Enhancing healthy neuronal activity using depolarizing ion channel therapeutics

Abstract

Provided herein are depolarizing ion channel therapeutics and methods of using the same for boosting retinal neuronal function.

Description

Attorney Docket No.: BIS-003 WOENHANCING HEALTHY NEURONAL ACTIVITY USING DEPOLARIZING ION CHANNEL THERAPEUTICS CROSS-REFERENCE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 737,227, filed December 20, 2024, which is incorporated by reference in its entirety herein.FIELD OF THE DISCLOSURE

[0002] This disclosure relates to depolarizing ion channel therapeutics for boosting retinal neuronal function, and methods of using the same.BACKGROUND

[0003] The human visual system is a complex and highly efficient network responsible for converting light into visual information that the brain can interpret. Central to this process are the retinal ganglion cells (RGCs), which are the primary neurons responsible for transmitting visual information from the retina to the brain. These neurons play a crucial role in the visual pathway, as they encode and relay signals through the optic nerve to various brain regions, including the lateral geniculate nucleus and the visual cortex. Enhancing the function of these neurons can significantly improve visual processing and overall visual acuity.

[0004] Improving visual processing and visual acuity in subjects that ordinally have normal vision can be important for subjects who must focus on visual cues, such as pilots, machinery operators and the like. In other subjects, strengthening the resilience of RGCs against damage can provide neuroprotective benefits, which may be relevant in conditions where normal RGCs are susceptible to injury or degeneration. Enhanced RGC function can help preserve the integrity of the visual pathway, thereby maintaining visual capabilities for a longer period.

[0005] Boosting RGC function can lead to the development of advanced visual prosthetics and therapies and offer new solutions for individuals in need of augmented vision.1IPTS / 200232923.1Attorney Docket No.: BIS-003 WO

[0006] The implications for boosting RGC in subjects that currently have normal RGCs using pharmacological treatments have not yet been appreciated or addressed. For example, boosting the neurons responsible for transmitting visual information from the retina to the brain holds significant potential for medical and technological advancements.SUMMARY

[0007] This disclosure relates to the discovery that, in patients with normal vision, administering a depolarizing ion channel therapeutic boosts retinal neuronal activity. As described herein and shown for example in the disclosed figures, treating subjects with a depolarizing ion channel (DIC) therapeutic can boost such activity. This disclosure, therefore, is directed in part to methods for enhancing or boosting retinal activity in a subject, e.g., methods to enhance visual acuity, night vision, and / or color vision, where the subject may have normal neuronal activity but may need to enhance such activity (for example, to facilitate machine operation, work at night, transport driving, tactical personnel, etc.).. For example, methods disclosed herein may include and / or result in invoking a facilitating mechanism in patients that allow normal circuits to boost 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 that pass signals from upstream circuits to downstream brain areas. The DIC boosts the activity of the projection neurons, so that signal from upstream circuits increases the projection neurons crossing a voltage threshold, which, in turn, allows them to fire action potentials and pass the information to downstream neurons.

[0008] For example, provided herein is a method of enhancing vision, and / or boosting retinal neuronal activity in a subject in need thereof without administration of light stimulation or activation by light, the method comprising administering to the subject 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 endogenous neural circuitry. Such disclosed methods do 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.

[0009] 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 2IPTS / 200232923.1Attorney Docket No.: BIS-003 WOchannel 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 potassium ion 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.

[0010] Also provided herein is a method of providing retinal neuroprotective benefits in a patient need thereof, e.g., ameliorating and / or delaying and / or substantially preventing the onset of a disease or disorder in a patient that is susceptible to a retinal disease or disorder, e.g., treating a patient that is a smoker, or will be or has been administered a neuronal degrading pharmaceutical, or a patient that has been identified as having a predisposition (but not yet suffering from) a disorder such as glaucoma, 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, 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.

[0011] Described herein, in certain embodiments, is a method of boosting retinal neuronal signals in a subject in need thereof without administration of light stimulation or activation by light, the method comprising administering to the subject an effective amount of a depolarizing ion channel therapeutic wherein the depolarizing ion channel therapeutic depolarizes neurons in a neural circuitry of the subject upon administration, and wherein the neurons are downstream of endogenous neural circuitry.

[0012] Described herein, in certain embodiments, is a method of enhancing night vision, vision acuity, and / or color perception in a subject in need thereof, the method comprising administering to the subject an effective amount of a depolarizing ion channel therapeutic.

[0013] In some embodiments, the subject does not have degenerated neural circuitry.

[0014] Described herein, in certain embodiments, is a method of enhancing neurotransmission from existing endogenous neural circuitry in a subject, the method3IPTS / 200232923.1Attorney Docket No.: BIS-003 WOcomprising administering to the subject an effective amount of a depolarizing ion channel therapeutic.

[0015] In some embodiments, the neurons are projection neurons.

[0016] In some embodiments, 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.

[0017] In some embodiments, administering to the subject 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. In some embodiments, the vector comprises an inducible promoter operably linked to the DNA or RNA polynucleotide. In some embodiments, the inducible promoter can modulate, activate, or deactivate expression of the depolarizing ion channel protein or fusion protein comprising a depolarizing ion channel protein.

[0018] In some embodiments, the DNA or RNA polynucleotide further encodes a gain control protein.

[0019] In some embodiments, the DNA or RNA polynucleotide is operably linked to a tissue- or cell type-specific promoter.

[0020] In some embodiments, 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. In some embodiments, the depolarizing ion channel protein is a ligand-gated ion channel protein. In some embodiments, the depolarizing ion channel protein is a voltage-gated ion channel protein. In some embodiments, the depolarizing ion channel protein is a mechanosensitive ion channel protein. In some embodiments, the depolarizing ion channel protein is a cyclic nucleotide-gated ion channel protein.

[0021] In some embodiments, the depolarizing ion channel protein is a light-activated ion channel.

[0022] In some embodiments, the depolarizing ion channel protein is a sodium ion channel protein, a potassium ion channel protein, or a calcium ion channel. In some embodiments, the depolarizing ion channel protein is a sodium ion channel protein. In some4IPTS / 200232923.1Attorney Docket No.: BIS-003 WOembodiments, the depolarizing ion channel protein is a potassium ion channel protein. In some embodiments, the depolarizing ion channel protein is a calcium ion channel.

[0023] In some embodiments, the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a DNA vector. In some embodiments, the vector is, an RNA vector. In some embodiments, the vector is a viral vector.

[0024] In some embodiments, the viral vector is selected from the group consisting of: an adenoviral vector, a retroviral vector, apoxviral vector, an adeno-associated viral (AAV) vector, a baculoviral vector, a herpes simplex viral vector, and a synthetic vector. In some embodiments, the viral vector is an adenoviral vector. In some embodiments, the viral vector is a retroviral vector. In some embodiments, the viral vector is a poxviral vector. In some embodiments, the viral vector is an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a baculoviral vector. In some embodiments, the viral vector is a herpes simplex viral vector. In some embodiments, the viral vector is a synthetic vector.

[0025] In some embodiments, 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. In some embodiments, the AAV vector has about 85% sequence identity to an AAV vector selected from the group consisting of an AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74 vector. In some embodiments, the AAV vector has about 90% sequence identity to an AAV vector selected from the group consisting of an AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74 vector. In some embodiments, the AAV vector has about 95% sequence identity to an AAV vector selected from the group consisting of an AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74 vector. In some embodiments, the AAV vector has about 99% sequence identity to an AAV vector selected from the group consisting of an AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrhlO, and AAVrh74 vector.

[0026] In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV2 vector. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV4 vector. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV5 vector. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV6 vector. In some embodiments, the AAV vector has about 85%, 90%,IPTS / 200232923.1Attorney Docket No.: BIS-003 WO95%, 99% or more sequence identity to an AAV7 vector. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV9 vector. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAVrhlO vector. In some embodiments, the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAVrh74 vector.

[0027] In some embodiments the retroviral vector is a y-retroviral vector or a lentiviral vector. In some embodiments the retroviral vector is a y-retro viral vector. In some embodiments the retroviral vector is a lentiviral vector.

[0028] In some embodiments, the DNA or RNA polynucleotide is operably linked to a constitutive promoter. In some embodiments, the DNA polynucleotide is operably linked to a constitutive promoter. In some embodiments, the RNA polynucleotide is operably linked to a constitutive promoter.

[0029] In some embodiments, the constitutive promoter is a CAG promoter.

[0030] In some embodiments, the AAV further comprises two inverted terminal repeats (ITRs). In some embodiments, 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 RNA polynucleotide and ITR2 is positioned 3' to the DNA or RNA polynucleotide to form a cassette comprising the structure ITR1-polynucleotide-ITR2.

[0031] In some embodiments, the two ITRs are AAV serotype 2 ITRs.

[0032] In some embodiments, the depolarizing ion channel therapeutic comprises an AAV2 vector comprising the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the depolarizing ion channel therapeutic comprises SEQ ID NO: 1. In some embodiments, 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 is a schematic depiction of exemplary methods described herein, in which treatment with a depolarizing ion channel (DIG) boosts activation of projection neurons and signaling to downstream targets..

[0034] FIG. 2 shows the boosting effect of a DIC therapeutic on neuronal activity, as measured by the amplitude of the Photopic Negative Response (PhNR) in electroretinogram 6IPTS / 200232923.1Attorney Docket No.: BIS-003 WO(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.

[0035] FIG. 3 indicates the dose dependent boosting effect of a DIC therapeutic on neuronal activity - i.e., the boosting increases with higher vector doses. 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 graphs show the raw ERG signals from two DIC therapeutic doses: 3.7 x1010vg / eye (low dose) (n = 2 eyes) and 1.7 x1011vg / eye (high dose) (n = 2 eyes). 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.

[0036] FIG. 4 shows the dose-dependent boosting effect of a DIC therapeutic measured as dose-dependent increases in membrane potential in cardiac myocytes. The scatter plot shows the maximal diastolic potential values (MDPs) (with mean ± SEM) recorded for each cell from each experimental group (i.e., three dose groups where cardiac myocytes were treated with AAV2 vectors containing a gene encoding a depolarizing ion channel protein fused with Green Fluorescent Protein (SEQ ID NO: 1)) and the control group (treated with a vector containing a gene encoding EGFP and that does not contain a DIC). Doses were expressed as Multiplicity of Infection (MOI) (i.e., the number of viral particles / cell) and were as follows: MOI1 was 4000 viral particles / cell, MO 12 was 20,000 viral particles / cell, and MOI3 was 100,000 viral particles / cell. The MOI for the eGFP control was 100,000 viral particles / cell. As depicted in the figure, the data showed a clear dose-dependent depolarization (p<0.05 and p<0.0001 for MOI2 and MOK, respectively, compared to the control).DETAILED DESCRIPTIONDefinitions

[0037] 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 7IPTS / 200232923.1Attorney Docket No.: BIS-003 WOcollected 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.

[0038] 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.

[0039] The articles “a" and “an” are used in this disclosure to refer to one or more than one (j.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.

[0040] The term “and / or” is used in this disclosure to mean either “and” or “or” unless indicated otherwise.

[0041] 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.

[0042] 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.

[0043] 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, AAV11. AAV12,8IPTS / 200232923.1Attorney Docket No.: BIS-003 WOAAV13, 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. 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 ITRs) 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 vims 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 vims. 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 9IPTS / 200232923.1Attorney Docket No.: BIS-003 WOothers. 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 long as 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.

[0044] 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.

[0045] 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.

[0046] 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 10IPTS / 200232923.1Attorney Docket No.: BIS-003 WOacceptable" 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.

[0047] “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.

[0048] 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.

[0049] It is to be understood that the present disclosure also provides pharmaceutical compositions including any DIC described herein in combination with at least one pharmaceutically acceptable excipient or carrier.

[0050] As used herein, the term “pharmaceutical composition” is a formulation containing the DICs 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 the 11IPTS / 200232923.1Attorney Docket No.: BIS-003 WOlike. 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.

[0051] 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.

[0052] 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.

[0053] 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 by including in the composition an agent which delays absorption, for example, aluminum 12IPTS / 200232923.1Attorney Docket No.: BIS-003 WOmonostearate and gelatin.

[0054] 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.

[0055] 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, polyanhydrides, 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.

[0056] 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.

[0057] 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 a mammal, 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.,13IPTS / 200232923.1Attorney Docket No.: BIS-003 WOcows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, non-human primates, and the like).

[0058] 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.

[0059] 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). 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 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, nourseothricin, or zeocin.

[0060] Without being bound by theory, the disclosure herein is based at least in part on 14IPTS / 200232923.1Attorney Docket No.: BIS-003 WOthe 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.

[0061] Described herein is a novel pharmacological mechanism, i.e., a depolarizing ion channel (DIC) therapeutic that boosts neuronal circuits, e.g., retinal circuits, and signaling 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). The DICs boost the activity of the projection neurons, so that signals from upstream circuits are boosted and 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 pathways, thereby boosting other neural functions.

[0062] 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.

[0063] 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 from weak 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.

[0064] As an example, to distinguish the methods described herein, embodiments are provided that may ameliorate vision losses associated with retinal degenerative diseases, such as the loss of color vision in a patient that may be susceptible to such loss. Also contemplated herein is a method of augmenting color vision in a subject in need thereof. 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 15IPTS / 200232923.1Attorney Docket No.: BIS-003 WOa 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 retina; DIC therapeutic treatment boosts the ganglion cells’ activity so the endogenous labeled line circuits boost the firing of the ganglion cells. Thus, the endogenous labeled line circuits are able to boost 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.

[0065] 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 boost color vision to patients or subjects, even though a disclosed DIC therapeutic itself may have no relation to color vision.

[0066] In another embodiment, methods described herein can boost high acuity vision to patients or subjects. Without being bound by theory, 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 e.g., normal upstream circuitry (e.g., stable 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 (chromophorebased, 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 that feed into the ganglion cells.

[0067] By the same logic, when used in the retina to boost color vision, there is no need to express exogenous chromophores with particular wavelength specificities to provide color16IPTS / 200232923.1Attorney Docket No.: BIS-003 WOsensitivity, as the methods described herein allow endogenous, partially degenerated color circuits to activate the ganglion cells, so natural color vision can re-emerge.

[0068] For example, methods described herein can be used enhance night vision in subject. Methods described herein, without being bound by theory, boost signals from circuits that operate at low light levels and may allow patients to see in much darker environments than was possible before treatment.Depolarizing Ion Channel Therapeutics

[0069] As described herein, a depolarizing ion channel therapeutic may be used as part of the disclosed methods for treating neurodegenerative diseases, including retinal degenerative diseases. In some embodiments, a DIC comprises: a) a depolarizing ion channel protein; or b) a DNA or RNA polynucleotide that encodes a depolarizing ion channel 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.

[0070] 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.

[0071] 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:17IPTS / 200232923.1Attorney Docket No.: BIS-003 WOMDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYCAGWIESRGTNGAQTASNVL QWLAAGFSILLLMFYAYQTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLYLAT GHRVQWERYAEWEETCPVICIHESNETGESNDYSRRTMGEEVSDIGTIVWGATSAM ATGYVKVIFFCEGECYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWEFFVSW GMFPIEFIEGPEGFGVESVYGSTVGHTIIDEMSKNCWGEEGHYERVEIHEHIEIHGDIR KTTKENIGGTEIEVETEVEDEAEAGAVNKGTGKMAEEISSATRSEFAAGGINPWPNPY HHEDMGCGGMTPTGECFSTEWWCDPSYGESDAGYGYCFVEATGGYEVVGVEKKQ AWEHSRGTPGEKIGAQVCQWIAFSIAIAEETFYGFSAWKATCGWEEVYVCCVEVEF VTEEIFKEFSSPATVYESTGNHAYCERYFEWEESCPVIEIRESNESGEKNDYSKRTMG EIVSCVGMIVFGMAAGEATDWEKWEEYIVSCIYGGYMYFQAAKCYVEANHSVPKG HCRMVVKEMAYAYFASWGSYPIEWAVGPEGEEKESPYANSIGHSICDIIAKEFWTFE AHHERIKIHEHIEIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTVVSKGEEDNMAIIKE FMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKEKVTKGGPEPFAWDIESPQFM YGSKAYVKHPADIPDYEKESFPEGFKWERVMNFEDGGVVTVTQDSSEQDGEFIYKV KERGTNFPSDGPVMQKKTMGWEASSERMYPEDGAEKGEIKQREKEKDGGHYDAEV KTTYKAKKPVQEPGAYNVNIKEDITSHNEDYT1VEQYERAEGRHSTGGMDEEYK (SEQ ID NO: 11);and MDYGGAESAVGREEEFVTNPVVVNGSVEVPEDQCYCAGWIESRGTNGAQTASNVE QWEAAGFSIEEEMFYAYQTWKSTCGWEEIYVCAIEMVKVIEEFFFEFKNPSMEYEAT GHRVQWERYAEWEETCPVISIHESNETGLSNDYSRRTMGEEVSDIGTIVWGATSAMA TGYVKVIFFCEGECYGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWEFFVSWG MFPIEFIEGPEGFGVESVYGSTVGHTIIDEMSKNCWGEEGHYERVEIHEHIEIHGDIRK TTKENIGGTEIEVETEVEDESEAGSVNKGTGKMAEEISSATRSEFAAGGINPWPNPYH HEDMGCGGMTPTGECFSTEWWCDPSYGLSDAGYGYCFVEATGGYEVVGVEKKQA WEHSRGTPGEKIGAQVCQWIAFSIAIAEETFYGFSAWKATCGWEEVYVCCVEVEFVT EEIFKEFSSPATVYESTGNHAYCERYFEWEESCPVIEIRESNESGEKNDYSKRTMGEIV SCVGMIVFGMAAGEATDWEKWEEYIVSCIYGGYMYFQAAKCYVEANHSVPKGHCR MVVKEMAYAYFASWGSYPIEWAVGPEGEEKESPYANSIGHSICEIIAKEFWTFEAHH ERIKIHEHIEIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDT (SEQ ID NO: 12).

[0072] In some embodiments, the DIC protein is a sodium ion channel.

[0073] 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.18IPTS / 200232923.1Attorney Docket No.: BIS-003 WO

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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 enhanced green fluorescent protein (EGFP). In some embodiments, the green fluorescent protein is Emerald. In some embodiments, the green fluorescent protein is superfolder GFP. In some embodiments, the green fluorescent protein is Azami Green. In 19IPTS / 200232923.1Attorney Docket No.: BIS-003 WOsome 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.

[0078] 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.

[0079] 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 be used include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors contain polynucleotide20IPTS / 200232923.1Attorney Docket No.: BIS-003 WOsequences 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.

[0080] 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.

[0081] 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.

[0082] 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 AAV serotypes 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 21IPTS / 200232923.1Attorney Docket No.: BIS-003 WOfrom 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.

[0083] Alternatively, as one of skill in the art will understand, a vector of the disclosure may be a a γ-retroviral vector or a lentiviral vector.

[0084] 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 poly adenylation 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 transgene expression in retinal cells.

[0085] 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 22IPTS / 200232923.1Attorney Docket No.: BIS-003 WOembodiments, 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.

[0086] 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 intra vitreal 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.

[0087] 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 promoter is 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.23IPTS / 200232923.1Attorney Docket No.: BIS-003 WO

[0088] 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.

[0089] 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.

[0090] 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.

[0091] For example, provided herein is protein that comprises a light-sensitive channel from e.g., Stigeoclonium helveticum, such as an optogenetic protein encoded by a nucleic acid with the nucleic acid sequence of SEQ ID NO: 4, below:ATGGAAACAGCCGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCG AAGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAG24IPTS / 200232923.1Attorney Docket No.: BIS-003 WOACGCTCACGGAGAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCT TCCCCCACATCAACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCC AGTGGTTCACCGTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAG CTTCAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTC AAGTGCTTCATCGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAG ACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCC CCGTGATCCTGATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCA AGCGGACCATGACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCA CAGCCGCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTT CTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCAC ACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCT TCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGG CCTGATCACACCTTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGC AAGAACACTTGGGGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCAC ATCCTGATCCACGGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAG AACATGGAGATCGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTG.

[0092] 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.

[0093] For example, the DIC fusion protein may be encoded by a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 2, below:ATGGAAACAGCCGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCG AAGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAG ACGCTCACGGAGAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCT TCCCCCACATCAACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCC AGTGGTTCACCGTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAG CTTCAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTC AAGTGCTTCATCGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAG ACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCC CCGTGATCCTGATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCA AGCGGACCATGACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCA CAGCCGCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTT25IPTS / 200232923.1Attorney Docket No.: BIS-003 WOCTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCAC ACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCT TCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGG CCTGATCACACCTTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGC AAGAACACTTGGGGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCAC ATCCTGATCCACGGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAG AACATGGAGATCGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTGGCGGC ACCGGTAGTAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCAT CCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGA GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATTTGCACCAC CGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTG CAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCG CCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCA ACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGC TGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGA ACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGG.

[0094] In some embodiments, the DIC fusion protein may have the amino acid sequence of SEQ ID NO: 3, below:METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAADAHGETSNATTAGADHGCFP HINHGTELQHKIAVGLQWFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVKCFIE LFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPVILIHLSNLTGLHEEYSKRTMTILV TDIGNIVWGITAAFTKGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGVCRKICKI MAYVFFCSWLMFPVMFIAGHEGLGLITPYTSGIGHLILDLISKNTWGFLGHHLRVKIH EHILIHGDIRKTTTINVAGENMEIETFVDEEEEGGVAAPVVAVSKGEELFTGVVPILVE LDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRY PDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK EDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPI GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK.

[0095] 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 26IPTS / 200232923.1Attorney Docket No.: BIS-003 WOnucleic 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.

[0096] 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 CTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCG GGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGA AAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCT GTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCT TCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTC TTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGG CAAAGAATTGGATCCGCCACCATGGAAACAGCCGCCACAATGACCCACGCCTTT ATCTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAGGCCTGCTGAGCGCCGCA GCAGTGGTGACACCAGCAGCAGACGCTCACGGAGAAACCTCTAACGCCACAACA GCCGGAGCCGATCACGGTTGCTTCCCCCACATCAACCACGGAACCGAGCTGCAG CACAAGATCGCAGTGGGACTCCAGTGGTTCACCGTGATCGTGGCTATCGTGCAGC TCATCTTCTACGGTTGGCACAGCTTCAAGGCCACAACCGGCTGGGAGGAGGTCTA CGTCTGCGTGATCGAGCTCGTCAAGTGCTTCATCGAGCTGTTCCACGAGGTCGAC AGCCCAGCCACAGTGTACCAGACCAACGGAGGAGCCGTGATTTGGCTGCGGTAC AGCATGTGGCTCCTGACTTGCCCCGTGATCCTGATCCACCTGAGCAACCTGACCG27IPTS / 200232923.1Attorney Docket No.: BIS-003 WOGACTGCACGAAGAGTACAGCAAGCGGACCATGACCATCCTGGTGACCGACATCG GCAACATCGTGTGGGGGATCACAGCCGCCTTTACAAAGGGCCCCCTGAAGATCC TGTTCTTCATGATCGGCCTGTTCTACGGCGTGACTTGCTTCTTCCAGATCGCCAAG GTGTATATCGAGAGCTACCACACCCTGCCCAAAGGCGTCTGCCGGAAGATTTGCA AGATCATGGCCTACGTCTTCTTCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATC GCCGGACACGAGGGACTGGGCCTGATCACACCTTACACCAGCGGAATCGGCCAC CTGATCCTGGATCTGATCAGCAAGAACACTTGGGGCTTCCTGGGCCACCACCTGA GAGTGAAGATCCACGAGCACATCCTGATCCACGGCGACATCCGGAAGACAACCA CCATCAACGTGGCCGGCGAGAACATGGAGATCGAGACCTTCGTCGACGAGGAGG AGGAGGGAGGAGTGGCGGCACCGGTAGTAGCAGTGAGCAAGGGCGAGGAGCTG TTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCAC AAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACC CTGAAGTTCATTTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGA CCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCA GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATC TTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGC GACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGC AACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATC ATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAAC ATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCC CTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTG ACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAGAATTCGAT ATCAAGCTTATCGATAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTG GTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCT TTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCC TGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGG TGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTG TCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCA TCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAA TTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTG CCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCC AGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTC28IPTS / 200232923.1Attorney Docket No.: BIS-003 WOGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATA CCGTCGACCCGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTT GGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGT GATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACA ACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTA AAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCTTCCTAGAGC ATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACA.

[0097] 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 is at 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.

[0098] In some embodiments, a AAV vector described herein comprises the nucleic acid sequence of SEQ ID NO: 1, below:GCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGC GACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGC29IPTS / 200232923.1Attorney Docket No.: BIS-003 WOCAACTCCATCACTAGGGGTTCCTTGTAGTTAATGATTAACCCGCCATGCTACTTAT CTACGTAGCCATGCTCTAGGAAGAGTACCATTGACGTCAATAATGACGTATGTTC CCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACG GTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCT TATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG GTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACC CCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGG GGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGG GGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGT TTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGC GGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGG CGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTG TTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTG CGGGGGGAGCGGCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGAC GGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTG CTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTT ATTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATCCGCCACCATGGAAACAGC CGCCACAATGACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCGAAGCCACAATT AGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCAGCAGACGCTCACGGA GAAACCTCTAACGCCACAACAGCCGGAGCCGATCACGGTTGCTTCCCCCACATC AACCACGGAACCGAGCTGCAGCACAAGATCGCAGTGGGACTCCAGTGGTTCACC GTGATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGGCACAGCTTCAAGGCCA CAACCGGCTGGGAGGAGGTCTACGTCTGCGTGATCGAGCTCGTCAAGTGCTTCAT CGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAGTGTACCAGACCAACGGAGG AGCCGTGATTTGGCTGCGGTACAGCATGTGGCTCCTGACTTGCCCCGTGATCCTG ATCCACCTGAGCAACCTGACCGGACTGCACGAAGAGTACAGCAAGCGGACCATG ACCATCCTGGTGACCGACATCGGCAACATCGTGTGGGGGATCACAGCCGCCTTTA CAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTCTACGGCGTGAC TTGCTTCTTCCAGATCGCCAAGGTGTATATCGAGAGCTACCACACCCTGCCCAAA GGCGTCTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTTCTTCTGCTCTTGGCT GATGTTCCCCGTGATGTTCATCGCCGGACACGAGGGACTGGGCCTGATCACACCT30IPTS / 200232923.1Attorney Docket No.: BIS-003 WOTACACCAGCGGAATCGGCCACCTGATCCTGGATCTGATCAGCAAGAACACTTGG GGCTTCCTGGGCCACCACCTGAGAGTGAAGATCCACGAGCACATCCTGATCCAC GGCGACATCCGGAAGACAACCACCATCAACGTGGCCGGCGAGAACATGGAGATC GAGACCTTCGTCGACGAGGAGGAGGAGGGAGGAGTGGCGGCACCGGTAGTAGC AGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCG ATGCCACCTACGGCAAGCTGACCCTGAAGTTCATTTGCACCACCGGCAAGCTGCC CGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGC CGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA GGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACC CGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC TACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAG GTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGAC CACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAAC CACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGAT CACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGTAAGAATTCGATATCAAGCTTATCGATAATCAACCTCTGGATTA CAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTAT GTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTC ATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCC CGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACT GGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCT CCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGG GCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCT TTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGC TACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGG CTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTT TGGGCCGCCTCCCCGCATCGATACCGTCGACCCGGGCGGCCGCTTCGAGCAGAC ATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAG CTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAG GGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAA31IPTS / 200232923.1Attorney Docket No.: BIS-003 WOATCGATAAGGATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTT AATCATTAACTACAAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGC GCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT GCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCA.Pharmaceutical Compositions and Routes of Administration

[0099] 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 acceptable carriers 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.

[0100] 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. Such32IPTS / 200232923.1Attorney Docket No.: BIS-003 WOformulation 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

[0101] Provided herein is a method of boosting activity of projection neurons in a subject, 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 endogenous neural circuitry upstream from the projection neurons, to boost the depolarization of 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 boosting neural function in the patient.

[0102] 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.

[0103] Also provided herein is a method of enhancing neurotransmission from existing, endogenous neural circuitry in a patient susceptible to a neural degenerative disease (e.g., retinal degenerative disease and / or other sensory degenerative disease) and in need of neuroprotection, the method comprising administering to the patient in need thereof an effective amount of a depolarizing ion channel therapeutic.

[0104] Also provided herein is a method of increasing the voltage or membrane potential in neurons receiving weak or partial inputs from a 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 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.33IPTS / 200232923.1Attorney Docket No.: BIS-003 WO

[0105] In some embodiments, contemplated subjects does not have a measurable loss of functioning neural circuits before administration of the DIC, but are need of vision or other sensory enhancement 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.

[0106] Contemplated methods include, administering to the subject 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.

[0107] In some embodiments, the effective amount of the DIC therapeutic comprises an amount ranging between about 1.0 x 1010vg / eye to about 1.0 x 1012vg / eye. In some embodiments, the effective amount of the DIC therapeutic comprises about 1.0 x 1010vg / eye, 1.5 x 1010vg / eye, 2.0 x 1010vg / eye, 2.5 x 1010vg / eye, 3.0 x 1010vg / eye, 3.5 x 1010vg / eye, 4.0 x 1010vg / eye, 4.5 x 1010vg / eye, 5.0 x 1010vg / eye, 5.5 x 1010vg / eye, 6.0 x 1010vg / eye, 6.5 x 1010vg / eye, 7.0 x 1010vg / eye, 7.5 x 1010vg / eye, 8.0 x 1010vg / eye, 8.5 x 1010vg / eye, 9.0 x 1010vg / eye, 9.5 x 1010vg / eye. In some embodiments, the effective amount of the DIC therapeutic comprises about 1.0 x 1011vg / eye, 1.5 x 10” vg / eye, 2.0 x 10” vg / eye, 2.5 x 10” vg / eye, 3.0 x 1011vg / eye, 3.5 x 1011vg / eye, 4. 0 x 10” vg / eye, 4.5 x 10” vg / eye, 5.0 x 10” vg / eye, 5.5 x 10” vg / eye, 6.0 x 10” vg / eye, 6. 5 x 10” vg / eye, 7.0 x 10” vg / eye, 7.5 x 10” vg / eye, 8.0 x 10” vg / eye, 8.5 x 10” vg / eye, 9. 0 x 10” vg / eye, 9.5 x 10” vg / eye. In some embodiments, the effective amount of the DIC therapeutic comprises about 1.0 x 1012vg / eye, 1.5 x 1012vg / eye, 2.0 x 1012vg / eye, 2.5 x 1012vg / eye, 3.0 x 1012vg / eye, 3.5 x 1012vg / eye, 4.0 x 1012vg / eye, 4.5 x 1012vg / eye, 5.0 x 1012vg / eye, 5.5 x 1012vg / eye, 6.0 x 1012vg / eye, 6.5 x 1012vg / eye, 7.0 x 1012vg / eye, 7.5 x 1012vg / eye, 8.0 x 1012vg / eye, 8.5 x 1012vg / eye, 9.0 x 1012vg / eye, 9.5 x 1012vg / eye. In some embodiments, the effective amount of the DIC therapeutic comprises about 3.7 x 1010vg / eye. In some embodiments, the effective amount of the DIC therapeutic comprises 3.7 x 1010vg / eye. In some embodiments, the effective amount of the DIC therapeutic comprises about 1.7 x 1011vg / eye. In some embodiments, the effective amount of the DIC therapeutic comprises 1.7 x 1011vg / eye.

[0108] 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 not34IPTS / 200232923.1Attorney Docket No.: BIS-003 WOinclude administration of light activation by light and / or without activation of exogenously-introduced de novo neural circuits.

[0109] Also provided herein is a method of ameliorating, or postponing the onset of 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. Another contemplated method includes a method of boosting retinal neuronal activity, or boosting other sensory neuronal activity (e.g., auditory, olfactory and / or gustatory) 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 endogenous neural circuitry.

[0110] Further contemplated herein are methods of ameliorating and / or delaying the onset of glaucoma, 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.

[0111] 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.

[0112] In some embodiments, contemplated methods of delaying the onset of a degenerative disease includes a degenerative disease selected from the group consisting of retinitis pigmentosa, choroidemia, Usher syndrome, and macular degeneration (and / or the night blindness associated e.g., with these disorders).IPTS / 200232923.1Attorney Docket No.: BIS-003 WO

[0113] In some embodiments, methods described herein are directed delaying the onset of 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.

[0114] 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.

[0115] 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

[0116] 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

[0117] The present example describes an exemplary depolarizing ion channel (DIC) therapeutic and methods of using and assessing it in normal subjects.36IPTS / 200232923.1Attorney Docket No.: BIS-003 WOMaterials and MethodsVector material

[0118] The vector injected into subjects (non-human primates) was a non-replicating, 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 polyadenylation sequence. The vector (SEQ NO: 1) was formulated in a balanced salt solution (BSS) supplemented with 0.014% Polysorbate 20.Results: Neuronal responses were increased in nonhuman primates following treatment with a DIC therapeutic

[0119] Three healthy nonhuman primates were intravitreally injected with an AAV2 vector that expresses a gene encoding a DIC with doses of 3.7 x 1010vg / 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.2 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. 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 2x10-5to 3x10-4mW / 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.3 indicates the dose-dependent boosting effect of this DIC therapeutic on neuronal activity. Example 2: Dose-Dependence of Depolarizing Ion Channel Therapeutic In CardiomyocytesMaterials and MethodsCell culture methods37IPTS / 200232923.1Attorney Docket No.: BIS-003 WO

[0120] Cardiomyocytes, derived from induced pluripotent stem cells, referred to as iPSC-CM, were thawed and then seeded onto glass coverslips precoated with fibronectin aiming for a cell density of 30,000 cells / spot per coverslip. The coverslips were placed in 3 cm plastic petri dishes for cell culturing and the cells were maintained in culture at 37 °C with media being changed every 48 hours. Four (4) days post seeding, the iPSC-CM were transduced using AAV2 vectors that carried a gene encoding either EGFP (Enhanced Green Fluorescent Protein) or ChronosFP (a depolarizing ion channel protein fused with Green Fluorescent Protein) (SEQ ID NO: 1). The vector DNA sequence contained AAV serotype 2 (AAV2) inverted terminal repeats (ITRs), a cytomegalovirus early enhancer / chicken beta actin (CAG) promoter (SEQ ID NO: 6), the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) (SEQ ID NO: 7), and an SV40 polyadenylation sequence (SEQ ID NO: 8). Different doses were used, expressed as Multiplicity of Infection (MOI) (i.e., the number of viral particles per cell). An MOI of 100,000 was used for the control (the EGFP vector), and 3 different MOIs were used for the ChronosFP vector (4000, 20,000, and 100,000). All manual patch clamp data were obtained between 10 and 13 days post seeding, corresponding to 6 and 9 days post transduction.Transduction methods

[0121] Four (4) days post seeding, the iPSC-CM were transduced using AAV2 encoding EGFP or ChronosFP. The transduction protocol that was employed was as follows: (1) The media volume in each petri dish was reduced to 1 mL; (2) 10 pL of virus was added to each petri dish; (3) the media was carefully mixed in each petri dish; (4) after two hours, 1.5 mL of fresh media was added to each dish; and (5) the cells were maintained in culture at 37 °C with media being changed every 48 hours.Manual patch clamp methods

[0122] Spontaneous action potentials were recorded using the current clamp configuration with an EPC9 amplifier. Recordings were made using glass patch pipettes with resistances ranging between 2.5 - 4.0 MQ. All recordings were made from spontaneously beating syncytial networks at ambient temperature. Recording were made from a covered rig to minimize the effect of ambient light on ChronosFP. A red filter was placed in the microscope’s light path to block blue light, thereby limiting the activation of ChronosFP. Moreover, as soon as the cell attached configuration was achieved the microscope’s lamp was turned off. After formation of the perforated patch clamp configuration, action potentials38IPTS / 200232923.1Attorney Docket No.: BIS-003 WOwere recorded in extracellular solution monitoring the resting membrane potential for 5-10 minutes. A minimum of eight replicates (N > 8) were collected for each group.Results: Dose-dependent depolarization response to DIC therapeutic

[0123] The results in FIG. 4 show clear dose-dependent depolarization, indicating that treatment with the DIC therapeutic produces a dose-dependent boosting effect in cardiomyocytes.INCORPORATION BY REFERENCE

[0124] The entire disclosure of each of the patent documents and scientific articles cited herein is incorporated by reference for all purposes.EQUIVALENTS

[0125] 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.39IPTS / 200232923.1Attorney Docket No.: BIS-003 WOSEQUENCE LISTING SEQ Description Sequence SequenceID typeNO1 AAV vector Nucleic GCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG Acid CAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCG GCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAG TGGCCAACTCCATCACTAGGGGTTCCTTGTAGTTAA TGATTAACCCGCCATGCTACTTATCTACGTAGCCAT GCTCTAGGAAGAGTACCATTGACGTCAATAATGAC GTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA TTGACGTCAATGGGTGGAGTATTTACGGTAAACTG CCCACTTGGCAGTACATCAAGTGTATCATATGCCAA GTACGCCCCCTATTGACGTCAATGACGGTAAATGG CCCGCCTGGCATTATGCCCAGTACATGACCTTATGG GACTTTCCTACTTGGCAGTACATCTACGTATTAGTC ATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTC TGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCC CAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC AGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCG CCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCG GGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCA GAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGA GGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAG CGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTC GCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCC CGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG TGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGT AATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTT CTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGA GGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGCTGT CCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGG CAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCG GCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCT TCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTA TTGTGCTGTCTCATCATTTTGGCAAAGAATTGGATC CGCCACCATGGAAACAGCCGCCACAATGACCCACG CCTTTATCTCAGCCGTGCCTAGCGCCGAAGCCACAA TTAGAGGCCTGCTGAGCGCCGCAGCAGTGGTGACA CCAGCAGCAGACGCTCACGGAGAAACCTCTAACGC CACAACAGCCGGAGCCGATCACGGTTGCTTCCCCC ACATCAACCACGGAACCGAGCTGCAGCACAAGATC GCAGTGGGACTCCAGTGGTTCACCGTGATCGTGGC TATCGTGCAGCTCATCTTCTACGGTTGGCACAGCTT CAAGGCCACAACCGGCTGGGAGGAGGTCTACGTCT GCGTGATCGAGCTCGTCAAGTGCTTCATCGAGCTGT TCCACGAGGTCGACAGCCCAGCCACAGTGTACCAGACCAACGGAGGAGCCGTGATTTGGCTGCGGTACAG 40IPTS / 200232923 1Attorney Docket No.: BIS-003 WOCATGTGGCTCCTGACTTGCCCCGTGATCCTGATCCA CCTGAGCAACCTGACCGGACTGCACGAAGAGTACA GCAAGCGGACCATGACCATCCTGGTGACCGACATC GGCAACATCGTGTGGGGGATCACAGCCGCCTTTAC AAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGG CCTGTTCTACGGCGTGACTTGCTTCTTCCAGATCGC CAAGGTGTATATCGAGAGCTACCACACCCTGCCCA AAGGCGTCTGCCGGAAGATTTGCAAGATCATGGCC TACGTCTTCTTCTGCTCTTGGCTGATGTTCCCCGTGA TGTTCATCGCCGGACACGAGGGACTGGGCCTGATC ACACCTTACACCAGCGGAATCGGCCACCTGATCCT GGATCTGATCAGCAAGAACACTTGGGGCTTCCTGG GCCACCACCTGAGAGTGAAGATCCACGAGCACATC CTGATCCACGGCGACATCCGGAAGACAACCACCAT CAACGTGGCCGGCGAGAACATGGAGATCGAGACCT TCGTCGACGAGGAGGAGGAGGGAGGAGTGGCGGC ACCGGTAGTAGCAGTGAGCAAGGGCGAGGAGCTGT TCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGG CGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGA CCCTGAAGTTCATTTGCACCACCGGCAAGCTGCCCG TGCCCTGGCCCACCCTCGTGACCACCCTGACCTACG GCGTGCAGTGCTTCAGCCGCTACCCCGACCACATG AAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGA AGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGG ACGACGGCAACTACAAGACCCGCGCCGAGGTGAAG TTCGAGGGCGACACCCTGGTGAACCGCATCGAGCT GAAGGGCATCGACTTCAAGGAGGACGGCAACATCC TGGGGCACAAGCTGGAGTACAACTACAACAGCCAC AACGTCTATATCATGGCCGACAAGCAGAAGAACGG CATCAAGGTGAACTTCAAGATCCGCCACAACATCG AGGACGGCAGCGTGCAGCTCGCCGACCACTACCAG CAGAACACCCCCATCGGCGACGGCCCCGTGCTGCT GCCCGACAACCACTACCTGAGCACCCAGTCCGCCC TGAGCAAAGACCCCAACGAGAAGCGCGATCACATG GTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACT CTCGGCATGGACGAGCTGTACAAGTAAGAATTCGA TATCAAGCTTATCGATAATCAACCTCTGGATTACAA AATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAAT GCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTC ATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTC TTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTG GCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCA CTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTT CCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGG CGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTG 41IPTS / 200232923 1Attorney Docket No.: BIS-003 WOGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTG CTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACG TCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCG GACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGG CCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGT CGGATCTCCCTTTGGGCCGCCTCCCCGCATCGATAC CGTCGACCCGGGCGGCCGCTTCGAGCAGACATGAT AAGATACATTGATGAGTTTGGACAAACCACAACTA GAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTT GTGATGCTATTGCTTTATTTGTAACCATTATAAGCT GCAATAAACAAGTTAACAACAACAATTGCATTCAT TTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTT TTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAA AATCGATAAGGATCTTCCTAGAGCATGGCTACGTA GATAAGTAGCATGGCGGGTTAATCATTAACTACAA GGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCT GCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAA AGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCC TCAGTGAGCGAGCGAGCGCGCAGCTGCA2 Sequence Nucleic ATGGAAACAGCCGCCACAATGACCCACGCCTTTAT encoding acid CTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAG optogenetic GCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCA protein GCAGACGCTCACGGAGAAACCTCTAACGCCACAAC AGCCGGAGCCGATCACGGTTGCTTCCCCCACATCA ACCACGGAACCGAGCTGCAGCACAAGATCGCAGTG GGACTCCAGTGGTTCACCGTGATCGTGGCTATCGTG CAGCTCATCTTCTACGGTTGGCACAGCTTCAAGGCC ACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGAT CGAGCTCGTCAAGTGCTTCATCGAGCTGTTCCACGA GGTCGACAGCCCAGCCACAGTGTACCAGACCAACG GAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGG CTCCTGACTTGCCCCGTGATCCTGATCCACCTGAGC AACCTGACCGGACTGCACGAAGAGTACAGCAAGCG GACCATGACCATCCTGGTGACCGACATCGGCAACA TCGTGTGGGGGATCACAGCCGCCTTTACAAAGGGC CCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTC TACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTG TATATCGAGAGCTACCACACCCTGCCCAAAGGCGT CTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTT CTTCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATC GCCGGACACGAGGGACTGGGCCTGATCACACCTTA CACCAGCGGAATCGGCCACCTGATCCTGGATCTGA TCAGCAAGAACACTTGGGGCTTCCTGGGCCACCAC CTGAGAGTGAAGATCCACGAGCACATCCTGATCCA CGGCGACATCCGGAAGACAACCACCATCAACGTGG CCGGCGAGAACATGGAGATCGAGACCTTCGTCGAC GAGGAGGAGGAGGGAGGAGTGGCGGCACCGGTAGTAGCAGTGAGCAAGGGCGAGGAGCTGTTCACCGGG 42IPTS / 200232923 1Attorney Docket No.: BIS-003 WOGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGT AAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCG AGGGCGATGCCACCTACGGCAAGCTGACCCTGAAG TTCATTTGCACCACCGGCAAGCTGCCCGTGCCCTGG CCCACCCTCGTGACCACCCTGACCTACGGCGTGCA GTGCTTCAGCCGCTACCCCGACCACATGAAGCAGC ACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTAC GTCCAGGAGCGCACCATCTTCTTCAAGGACGACGG CAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGC ATCGACTTCAAGGAGGACGGCAACATCCTGGGGCA CAAGCTGGAGTACAACTACAACAGCCACAACGTCT ATATCATGGCCGACAAGCAGAAGAACGGCATCAAG GTGAACTTCAAGATCCGCCACAACATCGAGG3 DIC fusion Amino METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAAD protein acid AHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQ sequence WFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVK CFIEEFHEVDSPATVYQTNGGAVIWERYSMWEETCPV IEIHESNETGEHEEYSKRTMTIEVTDIGNIVWGITAAFT KGPEKIEFFMIGEFYGVTCFFQIAKVYIESYHTEPKGV CRKICKIMAYVFFCSWEMFPVMFIAGHEGEGEITPYTS GIGHEIEDEISKNTWGFEGHHERVKIHEHIEIHGDIRKT TTINVAGENMEIETFVDEEEEGGVAAPVVAVSKGEEE FTGVVPIEVEEDGDVNGHKFSVSGEGEGDATYGKETE KFICTTGKEPVPWPTEVTTETYGVQCFSRYPDHMKQH DFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT EVNRIEEKGIDFKEDGNIEGHKEEYNYNSHNVYIMAD KQKNGIKVNFKIRHNIEDGSVQEADHYQQNTPIGDGP VEEPDNHYESTQSAESKDPNEKRDHMVEEEFVTAAGI TEGMDEEYK4 Sequence Nucleic ATGGAAACAGCCGCCACAATGACCCACGCCTTTAT encoding acid CTCAGCCGTGCCTAGCGCCGAAGCCACAATTAGAG optogenetic GCCTGCTGAGCGCCGCAGCAGTGGTGACACCAGCA protein GCAGACGCTCACGGAGAAACCTCTAACGCCACAAC AGCCGGAGCCGATCACGGTTGCTTCCCCCACATCA ACCACGGAACCGAGCTGCAGCACAAGATCGCAGTG GGACTCCAGTGGTTCACCGTGATCGTGGCTATCGTG CAGCTCATCTTCTACGGTTGGCACAGCTTCAAGGCC ACAACCGGCTGGGAGGAGGTCTACGTCTGCGTGAT CGAGCTCGTCAAGTGCTTCATCGAGCTGTTCCACGA GGTCGACAGCCCAGCCACAGTGTACCAGACCAACG GAGGAGCCGTGATTTGGCTGCGGTACAGCATGTGG CTCCTGACTTGCCCCGTGATCCTGATCCACCTGAGC AACCTGACCGGACTGCACGAAGAGTACAGCAAGCG GACCATGACCATCCTGGTGACCGACATCGGCAACA TCGTGTGGGGGATCACAGCCGCCTTTACAAAGGGC CCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTCTACGGCGTGACTTGCTTCTTCCAGATCGCCAAGGTG 43IPTS / 200232923 1Attorney Docket No.: BIS-003 WOTATATCGAGAGCTACCACACCCTGCCCAAAGGCGT CTGCCGGAAGATTTGCAAGATCATGGCCTACGTCTT CTTCTGCTCTTGGCTGATGTTCCCCGTGATGTTCATC GCCGGACACGAGGGACTGGGCCTGATCACACCTTA CACCAGCGGAATCGGCCACCTGATCCTGGATCTGA TCAGCAAGAACACTTGGGGCTTCCTGGGCCACCAC CTGAGAGTGAAGATCCACGAGCACATCCTGATCCA CGGCGACATCCGGAAGACAACCACCATCAACGTGG CCGGCGAGAACATGGAGATCGAGACCTTCGTCGAC GAGGAGGAGGAGGGAGGAGTG5 Optogenetic Amino METAATMTHAFISAVPSAEATIRGLLSAAAVVTPAAD protein acid AHGETSNATTAGADHGCFPHINHGTELQHKIAVGLQ WFTVIVAIVQLIFYGWHSFKATTGWEEVYVCVIELVK CFIELFHEVDSPATVYQTNGGAVIWLRYSMWLLTCPV ILIHLSNLTGLHEEYSKRTMTILVTDIGNIVWGITAAFT KGPLKILFFMIGLFYGVTCFFQIAKVYIESYHTLPKGV CRKICKIMAYVFFCSWLMFPVMFIAGHEGLGLITPYTS GIGHLILDLISKNTWGFLGHHLRVKIHEHILIHGDIRKT TTINVAGENMEIETFVDEEEEGGV6 CAG Nucleic TCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCA promoter acid TCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATT TATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGG GGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGG CGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAG GTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGA AAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCG GCCCTATAAAAAGCGAAGCGCGCGGCGGGCG7 WPRE Nucleic ATCAACCTCTGGATTACAAAATTTGTGAAAGATTG enhancer acid ACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTAT GTGGATACGCTGCTTTAATGCCTTTGTATCATGCTA TTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTA TAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTG GCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTG TGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTG CCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTT TCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCG CCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGT TGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAA TCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCA CCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCC CTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCG GCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTC GCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGG CCGCCTCCCCGC8 SV40 poly Nucleic TAAGATACATTGATGAGTTTGGACAAACCACAACTadenylation acid AGAATGCAGTGAAAAAAATGCTTTATTTGTGAAAT44IPTS / 200232923 1Attorney Docket No.: BIS-003 WO(poly(A)) TTGTGATGCTATTGCTTTATTTGTAACCATTATAAG element CTGCAATAAACAAGTT9 Expression Nucleic TGTAGTTAATGATTAACCCGCCATGCTACTTATCTA cassette acid CGTAGCCATGCTCTAGGAAGAGTACCATTGACGTC AATAATGACGTATGTTCCCATAGTAACGCCAATAG GGACTTTCCATTGACGTCAATGGGTGGAGTATTTAC GGTAAACTGCCCACTTGGCAGTACATCAAGTGTAT CATATGCCAAGTACGCCCCCTATTGACGTCAATGAC GGTAAATGGCCCGCCTGGCATTATGCCCAGTACAT GACCTTATGGGACTTTCCTACTTGGCAGTACATCTA CGTATTAGTCATCGCTATTACCATGGTCGAGGTGAG CCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCC TCCCCACCCCCAATTTTGTATTTATTTATTTTTTAAT TATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGG GGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGG GGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGC AGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAA AAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGC GCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCT CGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTAC TCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCT CCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTT GTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGG CTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTC GGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGG GGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGT GACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCA TGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGT GCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGA ATTGGATCCGCCACCATGGAAACAGCCGCCACAAT GACCCACGCCTTTATCTCAGCCGTGCCTAGCGCCGA AGCCACAATTAGAGGCCTGCTGAGCGCCGCAGCAG TGGTGACACCAGCAGCAGACGCTCACGGAGAAACC TCTAACGCCACAACAGCCGGAGCCGATCACGGTTG CTTCCCCCACATCAACCACGGAACCGAGCTGCAGC ACAAGATCGCAGTGGGACTCCAGTGGTTCACCGTG ATCGTGGCTATCGTGCAGCTCATCTTCTACGGTTGG CACAGCTTCAAGGCCACAACCGGCTGGGAGGAGGT CTACGTCTGCGTGATCGAGCTCGTCAAGTGCTTCAT CGAGCTGTTCCACGAGGTCGACAGCCCAGCCACAG TGTACCAGACCAACGGAGGAGCCGTGATTTGGCTG CGGTACAGCATGTGGCTCCTGACTTGCCCCGTGATC CTGATCCACCTGAGCAACCTGACCGGACTGCACGA AGAGTACAGCAAGCGGACCATGACCATCCTGGTGA CCGACATCGGCAACATCGTGTGGGGGATCACAGCC GCCTTTACAAAGGGCCCCCTGAAGATCCTGTTCTTCATGATCGGCCTGTTCTACGGCGTGACTTGCTTCTTC 45IPTS / 200232923 1Attorney Docket No.: BIS-003 WOCAGATCGCCAAGGTGTATATCGAGAGCTACCACAC CCTGCCCAAAGGCGTCTGCCGGAAGATTTGCAAGA TCATGGCCTACGTCTTCTTCTGCTCTTGGCTGATGTT CCCCGTGATGTTCATCGCCGGACACGAGGGACTGG GCCTGATCACACCTTACACCAGCGGAATCGGCCAC CTGATCCTGGATCTGATCAGCAAGAACACTTGGGG CTTCCTGGGCCACCACCTGAGAGTGAAGATCCACG AGCACATCCTGATCCACGGCGACATCCGGAAGACA ACCACCATCAACGTGGCCGGCGAGAACATGGAGAT CGAGACCTTCGTCGACGAGGAGGAGGAGGGAGGA GTGGCGGCACCGGTAGTAGCAGTGAGCAAGGGCGA GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCG AGCTGGACGGCGACGTAAACGGCCACAAGTTCAGC GTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGG CAAGCTGACCCTGAAGTTCATTTGCACCACCGGCA AGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCC TGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCG ACCACATGAAGCAGCACGACTTCTTCAAGTCCGCC ATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT CTTCAAGGACGACGGCAACTACAAGACCCGCGCCG AGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGC ATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGG CAACATCCTGGGGCACAAGCTGGAGTACAACTACA ACAGCCACAACGTCTATATCATGGCCGACAAGCAG AAGAACGGCATCAAGGTGAACTTCAAGATCCGCCA CAACATCGAGGACGGCAGCGTGCAGCTCGCCGACC ACTACCAGCAGAACACCCCCATCGGCGACGGCCCC GTGCTGCTGCCCGACAACCACTACCTGAGCACCCA GTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCG GGATCACTCTCGGCATGGACGAGCTGTACAAGTAA GAATTCGATATCAAGCTTATCGATAATCAACCTCTG GATTACAAAATTTGTGAAAGATTGACTGGTATTCTT AACTATGTTGCTCCTTTTACGCTATGTGGATACGCT GCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTA TGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTT GCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAG GCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGC AACCCCCACTGGTTGGGGCATTGCCACCACCTGTCA GCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATT GCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCG CTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACA ATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTC CTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGC GCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCA ATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGG CTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC 46IPTS / 200232923 1Attorney Docket No.: BIS-003 WOATCGATACCGTCGACCCGGGCGGCCGCTTCGAGCA GACATGATAAGATACATTGATGAGTTTGGACAAAC CACAACTAGAATGCAGTGAAAAAAATGCTTTATTT GTGAAATTTGTGATGCTATTGCTTTATTTGTAACCA TTATAAGCTGCAATAAACAAGTTAACAACAACAAT TGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATG TGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAA ATGTGGTAAAATCGATAAGGATCTTCCTAGAGCAT GGCTACGTAGATAAGTAGCATGGCGGGTTAATCAT TAACTACA1011 Optogenetic Amino MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYC protein acid AGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAY QTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLY LATGHRVQWLRYAEWLLTCPVICIHLSNLTGLSNDYS RRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLC YGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWL FFVSWGMFPILFILGPEGFGVLSVYGSTVGHTIIDLMS KNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEI EVETLVEDEAEAGAVNKGTGKMAELISSATRSLFAAG GINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSY GLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGT PGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWE EVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLR YFEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVG MIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAA KCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPI LWAVGPEGLLKLSPYANSIGHSICDIIAKEFWTFLAHH LRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDE DTVVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIE GEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFM YGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDG GVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQK KTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHY DAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTI VEQYERAEGRHSTGGMDELYK12 Optogenetic Amino MDYGGALSAVGRELLFVTNPVVVNGSVLVPEDQCYC protein acid AGWIESRGTNGAQTASNVLQWLAAGFSILLLMFYAY QTWKSTCGWEEIYVCAIEMVKVILEFFFEFKNPSMLY LATGHRVQWLRYAEWLLTCPVISIHLSNLTGLSNDYS RRTMGLLVSDIGTIVWGATSAMATGYVKVIFFCLGLC YGANTFFHAAKAYIEGYHTVPKGRCRQVVTGMAWL FFVSWGMFPIEFIEGPEGFGVLSVYGSTVGHT1IDEMS KNCWGLLGHYLRVLIHEHILIHGDIRKTTKLNIGGTEI EVETLVEDESEAGSVNKGTGKMAELISSATRSLFAAG GINPWPNPYHHEDMGCGGMTPTGECFSTEWWCDPSY GLSDAGYGYCFVEATGGYLVVGVEKKQAWLHSRGTPGEKIGAQVCQWIAFSIAIALLTFYGFSAWKATCGWE 47IPTS / 200232923 1Attorney Docket No.: BIS-003 WOEVYVCCVEVLFVTLEIFKEFSSPATVYLSTGNHAYCLR YFEWLLSCPVILIRLSNLSGLKNDYSKRTMGLIVSCVG MIVFGMAAGLATDWLKWLLYIVSCIYGGYMYFQAA KCYVEANHSVPKGHCRMVVKLMAYAYFASWGSYPI LWAVGPEGLLKLSPYANSIGHSICEIIAKEFWTFLAHH LRIKIHEHILIHGDIRKTTKMEIGGEEVEVEEFVEEEDEDTIPTS / 200232923 1

Claims

1. Attorney Docket No.: BIS-003 WO2.CLAIMS WHAT IS CLAIMED IS:

1. A method of boosting retinal neuronal signals in a subject in need thereof without administration of light stimulation or activation by light, the method comprising administering to the subject an effective amount of a depolarizing ion channel therapeutic wherein the depolarizing ion channel therapeutic depolarizes neurons in a neural circuitry of the subject upon administration, and wherein the neurons are downstream of endogenous neural circuitry.

2. A method of enhancing night vision, vision acuity, and / or color perception in a subject in need thereof, the method comprising administering to the subject an effective amount of a depolarizing ion channel therapeutic.

3. The method of any one of claims 1-2, wherein the subject does not have degenerated neural circuitry.

4. A method of enhancing neurotransmission from existing endogenous neural circuitry in a subject, the method comprising administering to the subject an effective amount of a depolarizing ion channel therapeutic.

5. The method of claim 1, wherein the neurons are projection neurons.

6. The method of any one of claims 1-5, wherein the depolarizing ion channel therapeutic comprises:9.a) a depolarizing ion channel protein; or10.b) a DNA or RNA polynucleotide encoding a depolarizing ion channel protein or a fusion protein comprising a depolarizing ion channel protein.

7. The method of any one of claims 1-6, wherein administering to the subject 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.12.4913.IPTS / 200232923 1 Attorney Docket No.: BIS-003 WO8. The method of claim 7, wherein the vector comprises an inducible promoter operably linked to the DNA or RNA polynucleotide.

9. The method of claim 10, 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.

10. The method of any one of claims 6-9, wherein the DNA or RNA polynucleotide further encodes a gain control protein.

11. The method of any one of claims 6-10, wherein the DNA or RNA polynucleotide is operably linked to a tissue- or cell type-specific promoter.

12. The method of any one of claims 6-11, 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.

13. The method of any one of claims 6-12, wherein the depolarizing ion channel protein is a light-activated ion channel.

14. The method of any one of claims 6-13 wherein the depolarizing ion channel protein is a sodium ion channel protein, a potassium ion channel protein, or a calcium ion channel.

15. The method of any one of claims 6-14, wherein the vector is a plasmid, a DNA vector, an RNA vector, a virion, or a viral vector.

16. The method of claim 15, wherein the vector is a viral vector.

17. The method of claim 16, 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.

18. The method of claim 19, wherein the viral vector is an AAV vector.25.5026.IPTS / 200232923 1 Attorney Docket No.: BIS-003 WO19. The method of claim 18, 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.

20. The method of claim 19, wherein the AAV vector has about 85%, 90%, 95%, 99% or more sequence identity to an AAV2 vector.

21. The method of claim 20, wherein the retroviral vector is a y-retro viral vector or a lentiviral vector.

22. The method of any one of claims 6-21, wherein the DNA or RNA polynucleotide is operably linked to a constitutive promoter.

23. The method of claim 22, wherein the constitutive promoter is a CAG promoter.

24. The method of claim any one of claims 19-23, 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 RNA polynucleotide and 1TR2 is positioned 3' to the DNA or RNA polynucleotide to form a cassette comprising the structure ITR1 -polynucleotide-ITR2.

25. The method of claim 24, wherein the two ITRs are AAV serotype 2 ITRs.

26. The method of any one of claims 19-25, 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: 1.35.5136.IPTS / 200232923 1

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