and methods of using the same for treating retinal and neurodegenerative diseases.

Description

DEPOLARIZING ION CHANNEL THERAPEUTICS FOR TREATING NEURODEGENERATIVE DISEASESCROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 669,909, filed July 11, 2024, U.S. Provisional Patent Application No. 63 / 716,479, filed November 5, 2024, U.S. Provisional Patent Application No. 63 / 728,536, filed December 5, 2024, and U.S. Provisional Patent Application No. 63 / 757,691, filed February 12, 2025, each of which is incorporated by reference in its entirety herein.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on June 30, 2025, is titled BIS-002WO_SL.xml and is 24,240 bytes in size.BACKGROUND

[0003] Neurodegenerative diseases affect millions of people worldwide. In these diseases, nerve cells stop functioning or being able to pass their signals to downstream neural systems. While there are some treatments to help relieve some of the symptoms associated with these diseases, there are currently no effective methods to slow down the progression and there are currently no cures.

[0004] A widely observed phenomenon of these diseases, which include Alzheimer’s disease, Parkinson’s disease, and retinitis pigmentosa; is that patients’ symptoms fluctuate. For example, patients undergo ‘good days’ and ‘bad days.’ For example, on good days, patients with Alzheimer’s disease show periods of cognitive clarity, allowing them to carry out some activities of daily living, such as dressing and attending to personal hygiene. However, just as the clarity comes on good days, it goes, and, on the bad days, the ability to carry out these activities is no longer present. Similarly, patients with Parkinson’s disease, on good days, are able to initiate movements and walk, but the ability then disappears, and on bad days, they are immobile. With respect to degenerative diseases of the retina, such as retinitis pigmentosa, patients also report these kind of fluctuations. For example, on gooddays, some patients report that they are able to see light and some edges of objects, above their background visual ‘noise,’ but this ability then disappears on ‘bad’ days.

[0005] Although these fluctuations are widely observed across many of the above described exemplary retinal and degenerative diseases, the implications for therapy have not yet been appreciated or addressed. Such degenerative diseases are in significant need of new therapy that can ameliorate or treat the disorder and symptoms thereof.SUMMARY

[0006] This disclosure relates to the surprising discovery that, in patients with the ability to perform tasks on good days but with loss of this performance on bad days, circuits in the degenerating neural system of the patients are still at least partially intact - just blocked in their ability to pass their signals forward to downstream targets. As described herein and shown for example in the disclosed figures, treating such patients with a depolarizing ion channel (DIC) therapeutic can ameliorate this problem and help alleviate such disorders. This disclosure, therefore, is directed in part to methods for treating neurodegenerative diseases in patients presenting with such symptom fluctuations. For example, methods disclosed herein may include and / or result in invoking a facilitating mechanism in patients that allow these degenerated circuits to sufficiently send signals to downstream neurons. Specifically, disclosed methods, as shown for example schematically in FIG. 1, introduce a DIC therapeutic into a set of projection neurons or ‘weak link neurons’ that are failing to pass signals from upstream circuits to downstream brain areas. The DIC boosts the activity of the projection neurons, so that weak signals from upstream circuits become sufficient to make the projection neurons cross a voltage threshold, which, in turn, allows them to fire action potentials and pass the information to downstream neurons. A key aspect of the disclosed methods is that, upon administration of a DIC therapeutic, the therapeutic acts to unblock a blocked pathway, and restores a neural function that was lost for the patient with the neurodegenerative disease.

[0007] For example, provided herein is a method of treating a neurodegenerative disease in a patient in need thereof without administration of light stimulation or activation by light, the method comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic wherein the depolarizing ion channel therapeutic depolarizes neurons in a neural circuitry of the patient upon administration, and wherein the neurons are downstreamof degenerated endogenous neural circuitry. Also provided herein is a method of inducing activity of projection neurons in a patient suffering from a neurodegenerative disease, the method comprising administering an effective amount of a depolarizing ion channel therapeutic wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from the projection neurons, to depolarize the projection neurons above a voltage threshold capable of eliciting one or more action potentials, thereby inducing activity of the projection neurons in the patient and restoring neural function in the patient.

[0008] A method of restoring neural function in a patient in need thereof suffering from a neurodegenerative disease is provided in an embodiment, comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream of neurons receiving the therapeutic, to depolarize the neurons above a voltage threshold capable of eliciting one or more action potentials, thereby restoring neural function in the patient.

[0009] In some embodiments, the degenerated endogenous neural circuitry of the patient being treated is insufficient to activate downstream neurons to a voltage threshold capable of eliciting an action potential before the administration of the depolarizing ion channel therapeutic, and / or a patient has degenerated neural circuitry but does not have a complete loss of functioning circuits before administration of the depolarizing ion channel therapeutic, wherein a disclosed method does not include administering to the patient light stimulation of the depolarizing ion channel or activation by light and / or wherein a disclosed method does not include light activation of exogenously-introduced de novo neural circuits.

[0010] Contemplated depolarizing ion channel therapeutics for use in the disclosed methods may comprise a depolarizing ion channel protein; or a DNA or RNA polynucleotide encoding a depolarizing ion channel protein or a fusion protein comprising a depolarizing ion channel protein. Such contemplated therapeutics may include a vector comprising a DNA or RNA polynucleotide encoding a depolarizing ion channel protein or a fusion protein comprising a depolarizing ion channel protein. Depolarizing ion channel protein may be selected for example from the group consisting of a ligand-gated ion channel protein, a voltage-gated ion channel protein, a mechanosensitive ion channel protein, and a cyclic nucleotide-gated ion channel protein, and / or e.g., a sodium ion channel protein, a potassiumion channel protein, or a calcium ion channel. A depolarizing ion channel therapeutic may comprise for example, an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9, or the depolarizing ion channel therapeutic comprises SEQ ID NO: 1.

[0011] Also provided herein is a method of treating a disease or disorder selected from the group consisting of amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, dementia including dementia associated with other neurodegenerative disorders or diseases, Charcot-Marie-Tooth disease, Huntington’s disease, a lysosomal storage disease, multiple system atrophy, tauopathies, and prion diseases in a human patient in need thereof, comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic, such as administering SEQ ID NO: 1, or administering an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 is a schematic depiction of exemplary methods described herein, in which treatment with a depolarizing ion channel (DIC) therapeutic restores the ability of degenerated neural circuits to activate projection neurons and allow them to signal to downstream targets.

[0013] FIG. 2 is a set of graphs showing the effect of a DIC therapeutic, as described therein, for restoring or increasing color vision in patients with retinitis pigmentosa. The graphs show the percentage of correct choices the patients made in a standard color test before and after treatment, as described herein. Each circle corresponds to a block of 10 trials.

[0014] FIG. 3A and FIG. 3B, are a set of graphs showing the effect of a DIC therapeutic, as described therein, for increasing visual acuity in patients with retinitis pigmentosa. Visual acuity was assessed using a standard visual acuity paradigm, where the letter E was presented in one of 4 orientations, and the letters were increasingly reduced in size; performance was measured as the percentage of correct identifications made. Each circle corresponds to a block of 10 or more trials (FIG. 3A). FIG. 3B shows the visual acuity scores for several patients before and after treatment.

[0015] FIG. 4 is a set of graphs showing the effect of a DIC therapeutic, as described therein, for increasing performance in a maze navigation task that tests for night vision (or night blindness thereof). Performance was measured by the time spent completing the maze as well as the number of mistakes that occurred in the process of completing it.

[0016] FIG. 5 shows the boosting effect of a DIC therapeutic on neuronal activity, as measured by the amplitude of the Photopic Negative Response (PhNR) in electroretinogram (ERG) recordings. The DIC was targeted to the retinal ganglion cells that produce the PhNR wave, and the amplitudes of the PhNR waves from DIC-treated eyes and untreated eyes in normal nonhuman primates are shown. The amplitudes from the DIC-treated eyes (open circles) are nearly twice the size of those from the untreated eyes (stars) (p<0.01 t-test, comparing the mean amplitude of the PhNRs from the DIC-treated eyes (n=6) with those from the untreated eyes (n=8) indicating that applying a DIC therapeutic to neurons boosts (or amplifies) their signals, even in normal animals. The vectors carry the same DIC as that described in Methods for FIGs. 2-4 (Examples), but at lower doses (3.7 x 1010vg / eye; 1.7x 1011vg / eye). Even at doses 10-fold lower than those used in the studies shown in FIGs. 2-4, the effects of the DIC therapeutic on neural activity is strong and clear. FIG. 5 shows the boosting effect of a DIC therapeutic on neuronal activity, as measured by the amplitude of the Photopic Negative Response (PhNR) in electroretinogram (ERG) recordings. The DIC was targeted to the retinal ganglion cells, the cells that produce the PhNR wave. The amplitudes of the PhNR waves from untreated and DIC-treated eyes in normal nonhuman primates are shown. The untreated eyes provide the baseline, that is, the PhNR responses from normal animals (stars). The PhNR responses from the DIC-treated eyes are superimposed (open circles). As shown, the responses from the DIC-treated eyes are nearly twice the size of those from the untreated eyes (p<0.01 t-test, comparing the mean amplitude of the PhNRs from the DIC-treated eyes (n=6) with those from the untreated eyes (n=8). These data support the notion that applying a DIC therapeutic to neurons boosts (or amplifies) their signals, even in normal animals. The vectors carry the same DIC (ChronosFP) as that described in Methods, but at lower doses. Thus, even with lower doses (10-fold lower than that used in the clinical studies shown in FIGs. 2-4), the effects of the DIC therapeutic on neural activity is strong and clear.

[0017] Note that ChronosFP contains a chromophore, but the chromophore was not activated, as the stimulating light used by the ERG device (Diagnosys, Inc) is red light (660 nm wavelength) at dim light levels (from 2xl0-5to 3 xl0‘4mW / mm2). The green absorbing chromophore on the DIC does not respond to this wavelength (Klapoetke et al, 2015, Figs 5B and C), even at 6 mW / mm2. Thus, the DIC was activating the cells without utilizing a chromophore mechanism.

[0018] FIG. 6A and FIG. 6B indicate the dose dependent boosting effect of a DIC therapeutic on neuronal activity - i.e., the boosting increases with higher vector doses. As in FIG. 5, the DIC therapeutic was targeted to the retinal ganglion cells and the effect on the cells was measured using the PhNR wave of the electroretinogram (ERG). FIG. 6B graphs show the raw ERG signals from two DIC therapeutic doses: 3.7 xlO10vg / mL (low dose) and 1.17 xlO11vg / mL (high dose). As shown, the PhNR wave was larger with the higher dose than with the lower dose indicating the DIC therapeutic boosted the activity of the ganglion cells, and the magnitude of the boost correlated with the dose delivered. FIG. 6A and FIG. 6B show that the boosting effect of a DIC therapeutic on neuronal activity is dose dependent - i.e., the boosting increases with higher vector doses. As in Fig 5, the DIC therapeutic was targeted to the retinal ganglion cells, and the effect on the cells was measured using the PhNR wave of the electroretinogram (ERG). The PhNR component of the ERG is shown in FIG. 6A. The graphs in FIG. 6B show the raw ERG signals from non human primate eyes at two DIC therapeutic doses: 3.7 xlO10vg / eye (n=2 eyes) and 1.17 xlO11vg / eye (n=2 eyes). As shown, the PhNR wave was larger with the higher dose than with the lower dose. Thus, the DIC therapeutic boosted the activity of the ganglion cells, and the magnitude of the boost correlated with the dose delivered.DETAILED DESCRIPTIONDefinitions

[0019] The features and other details of the disclosure will now be more particularly described. Certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

[0020] As used herein, “about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, “about” will mean up to plus or minus 10% of the particular value.

[0021] The articles “a” and “an” are used in this disclosure to refer to one or more than one (i.e., to at least one) of the grammatical object of the article, unless the context is inappropriate. By way of example, “an element” means one element or more than one element.

[0022] The term “and / or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.

[0023] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context. It is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited steps.

[0024] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present invention remain operable. Moreover, two or more steps or actions may be conducted simultaneously. The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention(s) unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of that provided by the present disclosure.

[0025] As used herein, the term “adeno-associated virus” (AAV) refers to a vector derived from an adeno-associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1 1, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.EB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11 , AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV vector selected fromthe group consisting of an AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74 vector. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV2 vector. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g., the rep and / or cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences promote the rescue, replication, and packaging of the AAV virion. Thus, an AAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional TTRs) of the virus. ITRs do not need to be the wild-type polynucleotide sequences and may be altered, e.g., by the insertion, deletion, or substitution of nucleotides, so long as the sequences provide for functional rescue, replication, and packaging. AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest (e.g., a vector having a gene that encodes a depolarizing ion channel protein of the disclosure) and a transcriptional termination region. The terms “adeno-associated virus inverted terminal repeats” and “AAV ITRs” refer to art-recognized regions flanking each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV rep coding region, provide for the efficient excision and integration of a polynucleotide sequence interposed between two flanking ITRs into a mammalian genome. The polynucleotide sequences of AAV ITR regions are known. As used herein, an “AAV ITR” does not necessarily include the wild-type polynucleotide sequence, which may be altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITR may be derived from any of several AAV serotypes, including without limitation AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.EB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16, among others. Furthermore, 5' and 3' ITRs which flank a selected polynucleotide sequence in an AAV vector need not be identical or derived from the same AAV serotype or isolate, so longas they function as intended, e.g., to allow for excision and rescue of the sequence of interest from a host cell genome or vector, and to allow integration of the heterologous sequence into the recipient cell genome when AAV Rep gene products are present in the cell. Additionally, AAV ITRs may be derived from any of several AAV serotypes, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV.PHP.EB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1 , AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV-TT, AAV-DJ8, or AAV.HSC16, among others.

[0026] Throughout the specification and claims, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated word or group of words but not the exclusion of any other word or group of words.

[0027] As used herein, the terms “effective amount,” “therapeutically effective amount,” and the like, when used in reference to a composition described herein, such as a vector having a gene that encodes a depolarizing ion channel protein, refer to a quantity sufficient to, when administered to a patient, including a mammal (e.g., a human), effect beneficial or desired results (e.g., expression of a depolarizing ion channel protein), which may include clinical results. For example, an effective amount of one or more composition described herein (e.g., a vector having a gene that encodes a depolarizing ion channel protein) may achieve expression of a protein of interest as compared to the expression of said protein without administration of the composition of interest. An “effective amount,” “therapeutically effective amount,” and the like, of a composition, such as a vector having a gene that encodes a depolarizing ion channel protein, also include an amount that results in a beneficial or desired result in a patient as compared to a control.

[0028] The term “pharmaceutically acceptable” means safe for administration to a mammal, such as a human. In some embodiments, a pharmaceutically acceptable composition is approved by a regulatory agency of the Federal government or a state government or is listed in the U.S. Pharmacopeia or any other generally recognized pharmacopeia for use in animals (e.g., humans). As used herein, the term “pharmaceutically acceptable” refers to those compounds, anions, cations, materials, compositions, carriers,and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0029] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. As used herein, the terms “heterologous promoter” and “heterologous control regions” refer to promoters and other control regions that are not normally associated with a particular nucleic acid in nature. For example, a “transcriptional control region heterologous to a coding region (e.g., a transgene)” is a transcriptional control region that is not normally associated with the coding region in nature.

[0030] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient,” as used herein, refer interchangeably to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions together with one or more pharmaceutically acceptable excipients.

[0031] It is to be understood that the present disclosure also provides pharmaceutical compositions including any depolarizing ion channel (DIC) therapeutic described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0032] As used herein, the term “pharmaceutical composition” is a formulation containing the DIC therapeutics of the present disclosure in a form suitable for administration to a patient. In one embodiment, the pharmaceutical composition is in bulk or in unit dosage form. The quantity of active ingredient in a unit dose of composition is an effective amount and is varied according to the particular treatment involved. One skilled in the art will appreciate that it is sometimes necessary to make routine variations to the dosage depending on the age and condition of the patient. The dosage will also depend on the route of administration. A variety of routes are contemplated, including intravitreally, oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and thelike. In some embodiments, the composition is administered intravitreally. In one embodiment, the active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that are required.

[0033] As used herein, the term “pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, nontoxic and neither biologically nor otherwise undesirable, and includes excipient that is acceptable for veterinary use as well as human pharmaceutical use. A “pharmaceutically acceptable excipient” as used in the specification and claims includes both one and more than one such excipient.

[0034] The pharmaceutical compositions containing active compounds of the present disclosure may be manufactured in a manner that is generally known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers including excipients and / or auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Of course, the appropriate formulation is dependent upon the route of administration chosen.

[0035] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Further, compositions may include isotonic agents, for example, sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0036] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0037] The active compounds can be prepared with pharmaceutically acceptable carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0038] As used herein, the term “promoter” refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of a transgene. Exemplary promoters suitable for use with the compositions and methods described herein are described herein, such as a CAG promoter. Additionally, the term “promoter” may refer to a synthetic promoter, such as a regulatory DNA sequence that doe does not occur naturally in a biological system. Synthetic promoters contain parts of naturally occurring promoters combined with polynucleotide sequences that do not occur in nature and can be optimized to express recombinant DNA.

[0039] A “subject” or “patient” may include any animal, including mammals, mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or non-human primates, and most preferably humans. The compositions and methods disclosed herein can relate to amammal, such as a human, but can also be other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals e.g., rats, mice, guinea pigs, non-human primates, and the like).

[0040] The terms “treat,” “treatment,” “treating,” and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in terms of partially or completely curing a disease and / or adverse effect attributed to the disease. The term “treatment” as used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) inhibiting the disease, e.g., preventing the disease from increasing in severity or scope; (b) relieving the disease, e.g., causing partial or complete amelioration of the disease; or (c) preventing relapse of the disease, e.g., preventing the disease from returning to an active state following previous successful treatment of symptoms of the disease or treatment of the disease.

[0041] As used herein, the term “vector” includes a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, or another suitable replicon (e.g., viral vector). In some embodiments, the viral vector is adenoviral vector, a retroviral vector, a poxviral vector, an adeno-associated viral (AAV) vector, a baculoviral vector, a herpes simplex viral vector, and a synthetic vector. A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous polynucleotides or proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 011026; incorporated herein by reference as it pertains to vectors suitable for the expression of a gene of interest. Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of heterologous nucleic acid materials (e.g., a vector having a gene that encodes a depolarizing ion channel protein) in a mammalian cell. Certain vectors that can be used for the expression of the genes described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of gene agents disclosed herein contain polynucleotide sequences that enhance the rate of translation of these polynucleotides or improve the stability or nuclear export of the RNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, an IRES, and polyA in order to direct efficient transcription of the gene carried on the expression vector. The expression vectorssuitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

[0042] Without being bound by theory, the disclosure herein is based at least in part on the observation that patients with neurodegenerative diseases often experience symptom fluctuations (e.g., the ability to perform tasks on ‘good days’ but not on ‘bad days,’ or during ‘good periods’ or ‘bad periods’), which led to the discovery of how retinal degenerative and other neurodegenerative diseases mechanistically lead to functional deficits, and how these diseases might be treated.

[0043] As described herein, symptom fluctuations of patients demonstrate that many circuits in a degenerating neural system are still at least partially intact, just unable to get their signals through to downstream neurons. Based upon this, as described herein is a novel treatment mechanism, i.e., a depolarizing ion channel (DIC) therapeutic that allows these degenerated circuits to get their signals through to downstream neurons. For example, the methods described herein, as exemplified schematically in FIG. 1, relate to introducing (e.g., expressing) DICs into a set of neurons (e.g., projection neurons) that are failing to pass signals from upstream circuits to downstream brain areas. The DICs boost the activity of the projection neurons, so that weak signals from upstream circuits become sufficient to make the projection neurons cross a voltage threshold, which, in turn, allows them to fire action potentials and pass the information on to downstream neurons. It can be appreciated that the DIC therapeutic of the disclosure can be applied to other receiving neurons (not just projection neurons) to facilitate transmission of other weak pathways, thereby restoring a neural function that was lost e.g., vision in a patient with a retinal disorder or movement in a patient with a motor neurodegenerative disease, respectively.

[0044] The methods described herein are distinguished from optogenetics. With prior methods involving optogenetics, neurons are directly activated by light-activating an optogenetic protein that is expressed in the neurons; these neurons in turn activate downstream targets. Activation in this way produces non-natural circuits, ones that are created de novo by the light-activation of the optogenetic protein.

[0045] In contrast, the methods described herein work without light-activation of a protein and instead neurons are depolarized by a DIC therapeutic to boost the signals fromweak incoming circuits (i.e., the circuits that feed into the neurons that have become too weak, as a result of disease, to drive the neurons to fire). The boost allows the neurons to fire action potentials and pass the signals on to downstream targets.

[0046] As an example, to distinguish the methods described herein, embodiments are provided that treat vision losses associated with retinal degenerative diseases, such as the loss of color vision. Human color vision is thought to be produced by a ‘labeled line’ system. Specifically, there are 3 types of cones, each of which has a peak absorption at a different wavelength (referred to as red, green, and blue cones); these cone cells, in turn, activate different downstream neurons, including, eventually, different ganglion cells (each of these three exemplary neural circuits is a different labeled line). Without being bound by theory, the way the brain knows which color was present is by identifying which ganglion cells fired. In canonical optogenetic-based methods, in order to produce color vision, multiple chromophores would need to be used, and each would have to be expressed in a different cell class, so the brain would be able to identify which color occurred. By contrast, in the methods described herein, this is not required. Disclosed methods instead utilize the endogenous labeled lines that are present in the degenerated retina but are too weak to get their signals through to the ganglion cells; DIC therapeutic treatment boosts the ganglion cells’ activity so the endogenous labeled line circuits are able to get the ganglion cells to fire. Thus, the endogenous labeled line circuits are able to get their signals to the brain. Likewise, the methods described herein do not create high resolution vision de novo but instead allow endogenous high resolution circuits to activate the ganglion cells and pass their signals through to the brain. In contrast, existing optogenetic or other chromophore-based methods create vision de novo and are, therefore, limited by the resolution of the cells such methods are expressed in, so, unlike the disclosed method, they cannot create the high resolution vision shown in FIG. 3.

[0047] In an embodiment, the introduction of a DIC therapeutic (by e.g., administration to a patient systemically) into the projection neurons of the retina e.g., the retinal ganglion cells) can restore color vision to patients with retinal degenerative diseases, even though a disclosed DIC therapeutic itself may have no relation to color vision. In this embodiment, the DIC therapeutics depolarizes ganglion cells, bringing them closer to their threshold to fire, which allows weak signals from incoming degenerated circuits to become sufficient to make the ganglion cells fire and send signals to the brain. If the degenerated endogenous neuralcircuits are carrying color information, then the patient regains color vision (e.g., the DIC therapeutic unblocks the color information channels). FIG. 2 shows the effectiveness of the method described herein for color vision.

[0048] In another embodiment, methods described herein can restore high acuity vision (or some high acuity vision) to patients who have lost it as a result of a retinal degenerative disease, as exemplified by the results shown in FIG. 3. Similar to the case with color vision, the DIC therapeutic treatment of the disclosure can bring high acuity vision back because the circuits for it are latent (z'.<?., still sufficiently present in the retina, just too weak to make the ganglion cells (the projection neurons) fire. Without being bound by theory, the ability of the DIC therapeutic treatment of the disclosure to depolarize the ganglion cells allows the weak signals from these circuits to be sufficient to make the ganglion cells fire and send signals to the brain. For example, as shown in FIG. 3, a patient who, before treatment, could only recognize letters on the top two lines of an eye chart, were able to see much small letters (letters that require high resolution vision to see) after treatment. Higher resolution vision becomes possible, at least in part, because circuits feeding into the ganglion cell have this capacity. If those circuits are too damaged however (z.<?., if the photoreceptors that provide high resolution information are already gone (due to the degenerative disease)), then DIC therapeutic treatment cannot restore high resolution information. The restoration of high acuity vision (or higher than the patient had before treatment) is able to occur using the methods described herein if the degenerated circuits that carry that information are still partially intact, just weak.

[0049] Further comparisons to existing methods: when ganglion cells are made active by activating an exogenous chromophore with light (at the excitation wavelength of the chromophore), the light activation overrides the upstream circuits, so information from upstream circuits is lost, and, instead, de novo circuits are created by the light activation. For clinical purposes, these methods have some desirability for patients who have little or no upstream circuitry (e.g., fully degenerated endogenous neural circuits that are no longer capable of functioning). By contrast, as described herein, disclosed methods are directed to treating patients who have some residual upstream circuitry (e.g., partially degenerated neural circuits), which the DIC therapeutic described herein can utilize to make the ganglion cells fire. Because the methods described herein utilize existing upstream circuitry, such disclosed methods can provide patients with much higher visual resolution than existing methods(chromophore-based, optogenetic, electrode-based, etc.). When used in the retina, the methods described herein are not limited to the resolution of the ganglion cells, but limited to the resolution of upstream circuits lhal / ccd into the ganglion cells.

[0050] By the same logic, when used in the retina to restore color vision, there is no need to express exogenous chromophores with particular wavelength specificities to provide color sensitivity, as the methods described herein allow endogenous, partially degenerated color circuits to activate the ganglion cells, so natural color vision can re-emerge.

[0051] For example, methods described herein can be used to combat the widely- experienced night blindness that occurs in patients with retinal degenerative diseases, such as retinitis pigmentosa, choroidemia, Ushers syndrome, and macular degeneration. Methods described herein, without being bound by theory, boost signals from circuits that operate at low light levels and allow patients to see in much darker environments than was possible before treatment, the results of which are exemplified in FIG. 4.

[0052] As yet another example, if, for a given patient with a neurodegenerative disease, the underlying deficit is not the complete loss of functioning circuits, but rather a degeneration of the patient’s existing, endogenous circuits such that they are insufficient to depolarize their downstream neurons (e.g., projection neurons) to action potential threshold, then, in such a patient population, the methods described herein can be effective for treating a neurodegenerative disease.

[0053] For example, for a patient with a retinal degenerative disease with only about 10% of their cone photoreceptors remaining, the degenerated endogenous retinal circuitry that carries the cone information will produce very weak signals, even if the structure of the circuitry is still largely intact (e.g., the circuit still produces center / surround antagonism but with only 10% of the normal number of cones contributing to the center and 10% of the normal number contributing to the surround). If the signals from these degenerated endogenous circuits are too weak to make the downstream (e.g., projection) neurons fire, then color information never reaches the brain. In such exemplary patient populations, the methods described herein, can boost the impact of the weak circuits so their signals can be propagated to downstream neurons. As noted, patients with degenerated neural circuitry that no longer functions well enough (e.g., degeneration is severe e.g., no underlying circuitry remaining or too few cones remaining) to provide enough depolarization, when combined with a voltage provided by DIC of the disclosure, to make the ganglion cells fire actionpotentials, are not contemplated for use in the disclosed methods; such treatment will not be effective in such patient populations.Depolarizing Ion Channel Therapeutics

[0054] As described herein, a depolarizing ion channel (DIC) therapeutic may be used as part of the disclosed methods for treating neurodegenerative diseases, including retinal degenerative diseases. In some embodiments, a DIC therapeutic comprises: a) a depolarizing ion channel (DIC) protein; or b) a DNA or RNA polynucleotide that encodes a depolarizing ion channel (DIC) protein or a fusion protein comprising a depolarizing ion channel protein. In some embodiments, the DNA or RNA polynucleotide further encodes a gain control protein.

[0055] In some embodiments, a “depolarizing ion channel protein” or “DIC” protein of the disclosure is any ion channel protein that facilitates depolarization of a cell. For example, DIC of the disclosure can be selected from the group consisting of a ligand-gated ion channel protein (e.g., a cationic cys-loop receptor or anionic cys-loop receptor), voltage-gated ion channel protein e.g., sodium channel protein, calcium channel protein, potassium channel protein, chloride channel protein, and proton channel protein), mechanosensitive ion channel protein e.g.,, cation-selective mechanosensitive channel protein, anion mechanosensitive channel protein, and non-selective mechanosensitive ion channel protein), cyclic nucleotide- gated ion channel protein, channelrhodopsins, halorhodopsins, and archaerhodopsins.

[0056] In some embodiments, the depolarizing ion channel protein is a light-activated ion channel (e.g., an optogenetic protein or a synthetically-adapted light-activated ion channel e.g., a nicotinic acetylcholine receptor, light- activated potassium channel, or cyclic nucleotide-gated potassium ion channel). In some embodiments, a light-activated ion channel is an optogenetic protein described herein or any suitable optogenetic protein now known or later discovered. For example, in some embodiments, a contemplated DIC protein described herein can be a optogenetic protein described in e.g., U.S. Patent No. 11,180, 537, which is incorporated herein by reference, e.g., an optogenetic protein comprising the amino acid sequence of SEQ ID NO: 11 or 12, below:MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVL QWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLAT GHRVQWLRYAEWLLTCPVICIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDEAEAGAVNKGTGKMAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTVVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK(SEQ ID NO: 11); andMDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGW1ESRGTNGAQTASNVLQWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLATGHRVQWLRYAEWLLTCPVISIHLSNLTGLSNDYSRRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLCYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWLFFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMSKNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEIEVETLVEDESEAGSVNKGTGKMAELISSATRSLFAAGGINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWEEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLRYFEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVGMIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPILWAVGPEGLLKLSPYANSIGHSICEIIAKEFWTFLAHHLRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDT (SEQ ID NO: 12).

[0057] In some embodiments, the DIC protein is a sodium ion channel.

[0058] In some embodiments, a DIC described herein is fused to a reporter protein, e.g. where a reporter protein is fused to the 3' end of the DIC.

[0059] A reporter protein may include, for example, a fluorescent protein, a luciferase, beta-galactosidase, alkaline phosphatase, beta-lactamase, a protein or enzyme which confers resistance to cytotoxic substances or to minimal medium, a cytotoxic or pro-apoptotic protein, or a protein which modifies the growth or morphology of the cell in which they are expressed. For example, in some embodiments, the reporter protein fused to a DIC is a fluorescent protein, e.g., luciferase and / or the reporter protein may be fused to a DIC that includes one of: beta-galactosidase, alkaline phosphatase, or beta-lactamase. In some embodiments, the reporter protein fused to a DIC is a protein or enzyme which confers resistance to cytotoxic substances or to minimal medium, e.g., a cytotoxic or pro-apoptotic protein and / or protein which modifies the growth or morphology of the cell in which they are expressed.

[0060] Fluorescent proteins of the disclosure may be any suitable fluorescent protein, such as a green fluorescent protein a blue fluorescent protein, a cyan fluorescent protein, a yellow fluorescent protein, an orange fluorescent protein, or a red fluorescent protein. For example, the reporter protein is a green fluorescent protein.

[0061] Exemplary fluorescent proteins maybe selected from green fluorescent protein (GFP) (e.g., with an excitation maximum 395 / 475 nm, emission maximum 509 nm and relative brightness (e.g., % of EGFP 48%), as well as green fluorescent proteins such as EFTP, Emerald, superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, AcGFP, ZsGreen, or T-Sapphire, blue fluorescent proteins such as EBFP, EBFP2, Azurite, mTagBFP, cyan fluorescent proteins such as ECFP, mECFP, cerulean, mTurqoise, CyPet, AmCyanl, Midori-Ishi Cyan, TagCFP, mTFPl(Teal), yellow fluorescent proteins such as EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellowl or mBanana; Orange Fluorescent Proteins such as Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express (Tl), DsRed- Monomer, mTangerine; Red Fluorescent Proteins such as mRuby, mApple, mStrawberry, AsRed2, mRFPl, JRed, mCherry, HcRedl, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, or AQ143.

[0062] In some embodiments, the fluorescent is a green fluorescent protein. For example, in some embodiments, the green fluorescent protein is GFP. In some embodiments, the green fluorescent protein is EGFP. In some embodiments, the green fluorescent protein is Emerald. In some embodiments, the green fluorescent protein is superfolder GFP. In someembodiments, the green fluorescent protein is Azami Green. In some embodiments, the green fluorescent protein is mWasabi. In some embodiments, the green fluorescent protein is TagGFP. In some embodiments, the green fluorescent protein is TurboGFP. In some embodiments, the green fluorescent protein is AcGFP. In some embodiments, the green fluorescent protein is ZsGreen. In some embodiments, the green fluorescent protein is T- Sapphire.

[0063] In certain embodiments methods disclosed here comprise administering to the patient an effective amount of a depolarizing ion channel therapeutic that comprises a DIC or encodes a DIC and includes a vector having a gene that encodes a depolarizing ion channel protein or that encodes a fusion protein comprising a depolarizing ion channel protein, e.g., where vector comprises an inducible promoter operably linked to the gene. Contemplated inducible promoters can modulate, activate, or deactivate expression of the depolarizing ion channel protein.

[0064] Effective intracellular concentrations of a gene disclosed herein may be achieved via the stable expression of a vector encoding a gene e.g., by integration into the nuclear or mitochondrial genome of a mammalian cell), such as a gene that expresses a DIC, as described herein. In order to introduce such a gene into a mammalian cell, the gene can be incorporated into a vector. Vectors can be introduced into a cell by a variety of methods, including transformation, transfection, direct uptake, projectile bombardment, and by encapsulation of the vector in a liposome. Examples of suitable methods of transfecting or transforming cells are calcium phosphate precipitation, electroporation, microinjection, infection, lipofection, and direct uptake. The genes that encode a DIC disclosed herein can also be introduced into a mammalian cell by targeting a vector containing a polynucleotide encoding such a gene to cell membrane phospholipids. For example, vectors can be targeted to the phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to a VSV-G protein, a viral protein with affinity for all cell membrane phospholipids. Stable expression of an exogenous polynucleotide in a mammalian cell can be achieved by integration of the polynucleotide containing the gene into the nuclear genome of the mammalian cell. Expression vectors for use in the compositions and methods described herein contain a polynucleotide sequence that encodes a gene as well as, e.g., additional sequence elements used for the expression of these genes and / or the integration of these polynucleotide sequences into the genome of a mammalian cell. Certain vectors that can beused include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5' and 3' UTR regions, an internal ribosomal entry site (IRES), and polyA in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, and nourseothricin.

[0065] Genes described herein can be incorporated into recombinant AAV (rAAV) vectors in order to facilitate their introduction into a cell, such as a target cell, and / or for administration. rAAV vectors useful in the conjunction with the compositions and methods described herein include recombinant nucleic acid constructs that contain (1) a gene; and (2) nucleic acids that facilitate expression of the heterologous genes. The viral nucleic acids may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional inverted terminal repeat (ITR)s) of the DNA into a virion. Such rAAV vectors may also contain marker or reporter genes.

[0066] Useful rAAV vectors include those having one or more of the naturally -occurring AAV genes deleted in whole or in part, but retain functional flanking ITR sequences. The AAV ITRs may be of any serotype e.g., derived from AAV serotype 2 or 5) suitable for a particular application. In some embodiments, the AAV comprises two ITRs, wherein the two ITRs comprise a first ITR (ITR1) and a second ITR (ITR2), wherein ITR1 is position 5' to the polynucleotide encoding a DIC fusion protein and ITR2 is position 3' to the polynucleotide to form a cassette comprising the structure ITR1-DIC fusion protein- ITR2, for example the two ITRS are AAV serotype 2 ITRs.

[0067] The genes (e.g., a gene encoding a DIC) described herein can be incorporated into a rAAV virion in order to facilitate introduction of the nucleic acid or vector into a cell. The capsid proteins of AAV compose the exterior, non-nucleic acid portion of the virion and are encoded by the AAV Cap gene. The Cap gene encodes three viral coat proteins, VP1, VP2, and VP3, which are required for virion assembly. rAAV virions useful in conjunction with the compositions and methods described herein include those derived from a variety of AAVserotypes including AAV 1, 2, 3, 4, 5, 6, 7, 8, and 9. Also useful in conjunction with the compositions and methods described herein are pseudotyped rAAV vectors. Pseudotyped vectors include AAV vectors of a given serotype pseudotyped with a capsid gene derived from a serotype other than the given serotype (e.g., AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, among others). For example, a representative pseudotyped vector is an AAV2 vector encoding a therapeutic protein pseudotyped with a capsid gene derived from AAV serotype 8 or AAV serotype 9.For example, in certain embodiments, AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions. For example, suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types. Other rAAV virions that can be used in methods of the invention include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling.

[0068] Alternatively, as one of skill in the art will understand, a vector of the disclosure may be a ^-retroviral vector or a lentiviral vector.

[0069] A contemplated vector may include appropriate expression control sequences including, but not limited to, transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance protein processing and / or secretion. For eukaryotic cells, expression control sequences typically include a promoter; an enhancer; such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc.; and a polyadenylation sequence which may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene sequences and before the 3' ITR sequence. In one embodiment, the bovine growth hormone polyA is used. Another regulatory component of the vector useful is an internal ribosome entry site (IRES). An IRES sequence, or other suitable systems may be used to produce more than one polypeptide from a single gene transcript. An IRES (or other suitable sequence) is used to produce a protein that contains more than one polypeptide chain or to express two different proteins from or within the same cell. An example of an IRES is the poliovirus IRES, which supports trans gene expression in retinal cells.

[0070] The selection of the promoter to be employed in the vector may be made from among a wide number of constitutive or inducible promoters that can express the selected DIC in a cell e.g., a neuron or an ocular cell. In some embodiments, the vector comprises an inducible promoter operably linked to a gene encoding a DIC of the disclosure. In some embodiments, the vector comprises a constitutive promoter operably linked to a gene encoding a DIC of the disclosure. In some embodiments, the inducible promoter can modulate, activate, or deactivate expression of the DIC. In one embodiment, the promoter is cell-specific. The term “cell-specific” means that the particular promoter selected for the recombinant vector can direct expression of the selected DIC in a particular cell type. In an embodiment, the promoter is specific for expression of the DIC in projection neurons. In an embodiment, the promoter is specific for expression of the DIC in retinal ganglion cells. In an embodiment, the promoter is specific for expression of the DIC in bipolar cells. For example, the DIC may be expressed in retinal ganglion cells via a retinal ganglion cell-specific gene promoter, for example, Thy-I.

[0071] The architecture of the ganglion cell layer (GCL) of the primate retina may also allow for targeting of specific cell types, using, for example, mechanical means. Ganglion cell bodies lie within the GCL. Near the fovea, the GCL is at its maximal thickness, and contains several layers of cell bodies. The cell bodies of different retinal ganglion cell types lie in different positions (e.g., on-type ganglion cells lie more vitreally, as observed by multielectrode recording), which may allow them to be preferentially targeted (for example, by intravitreal administration of a viral vector e.g., an AAV that expresses a DIC). Selective targeting to on-type cells may be achieved even with a contemplated vector that includes a non-specific promoter (e.g., a CAG promoter) because the cells lie closer to the retina’s surface (i.e., vitreally), and AAVs do not penetrate the retina well when delivered by intravitreal injection.

[0072] Examples of constitutive promoters which may be included in a vector contemplated herein are, without limitation, a CAG promoter, CMV immediate early enhancer / chicken- actin (CA) promoter-exon 1-intron 1 element, a RSV LTR promoter / enhancer, a SV40 promoter, a CMV promoter, a 381 bp CMV immediate early gene enhancer, a dihydrofolate reductase promoter, a phosphoglycerol kinase (PGK) promoter, and a 578 bp CBA promoter-exonl-intronl. For example, a contemplated promoteris a CAG promoter. In some embodiments, the CAG promoter has the nucleic acid sequence of SEQ ID NO: 6, below:TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCC CCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGG GGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGG GCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTT CCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGG CGGGCG.

[0073] In some embodiments, vectors contemplated herein include an enhancer, such as, for example, a WPRE enhancer. In some embodiments, a WPRE enhancer has the nucleic acid sequence of SEQ ID NO: 7, below:ATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTG CTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTT ATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGC TGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGC CCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCG GGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCG CGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCC GCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGAC GAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC.

[0074] In some embodiments, vectors contemplated herein include a poly adenylation (poly(A)) element, such as, for example, an SV40 poly(A). In some embodiments, a SV40 poly(A) has the nucleic acid sequence of SEQ ID NO: 8, below:TAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAAT GCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGC AATAAACAAGTT.

[0075] DIC of the disclosure include, in some embodiments, an optogenetic protein or other light-gated ion channels or pumps, such as but not limited to SEQ ID NO: 5 below:METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFP HINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIE LFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILV TDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKI MAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIH EHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGV.

[0076] For example, provided herein is protein that comprises a light-sensitive channel from e.g., Stigeoclonium helveiicum. such as an optogenetic protein encoded by a nucleic acid with the nucleic acid sequence of SEQ ID NO: 4, below: ATGGAAACAGCCGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAG ACGCTCACGGAGAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCT TCCCCCACATCAACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCC AGTGGTTCACCGTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAG CTTCAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTC AAGTGCTTCATCGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAG ACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCC CCGTGATCCTGATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCA AGCGGACCATGACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCA CAGCCGCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTT CTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCAC ACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCT TCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGG CCTGATCACACCTTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGC AAGAACACTTGGGGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCAC ATCCTGATCCACGGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAG AACATGGAGATCGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTG.

[0077] Contemplated DIC fusion proteins may be encoded by a nucleic acid sequence that is at least 94% identical to the nucleic acid molecule of SEQ ID NO: 2. In some embodiments, the DIC fusion protein is encoded by nucleic acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid molecule of SEQ ID NO: 2.

[0078] For example, the DIC fusion protein may be encoded by a nucleic acid moleculehaving the nucleic acid sequence of SEQ ID NO: 2, below:ATGGAAACAGCCGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAGACGCTCACGGAGAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCTTCCCCCACATCAACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCCAGTGGTTCACCGTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAGCTTCAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTCAAGTGCTTCATCGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAGACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCCCCGTGATCCTGATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCAAGCGGACCATGACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCACAGCCGCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTCTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCACACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCTTCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGGCCTGATCACACCTTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGCAAGAACACTTGGGGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCACATCCTGATCCACGGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAGAACATGGAGATCGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTGGCGGCACCGGTAGTAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATTTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG.

[0079] In some embodiments, the DIC fusion protein may have the amino acid sequence of SEQ ID NO: 3, below:METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIE LFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILV TDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKI MAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIH EHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGVAAPVVAVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRY PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK EDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPI GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0080] In some embodiments, a composition of the disclosure includes an AAV2 vector having a gene that expresses a DIC fusion protein (e.g., a nucleic acid encoding a protein having 98% or 99% identity to the amino acid sequence of SEQ ID NO: 3). For example, in some embodiments, a composition of the disclosure includes an AAV2 vector having a nucleic acid encoding a protein having 98% identity to the amino acid sequence of SEQ ID NO: 3. In some embodiments, a composition of the disclosure includes an AAV2 vector having a nucleic acid encoding a protein having 99% identity to the amino acid sequence of SEQ ID NO: 3.

[0081] A vector contemplated herein may include an expression cassette having, for example, the nucleic acid sequence of SEQ ID NO: 9, below: TGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAA GAGTACCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGAC TTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTA CATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAAT GGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCA GTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTC TGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATT TTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCA GGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGC GGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCG GCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGC GCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCT CTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCCGCCACCATGGAAACAGCCGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAGACGCTCACGGAGAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCTTCCCCCACATCAACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCCAGTGGTTCACCGTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAGCTTCAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTCAAGTGCTTCATCGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAGACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCCCCGTGATCCTGATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCAAGCGGACCATGACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCACAGCCGCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTCTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCACACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCTTCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGGCCTGATCACACCTTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGCAAGAACACTTGGGGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCACATCCTGATCCACGGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAGAACATGGAGATCGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTGGCGGCACCGGTAGTAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATTTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAAC ATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCC CTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG ACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTCGAT ATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCT TTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCC TGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGG TGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTG TCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCA TCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAA TTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTG CCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCC AGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTC GCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATA CCGTCGACCCGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTT GGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGT GATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACA ACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTA AAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCTTCCTAGAGC ATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACA.

[0082] Vectors contemplated herein include an AAV2, wherein the AAV2 includes a DNA polynucleotide encoding a DIC fusion protein or an RNA equivalent thereof. In some embodiments, the protein is encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 4 or a protein having the amino acid sequence of SEQ ID NO: 5 fused to a reporter protein. In some embodiments, the reporter protein is GFP. For example, a DIC of the disclosure is exemplified by an AAV vector that has a nucleic acid sequence that is at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to the nucleic acid molecule of SEQ ID NO: 1 . For example, in some embodiments, the AAV vector has a nucleic acid sequence that is at least 91%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that isat least 92%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 93%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 94%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 95%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 96%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 97%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 98%, identical to the nucleic acid molecule of SEQ ID NO: 1. In some embodiments, the AAV vector has a nucleic acid sequence that is at least 99%, identical to the nucleic acid molecule of SEQ ID NO: 1 [also referred to as B SOI].

[0083] In some embodiments, a AAV vector described herein comprises the nucleic acid sequence of SEQ ID NO: 1, below:GCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGC GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGC CAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTAT CTACGTAGCCATGCTCTAGGAAGAGTACCATTGACGTCAATAATGACGTATGTTC CCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACG GTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACC CCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGG GGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGG GGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGT TTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGC GGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGG CGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTG TTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCCGCCACCATGGAAACAGCCGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAGACGCTCACGGAGAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCTTCCCCCACATCAACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCCAGTGGTTCACCGTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAGCTTCAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTCAAGTGCTTCATCGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAGACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCCCCGTGATCCTGATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCAAGCGGACCATGACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCACAGCCGCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTCTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCACACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCTTCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGGCCTGATCACACCTTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGCAAGAACACTTGGGGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCACATCCTGATCCACGGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAGAACATGGAGATCGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTGGCGGCACCGGTAGTAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATTTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGAT CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGTAAGAATTCGATATCAAGCTTATCGATAATCAACCTCTGGATTA CAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTAT GTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTC ATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCC CGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACT GGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGG GCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCT TTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGC TACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGG CTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTT TGGGCCGCCTCCCCGCATCGATACCGTCGACCCGGGCGGCCGCTTCGAGCAGAC ATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAG CTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG GGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAA ATCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTT AATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCA.Pharmaceutical Compositions and Routes of Administration

[0084] Any one of the DIC therapeutics described herein, such as a vector having a gene that encodes a DIC protein, can be formulated into pharmaceutical compositions for administration to a mammalian (e.g., a human) patient in a biologically compatible form suitable for administration in vivo. The compositions disclosed herein may be formulated in any suitable vehicle for delivery to a patient e.g., a human). For instance, they may be formulated in a pharmaceutically acceptable suspension, dispersion, solution, or emulsion. Suitable mediums include saline and liposomal preparations. Pharmaceutically acceptablecarriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Recombinant human album (rAlbumin Human NF RECOMBUMIN® Prime) may also be used as a stabilizer with an AAV vector (Albumedix, Nottingham UK). Examples of nonaqueous solvents are propylene glycol; polyethylene glycol; vegetable oils, such as olive oil; and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. A colloidal dispersion system may also be used for targeted gene delivery. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes. The compositions described herein may be used in the form of the free base, in the form of salts, solvates, and as prodrugs. All forms are within the methods described herein.

[0085] A DIC protein and a promoter e.g., a tissue- or cell type-specific promoter for use in e.g., target ocular cells may be assessed for contamination by conventional methods and then formulated into a pharmaceutical composition intended for e.g., retinal injection. A vector (e.g., an AAV vector) of the disclosure can be administered subcutaneously, intradermally, intravenously, intraperitoneally, via inhalation, nasally, orally, intramuscularly, intracranially, via intrapulmonary route, via ophthalmic route, parenterally, rectally, vaginally, via transmucosal route, intravitreal, retinal, or subretinal injection. Such formulation involves the use of a pharmaceutically and / or physiologically acceptable vehicle or carrier, particularly one suitable for the preferred route of administration, such as buffered saline or other buffers, e.g., HEPES, to maintain pH at appropriate physiological levels.Methods of the Treatment

[0086] Provided herein is a method of inducing activity of projection neurons in a patient suffering from a neurodegenerative disease, the method comprising administering an effective amount of a depolarizing ion channel therapeutic, wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from the projection neurons, to depolarize the projection neurons above a voltage threshold capable of eliciting one or more action potentials, thereby inducing activityof the projection neurons in the patient and restoring neural function in the patient.

[0087] Also provided herein is a method of restoring neural function in a patient in need thereof suffering from a neurodegenerative disease, the method comprising administering an effective amount of a depolarizing ion channel therapeutic such that it interacts with a patient’s neurons, wherein the effective amount is sufficient, when combined with a voltage produced in the neurons by incoming signals from degenerated neural circuitry upstream of the neurons, to make the neurons fire one or more action potentials, thereby restoring neural function in the patient.

[0088] Also provided herein is a method of enhancing neurotransmission from existing, degenerated endogenous neural circuitry in a patient having a retinal degenerative disease in need thereof, the method comprising administering to the patient in need thereof an effective amount of a depolarizing ion channel therapeutic, wherein the neurons receiving the transmission from the existing, endogenous degenerated neural circuits become capable of firing one or more action potentials.

[0089] Also provided herein is a method of increasing the voltage or membrane potential in neurons receiving weak or partial inputs from a degenerated endogenous neural circuit in a patient, the method comprising administering to the patient in need thereof an effective amount of a depolarizing ion channel therapeutic, wherein the membrane potential of the neurons, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from the neurons, is enhanced and the action potential firing rate of said neurons is greater than the action potential firing rate in said neurons prior to administration of the depolarizing ion channel therapeutic.

[0090] Also provided herein is a method of depolarizing neurons receiving weak or partial inputs from a degenerated neural circuit in a patient in need thereof, the method comprising administering to the patient in need thereof an effective amount of a depolarizing ion channel therapeutic, wherein upon administration of the depolarizing ion channel, the neurons that receive the therapeutic become able to be depolarized and, this depolarization, when combined with a voltage provided by degenerated neural circuitry upstream from the neurons, transmit a greater quantity of action potentials than said neurons prior to administration of the depolarizing ion channel therapeutic.

[0091] In some embodiments, the degenerated neural circuitry of a contemplated patient prior to an administration step in a method disclosed herein is insufficient to activate neurons to a voltage threshold capable of eliciting an action potential before the administration.

[0092] In some embodiments, the patient does not have a complete loss of functioning neural circuits before administration of the DIC, wherein the method does not include administering to the patient light stimulation or activation by light and / or wherein the method does not include activation of exogenously-introduced de novo neural circuits. It can be appreciated that neurons subject to the contemplated methods may be projection neurons.

[0093] Contemplated methods include, administering to the patient an effective amount of a DIC therapeutic that comprises a vector having a gene that encodes a DIC protein or that encodes a fusion protein comprising a DIC protein.

[0094] In some embodiments, the method is without or does not include a specific light stimulation of the DIC therapeutic, or without activation and / or without activation of exogenously-introduced de novo neural circuits. In some embodiments, the method does not include administration of light activation by light and / or without activation of exogenously- introduced de novo neural circuits.

[0095] Also provided herein is a method of treating a neurodegenerative disease in a patient in need thereof without specific administration of light stimulation, without activation of an administered therapeutic by light, and / or without activation of exogenously-introduced de novo neural circuits, the method comprising administering to the patient an effective amount of a DIC therapeutic, wherein upon administration the DIC depolarizes neurons in the neural circuity that are downstream of degeneration endogenous neural circuity of the patient.

[0096] Another contemplated method includes a method of treating a neurodegenerative disease in a patient in need thereof without administration of light stimulation or activation by light and / or without activation of exogenously-introduced de novo neural circuits, the method comprising administering to the patient an effective amount of a DIC therapeutic wherein the DIC depolarizes neurons in the neural circuitry of the patient upon administration, and wherein the neurons are downstream of degenerated endogenous neural circuitry.

[0097] Also provided herein is a method of treating a patient in need thereof suffering from a neurodegenerative disease and having variable symptom expression or fluctuating symptom intervals, the method comprising administering an effective amount of a DIC therapeutic wherein the effective amount is sufficient, when combined with a voltageprovided by degenerated endogenous neural circuitry upstream from neurons to depolarize the neurons above a voltage threshold capable of eliciting one or more action potentials.

[0098] Further contemplated herein are methods of treating amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, dementia including dementia associated with other neurodegenerative disorders or diseases, a retinal degenerative disease, Charcot-Marie-Tooth disease, Huntington’s disease, retinitis pigmentosa, a lysosomal storage disease, multiple system atrophy, tauopathies, and / or prion diseases in a human patient in need thereof, comprising administering an effective amount of an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9. Dementia may be for example, Lewy body dementia. Lysosomal storage diseases may be selected from the group consisting of Gaucher disease, Tay-Sachs disease, and Hurler syndrome.

[0099] Since methods described herein are, in some embodiments, a gene therapy, such embodiments may require only a single dose (e.g., a single injection), as the vector will continue to express the DIC, making it a low burden treatment for patients.

[0100] In some embodiments, a contemplated degenerative disease is selected from the group consisting of retinitis pigmentosa, choroidemia, Usher syndrome, and macular degeneration (and / or the night blindness associate e.g., with these disorders). For example, methods described herein, without being bound by theory, boost signals from circuits that operate at low light levels and allows patients to see in much darker environments than was possible before treatment, the results of which are exemplified in FIG. 4

[0101] Patients may have fluctuating symptom intervals, with e.g., about one day to about 1 year or more remission intervals (or about 1-8 hours), preceded and / or followed by an active symptom interval of about 1 day to about 1 year, or about 1-8 hours. Exemplary fluctuating symptoms may be one or more of: loss of or altered voluntary motor function, pain, fatigue, cognitive dysfunction, and altered sensory function. Criteria for diagnosing the neurodegenerative disease or the fluctuating symptom can be based on, for example, a DSM- 5 criteria.

[0102] In some embodiments, methods described herein are applied to Parkinson’s disease, which affects 500,000 people in the U.S. alone. The disease develops when dopamine neurons that connect the substantia nigra to the striatum degenerate. The degeneration leads to movement difficulties, shaking, stiffness, and problems with balance and coordination. As the disease progress, patients have increasing difficulty walking andtalking. Current treatments focus on increasing dopamine release from neurons in substantia nigra. The methods described herein can be used instead of or in conjunction with other therapeutic interventions. For example, instead of focusing on increasing release by presynaptic neurons, the methods described herein target the cells receiving the dopamine input (e.g., in the striatum). The DIC treatment described herein, without being bound by theory, boosts the activity of these cells, so the incoming circuits are more effective in driving them (and medications that increase release are more effective).

[0103] Methods described herein may be used to treat multiple sclerosis (MS), which affects as estimated 1 million people in the U.S.. MS is an autoimmune disease where the immune system attacks the myelin sheath that insulates nerve fibers in the central nervous system. MS symptoms vary from patientto patient, depending on which areas of the nervous system are undergoing the demyelination. Demyelination interferes with communication between the brain and the rest of the body or between different brain areas. A common and debilitating symptom in MS is blurring or loss of vision as a result of demyelination of the optic nerve. Loss of conduction through the nerve is diminished (the nerve is unable to fire or the firing rate is reduced), so visual information is unable to reach the brain (in particular, the lateral geniculate nucleus and the superior colliculus). This is referred to as optic neuritis. If the demyelination affects motor neurons, patients with MS experience weakness, urinary difficulties, walking unsteadiness, stiffness in the arms or legs, and a reduction in the ability to move their eyes. If the demyelination affects sensory neurons, patients with MS experience tingling, numbness, and loss of coordination and balance. Depression and fatigue are common in patients with MS. Existing treatments for MS canonically focus on suppressing the immune response, such as through corticosteroids and plasma exchange (plasmapheresis). The methods described herein offer a different approach that can be used instead of or in conjunction with other (e.g., optogenetic) approaches. The fact that patients with MS often go into periods of remission or have fluctuating symptoms indicates that many neural circuits remain largely intact, albeit degenerated (at least at the early stages); the circuits are unable or just too weak to send their signals through to downstream targets due to the demyelination of their projection neuron or other connecting neurons (e.g., too degenerated). In these patient populations, a method described herein can be used, including administration of a DIC therapeutic to the neurons that receive input from the projection neurons, since it is the projection neurons that are not firing sufficiently (e.g., degenerated). The weak signals fromthe projection neurons can be boosted by the methods described herein so that e.g., administering a DIC therapeutic described herein to postsynaptic targets of the projection neurons allows the weak signals from the projection neurons to be sufficient to make the postsynaptic cells fire. Alternatively, methods described herein including administration of a DIC therapeutic can be administered directly to the projection neurons, as a way to boost their firing rate to overcome the loss of insulation from the demyelination.

[0104] In some embodiments, methods described herein are used to treat Charcot-Marie- Tooth disease (CMT), a relatively common (1 in 2500 people) inherited nerve disease that causes abnormalities in the nerves that supply the feet, legs, hands, and arms. CMT affects both motor and sensory nerves, and, in some embodiments, DIC therapeutics of the disclosure boost neurons post-synaptic to the motor or sensory nerves, so that weak signals from the affected nerves can be sufficient to make the post-synaptic cells fire one or more action potentials.

[0105] In some embodiments, methods of the disclosure are used to treat Alzheimer’s disease (AD), which affects about 1 in 9 people age 65 and older. Patients with AD commonly show symptom fluctuations, where they have ‘good days’ and ‘bad days’ or ‘good periods’ and ‘bad periods’ within a day. During the good days, patients with AD have periods of cognitive clarity, which allows them to carry out some of the activities of daily living, such as dressing and attending to personal hygiene. The presence of these good days or good periods implies that the neural circuits that underlie the behaviors performed during these times are at least partially intact, making the them amenable to the DIC treatment described herein. Current treatments for AD focus largely on increasing cholinergic output, but a DIC therapeutic described herein can be used, in some embodiments, to target neurons postsynaptic to the cholinergic cells, thereby serving as an alternative approach or an approach used in conjunction with approaches that increase cholinergic output. Methods described herein, without being bound by theory, can boost the activity of the post-synaptic cells, making them fire and allowing the signals from the cholinergic cells to be passed to downstream targets. A disclosed method would allow a reconnecting of the circuits that have deteriorated due to the disease and stabilize the activity in the patient, so the patient stays in the good periods.

[0106] In some embodiments, methods described herein are used to treat a lysosomal storage disease (LSD). LSD consist of a group of more than 70 inherited metabolic disorderswhere lysosomes have aberrant function. Briefly, lysosomes are sacs of enzymes inside cells that break down large molecules and send the fragments to other parts of the cell for recycling. When lysosomes are defective, the large molecules (such as complex lipids, glycoproteins, and glycosaminoglycans) build up in cells and become toxic, leading to a wide range of clinical symptoms. While the individual disorders are rare, as a group, they affect many people; an estimated 1 in 5000 live births worldwide. Gaucher disease, Tay-Sachs disease (in Ashkenazi Jews), and Hurler syndrome (in Scandinavians and Russians) are types of lysosomal storage disease, among others. One of skill in the art will appreciate that any now known or later discovered LSD are contemplated by the disclosure. In LSDs, many of the symptoms are neurological. Current treatments for LSDs include, without limitation, replacing deficient enzymes, as well as small molecule therapies. As contemplated herein, the methods described herein, can be used in combination with enzyme replacing therapies (e.g., administering to the patient a replacement deficient enzyme or analog thereof) or small molecule therapies by, without being bound by theory, boosting post-synaptic neurons in degenerated neural circuits so that signals from upstream circuits (weakened by loss of neurons) become sufficient to make the post-synaptic neurons fire action potentials and pass their signals to downstream targets.

[0107] In some embodiments, methods described herein are directed to treating retinal diseases. Contemplated retinal degenerative diseases include retinitis pigmentosa (RP), age- related macular degeneration, Usher syndrome, Stargardt macular dystrophy, Leber congenital amaurosis, and Bardet-Biedl syndrome.

[0108] Retinitis pigmentosa includes autosomal recessive inherited retinitis pigmentosa as well as autosomal dominant inherited retinitis pigmentosa and X-chromosome recessive inherited retinitis pigmentosa. The most common retinitis pigmentosa is the type showing autosomal recessive inheritance, which accounts for about 35% of the total number of patients diagnosed with RP. The next most common is the type showing autosomal dominant inheritance, which accounts for about 10% of the total number of patients diagnosed with RP. The least common is the type showing X-linked inheritance (X-chromosome recessive inheritance), which accounts for about 5% of the total number of patients diagnosed with RP.

[0109] Diseases in which retinal degeneration occurs as a complication are also contemplated in the disclosure herein. Such diseases include: Snowflake vitreoretinal degeneration; Choroidal neovasculatization caused by adult- onset foveomacular dystrophy;Bietti crystalline comeoretinal dystrophy; and diabetic retinopathy. A non-limiting list of diseases in which retinal degeneration occurs as a symptom include: Aceruloplasminemia; Adrenoleukodystrophy; Alstrom disease; Alstrom Syndrome; Asphyxiating Thoracic Dystrophy; Bonneman-Meinecke-Reich syndrome; Bonnemann-Meinecke-Reich syndrome; CDG syndrome type IA; Chorioretinopathy dominant form - microcephaly; Choroideremia - hypopituitarism; Congenital disorder of glycosylation type IA; Congenital Disorders of Glycosylation Type la; Cystinosis; Hypotrichosis, syndactyly and retinal degeneration; Jeune syndrome; Mucolipidosis IV; Mucolipidosis type 4; Mucopolysaccharidoses; Muscle-eye- brain syndrome; Neonatal ALD; Olivopontocerebellar atrophy type 3; Osteopetrosis, autosomal recessive 4; Pigmentary retinopathy; Pseudoadrenoleukodystrophy; Retinoschisis, X-linked; Retinoschisisl, X-linked, Juvenile; Santavuori Disease; Spastic paraplegia, autosomal recessive; and Werner syndrome. The present methods can be used to treat a patient who has RP.

[0110] The compositions described herein may be used in a method of treatment, for example, in a method of improving light sensitivity in a patient in need thereof. Such treatment may, for example, obtain a desired therapeutic effect in terms of partially or completely curing a disease and / or adverse effect attributed to the disease. Methods to measure clinical efficacy include evaluation of visual acuity, color perception, maze navigation, object recognition, the ability to count fingers, flash visual evoked response (VEP), the pupillary light reflex (PLR), electroretinogram (ERG; including bilateral full-field ERG), and nystagmus testing. International Society for Clinical Electrophysiology of Vision standard guideline may be followed for the analyses. Pupil responses may be recorded simultaneously in both eyes. Nystagmus may be characterized qualitatively and quantitatively by analysis of motion paths in videos taken at baseline and at various desired time points post- treatment. Interpupillary distances may be measured directly from video frames.EXAMPLES

[0111] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the disclosure in any way.Example 1: Depolarizing Ion Channel Therapeutic For Treating a Retinal Disease

[0112] The present example describes an exemplary depolarizing ion channel (DIC) therapeutic and methods of using and assessing it for treating a retinal disease: retinitis pigmentosa.Materials and MethodsVector material

[0113] The vector injected into patients having retinitis pigmentosa was a nonreplicating, Rep- and Cap gene-deleted, recombinant adeno-associated virus (rAAV) vector expressing a depolarizing ion channel gene fused with Green Fluorescent Protein (ChronosFP). The vector DNA contained AAV serotype 2 (AAV2) inverted terminal repeats (ITRs) and was packaged in an AAV2 capsid. The vector DNA also contained a cytomegalovirus early enhancer / chicken beta actin (CAG) promoter, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and an SV40 poly adenylation sequence. The vector (SEQ NO: 1) was formulated in a balanced salt solution (BSS) supplemented with 0.014% Polysorbate 20.Vector injection

[0114] Retinitis pigmentosa patients were injected with a vector dose of 1.2 x 1012vector genomes / eye, injected intravitreally in a 100 pL volume.Acuity testing

[0115] Retinitis pigmentosa patients were tested using tumbling E optotypes in the In Sight Acuity System (Veatch Opthalmic Instruments, AZ, USA) at a distance of 3 feet. On each trial, either a single optotype or a row of optotypes was presented depending on the size of the letter and in accordance with the manufacturer software. Visual acuity testing of the study eye started at the 20 / 400 line, and moved to 20 / 200, 20 / 150, 20 / 100, 20 / 80, 20 / 70, 20 / 60, 20 / 50, 20 / 40, 20 / 30, 20 / 25, and 20 / 20 lines, respectively. For each optotype size, the patient was presented with 10-12 instances of the optotype, each chosen at random. If, for a given size, the patient’s accuracy after the 10-12 trials was above chance (which was 25% for the 4 orientations of the letter E), the test progressed to the next, more challenging, line.Visual acuity was recorded as the last line at which the patient performed at equal to or betterthan 62.5% correct (a standard threshold - halfway between chance (25%) and a perfect score (100%)).Color Testing

[0116] Retinitis pigmentosa patients were tested with a red, green, and blue (RGB) color test. On each trial of the test, a shape that varied in color (red, green, or blue) was presented. The colors were equally luminant with respect to each other, chosen at random, and each color occurred with equal probability. If the patient could not see the colors and was guessing, their performance was at chance (33%). The trials were presented in blocks of 10.Maze Navigation Testing

[0117] Retinitis pigmentosa patients were tested on their ability to navigate a maze, which was constructed by placing gray tiles (12”xl2”) on a black mat (16’xl2’). The task was to maneuver through the maze without stepping on the gray tiles (the obstacles). Patients were tested at a range of light levels, from 1.7 lux to 96 lux. Patients were dark- adapted for 20 minutes before performing maze navigation at the dim light levels. Light levels were chosen to approximate different real-life situations that patients were likely to encounter. Light levels were measured using an NIST-calibrated light meter (Extech EA33) at 9 different locations on the maze. Variance (square of standard deviation) of luminance was less than 10% of the mean at each light level, with an average variance of 3.4% of the mean light level. As expected, light levels did not vary significantly across different maze conditions, since all configurations were constructed using the same number of gray tiles. Each patient was tested on several mazes at each light level, with the results in the dim light condition shown in FIG. 4.Results

[0118] Patients with retinitis pigmentosa gained the ability to recognize colors following treatment with a DIC therapeutic as shown in FIG. 2. The patients were presented with a standard color test before and after treatment, which consisted of an intravitreal injection of an AAV2 vector that encodes the gene for a DIC (SEQ ID NO: 1). Only the study eye was tested and patient’s other eye was covered with an eye patch. Briefly, the test was as follows: on each trial, a shape that varied in color (red, green, or blue) was presented. The color waschosen at random, and each color occurred with equal probability. If the patient could not see the colors and was guessing, his / her performance would be at chance, i.e., 33%. As shown in the leftmost panel of FIG. 2, before treatment, Patient l ’s performance was at chance or below. After treatment, he could recognize red and blue about 80% to 90% of the time and showed a substantial improvement in recognizing green. Each circle corresponds to a block of 10 trials. Patient 2’s results are shown in the middle panel of FIG 2. Before treatment, the patient had some ability to distinguish color (particularly blue and green from red). After treatment, his ability to distinguish all 3 colors approached 100%. As with the leftmost panel of Patient 1, each circle corresponds to a block of 10 trials. Patient 3’s results are shown in the rightmost panel of FIG. 2. Before treatment, this patient was able to recognize green and blue, but not red. After treatment, he gained the ability to see red: that is, without being bound by theory, the scaffolding of the red circuits in his degenerated retina were sufficiently intact, so that, with the boost from the DIC treatment, the signals from the red circuits were able to drive the ganglion cells to fire and send signals to the brain. The extent to which the DIC therapeutic worked depended on what remaining circuitry was available to amplify (or boost) and pass through to downstream neurons.Patients with retinitis pigmentosa gained in visual acuity following treatment with a DIC therapy

[0119] As in FIG. 2, the treatment consisted of an intravitreal injection of an AAV2 vector that expresses a gene encoding a DIC (SEQ ID NO: 1). Visual acuity was tested on the study eye only (the other eye was covered with an eye patch) using a standard rotated E paradigm. Briefly, the patients were presented with rows of Es that varied in orientation (up, down, right, and left), and each successive row contained smaller Es following the standard Snellen fractions (20 / 200, 20 / 150, 20100, etc.). The Es were presented in blocks, and multiple blocks were presented so that the patient was tested with 10-12 Es at each size. FIG. 3 A shows s patient’ s performance both before and after treatment. Before treatment the patient had 20 / 150 vision (meaning the patient could only see letters one line below the ‘big E’ on an eye chart). Following treatment, the visual acuity of the patient was substantially increased. He was able to see down to line 20 / 70 on the eye chart after 3 months and down to line 20 / 40 after 6 months, which is close to normal vision. The figure shows his performance at 6 months. FIG. 3B shows acuity results before treatment and after treatment for 6 patients,expressed as logMAR on the left and Snellen fraction on the right. Without being bound by theory, the reason the method described herein were successful in substantially increasing the patient’s visual acuity was because the patient’s retina still contained much of the circuitry for high acuity vision: that is, while their photoreceptors had degenerated, the scaffolding of circuitry that produces high acuity vision appeared to still be sufficiently intact (e.g., endogenous neuronal circuitry was present, albeit degenerated). In other words, before treatment, the circuits were latent, just too weak to drive the ganglion cells to threshold, leaving the patient blind to anything but the largest letters.Treatment with a DIC therapeutic reduced night blindness in patients with retinitis pigmentosa

[0120] The treatment consisted of an intravitreal injection of an AAV2 vector that expresses a gene encoding a DIC (SEQ ID NO: 1). Patients were tested on a maze navigation task. In each case, the patient used only the study eye and the untreated eye was covered with an eye patch. The patients were tested on several mazes before and after treatment, with each maze having a different configuration (but drawn from a set of mazes with the same statistics (same number of turns, etc.)). Performance was measured as the time to complete the maze and the number of errors (the number of times the patient stepped on a gray tile). As shown in FIG. 4, the DIC treatment substantially improved patient navigation. After treatment, both patients were able to see sufficiently in the dim light conditions (1 .7 lux, comparable to the lighting in a dim restaurant) to make it through the mazes in half the time after treatment than before, and with many fewer errors (in some runs, zero errors). While the DIC expressed by the AAV2 had a chromophore attached to it, the DIC component, not the chromophore, produced the results described herein, as the light levels used in the experiments shown in FIGS. 2-4 were more than 10,000 times lower than the minimal light level needed to activate the chromophore (Klapoetke et al, 2015). For the color vision and acuity experiments, the light levels used in the experiments were 10,000 times lower, and for the night vision experiments, the light levels used in the experiments was 66,000 times lower than the minimal light level needed to activate the chromophore. Thus, these examples do not use a chromophore mechanism.Neuronal responses were increased in nonhuman primates following treatment with a DIC therapeutic

[0121] Three healthy nonhuman primates were intravitreally injected with an AAV2 vector that expresses a gene encoding a DIC as in FIGs. 2-4 with doses of 3.7 x IO10vg / eye (n=4 eyes) and 1.17 x 1011vg / eye (n=2 eyes). Four other nonhuman primates were used as controls (untreated) (n=8 eyes). FIG. 5 shows the boosting effect of a DIC therapeutic on neuronal activity, as measured by the amplitude of the Photopic Negative Response (PhNR) in electroretinogram (ERG) recordings. Responses from the DIC-treated eyes are nearly twice the size of those from the untreated eyes (p<0.01 t-test, comparing the mean amplitude of the PhNRs from the DIC-treated eyes (n=6) with those from the untreated eyes (n=8) indicating that applying a DIC therapeutic to neurons boosts their signals even in normal animals, and even at lower doses (10-fold lower than that used in the studies shown in FIGs. 2-4 with patients). The chromophore on the DIC was not activated, as the stimulating light used by the ERG device (Diagnosys, Inc) was dim red light (660 nm wavelength) at 2xl0-5to 3xl0-4mW / mm2, and the green-absorbing chromophore on the DIC (Chronos) does not respond to this wavelength, even with very bright light (6 mW / mm2), as shown in Klapoetke et al (2015), and therefore the DIC was activating the cells without utilizing a chromophore mechanism. FIG. 6B indicates the dose-dependent boosting effect of this DIC therapeutic on neuronal activity.INCORPORATION BY REFERENCE

[0122] The entire disclosure of each of the patent documents and scientific articles cited herein is incorporated by reference for all purposes.EQUIVALENTS

[0123] The disclosure can be embodied in other specific forms without departing from the essential characteristics thereof. The foregoing embodiments therefore are to be considered illustrative rather than limiting on the disclosure described herein. The scope of the disclosure is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT IS CLAIMED IS:

1. A method of treating a neurodegenerative disease in a patient in need thereof without administration of light stimulation or activation by light, the method comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic wherein the depolarizing ion channel therapeutic depolarizes neurons in a neural circuitry of the patient upon administration, and wherein the neurons are downstream of degenerated endogenous neural circuitry.

2. A method of inducing activity of projection neurons in a patient suffering from a neurodegenerative disease, the method comprising administering an effective amount of a depolarizing ion channel therapeutic wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from the projection neurons, to depolarize the projection neurons above a voltage threshold capable of eliciting one or more action potentials, thereby inducing activity of the projection neurons in the patient and restoring neural function in the patient.

3. A method of restoring neural function in a patient in need thereof suffering from a neurodegenerative disease, the method comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream of neurons receiving the therapeutic, to depolarize the neurons above a voltage threshold capable of eliciting one or more action potentials, thereby restoring neural function in the patient.

4. The method of claim 2 or 3, wherein the degenerated endogenous neural circuitry of the patient is insufficient to activate downstream neurons to a voltage threshold capable of eliciting an action potential before the administration of the depolarizing ion channel therapeutic.

5. The method of any one of claims 1-4, wherein the patient has degenerated neural circuitry but does not have a complete loss of functioning circuits before administration of the depolarizing ion channel therapeutic, wherein the method does not include administering to the patient light stimulation of the depolarizing ion channel or activation by light and / orwherein the method does not include light activation of exogenously-introduced de novo neural circuits.

6. A method of treating a patient suffering from a neurodegenerative disease and having variable symptom expression or fluctuating symptom intervals, the method comprising administering an effective amount of a depolarizing ion channel therapeutic to the patient, wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from neurons receiving the therapeutic, to depolarize the neurons above a voltage threshold capable of eliciting one or more action potentials.

7. A method of enhancing neurotransmission from existing degenerated endogenous neural circuitry in a patient having a retinal degenerative disease and in need of treatment, the method comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic, wherein downstream neurons of the patient receiving neurotransmission from the existing degenerated endogenous neural circuits become capable of firing action potentials.

8. The method of any one of claims 1-7, wherein the depolarizing ion channel therapeutic is nucleic acid sequence of SEQ ID NO: 1.

9. A method of increasing activity of neurons receiving weak or partial inputs from a degenerated endogenous neural circuit in a patient in need thereof, the method comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic, wherein upon administration of the depolarizing ion channel therapeutic, the activity of the neurons, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from the neurons, is enhanced and the number of action potentials generated by the neurons is greater than the number of action potentials in the neurons prior to administration of the depolarizing ion channel therapeutic.

10. A method of depolarizing neurons receiving weak or partial inputs from a degenerated endogenous neural circuit in a patient in need thereof, the method comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic, wherein upon administration of the depolarizing ion channel therapeutic, the patient’s neurons become capable of being depolarized and, when combined with a voltage provided by degeneratedendogenous neural circuitry upstream from the neurons transmit a greater quantity of action potentials than the patient’ s neurons were able to provide prior to administration of the depolarizing ion channel therapeutic.

11. The method of any one of claims 1-9, wherein the neurons are projection neurons.

12. The method of any one of claims 1-7 and 9-11, wherein the depolarizing ion channel therapeutic comprises: a) a depolarizing ion channel protein; or b) a DNA or RNA polynucleotide encoding a depolarizing ion channel protein or a fusion protein comprising a depolarizing ion channel protein.

13. The method of any one of claims 1-7 and 9-12, wherein administering to the patient an effective amount of the depolarizing ion channel therapeutic comprises administering a vector comprising a DNA or RNA polynucleotide encoding: a depolarizing ion channel protein or a fusion protein comprising a depolarizing ion channel protein.

14. The method of claim 13, wherein the vector comprises an inducible promoter operably linked to the DNA or RNA polynucleotide.

15. The method of claim 14, wherein the inducible promoter can modulate, activate, or deactivate expression of the depolarizing ion channel protein or fusion protein comprising a depolarizing ion channel protein.

16. The method of any one of claims 12-15, wherein the DNA or RNA polynucleotide further encodes a gain control protein.

17. The method of any one of claims 12-16, wherein the DNA or RNA polynucleotide is operably linked to a tissue- or cell type-specific promoter.

18. The method of any one of claims 12-17, wherein the depolarizing ion channel protein is selected from the group consisting of a ligand-gated ion channel protein, a voltage-gated ion channel protein, a mechanosensitive ion channel protein, and a cyclic nucleotide-gated ion channel protein.

19. The method of any one of claims 12-18, wherein the depolarizing ion channel protein is a light-activated ion channel.

20. The method of any one of claims 12-19, wherein the depolarizing ion channel protein is a sodium ion channel protein, a potassium ion channel protein, or a calcium ion channel.

21. The method of any one of claims 13-20, wherein the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector.

22. The method of claim 21, wherein the viral vector is selected from the group consisting of: an adenoviral vector, a retroviral vector, a poxviral vector, an adeno-associated viral (AAV) vector, a baculoviral vector, a herpes simplex viral vector, and a synthetic vector.

23. The method of claim 22, wherein the viral vector is an AAV vector.

24. The method of claim 23, wherein the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV vector selected from the group consisting of an AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74 vector.

25. The method of claim 24, wherein the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV2 vector.

26. The method of claim 22, wherein the retroviral vector is a y-retroviral vector or a lentiviral vector.

27. The method of any one of claims 11-26, wherein the DNA or RNA polynucleotide is operably linked to a constitutive promoter.

28. The method of claim 27, wherein the constitutive promoter is a CAG promoter.

29. The method of claim any one of claims 23-25 and 27-28, wherein the AAV further comprises two inverted terminal repeats (ITRs), wherein the two ITRs comprise a first ITR (ITR1) and a second ITR (ITR2), wherein ITR1 is positioned 5' to the DNA or RNApolynucleotide and ITR2 is positioned 3' to the DNA or RNA polynucleotide to form a cassette comprising the structure ITRl-polynucleotide-ITR2.

30. The method of claim 29, wherein the two ITRs are AAV serotype 2 ITRs.

31. The method of any one of claims 23-25 and 27-30, wherein the depolarizing ion channel therapeutic comprises an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9, or the depolarizing ion channel therapeutic comprises SEQ ID NO: 1.

32. The method of any one of claims 1-6 and 11-31, wherein the neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis; multiple sclerosis; Parkinson’s disease; Alzheimer’s disease; a dementia, including dementia associated with other neurodegenerative disorders or diseases; a retinal degenerative disease; Charcot-Marie-Tooth disease; Huntington's disease; retinitis pigmentosa; a lysosomal storage disease; multiple system atrophy; a tauopathy; and a prion disease.

33. The method of claim 32, wherein the retinal degenerative disease is selected from the group consisting of: retinitis pigmentosa, choroidemia, Usher syndrome, and macular degeneration.

34. The method of claim 32, wherein the dementia is Lewy body dementia.

35. The method of claim 32, wherein the lysosomal storage disease is selected from the group consisting: of Gaucher disease, Tay-Sachs disease, and Hurler syndrome.

36. The method of claim 35, wherein the method further comprises administering to the patient a replacement deficient enzyme or analog thereof.

37. The method of any one of claims 6 and 11-36, wherein the fluctuating symptom intervals are a about one day to about 1 year or more remission interval preceded and / or followed by an active symptom interval of about 1 day to about 1 year.

38. The method of any one of claims 6 and 11-37, wherein the fluctuating symptoms comprise one or more of: loss of or altered voluntary motor function, pain, fatigue, cognitive dysfunction, and altered sensory function.

39. The method of any one of claims 1-38, wherein the neurodegenerative disease or the fluctuating symptoms are diagnosed by a DSM-5 criteria.

40. A method of treating a disease or disorder selected from the group consisting of amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, dementia including dementia associated with other neurodegenerative disorders or diseases, Charcot-Marie-Tooth disease, Huntington’s disease, a lysosomal storage disease, multiple system atrophy, tauopathies, and prion diseases in a human patient in need thereof, comprising administering to the patient an effective amount of a depolarizing ion channel therapeutic.

41. A method of treating a disease or disorder selected from the group consisting of: amyotrophic lateral sclerosis; multiple sclerosis; Parkinson’s disease; Alzheimer’s disease; dementia, Charcot-Marie-Tooth disease; Huntington’s disease; a lysosomal storage disease; multiple system atrophy; a tauopathy, and a prion disease in a human patient in need thereof, the method comprising administering to the patient an effective amount of an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9.

42. A method of treating a disease or disorder selected from the group consisting of: amyotrophic lateral sclerosis; multiple sclerosis; Parkinson’s disease; Alzheimer’s disease; dementia; Charcot-Marie-Tooth disease; Huntington’s disease; a lysosomal storage disease; multiple system atrophy; a tauopathy, and a prion disease, in a human patient in need thereof, the method comprising administering to the patient an effective amount of the nucleic acid sequence of SEQ ID NO: 1.

43. A method of treating a neurodegenerative disease in a patient in need thereof without administration of light stimulation or activation by light, the method comprising administering to the patient an effective amount of nucleic acid sequence of SEQ ID NO: 1, wherein SEQ ID NO: 1 depolarizes neurons in a neural circuitry of the patient upon administration, and wherein the neurons are downstream of degenerated endogenous neural circuitry.

44. A method of inducing activity of projection neurons in a patient suffering from a neurodegenerative disease, the method comprising administering an effective amount of nucleic acid sequence of SEQ ID NO: 1, wherein the effective amount is sufficient, whencombined with a voltage provided by degenerated endogenous neural circuitry upstream from the projection neurons, to depolarize the projection neurons above a voltage threshold capable of eliciting one or more action potentials, thereby inducing activity of the projection neurons in the patient and restoring neural function in the patient.

45. A method of restoring neural function in a patient in need thereof suffering from a neurodegenerative disease, the method comprising administering to the patient an effective amount of nucleic acid sequence of SEQ ID NO: 1, wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream of neurons receiving the therapeutic, to depolarize the neurons above a voltage threshold capable of eliciting one or more action potentials, thereby restoring neural function in the patient.

46. The method of claim 44 or 45, wherein the degenerated endogenous neural circuitry of the patient is insufficient to activate downstream neurons to a voltage threshold capable of eliciting an action potential before the administration of SEQ ID NO: 1 .

47. A method of treating a patient suffering from a neurodegenerative disease and having variable symptom expression or fluctuating symptom intervals, the method comprising administering an effective amount of an effective amount of nucleic acid sequence of SEQ ID NO: 1 to the patient, wherein the effective amount is sufficient, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from neurons receiving the therapeutic, to depolarize the neurons above a voltage threshold capable of eliciting one or more action potentials.

48. A method of enhancing neurotransmission from existing degenerated endogenous neural circuitry in a patient having a retinal degenerative disease and in need of treatment, the method comprising administering to the patient an effective amount of nucleic acid sequence of SEQ ID NO: 1, wherein downstream neurons of the patient receiving neurotransmission from the existing degenerated endogenous neural circuits become capable of firing action potentials.

49. A method of depolarizing neurons receiving weak or partial inputs from a degenerated endogenous neural circuit in a patient in need thereof, the method comprising administering to the patient an effective amount of a an effective amount of nucleic acid sequence of SEQID NO: 1, wherein upon administration of SEQ ID NO: 1, the patient’s neurons become capable of being depolarized and, when combined with a voltage provided by degenerated endogenous neural circuitry upstream from the neurons transmit a greater quantity of action potentials than said neurons prior to administration of SEQ ID NO: 1.

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