WO2023097186A1

WO2023097186A1 - Compositions and methods for enhancing visual function

Info

Publication number: WO2023097186A1
Application number: PCT/US2022/080257
Authority: WO
Inventors: Ehud Y. Isacoff; Amy E. HOLT; Johannes BROICHHAGEN
Original assignee: The Regents Of The University Of California; Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V.
Priority date: 2021-11-24
Filing date: 2022-11-21
Publication date: 2023-06-01

Abstract

The present disclosure provides a conjugate comprising an affinity agent, a branched linker, and a plurality of photoisomerizable regulators. The present disclosure provides compositions comprising the conjugate, as well as devices comprising the compositions. The present disclosure provides methods for enhancing visual function, the methods comprising administering the conjugate to an individual in need thereof.

Description

COMPOSITIONS AND METHODS FOR ENHANCING VISUAL FUNCTION

CROSS -REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/283,022, filed November 24, 2021, which application is incorporated herein by reference in its entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “BERK- 454WO_SEQ_LIST” created on November 17, 2022 and having a size of 46 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under Grant No. EY018241 awarded by the National Institutes of Health. The government has certain rights in the invention.

INTRODUCTION

[0004] Retinitis pigmentosa (RP) results in blindness due to degeneration of photoreceptors, but spares other retinal cells, leading to the hope that expression of light-activated signaling proteins in the surviving cells could restore vision. While most efforts have employed light-activated ion channels, light-activated G-protein coupled receptors (GPCRs), such as the opsins of photoreceptor cells, represent an attractive alternative being native to the retina and functioning with high sensitivity, and possibly at low expression, because they activate channels downstream of an amplifying signal cascade. Indeed, recently ectopic expression of rhodopsin or melanopsin was shown to restore light responses under dim light. However, outside of photoreceptor cells, rhodopsin generates slow light responses and melanopsin generates even slower ones. In the case of rhodopsin, the kinetics are already too slow to support patterned vision, even with an immobile visual stimulus.

[0005] There is a need in the art for compositions and methods for enhancing visual function.

SUMMARY

[0006] The present disclosure provides a conjugate comprising an affinity agent, a branched linker, and a plurality of photoisomerizable regulators. The present disclosure provides compositions comprising the conjugate, as well as devices comprising the compositions. The present disclosure provides methods for enhancing visual function, the methods comprising administering the conjugate to an individual in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIG. 1A-1E provides a schematic depiction of a design of multi-branched 9xBGAGi2,46o for photo-activation of SNAP-mGluR2.

[0008] FIG. 2A-2I depict the function of lx, 4x and 9x branched versions of BGAGi2,46o on SNAP- mGluR2 in retinal ganglion cells (RGCs) of the rdl mouse isolated retina as measured on multi-electrode array (MEA), as compared to that of untreated retina of sighted wildtype mouse.

[0009] FIG. 3A-3B depict the responses to light pulses of differing duration in rdl mouse isolated retina MEA recordings when 4x or 9xBGAGi2,46o is attached to SNAP-mGluR2 expressed in RGCs (FIG. 3A); and sensitivity of light avoidance with different BGAGs and duration of restoration in beta cyclodextrin (FIG. 3B).

[0010] FIG. 4 depicts data showing that 9xBGAGi246o confers higher light sensitivity than 4xBGAGi246o in restored light avoidance behavior.

[0011] FIG. 5 depicts data showing that 9xBGAGi246o is ~10-fold more potent than 4xBGAGi246o- [0012] FIG. 6A-6B depict equally effective restoration of high-acuity line pattern discrimination by 4xBGAGi246o and 9xBGAGi246o-

[0013] FIG. 7 depicts a synthetic scheme.

[0014] FIG. 8 provides the structure of 9xBGAGi246o-

[0015] FIG. 9A-9I provide nucleotide sequences of suitable promoters (SEQ ID NOs: 20 to 28, respectively).

[0016] FIG. 10A-10D provide amino acid sequences of SNAP-mGluR2 fusion polypeptides (SEQ ID NOs: 29, 31, 32 and 31, respectively).

[0017] FIG. 11A-11B provide the nucleotide sequence encoding ChrimsonR (FIG. 11 A, SEQ ID NO:35) and the amino acid sequence of ChrimsonR (FIG. 11B, SEQ ID NO:36).

DEFINITIONS

[0018] The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art recognized meanings.

[0019] The term “alkyl” refers to a monoradical branched or unbranched saturated hydrocarbon chain, e.g., having from 1 to 40 carbon atoms, from 1 to 10 carbon atoms, or from 1 to 6 carbon atoms. This term is exemplified by groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, n-hexyl, n- decyl, tetradecyl, and the like. [0020] The term “substituted alkyl” refers to an alkyl group as defined above wherein one or more carbon atoms in the alkyl chain have been optionally replaced with a heteroatom such as -O-, -S(O)_n- (where n is 0 to 2), -NR- (where R is hydrogen or alkyl) and having from 1 to 5 substituents selected from the group consisting of alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-aryl, -SO-heteroaryl, - SCh-alkyl, -SCh-aryl, -SCh-heteroaryl, and -NR^aRb, wherein R^a and R^b may be the same or different and are chosen from hydrogen, optionally substituted alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl and heterocyclic.

[0021] The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl” refers to the groups R^aNHR^b- where R^a is alkyl group as defined above and R^b is alkylene, alkenylene or alkynylene group as defined above.

[0022] The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and -substituted alkylene-aryl where alkylene, substituted alkylene and aryl are defined herein.

[0023] The term “alkoxy” refers to the groups alkyl-O-, alkenyl-O-, cycloalkyl-O-, cycloalkenyl-O-, and alkynyl-O-, where alkyl, alkenyl, cycloalkyl, cycloalkenyl, and alkynyl are as defined herein.

[0024] The term “substituted alkoxy” refers to the groups substituted alkyl-O-, substituted alkenyl-O-, substituted cycloalkyl-O-, substituted cycloalkenyl-O-, and substituted alkynyl-O- where substituted alkyl, substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyl and substituted alkynyl are as defined herein.

[0025] The term “haloalkoxy” refers to the groups alkyl-O- wherein one or more hydrogen atoms on the alkyl group have been substituted with a halo group and include, by way of examples, groups such as trifluoromethoxy, and the like.

[0026] The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl, alkylene-O-substituted alkyl, substituted alkylene-O-alkyl, and substituted alkylene-O-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0027] The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl, alkylene-S-substituted alkyl, substituted alkylene-S-alkyl and substituted alkylene-S-substituted alkyl wherein alkyl, substituted alkyl, alkylene and substituted alkylene are as defined herein.

[0028] The term “alkenyl” refers to a monoradical of a branched or unbranched unsaturated hydrocarbon group having from 2 to 40 carbon atoms, from 2 to 10 carbon atoms, or from 2 to 6 carbon atoms and having at least 1 site (e.g., from 1-6 sites) of vinyl unsaturation. [0029] The term “substituted alkenyl” refers to an alkenyl group as defined above having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SCh-alkyl, -SO2- substituted alkyl, -SCh-aryl and -SCh-heteroaryl.

[0030] The term “alkynyl” refers to a monoradical of an unsaturated hydrocarbon having from 2 to 40 carbon atoms, from 2 to 20 carbon atoms, or from 2 to 6 carbon atoms and having at least 1 site (e.g., from 1-6 sites) of acetylene (triple bond) unsaturation.

[0031] The term “substituted alkynyl” refers to an alkynyl group as defined above having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2- substituted alkyl, -SO2-aryl, and -SO2-heteroaryl.

[0032] The term “acyl” refers to the groups HC(O)-, alkyl-C(O)-, substituted alkyl-C(O)-, cycloalkyl- C(O)-, substituted cycloalkyl-C(O)-, cycloalkenyl-C(O)-, substituted cycloalkenyl-C(O)-, aryl-C(O)-, heteroaryl-C(O)- and heterocyclic-C(O)- where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0033] The term “acylamino” or “aminocarbonyl” refers to the group -C(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, heterocyclic or where both R groups are joined to form a heterocyclic group (e.g., morpholino) wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0034] The term “aminoacyl” refers to the group -NRC(O)R where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0035] The term “aminoacyloxy” or “alkoxycarbonylamino” refers to the group -NRC(O)OR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl, and heterocyclic are as defined herein. [0036] The term “acyloxy” refers to the groups alkyl-C(O)O-, substituted alkyl-C(O)O-, cycloalkyl- C(O)O-, substituted cycloalkyl-C(O)O-, aryl-C(O)O-, heteroaryl-C(O)O-, and heterocyclic-C(O)O- wherein alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl, and heterocyclic are as defined herein.

[0037] The term “aryl” refers to an unsaturated aromatic carbocyclic group of from 6 to 20 carbon atoms having a single ring (e.g., phenyl) or multiple condensed (fused) rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, naphthyl and the like. Unless otherwise constrained by the definition for the aryl substituent, such aryl groups can optionally be substituted with from 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SCh-alkyl, - SCE-substituted alkyl, -SCh-aryl, -SCh-heteroaryl and trihalomethyl.

[0038] The term “aryloxy” refers to the group aryl-O- wherein the aryl group is as defined above including optionally substituted aryl groups as also defined herein.

[0039] The term “amino” refers to the group -NH2.

[0040] The term “substituted amino” refers to the group -NRR where each R is independently selected from the group consisting of hydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl, substituted alkynyl, aryl, heteroaryl, and heterocyclic provided that both R’s are not hydrogen.

[0041] The term “carboxyalkyl” or “alkoxycarbonyl” refers to the groups “-C(O)O-alkyl”, “-C(O)O- substituted alkyl”, “-C(O)O-cycloalkyl”, “-C(O)O-substituted cycloalkyl”, “-C(O)O-alkenyl”, “-C(O)O- substituted alkenyl”, “-C(O)O-alkynyl” and “-C(O)O-substituted alkynyl” where alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl, substituted alkenyl, alkynyl and substituted alkynyl alkynyl are as defined herein.

[0042] The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 20 carbon atoms having a single cyclic ring or multiple condensed rings. Such cycloalkyl groups include, by way of example, single ring structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, and the like, or multiple ring structures such as adamantanyl, and the like.

[0043] The term “substituted cycloalkyl” refers to cycloalkyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SCh-alkyl, -SCh-substituted alkyl, -SCk-aryl and -SCE-heteroaryl.

[0044] The term “cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 20 carbon atoms having a single cyclic ring and at least one point of internal unsaturation. Examples of suitable cycloalkenyl groups include, for instance, cyclobut-2-enyl, cyclopent-3-enyl, cyclooct-3-enyl, and the like.

[0045] The term “substituted cycloalkenyl” refers to cycloalkenyl groups having from 1 to 5 substituents, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2- substituted alkyl, -SO2-aryl and -SO2-heteroaryl.

[0046] The term “halo” or “halogen” refers to fluoro, chloro, bromo and iodo.

[0047] The term “heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms and 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur within at least one ring (if there is more than one ring). Unless otherwise constrained by the definition for the heteroaryl substituent, such heteroaryl groups can be optionally substituted with 1 to 5 substituents, or from 1 to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substituted alkoxy, substituted alkenyl, substituted alkynyl, substituted cycloalkyl, substituted cycloalkenyl, amino, substituted amino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl, carboxylalkyl, cyano, halo, nitro, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy, substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, -SO-alkyl, -SO- substituted alkyl, -SO-aryl, -SO-heteroaryl, -SO2-alkyl, -SO2-substituted alkyl, -SO2-aryl and -SO2- heteroaryl, and trihalomethyl.

[0048] The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl where alkylene and heteroaryl are defined herein. Such heteroaralkyl groups are exemplified by pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

[0049] The term “heteroaryloxy” refers to the group heteroaryl-O-.

[0050] The term “heterocycle” or “heterocyclic” refers to a monoradical saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 40 carbon atoms and from 1 to 10 hetero atoms, e.g., from 1 to 4 heteroatoms, selected from nitrogen, sulfur, phosphorus, and/or oxygen within the ring. Unless otherwise constrained by the definition for the heterocyclic substituent, such heterocyclic groups can be optionally substituted with 1 to 5, or from 1 to 3 substituents, selected from alkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclic, heterocyclooxy, hydroxyamino, alkoxyamino, nitro, -SO-alkyl, -SO-substituted alkyl, -SO- aryl, -SO-heteroaryl, -SCh-alkyl, -SCh-substituted alkyl, -SCh-aryl and -SCE-heteroaryl.

[0051] Examples of nitrogen heteroaryls and heterocycles include, but are not limited to, pyrrole, thiophene, furan, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, pyrrolidine, piperidine, piperazine, indoline, morpholine, tetrahydrofuranyl, tetrahydrothiophene, and the like as well as N-alkoxy-nitrogen containing heterocycles.

[0052] The term “heterocyclooxy” refers to the group heterocyclic-O-.

[0053] The term “heterocyclothio” refers to the group heterocyclic-S-.

[0054] The term “heterocyclene” refers to the diradical group formed from a heterocycle, as defined herein, and is exemplified by the groups 2,6-morpholino, 2,5-morpholino and the like.

[0055] The term “heteroarylamino” refers to a 5 membered aromatic ring wherein one or two ring atoms are N, the remaining ring atoms being C. The heteroarylamino ring may be fused to a cycloalkyl, aryl or heteroaryl ring, and it may be optionally substituted with one or more substituents, e.g., one or two substituents, selected from alkyl, substituted alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, halo, cyano, acyl, amino, substituted amino, acylamino, -OR (where R is hydrogen, alkyl, alkenyl, cycloalkyl, acyl, aryl, heteroaryl, aralkyl, or heteroaralkyl), or -S(O)_nR where n is an integer from 0 to 2 and R is hydrogen (provided that n is 0), alkyl, alkenyl, cycloalkyl, amino, heterocyclo, aryl, heteroaryl, aralkyl, or heteroaralkyl.

[0056] The term “heterocycloamino” refers to a saturated monovalent cyclic group of 4 to 8 ring atoms, wherein at least one ring atom is N and optionally contains one or two additional ring heteroatoms selected from the group consisting of N, O, or S(O)n (where n is an integer from 0 to 2), the remaining ring atoms being C, where one or two C atoms may optionally be replaced by a carbonyl group. The heterocycloamino ring may be fused to a cycloalkyl, aryl or heteroaryl ring, and it may be optionally substituted with one or more substituents, e.g., one or two substituents, selected from alkyl, substituted alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl, halo, cyano, acyl, amino, substituted amino, acylamino, -OR (where R is hydrogen, alkyl, alkenyl, cycloalkyl, acyl, aryl, heteroaryl, aralkyl, or heteroaralkyl), or -S(O)_nR [where n is an integer from 0 to 2 and R is hydrogen (provided that n is 0), alkyl, alkenyl, cycloalkyl, amino, heterocyclo, aryl, heteroaryl, aralkyl, or heteroaralkyl].

[0057] The term “oxyacylamino” or “aminocarbonyloxy” refers to the group -OC(O)NRR where each R is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl, or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl and heterocyclic are as defined herein.

[0058] The term “thiol” refers to the group -SH.

[0059] The term “thioalkoxy” or “alkylthio” refers to the group -S-alkyl.

[0060] The term “substituted thioalkoxy” refers to the group -S-substituted alkyl.

[0061] The term “thioaryloxy” refers to the group aryl-S- wherein the aryl group is as defined above including optionally substituted aryl groups also defined above.

[0062] The term “thioheteroaryloxy” refers to the group heteroaryl-S- wherein the heteroaryl group is as defined above including optionally substituted aryl groups as also defined above.

[0063] As to any of the above groups which contain one or more substituents, it is understood, of course, that such groups do not contain any substitution or substitution patterns which are sterically impractical and/or synthetically non-feasible. In addition, the compounds of the embodiments include all stereochemical isomers arising from the substitution of these compounds.

[0064] The term “pharmaceutically-acceptable salt” refers to salts which retain biological effectiveness and are not biologically or otherwise undesirable. In many cases, the compounds of the embodiments are capable of forming acid and/or base salts by virtue of the presence of amino and/or carboxyl groups or groups similar thereto.

[0065] Pharmaceutically acceptable base addition salts can be prepared from inorganic and organic bases. Salts derived from inorganic bases, include by way of example only, sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, such as alkyl amines, dialkyl amines, trialkyl amines, substituted alkyl amines, di( substituted alkyl) amines, tri(substituted alkyl) amines, alkenyl amines, dialkenyl amines, trialkenyl amines, substituted alkenyl amines, di( substituted alkenyl) amines, tri(substituted alkenyl) amines, cycloalkyl amines, di(cycloalkyl) amines, tri(cycloalkyl) amines, substituted cycloalkyl amines, disubstituted cycloalkyl amine, trisubstituted cycloalkyl amines, cycloalkenyl amines, di(cycloalkenyl) amines, tri(cycloalkenyl) amines, substituted cycloalkenyl amines, disubstituted cycloalkenyl amine, trisubstituted cycloalkenyl amines, aryl amines, diaryl amines, triaryl amines, heteroaryl amines, diheteroaryl amines, triheteroaryl amines, heterocyclic amines, diheterocyclic amines, triheterocyclic amines, mixed di- and tri-amines where at least two of the substituents on the amine are different and are selected from the group consisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, heteroaryl, heterocyclic, and the like. Also included are amines where the two or three substituents, together with the amino nitrogen, form a heterocyclic or heteroaryl group. Examples of suitable amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine, tri(iso-propyl) amine, tri(n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine, and the like.

[0066] Pharmaceutically acceptable acid addition salts may be prepared from inorganic and organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.

[0067] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.

[0068] A polypeptide has a certain percent “sequence identity” to another polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. Sequence similarity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using the methods and computer programs, including BLAST, available over the world wide web at ncbi.nlm.nih.gov/BLAST/. Another alignment algorithm is FASTA, available in the Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA, a wholly owned subsidiary of Oxford Molecular Group, Inc. Other techniques for alignment are described in Methods in Enzymology, vol. 266: Computer Methods for Macromolecular Sequence Analysis (1996), ed. Doolittle, Academic Press, Inc., a division of Harcourt Brace & Co., San Diego, California, USA. Of particular interest are alignment programs that permit gaps in the sequence. The Smith- Waterman is one type of algorithm that permits gaps in sequence alignments. See Meth. Mol. Biol. 70: 173-187 (1997). Also, the GAP program using the Needleman and Wunsch alignment method can be utilized to align sequences. See J. Mol. Biol. 48: 443-453 (1970)

[0069] Of interest is the BestFit program using the local homology algorithm of Smith Waterman (Advances in Applied Mathematics 2: 482-489 (1981) to determine sequence identity. The gap generation penalty will generally range from 1 to 5, usually 2 to 4 and in many embodiments will be 3. The gap extension penalty will generally range from about 0.01 to 0.20 and in many instances will be 0.10. The program has default parameters determined by the sequences inputted to be compared. The sequence identity can be determined using the default parameters determined by the program. This program is available also from Genetics Computing Group (GCG) package, from Madison, Wisconsin, USA.

[0070] Another program of interest is the FastDB algorithm. FastDB is described in Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149, 1988, Alan R. Liss, Inc. Percent sequence identity is calculated by FastDB based upon the following parameters: [0071] Mismatch Penalty: 1.00;

[0072] Gap Penalty: 1.00;

[0073] Gap Size Penalty: 0.33; and

[0074] Joining Penalty: 30.0.

[0075] The term “linker” or “linkage” refers to a linking moiety that connects two groups and has a backbone of 100 atoms or less in length. A linker or linkage may be a covalent bond that connects two groups or a chain of between 1 and 100 atoms in length, for example a chain of 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20 or more carbon atoms in length, where the linker may be linear, branched, cyclic or a single atom. In some cases, the linker is a branched linker that refers to a linking moiety that connects three or more groups. In certain cases, one, two, three, four or five or more carbon atoms of a linker backbone may be optionally substituted with a sulfur, nitrogen or oxygen heteroatom. In some cases, the linker backbone includes a linking functional group, such as an ether, thioether, amino, amide, sulfonamide, carbamate, thiocarbamate, urea, thiourea, ester, thioester or imine. The bonds between backbone atoms may be saturated or unsaturated, and in some cases not more than one, two, or three unsaturated bonds are present in a linker backbone. The linker may include one or more substituent groups, for example with an alkyl, aryl or alkenyl group. A linker may include, without limitations, polyethylene glycol; ethers, thioethers, tertiary amines, alkyls, which may be straight or branched, e.g., methyl, ethyl, n-propyl, 1 -methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t-butyl), and the like. The linker backbone may include a cyclic group, for example, an aryl, a heterocycle or a cycloalkyl group, where 2 or more atoms, e.g., 2, 3, or 4 atoms, of the cyclic group are included in the backbone. A linker may be cleavable or non-cleavable. In some cases, the linker is a branched linker, such as a branched linker as described herein.

[0076] The terms “polyethylene oxide”, “PEG”, “polyethylene glycol” and “PEG” are used interchangeably and refer to a polymeric group including a chain described by the formula — (CH2-CH2— O— )_n- or a derivative thereof. In some embodiments, “n” is 5000 or less, such as 1000 or less, 500 or less, 200 or less, 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, such as 3 to 15, or 10 to 15.

[0077] The term “retinal cell” can refer herein to any of the cell types that comprise the retina, such as retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; and photoreceptor cells including rods and cones.

[0078] The terms “antibodies” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies that retain specific binding to antigen (e.g., to a target ligand-binding polypeptide), including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies (scAb), single domain antibodies (sdAb), single domain heavy chain antibodies, a single domain light chain antibodies, nanobodies, bi-specific antibodies, multispecific antibodies, and fusion proteins comprising an antigen-binding (also referred to herein as antigen binding) portion of an antibody and a non-antibody protein. Also encompassed by the term are Fab’, Fv, F(ab’)2, and or other antibody fragments that retain specific binding to antigen, and monoclonal antibodies.

[0079] The term “nanobody” (Nb), as used herein, refers to the smallest antigen binding fragment or single variable domain (VHH) derived from naturally occurring heavy chain antibody and is known to the person skilled in the art. They are derived from heavy chain only antibodies, seen in camelids. In the family of “camelids” immunoglobulins devoid of light polypeptide chains are found. “Camelids” comprise old world camelids (Camelus bactrianus and Camelus dromedarius) and new world camelids (for example, Llama paccos, Llama glama, Llama guanicoe and Llama vicugna'). A single variable domain heavy chain antibody is referred to herein as a nanobody or a VHH antibody.

[0080] Cartilaginous fishes also have heavy-chain antibodies (IgNAR; “immunoglobulin new antigen receptor”), from which single-domain antibodies called VNAR fragments can be obtained. Thus, in some cases, an affinity agent is an IgNAR.

[0081] “Antibody fragments” comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 8(10): 1057-1062); domain antibodies (dAb; Holt et al. (2003) Trends Biotechnol. 21:484); single-chain antibody molecules; and multi-specific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen combining sites and is still capable of cross-linking antigen. Antibody fragments include, e.g., scFv, sdAb, dAb, Fab, Fab’, Fab’2, F(ab’)2, Fd, Fv, Feb, and SMIP. Examples of sdAb are a camelid VHH and a cartilaginous fish VNAR. [0082] “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and - binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three complementarity determining regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0083] “Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994f

[0084] The term “diabodies” refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0085] As used herein, the terms “treatment,” “treating,” and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. “Treatment,” as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; (c) relieving the disease, i.e., causing regression of the disease; and (d) replacing a lost function that results from the disease.

[0086] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, e.g., a rat or a mouse. In some cases, the individual is a lagomorph, e.g., a rabbit. [0087] The term “retinal cell” can refer herein to any of the cell types that comprise the retina, such as retinal ganglion cells; amacrine cells; horizontal cells; bipolar cells; photoreceptor cells including rods and cones; Muller glial cells; astrocytes (e.g., a retinal astrocyte); and retinal pigment epithelium.

[0088] “AAV” is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or “rAAV vector”). The term “AAV” includes AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. See, e.g., Mori et al. (2004) Virology 330:375. The term “AAV” also includes chimeric AAV. “Primate AAV” refers to AAV isolated from a primate, “non- primate AAV” refers to AAV isolated from a non-primate mammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc.

[0089] An “rAAV vector” as used herein refers to an AAV vector comprising a polynucleotide not of AAV origin (i.e., a polynucleotide heterologous to AAV), e.g., where the heterologous polynucleotide comprises a nucleotide sequence encoding a gene product (a polypeptide or a polynucleotide) of interest for the genetic transformation of a cell. In general, the heterologous polynucleotide is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). The term rAAV vector encompasses both rAAV vector particles and rAAV vector plasmids.

[0090] An “AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “rAAV vector particle” or simply an “rAAV vector”. Thus, production of rAAV particle necessarily includes production of rAAV vector, as such a vector is contained within an rAAV particle.

[0091] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0092] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0093] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0094] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of such conjugates and reference to “the photoisomerizable moiety” includes reference to one or more photoisomerizable moieties and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0095] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such subcombination was individually and explicitly disclosed herein.

[0096] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0097] The present disclosure provides a conjugate comprising: a) an affinity agent that specifically binds: i) a target ligand-binding polypeptide; or ii) a polypeptide that binds to a target ligandbinding polypeptide; b) a branched linker; and c) a plurality of photoisomerizable regulators, wherein each of the photoisomerizable regulators independently comprises: i) a photoisomerizable group comprising an azobenzene moiety; and ii) a ligand that binds to the target ligand-binding polypeptide. The present disclosure provides systems and compositions comprising a conjugate of the present disclosure. The present disclosure provides methods of using a conjugate of the present disclosure to modulate activity of a target polypeptide, and to modulate activity of a target cell or cell population.

PHOTOSWITCH CONJUGATES

[0098] The present disclosure provides a conjugate comprising: a) an affinity agent that specifically binds: i) a target ligand-binding polypeptide; or ii) a polypeptide that binds to a target ligandbinding polypeptide; b) a branched linker; and c) a plurality of photoisomerizable regulators, wherein each of the photoisomerizable regulators independently comprises: i) a photoisomerizable group comprising an azobenzene moiety; and ii) a ligand that binds to the target ligand-binding polypeptide. A conjugate of the present disclosure is also referred to herein as an “affinity-tagged photoswitch.” A photoisomerizable regulator is also referred to herein as a “photoswitch.”

[0099] A conjugate of the present disclosure modulates activity of a target ligand-binding polypeptide. Each of the photoisomerizable regulators in the conjugate can independently interact with the target ligand-binding polypeptide, and the ligand present in each of the photoisomerizable regulators is capable of binding to the ligand-binding site in the target ligand-binding polypeptide in a manner that is controlled by light. Depending on factors such as the ligand, the design of the photoisomerizable regulator, and the wavelength of light, a conjugate of the present disclosure can increase or decrease activity of the target ligand-binding polypeptide, can modulate (increase or decrease) its sensitivity to other stimuli, can stabilize the target ligand-binding polypeptide in a particular conformation, or can induce a conformational change in the target ligand-binding polypeptide.

[00100] The affinity agent present in a conjugate of the present disclosure binds to a target ligand-binding polypeptide, and thereby brings the ligands present in the conjugate into proximity with the target ligand-binding polypeptide such that one of the ligands can bind, in a light-dependent manner, to the ligand-binding site in the target ligand-binding polypeptide. When a conjugate of the present disclosure binds to a target ligand-binding polypeptide, the target ligand-binding polypeptide becomes a light-regulated polypeptide. [00101] A change in the wavelength and/or intensity of light (AX) to which the light-regulated polypeptide is exposed results in a change in ligand binding to a ligand-binding site of the light-regulated polypeptide, e.g., results in a change in binding of one of the ligand portions of a conjugate of the present disclosure to the ligand-binding site of the light-regulated polypeptide. A “change in the wavelength of light to which the light-regulated polypeptide is exposed” includes: 1) a change from Xi to X2; 2) a change from X2 to Xi; 3) a change from Xi to darkness (no light); and 4) a change from darkness to Xi. Repetitive changing from Xi to X2, then from X2 to Xi, and back, e.g., switching from a first wavelength to a second wavelength, and back again repeatedly, is also contemplated. Repetitive changing from light to darkness, from darkness to light, etc., is also contemplated.

[00102] As indicated above, a conjugate of the present disclosure includes: a) an affinity agent; b) a branched linker; and c) a plurality of photoisomerizable regulators. In conjugates according to the present disclosure, the branched linker connects the affinity agent to the photoisomerizable regulators. For example, the branched linker can be a linker between the affinity agent and the photoisomerizable regulators, where the branched linker includes a plurality of arms. Each arm of the branched linker can include a photoisomerizable regulator. For instance, each arm of the branched linker can be attached to its respective photoisomerizable regulator, such that each arm of the conjugate has a photoisomerizable regulator. Thus, embodiments of the conjugates of the present disclosure can include photoisomerizable regulators connected to the affinity agent through the branched linker. Each of the photoisomerizable regulators in the conjugate includes a ligand that binds to the target ligand-binding polypeptide.

Correspondingly, conjugates of the present disclosure can include a plurality of ligands connected to the affinity agent through the branched linker. For example, in some embodiments, a branched linker can include a plurality of arms, where each arm is connected to a photoisomerizable regulator, which in turn are each connected to a ligand. In these embodiments, the affinity agent is connected to each of the photoisomerizable regulators, and thus connected to each of the ligands, through separate arms of the branched linker.

[00103] In some embodiments, a conjugate of the present disclosure can include nine photoisomerizable regulators. As such, in these embodiments, a conjugate of the present disclosure includes: a) an affinity agent; b) a branched linker; and c) nine photoisomerizable regulators. In conjugates according to the present disclosure, the branched linker connects the affinity agent to the nine photoisomerizable regulators. For example, the branched linker can be a linker between the affinity agent and the nine photoisomerizable regulators, where the branched linker includes nine arms. Each arm of the branched linker can include a photoisomerizable regulator. For instance, each arm of the branched linker can be attached to its respective photoisomerizable regulator, such that each arm of the conjugate has a photoisomerizable regulator. Thus, embodiments of the conjugates of the present disclosure can include nine photoisomerizable regulators connected to the affinity agent through the branched linker. Each of the nine photoisomerizable regulators in the conjugate includes a ligand that binds to the target ligand-binding polypeptide. Correspondingly, conjugates of the present disclosure can include nine ligands connected to the affinity agent through the branched linker. For example, in some embodiments, a branched linker can include nine arms, where each arm is connected to a photoisomerizable regulator, which in turn are each connected to a ligand. In these embodiments, the affinity agent is connected to each of the nine photoisomerizable regulators, and thus connected to each of the nine ligands, through separate arms of the branched linker.

[00104] In some cases, the change in wavelength (from Xi to X2; from light to darkness; or from darkness to light) results in a change in binding of one of the ligands to a ligand-binding site. As used herein, a “change in binding of a ligand to a ligand-binding site” or “change in binding of one of the ligands to a ligand binding site” includes increased binding and decreased binding. As used herein, “increased binding” includes one or more of: an increased probability of binding of one of the ligands in the photoisomerizable regulators in the conjugate to the ligand-binding site; an increased binding affinity of one or more of the ligands for the ligand-binding site; an increased local concentration of the ligands at the ligand-binding site; and an increased occupancy of one of the ligands in the ligand-binding site. As used herein, “decreased binding” includes one or more of: a decreased probability of binding of one of the ligands in the photoisomerizable regulators in the conjugate to the ligand-binding site; a decreased binding affinity of one or more of the ligands for the ligand-binding site; a decreased local concentration of the ligands at the ligand-binding site; and a decreased occupancy of one of the ligands in the ligandbinding site. As used herein, the term “change in wavelength” to which a conjugate of the present disclosure regulator is exposed, or to which a receptor/synthetic light regulator complex is exposed, refers to a change in wavelength from Xi to X2; a change from light to darkness; or a change from darkness to light. An increase in binding includes an increase of from about 10% to about 20%, from about 20% to about 50%, from about 50% to about 2-fold, from about 2-fold to about 5-fold, from about 5-fold to about 10-fold, from about 10-fold to about 50-fold, from about 50-fold to about 10²-fold, from about 10²-fold to about 10⁴-fold, from about 10⁴-fold to about 10⁶-fold, from about 10⁶-fold to about 10⁸- fold, or a greater than 10⁸-fold increase in binding. A decrease in binding includes a decrease of from about 5% to about 10% to about 20% to about 30%, from about 30% to about 40%, from about 40% to about 50%, from about 50% to about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, or from about 90% to 100% decrease in binding.

[00105] For example, in some cases, the ligands of the conjugate have a first probability of binding to the ligand binding site at a first wavelength of light; the ligands have a second probability of binding to the ligand binding site at a second wavelength of light; and the second probability is lower than the first probability. In other cases, the ligands of the conjugate have a first probability of binding to the ligand binding site at a first wavelength of light; the ligands have a second probability of binding to the ligand binding site at a second wavelength of light; and the second probability is higher than the first probability. In other cases, the ligands of the conjugate have a first probability of binding to the ligand binding site when exposed to light; the ligands have a second probability of binding to the ligand binding site in the absence of light (e.g., in darkness); and the second probability is lower than the first probability. In other cases, the ligands of the conjugate have a first probability of binding to the ligand binding site when exposed to light; the ligands have a second probability of binding to the ligand binding site in the absence of light and the second probability is higher than the first probability.

[00106] In some embodiments, because a conjugate of the present disclosure includes a plurality of photoisomerizable regulators, and thus includes a plurality of ligands, the probability of a ligand binding to the ligand site is higher than a conjugate that has fewer photoisomerizable regulators and corresponding ligands. Since the conjugate of the present disclosure includes a plurality of photoisomerizable regulators, the local concentration of ligands in proximity to the ligand binding site may be higher as compared to a conjugate that has fewer photoisomerizable regulators and corresponding ligands. The increased local concentration of ligands of the conjugates of the present disclosure near the ligand binding site may result in an increase in binding as described above.

[00107] For example, in some embodiments, a conjugate of the present disclosure includes nine photoisomerizable regulators, and thus includes nine ligands. As such, the probability of a ligand binding to the ligand site is higher than a conjugate that has less than nine (e.g., one, two or four) photoisomerizable regulators and corresponding ligands. Since some embodiments of the conjugate of the present disclosure can include nine photoisomerizable regulators, the local concentration of ligands in proximity to the ligand binding site may be higher as compared to a conjugate that has less than nine (e.g., one, two or four) photoisomerizable regulators and corresponding ligands. The increased local concentration of ligands of the conjugates of the present disclosure near the ligand binding site may result in an increase in binding as described above.

[00108] The local concentration of the ligands of a conjugate of the present disclosure at the ligand binding site in a light-regulated polypeptide is high. For example, the local concentration of the ligands of a conjugate of the present disclosure at the ligand binding site in a subject light-regulated polypeptide ranges from about 500 nM to about 50 mM, e.g., from about 500 nM to about 10 mM, from about 750 nM to about 1 mM, from about 1 pM to about 750 pM, from about 10 pM to about 500 pM, from about 10 pM to about 250 pM, from about 50 pM to about 200 pM, or from about 50 pM to about 150 pM, such as about 100 pM. In some embodiments, the local concentration of the ligands of a conjugate of the present disclosure at the ligand binding site in a subject light-regulated polypeptide ranges from about 500 nM to about 50 mM, e.g., from about 500 nM to about 750 nM, from about 750 nM to about 1 mM, from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, from about 10 mM to about 20 mM, from about 20 mM to about 30 mM, or from about 30 mM to about 50 mM. Change in wavelength resulting in binding of a ligand to the ligand-binding site or higher affinity ligand binding to ligand-binding site

[00109] In some cases, a change in the wavelength of light to which a light-regulated polypeptide is exposed results in an increase in binding affinity of one or more of the ligands of a conjugate of the present disclosure for a ligand-binding site the light-regulated polypeptide. For example, in some cases, a change in wavelength of light to which a light-regulated polypeptide is exposed results in an at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 25-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 10³-fold, at least about 5 x 10³-fold, at least about 10⁴-fold, at least about 5 x 10⁴-fold, or greater, increase in binding affinity.

[00110] Where the ligand is an agonist, the change in wavelength will in some cases result in activation of a light-regulated polypeptide. Where the ligand is an agonist, the change in wavelength will in some cases result in desensitization of a light-regulated polypeptide. Conversely, where the ligand is an antagonist, the change in wavelength will in some cases result in a block of activation of a light- regulated polypeptide, e.g., block of the ability to activate a light-regulated polypeptide with free agonist. Where the ligand is a blocker (e.g., a pore blocker of an ion channel, or an interaction domain that binds to other biological macromolecules such as polypeptides or nucleic acids), the change in wavelength will in some cases result in block of polypeptide activity.

[00111] Expressed another way, where the ligand is an agonist, and where a change in the wavelength of light to which a light-regulated polypeptide is exposed results in a higher binding affinity of the ligand moiety of the conjugate to the ligand-binding site of the light-regulated polypeptide, the change in wavelength results in transition from an inactive state to an active state, or to a desensitized state. Where the ligand is an antagonist, the change in wavelength results in transition from a responsive state to an unresponsive state. Where the ligand is a blocker, the change in wavelength results in transition from an active state to an inactive state.

Change in wavelength resulting in removal of a ligand from ligand-binding site, or reduced binding affinity

[00112] In some cases, a change in the wavelength of light to which a light-regulated polypeptide is exposed results in removal of a ligand of a conjugate of the present disclosure from a ligand-binding site of the light-regulated polypeptide, e.g., the ligand is not bound to the ligand-binding site. In some cases, a change in the wavelength of light to which the light-regulated polypeptide is exposed results in reduced binding affinity of one or more of the ligands of conjugate of the present disclosure for a ligandbinding site of the light-regulated polypeptide, e.g., the ligand has reduced binding affinity for the ligand-binding site. For example, in some cases, a change in the wavelength of light to which a light- regulated polypeptide is exposed results in a reduction of binding affinity of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or more.

[00113] Where the ligand is an agonist, the change in wavelength will in some cases result in activation of a light-regulated polypeptide. Where the ligand is an agonist, the change in wavelength will in some cases result in deactivation of a light-regulated polypeptide. Where the ligand is an agonist, the change in wavelength will in some cases result in recovery from desensitization of the light-regulated polypeptide. Conversely, where the ligand is an antagonist, the change in wavelength will in some cases result in occupancy of the ligand binding site and a reduction in background activity of the polypeptide, or, alternatively, in loss of activation by physiological stimuli. Where the ligand is an antagonist, the change in wavelength will in some cases result in removal of antagonism to permit activation by physiological stimuli. Where the ligand is a negative allosteric modulator, the change in wavelength that causes binding can result in increased sensitivity to or efficacy of another stimulus. Where the ligand is a positive allosteric modulator, the change in wavelength that causes binding can result in decreased sensitivity to or efficacy of another stimulus. In some cases, the ligand binding site will be a modulatory site where binding by the ligand increases or decreases the sensitivity to or efficacy of another stimulus, so that light regulates this process by controlling the binding of the photoswitched regulator. In some cases, the ligand is a blocker of an active site of the polypeptide (e.g., a pore blocker of an ion channel, or an interaction domain that binds to other biological macromolecules such as polypeptides or nucleic acids, or a blocker of an enzyme active site), and the change in wavelength results in block or relief of block in polypeptide activity to prevent or permit the receptor to function normally.

[00114] Expressed another way, where the ligand is an agonist, and where a change in the wavelength of light to which the light-regulated polypeptide is exposed results in removal (or nonbinding) of the ligand of conjugate of the present disclosure from the ligand-binding site of the light- regulated polypeptide, the change in wavelength results in transition from a more active state to a less active state, or from a desensitized state to a responsive state. Where the ligand is a negative allosteric modulator, the change in wavelength that causes un-binding results in increased sensitivity to or efficacy of another stimulus. Where the ligand is a positive allosteric modulator, the change in wavelength that causes un-binding results in decreased sensitivity to or efficacy of another stimulus. Where the ligand is an antagonist, the change in wavelength that causes un-binding results in transition from an unresponsive state to a responsive state or from an inactive state to a state with some background “basal” (unliganded) activity. Where the ligand is a blocker, the change in wavelength that causes un-binding results in transition from an inactive state to an active state.

Affinity agents

[00115] The affinity agent present in a conjugate of the present disclosure targets the photoisomerizable regulator to a target ligand-binding polypeptide, by binding directly to the target ligand-binding polypeptide or by binding to a polypeptide that binds to the target ligand-binding polypeptide, or by binding to a fusion partner expressed in fusion with the target ligand binding polypeptide.

[00116] In some cases, the affinity agent binds specifically to a target ligand-binding polypeptide. Thus, for example, in some cases, the affinity agent binds to a target ligand-binding polypeptide with an affinity of at least 10⁶ M, at least 10⁷ M, at least 10 ⁸ M, at least 10⁹ M, or at least 10 ¹⁰ M. In some cases, the affinity agent binds directly to the target ligand-binding polypeptide. In some cases, the affinity agent binds to a polypeptide that binds to the target ligand-binding polypeptide.

[00117] Suitable affinity agents include, but are not limited to, agents that bind to self-labelling polypeptides; antibodies; aptamers; peptides; and small molecules.

Agents that bind to self-labelling polypeptides

[00118] Suitable affinity agents include nucleoside base derivatives. In some cases, the nucleoside base of the nucleoside base derivative is selected from guanine, cytosine, uracil, thymine, xanthine, and hypoxanthine. For example, the nucleoside base of the nucleoside base derivative can be guanine, xanthine or hypoxanthine. In some cases, the nucleoside base of the nucleoside base derivative is guanine. In other instances, the nucleoside base of the nucleoside base derivative can be cytosine, thymine or uracil. In some cases, the nucleoside base of the nucleoside base derivative is cytosine. The nucleoside base can be derivatized to provide the nucleoside base derivative of the affinity agent of a conjugate of the present disclosure. In some cases, the nucleoside base derivative of the affinity agent is a benzylnucleoside base, such as benzylguanine or benzylcytosine. In some cases, the affinity agent is benzylguanine. In embodiments where the affinity agent is benzylguanine, the benzylguanine affinity agent may provide for covalent binding to a SNAP tag. In some cases, the affinity agent is benzylcytosine. In embodiments where the affinity agent is benzylcytosine, the benzylcytosine affinity agent may provide for covalent binding to a CLIP tag. In some cases, the affinity agent is a chloropyrimidine; a chloropyrimidine can bind to a SNAP tag.

[00119] Suitable affinity agents also include alkyl derivatives, such as haloalkyl derivatives where one or more hydrogen atoms in an alkyl or alkyl derivative is replaced by a halogen, e.g., fluoro, chloro, or bromo. In some cases, the haloalkyl derivative is a fluoroalkane. In some cases, the haloalkyl derivative is a chloroalkane. In some cases, the haloalkyl derivative is a bromoalkane. In some cases, the affinity agent is chloroalkane, such as QCCFLMOCILCILh. In embodiments where the affinity agent is chloroalkane, the chloroalkane affinity agent may provide for covalent binding to a HALO tag.

Antibodies

[00120] In some cases, an affinity agent present in a conjugate of the present disclosure is an antibody. In some cases, an antibody suitable for inclusion in a conjugate of the present disclosure binds to a target ligand-binding polypeptide. Examples of target ligand-binding polypeptides are provided below. An antibody suitable for inclusion in a conjugate of the present disclosure does not inhibit binding of the ligand present in the photoisomerizable regulator to the target ligand-binding polypeptide. Generally, an antibody suitable for inclusion in a conjugate of the present disclosure does not substantially alter activity of the target ligand-binding polypeptide. In some cases, the affinity agent is a single-chain Fv (scFv). In some cases, the affinity agent is a nanobody.

[00121] In some cases, the affinity agent is an antibody that binds specifically to a target ligandbinding polypeptide, where the target ligand-binding polypeptide is selected from a transcription regulator, an ion channel, a cation channel, a ligand-gated ion channel, a voltage-gated ion channel, a quorum sensor, a pheromone receptor, a neurotransmitter receptor, a G-protein-coupled receptor, and an enzyme.

[00122] In some cases, the affinity agent is an antibody that binds specifically to a target ligandbinding polypeptide, where the target ligand-binding polypeptide is selected from a potassium channel, a sodium channel, or a calcium channel.

[00123] In some cases, the affinity agent is an antibody that binds specifically to a target ligandbinding polypeptide, where the target ligand-binding polypeptide is selected from a glutamate receptor, a metabotropic glutamate receptor (mGluR), an ionotropic glutamate receptor (e.g., a kainate receptor; an AMPA receptor; an NMDA receptor), an ionotropic nicotinic acetylcholine receptor, an ionotropic GABA-A receptor, a metabotropic GABA-B receptor, a metabotropic dopamine receptor, an ionotropic purinergic P2X receptor, a metabotropic purinergic P2Y receptor, a metabotropic serotonin receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, a cation channel, a potassium channel, a calcium channel, a sodium channel, a proton channel, an anion channel, and a chloride channel. In some cases, the affinity agent is an antibody that binds specifically to a metabotropic glutamate receptor, where metabotropic glutamate receptors include, e.g., mGluR2, mGluR3, mGluR5, mGluR6, and the like.

[00124] As one non-limiting example, the affinity agent is a scFv that binds specifically to mGluR2. As another non-limiting example, the affinity agent is a nanobody that binds specifically to mGluR2. As another non-limiting example, the affinity agent is a scFv that binds specifically to mGluR3. As another non-limiting example, the affinity agent is a nanobody that binds specifically to mGluR3. As another non-limiting example, the affinity agent is a scFv that binds specifically to mGluR5. As another non-limiting example, the affinity agent is a nanobody that binds specifically to mGluR5. As another non-limiting example, the affinity agent is a scFv that binds specifically to mGluR6. As another non-limiting example, the affinity agent is a nanobody that binds specifically to mGluR6. Small molecules

[00125] Small molecules that are suitable for use as affinity agent in a conjugate of the present disclosure include small molecules having a molecular weight of less than 2 kDa, less than 1 kDa, less than 500 Daltons, less than 250 Daltons, less than 200 Daltons, less than 100 Daltons, less than 75 Daltons, or less than 50 Daltons. For example, a small molecule that is suitable for use as affinity agent in a conjugate of the present disclosure can have a molecular weight of from 10 Daltons to 2 kDa, e.g., from 10 Daltons to 25 Daltons, from 25 Daltons to 50 Daltons, from 50 Daltons to 100 Daltons, from 100 Daltons to 150 Daltons, from 150 Daltons to 250 Daltons, from 250 Daltons to 500 Daltons, from 500 Daltons to 1 kDa, or from 1 kDa to 2 kDa.

[00126] A small molecule that is suitable for use as affinity agent in a conjugate of the present disclosure is generally not a ligand for a target ligand-binding polypeptide. A small molecule that is suitable for use as affinity agent in a conjugate of the present disclosure generally binds to the target ligand-binding polypeptide at a site other than the site at which the ligand binds, and does not substantially inhibit binding of the ligand to the target ligand-binding polypeptide.

Aptamers

[00127] Aptamers that are suitable for use as affinity agent include RNA aptamers, DNA aptamers, and peptide aptamers. An aptamer suitable for inclusion in a conjugate of the present disclosure does not inhibit binding of the ligand present in the photoisomerizable regulator to the target ligand-binding polypeptide. Generally, an aptamer suitable for inclusion in a conjugate of the present disclosure does not substantially alter activity of the target ligand-binding polypeptide.

[00128] Nucleic acid aptamers can have a length of from about 10 nucleotides to about 200 nucleotides, e.g., from 10 nucleotides (nt) to 15 nt, from 10 nt to 15 nt, from 15 nt to 20 nt, from 20 nt to 25 nt, from 25 nt to 50 nt, from 50 nt to 75 nt, form 75 nt to 100 nt, from 100 nt to 150 nt, or from 150 nt to 200 nt. Nucleic acid aptamers can have a length of from about 10 nucleotides to about 50 nucleotides. Nucleic acid aptamers can have a length of from about 10 nucleotides to about 25 nucleotides.

[00129] A DNA aptamer can be prepared using any known method. For example, a DNA- SELEX method can be used. In the SELEX method, by setting strict selection conditions by increasing the number of rounds or using a competing substance, an aptamer exhibiting a stronger binding potential for a target polypeptide is concentrated and selected. Hence, by adjusting the number of rounds of SELEX and/or changing the competitive condition, aptamers with different binding forces, aptamers with different binding modes, and aptamers with the same binding force or binding mode but different base sequences can be obtained. The SELEX method comprises a process of amplification by polymerase chain reaction; by causing a mutation by using manganese ions and the like in the process, it is possible to perform SELEX with higher diversity. Aptamers specific for a polypeptide (or portion of a polypeptide) can be produced using standard techniques, such as, for example, those described in Ogawa, A., et al., Bioorg. Med. Chem, Lett, 14: 4001-4004, 2004; and Jayasena, S. D., Clinical Chemistry 45: 1628-1650, 1999.

[00130] A nucleic acid aptamer can include naturally-occurring nucleotides, and may also include non-naturally-occurring nucleotides. DNA aptamers that include only naturally-occurring nucleotides include DNA aptamers composed of deoxyribonucleotides having any of the natural bases adenine, guanine, cytosine, and thymine. RNA aptamers that include only naturally-occurring nucleotides include RNA aptamers composed of RNAs composed of ribonucleotides having any of the natural bases adenine, guanine, cytosine, and uracil. A non-naturally-occurring nucleotide comprises a non-naturally occurring base, a phosphate group, and a sugar. A non-naturally-occurring base (or “artificial base”) refers to an artificially constructed base analog having properties similar to those of the natural base constituting the natural nucleotide and that can form artificial base pairing with its partner base analog (referred to as a “complementary artificial base”), as in the natural base. The term “artificial base pairing” refers to base pairing formed between a pair of complementary artificial bases, as in a pair of complementary natural bases adenine and thymine, adenine and uracil, or guanine and cytosine. Artificial base pairing includes a chemical bond via a hydrogen bond found in the base pairing between natural bases, a physical bond via the molecular structure-based association between artificial bases, and stacking effects via hydrophobic interaction.

[00131] Aptamers can be modified to comprise one or more moieties such as: a 2’-O-methyl moiety; a 2’-NH2 moiety; and the like.

[00132] Aptamers that bind a variety of polypeptides are known in the art. For example, an aptamer database is available on the internet at www(dot)aptagen(dot)com/aptamer-index/aptamer-list. In addition, as noted above, those skilled in the art can readily design aptamers that bind a target ligandbinding polypeptide of interest.

Branched linkers

[00133] As noted above, a conjugate of the present disclosure includes: a) an affinity agent; b) a branched linker; and c) a plurality of photoisomerizable regulators. A branched linker is a linker that connects the affinity agent to the plurality of photoisomerizable regulators of the conjugate. As such, the branched linker can be connected to the affinity agent and can include a plurality of arms, each independently comprising a photoisomerizable regulator. For instance, the branched linker can be connected to the affinity agent at a first end and can include two or more branching points that connect to the plurality of arms, where each of the arms is independently connected to a photoisomerizable regulator. In some cases, the branched linker includes two or more branching points, such as 3 or more branching points, 4 or more branching points, 5 or more branching points, 6 or more branching points, 7 or more branching points, 8 or more branching points, or 9 or more branching points. A branching point is a position on the branched linker where the linker is attached to two or more branches or arms. In some cases, a branching point is a position on the branched linker where the linker is attached to two branches or arms. In some cases, a branching point is a position on the linker where the linker is attached to three branches or arms.

[00134] For example, in some embodiments, a conjugate of the present disclosure includes nine photoisomerizable regulators. As such, in these embodiments, the conjugate includes: a) an affinity agent; b) a branched linker; and c) nine photoisomerizable regulators. In these cases, the branched linker is a linker that connects the affinity agent to the nine photoisomerizable regulators of the conjugate. As such, the branched linker can be connected to the affinity agent and can include nine arms, each independently comprising a photoisomerizable regulator. For instance, the branched linker can be connected to the affinity agent at a first end and can include two or more branching points that connect to the nine arms, where each of the arms is independently connected to a photoisomerizable regulator.

[00135] Furthermore, as described above, each of the plurality of photoisomerizable regulators of the conjugate of the present disclosure includes a photoisomerizable group. As such, the branched linker can include a plurality of arms, where each of the arms is independently connected to a photoisomerizable group. Thus, conjugates of the present disclosure include a plurality of photoisomerizable groups. In addition, as described above, each of the plurality of photoisomerizable regulators of the conjugate of the present disclosure includes a ligand that binds to the target ligandbinding polypeptide. As such, the branched linker can include a plurality of arms, where each of the arms independently comprises a ligand. Thus, conjugates of the present disclosure include a plurality of ligands.

[00136] For example, in some instances, as described above, the conjugate may include nine photoisomerizable regulators. Thus, in these embodiments, each of the nine photoisomerizable regulators of the conjugate of the present disclosure includes a photoisomerizable group. As such, the branched linker can include nine arms, where each of the nine arms is independently connected to a photoisomerizable group. Thus, conjugates of the present disclosure include nine photoisomerizable groups. In addition, as described above, each of the nine photoisomerizable regulators of the conjugate of the present disclosure includes a ligand that binds to the target ligand-binding polypeptide. As such, the branched linker can include nine arms, where each of the nine arms independently comprises a ligand. Thus, conjugates of the present disclosure include nine ligands.

[00137] Branched linkers of the conjugates of the present disclosure can be any suitable branched linker that connects the affinity agent to the photoisomerizable regulators. The branched linker may include suitable functional groups that provide for convenient linking chemistry to the affinity agent and respective photoisomerizable regulators, photoisomerizable groups, and ligands. For instance, any type of functional group may be used to connect the branched linker to the affinity agent and/or to the photoisomerizable regulators, photoisomerizable groups, and ligands, such as, but not limited to, amide, ether, amine, ester, ketone, and carboxy functional groups, and the like. In some embodiments, amide functional groups provide for convenient linking chemistry between the branched linker and the affinity agent and/or the photoisomerizable regulators.

[00138] In some instances, the branched linker comprises a branching moiety of Formula (BL): -C(O)NH-[C[(CH₂)nC(O)NH]₃]x- (Formula (BL)), wherein n is an integer from 1 to 6, and x is an integer from 1 to 50.

[00139] In some instances, n is an integer from 1 to 6. For example, in some embodiments n is 1. In some embodiments, n is 2. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

[00140] In some instances, x is an integer from 1 to 50. For example, in some embodiments x is 1. In some embodiments, x is 4. In some embodiments, x is 13. In some embodiments, x is 40.

[00141] In some instances, the branched linker comprises a branching moiety that connects to nine arms. For example, the branching moiety can have the Formula (BL2): -C(O)NH-C[CH2CH₂C(O)NH]3-[C[CH2CH₂C(O)NH]3]3- (Formula (BL2)).

[00142] In some cases, the affinity agent is attached to the branched linker of Formula (BL) or (BL2) on the left side of the branching moiety, i.e., attached to the amide group on the left side of the branching moiety. As discussed above, branched linkers of the present disclosure can include a plurality of arms (e.g., nine arms), where each arm is connected to a separate photoisomerizable regulator, and thus the branched linker is attached to a plurality of photoisomerizable regulators (e.g., nine photoisomerizable regulators).

[00143] For example, in some embodiments, the branching moiety of the branched linker of Formula (BL) or (BL2) has the following structure:

wherein each R represents an arm of the branched linker.

[00144] As described herein, each arm, R, of the branched linker includes a photoisomerizable regulator. In addition, each photoisomerizable regulator includes a photoisomerizable group and a ligand, as described herein. In some cases, the affinity agent is attached to the branching moiety of Formula (BL) or (BL2) on the left side of the branching moiety, i.e., attached to the amide group on the left side of the branching moiety (indicated by the wavy line).

Photoisomerizable regulators

[00145] As noted above, a photoisomerizable regulator present in a conjugate of the present disclosure comprises: i) a photoisomerizable group; and ii) a ligand that binds to the target ligandbinding polypeptide.

Photoisomerizable groups

[00146] Photoisomerizable groups are known in the art, and any known photoisomerizable group can be included in the photoisomerizable regulator present in a conjugate of the present disclosure.

Suitable photoisomerizable groups include, but are not limited to, azobenzene, cyclic azobenzenes and azoheteroarenes and derivatives thereof; spiropyran and derivatives thereof; triphenyl methane and derivatives thereof; 4,5-epoxy-2-cyclopentene and derivatives thereof; fulgide and derivatives thereof; thioindigo and derivatives thereof; diarylethene and derivatives thereof; diallylethene and derivatives thereof; overcrowded alkenes and derivatives thereof; and anthracene and derivatives thereof. In some cases, a suitable photoisomerizable group is a photoisomerizable group as shown in the examples herein. [00147] Suitable spiropyran derivatives include, but are not limited to, 1,3,3- trimethylindolinobenzopyrylospiran; 1 ,3,3-trimethylindolino-6’ -nitrobenzopyrylospiran; 1 ,3,3- trimethylindolino-6’-bromobenzopyrylospiran; l-n-decyl-3,3-dimethylindolino-6’- nitrobenzopyrylospiran; 1 -n-octadecy- 1 -3,3-dimethylindolino-6’ -nitrobenzopyrylospiran; 3 ’ ,3 ’ -dimethyl- 6-nitro- 1 ’ - [2-(phenylcarbamoyl)ethyl] spiro; [2H- 1 -benzopyran-2,2 ’ -indoline] ; 1 ,3 ,3-trimetnylindolino- 8’-methoxybenzopyrylospiran; and 1,3,3-trimethylindolino-P-naphthopyrylospiran. Also suitable for use is a merocyanine form corresponding to spiropyran or a spiropyran derivative.

[00148] Suitable triphenylmethane derivatives include, but are not limited to, malachite green derivatives, specifically, there can be mentioned, for example, bis[dimethylamino)phenyl] phenylmethanol, bis[4-(diethylamino)phenyl]phenylmethanol, bis[4- (dibuthylamino)phenyl]phenylmethanol and bis[4-(diethylamino)phenyl]phenylmethane.

[00149] Suitable 4,5-epoxy-2-cyclopentene derivatives include, for example, 2,3-diphenyl-l- indenone oxide and 2’,3’-dimethyl-2,3-diphenyl-l-indenone oxide.

[00150] Suitable azobenzene moieties include, e.g., compounds having an azobenzene group crosslinked to a side chain. For example, in some cases, an azobenzene moiety includes 4- carboxyazobenzene that has an ester bond to the hydroxyl group of polyvinyl alcohol, or 4- carboxyazobenzene that has an amide bond to the amino group of polyallylamine. Also suitable are azobenzene compounds having azobenzene groups in the main chain, for example, those formed by an ester bond between bis(4-hydroxyphenyl)dimethylmethane (also referred to as bisphenol A) and 4,4’- dicarboxyazobenzene or by an ester bond between ethylene glycol and 4,4’ -dicarboxyazobenzene.

[00151] Suitable cyclic azobenzene and azoheteroarene compounds which can be adapted for use in the subject conjugates and photoisomerizable regulators include, but are not limited to, 11,12- dihydrodibenzo[c,g] [1 ,2]diazocine-5-oxide,

, heterodiazocines, such as those photoswitches described by Hammerich et al. J. Am. Chem. Soc., 2016, 138 (40), pp 13111-13114), and azoheteroarene photoswitches such as 3-pyrazoles (3pzH or 3pzMe), 5- pyrazoles (5pzH or 5pzMe), 3-pyrrroles (3pyH or 3pyMe), triazole and tetrazoles (tet or 3tri) as describes by Calbo et al. J. Am. Chem. Soc., 2017, 139 (3), pp 1261-1274, the disclosures of which are herein incorporated by reference.

[00152] Suitable fulgide derivatives include, but are not limited to, isopropylidene fulgide and adamantylidene fulgide.

[00153] Suitable diallylethene derivatives include, for example, l,2-dicyano-l,2-bis(2,3,5- trimethyl-4-thienyl)ethane; 2,3-bis(2,3,5-trimethyl-4-thiethyl) maleic anhydride; l,2-dicyano-l,2- bis(2,3,5-trimethyl-4-selenyl)ethane; 2,3-bis(2,3,5-trimethyl-4-selenyl) maleic anhydride; and 1,2- dicyano-l,2-bis(2-methyl-3-N-methylindole)ethane.

[00154] Suitable diarylethene derivatives include but are not limited to, substituted perfluorocylopentene-bis-3-thienyls and bis-3-thienylmaleimides.

[00155] Suitable overcrowded alkenes include, but are not limited to, cis-2-nitro-7- (dimethylamino)-9-(2’ ,3’ -dihydro- 1 ’ //-naphtho [2, 1 -b]thiopyran- 1 ’ -ylidene)-9//-thioxanthene and trans- dimethyl-[ 1 -(2-nitro-thioxanthen-9-ylidene)-2,3-dihydro- l//-benzo[f]thiochromen-8-yl] amine.

Overcrowded alkenes are described in the literature. See, e.g., terWiel et al. (2005) Org. Biomol. Chem. 3:28-30; and Geertsema et al. (1999) Angew. Chem. Int. Ed. Engl. 38:2738.

[00156] Other suitable photoisomerizable groups include, e.g., reactive groups commonly used in affinity labeling, including diazoketones, aryl azides, diazerenes, and benzophenones.

[00157] In some instances, the photoisomerizable group of the conjugate (e.g., as defined herein) is an azobenzene (e.g., an azobenzene photoswitch) of the following Formula 1:

wherein:

R¹ and R⁶ are one or more optional substituents selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, -NR¹⁰Rⁿ, -NR¹²C(O)R¹³, -NR¹²C(O)OR¹³, -NR¹²C(O)NR¹²R¹³, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20 aryl, heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C410 cycloalkenyl, substituted C_4-10 cycloalkenyl, cyano, halo, - OR¹⁰, -C(O)OR¹⁰, -SR¹⁰, -S(O)R¹⁰, -S(O)₂R¹⁰; x is an integer from 1 to 5; y is an integer from 1 to 5; and wherein R¹⁰-R¹³ are as defined below, or a pharmaceutically acceptable salt thereof. [00158] In some cases, a photoisomerizable group present in a conjugate of the present disclosure is a compound of Formula 2: wherein:

Q¹ is -CH₂- or -C(=O)-;

Q² is a ligand (or a label or reactive group or second affinity agent), as described according to the present disclosure; each R¹ is independently selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, -NR¹⁰Rⁿ, -NR¹²C(O)R¹³, -NR¹²C(O)OR¹³, -NR¹²C(O)NR¹²R¹³, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20 aryl, heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino, C_{4- 10} cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, substituted C_4-10 cycloalkenyl, cyano, halo, -OR¹⁰, -C(O)OR¹⁰, -SR¹⁰, - S(O)R¹⁰, -S(O)₂R¹⁰; w is an integer from 1 to 10; x is an integer from 1 to 5; y is an integer from 1 to 4;

R² is selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20 aryl, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, and substituted C_4-10 cycloalkenyl; each R⁶ is independently selected from hydrogen, C_1-10 alkyl, substituted C_{1- 10}kyl, - NR¹⁰Rⁿ, -NR¹²C(O)R¹³, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20 aryl, heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, substituted C_4-10 cycloalkenyl, cyano, halo, -OR¹⁰, -C(O)OR¹⁰, -SR¹⁰, -S(O)R¹⁰, -S(O)₂R¹⁰;

R¹⁰ and R¹¹ are each independently selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20 aryl, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, and substituted C_4-10 cycloalkenyl;

R¹² is selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20 aryl, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, and substituted C_4-10 cycloalkenyl; and

R¹³ is selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, Cg-Cio aryl, substituted C_6-20 aryl, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_{4- 10} cycloalkenyl, substituted C_{4- 10} cycloalkenyl, -CH₂- N(CH₂CH₃)₃ ⁺, and -CH₂-SO₃ . or a pharmaceutically acceptable salt thereof.

[00159] In certain embodiments of Formula 2, Q¹ is -CH₂-. In certain embodiments of

Formula 2, Q¹ is -C(=O)-.

[00160] In some instances of Formula 2, one of R¹ is linked via a linker to an affinity agent (e.g., as described herein). In some cases, the linker includes a branched linker (e.g., as described herein).

[00161] In some instances, R² is hydrogen.

[00162] In some instances, each R⁶ is hydrogen.

[00163] In some cases, a photoisomerizable group present in a conjugate of the present disclosure is a compound of Formula 3:

wherein

Q² is a ligand (or a label or reactive group or second affinity agent), as described according to the present disclosure; each R¹ is independently selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, -NR¹⁰Rⁿ, -NR¹²C(O)R¹³, -NR¹²C(O)OR¹³, -NR¹²C(O)NR¹²R¹³, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C₆.₂₀ aryl, substituted C₆.₂₀ aryl, heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino, C_{4- 10} cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, substituted C_4-10 cycloalkenyl, cyano, halo, -OR¹⁰, -C(O)OR¹⁰, -SR¹⁰, - S(O)R¹⁰, -S(O)₂R₁₀; w is an integer from 1 to 10; x is an integer from 1 to 5; y is an integer from 1 to 4;

R² is selected from hydrogen, C_{1- 10}kyl, substituted C_{1- 10}kyl, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6.-10 aryl, C_4-10 cycloalkyl, substituted C_{4- 10} cycloalkyl, C_{4- 10} cycloalkenyl, and substituted C_{4- 10} cycloalkenyl; each R⁶ is independently selected from hydrogen, C_{1- 10}kyl, substituted C_1-10 alkyl, - NR^1OR11, -NR¹²C(O)R¹³, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20 aryl, heteroaryl, heterocyclic, heterocyclooxy, heterocyclothio, heteroarylamino, heterocycloamino, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, substituted C_4-10 cycloalkenyl, cyano, halo, -OR¹⁰, -C(O)OR¹⁰, -SR¹⁰, -S(O)R¹⁰, -S(O)₂R¹⁰;

R¹⁰ and R¹¹ are each independently selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C_6-20 aryl, substituted C_6-20aryl, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, and substituted C_4-10 cycloalkenyl;

R¹³ is selected from hydrogen, C_1-10 alkyl, substituted C_1-10 alkyl, C_2-10 alkenyl, substituted C_2-10 alkenyl, C_2-10 alkynyl, substituted C_2-10 alkynyl, C₆-C₁₀ aryl, substituted C_6-20 aryl, C_4-10 cycloalkyl, substituted C_4-10 cycloalkyl, C_4-10 cycloalkenyl, substituted C_4-10 cycloalkenyl, -CH2- N(CH₂CH₃)³⁺, and -CH₂-SO₃- ; or a pharmaceutically acceptable salt thereof.

[00164] In some instances of Formula 3, one of the R¹ groups is linked via a linker to an affinity agent (e.g., as described herein). In some cases, the linker includes a branched linker (e.g., as described herein).

[00165] In some instances, R² is hydrogen.

[00166] In some instances, each R⁶ is hydrogen.

[00167] In some cases, a photoisomerizable group present in a conjugate of the present disclosure is a compound of Formula 4:

Q² is a ligand (or a label or reactive group or second affinity agent), as described according to the present disclosure; w is an integer from 1 to 10;

R¹ is selected from hydrogen, C_1-10 alkyl, -NR¹⁰Rⁿ, -NR¹²C(O)R¹³, -NR¹²C(O)OR¹³ and -NR¹²C(O)NR¹²R¹³;

R² is hydrogen or C_1-10 alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and C_1-10 alkyl; R¹² is hydrogen or C_{1- 10}kyl; and

R¹³ is selected from hydrogen, C_1-10 alkyl, C_1-8 alkenyl, C_6-10 aryl, and substituted C_1-10 alkyl, or a pharmaceutically acceptable salt thereof.

[00168] In certain embodiments of Formula 4, R¹ is C_1-10 alkyl, such as C_1-8 alkyl, e.g., C_1-6 alkyl, C_1-5 alkyl or C_1-4 alkyl. In some embodiments of Formula 4, R¹ is C_1-4 alkyl.

[00169] In certain embodiments of Formula 4, R¹ is -NR¹⁰Rⁿ.

[00170] In certain embodiments of Formula 4, R¹ is -NR¹²C(O)R¹³.

[00171] In certain embodiment, R² is H.

[00172] In some instances of Formula 4, the R¹ group is linked via a linker to an affinity agent (e.g., as described herein). In some cases, the linker includes a branched linker (e.g., as described herein). For example, in embodiments where R¹ is -NR¹⁰R¹¹, the R¹ group can be linked via a linker to an affinity agent through either the R¹⁰ group or the R¹¹ group. In other cases, where R¹ is -NR¹²C(O)R¹³, the R¹ group can be linked via a linker to an affinity agent through the R¹³ group.

[00173] In some instances of Formulae 2, 3 or 4, Q² is a ligand, as described according to the present disclosure. In some instances of Formulae 2, 3 or 4, Q² is a label, as described according to the present disclosure. For example, the label can be a detectable label, such as a fluorophore, as described herein. In some instances of Formulae 2, 3 or 4, Q² is a reactive group, as described according to the present disclosure. In some instances of Formulae 2, 3 or 4, Q² is a second affinity agent, as described according to the present disclosure.

[00174] In some cases, a photoisomerizable group present in a conjugate of the present disclosure is an azobenzene moiety as shown below:

where the wavy lines indicate the attachment points to the rest of the conjugate. For instance, the wavy line on the left side of the azobenzene moiety may indicate the attachment point to a linker (e.g., a branched linker as described herein) and the wavy line on the right side of the azobenzene moiety may indicate the attachment point to a ligand as described herein.

[00175] In some cases, the photoisomerizable group includes an azobenzene moiety, such azobenzene photoisomerizable groups are described in WO 2019/060785, the disclosure of which is incorporated herein by reference in its entirety. In some cases, the photoisomerizable group comprises azobenzene 460.

Ligands

[00176] As noted above, a photoisomerizable regulator present in a conjugate of the present disclosure comprises: i) a photoisomerizable group; and ii) a ligand that binds to the target ligandbinding polypeptide.

[00177] As used herein, the term “ligand” refers to a molecule (e.g., a small molecule, a peptide, or a protein) that binds to a polypeptide and effects a change in an activity of the polypeptide, and/or effects a change in conformation of the polypeptide, and/or affects binding of another polypeptide to the polypeptide, or affects the impact of another ligand on the polypeptide. Ligands include agonists, partial agonists, inverse agonists, antagonists, allosteric modulators, and blockers.

[00178] In some cases, the ligand is a naturally-occurring ligand. In other cases, the ligand is a synthetic ligand. In some cases, the ligand is an endogenous ligand. In some cases, the ligand is an agonist. In some cases, the ligand is an inverse agonist. In other cases, the ligand is a partial agonist. In other cases, the ligand is an antagonist. In other cases, the ligand is an allosteric modulator. In other cases, the ligand is a blocker. The term “antagonist” generally refers to an agent that binds to a ligandbinding polypeptide and inhibits the binding of the ligand-binding polypeptide. An “antagonist” may be an agent that binds to or near the orthosteric site (same site where an agonist binds) or an allosteric site but does not activate the ligand-binding polypeptide; instead, the antagonist generally excludes binding by an agonist or hinders activation by the agonist and thus prevents or hinders activation. An “allosteric modulator” may be an agent that binds to an allosteric site away from an orthosteric ligand binding site where binding of an allosteric ligand either decreases the sensitivity to or efficacy of an orthosteric ligand (negative allosteric modulator) or increases the sensitivity to or efficacy of an orthosteric ligand (positive allosteric modulator). The term “blocker” refers to an agent that acts directly on the active site, pore, or allosteric site. Ligands suitable for use herein bind reversibly to a ligand-binding site of a ligandbinding polypeptide.

[00179] The ligand is selected based in part on the target ligand-binding polypeptide, and the desired effect on the target ligand-binding polypeptide. For example, a ligand for a hormone-binding transcription factor will in some cases be a hormone, or a synthetic analog of the hormone, or a ligand that interferes with or modulates positively or negatively hormone binding or action. A ligand for a tetracycline transactivator will in some cases be tetracycline or a synthetic analog thereof. A ligand for an enzyme will in some cases be a synthetic agonist or antagonist of the enzyme. In some cases, a ligand will block the ligand-binding site. A ligand for an enzyme or ion channel will in some case be a blocker of the enzyme active site or ion channel pore. A ligand for a ligand-gated ion channel or a G protein coupled receptor or other membrane associated or soluble receptors will in some cases be a naturally- occurring ligand, or a synthetic version of the ligand, e.g., a synthetic analog of the ligand, or a ligand that interferes with or modulates positively or negatively the binding or action of that ligand.

[00180] In some cases, a ligand is a small molecule ligand. Small molecule ligands can have a molecular weight in a range of from about 50 daltons to about 3000 daltons, e.g., from about 50 daltons to about 75 daltons, from about 75 daltons to about 100 daltons, from about 100 daltons to about 250 daltons, from about 250 daltons to about 500 daltons, from about 500 daltons to about 750 daltons, from about 750 daltons to about 1000 daltons, from about 1000 daltons to about 1250 daltons, from about 1250 daltons to about 1500 daltons, from about 1500 daltons to about 2000 daltons, from about 2000 daltons to about 2500 daltons, or from about 2500 daltons to about 3000 daltons.

[00181] In other cases, a ligand is a peptide ligand. Peptide ligands can have a molecular weight in a range of from about 1 kDa to about 20 kDa, e.g., from about 1 kDa to about 2 kDa, from about 2 kDa to about 5 kDa, from about 5 kDa to about 7 kDa, from about 7 kDa to about 10 kDa, from about 10 kDa to about 12 kDa, from about 12 kDa to about 15 kDa, or from about 15 kDa to about 20 kDa.

Peptide ligands can have a length of from 2 amino acids to 20 amino acids, e.g., a peptide ligand can have a length of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Peptide ligands can have a length of from 2 amino acids to 5 amino acids, from 5 amino acids to 10 amino acids, from 10 amino acids to 15 amino acids, or from 15 amino acids to 20 amino acids. Peptide ligands can be longer than 20 amino acids, e.g., up to 200 amino acids.

[00182] Suitable ligands include, but are not limited to, ligands that block or activate the function of a ligand-binding protein, where ligand-binding proteins include ion and macromolecule permeant channels; receptors (including, but not limited to, ionotropic receptors that bind transmitters; ionotropic receptors that bind hormones; metabotropic receptors and other G protein coupled receptors (including but not limited to mGluR receptors, such as mGluR2, mGluR3, mGluR5 and mGluR6); receptor tyrosine kinases; growth factor receptors; and other membrane receptors that signal by binding to soluble or membrane-bound or extracellular small molecules or proteins); transporters (including but not limited to ion transporters, organic molecule transporters, peptide transporters, and protein transporters); enzymes (including but not limited to kinases; phosphatases; ubiquitin ligases; acetylases; oxo-reductases; lipases; enzymes that add lipid moieties to proteins or remove them; proteases; and enzymes that modify nucleic acids, including but not limited to ligases, helicases, topoisomerases, and telomerases); motor proteins (including kinesins, dyenins and other microtobule-based motors, myosins and other actin-based motors, DNA and RNA polymerases and other motors that track along polynucleotides); scaffolding proteins; adaptor proteins; cytoskeletal proteins; and other proteins that localize or organize protein domains and superstructures within cells.

[00183] Suitable ligands include, but are not limited to, ligands that function as general anesthetics; ligands that function as local anesthetics; ligands that function as analgesics; synthetic and semi-synthetic opioid analgesics (e.g., phenanthrenes, phenylheptylamines, phenylpiperidines, morphinans, and benzomorphans) where exemplary opioid analgesics include morphine, oxycodone, fentanyl, pentazocine, hydromorphone, meperidine, methadone, levorphanol, oxymorphone, levallorphan, codeine, dihydrocodeine, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, and pentazocine; ionotropic glutamate receptor agonists and antagonists, e.g., N-methyl-D-aspartate (NMD A) receptor agonists, antagonists, and allosteric modulators, kainate (KA) receptor agonists and antagonists, and allosteric modulators, a- amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMP A) receptor agonists and antagonists and allosteric modulators, and metabotropic glutamate receptor agonists and antagonists and allosteric modulators; non-opioid analgesics, e.g., acetylsalicylic acid, choline magnesium trisalicylate, acetaminophen, ibuprofen, fenoprofen, diflusinal, and naproxen; muscarinic receptor agonists; muscarinic receptor antagonists; acetylcholine receptor agonists; acetylcholine receptor antagonists; serotonin receptor agonists; serotonin receptor antagonists; enzyme inhibitors; a benzodiazepine, e.g. chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam; a barbiturate sedative, e.g. amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobartital, secobarbital, talbutal, theamylal, or thiopental; an Hi antagonist having a sedative action, e.g. diphenhydramine, pyrilamine, promethazine, chlorpheniramine, or chlorcyclizine; an NMDA receptor antagonist, e.g. dextromethorphan ((+)-3-hydroxy-N- methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid, budipine, topiramate, neramexane, or perzinfotel; an alpha-adrenergic, e.g. doxazosin, tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, phentolamine, terazasin, prazasin or 4-amino-6,7-dimethoxy-2- (5-methane-sulfonamido-l,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl) quinazoline; a tricyclic antidepressant, e.g. desipramine, imipramine, amitriptyline, or nortriptyline; an anticonvulsant, e.g. carbamazepine, lamotrigine, topiratmate, or valproate; a tachykinin (NK) antagonist, particularly an NK- 3, NK-2 or NK-1 antagonist, e.g. (a-R,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,l l-tetrahydro-9- methyl-5-(4-methylphenyl)-7H-[l,4]diazocino[2,l-g][l,7]-naphthyridine-6-13-dione (TAK-637), 5- [[(2R,3S)-2-[(lR)-l-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]- l,2-dihydro-3H-l,2,4-triazol-3-one (MK-869), aprepitant, lanepitant, dapitant or 3-[[2-methoxy-5- (trifluoromethoxy)phenyl] -methylamino] -2-phenylpiperidine (2S,3S); a muscarinic antagonist, e.g. oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin, solifenacin, temiverine, or ipratropium; a cyclooxygenase-2 (COX-2) selective inhibitor, e.g. celecoxib, rofecoxib, parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib; a vanilloid receptor agonist (e.g. resinferatoxin) or antagonist (e.g. capsazepine); a beta-adrenergic such as propranolol; a 5-HT receptor agonist or antagonist, e.g., a 5-HTiB/iD agonist such as eletriptan, sumatriptan, naratriptan, zolmitriptan or rizatriptan; a 5-HT2A receptor antagonist such as R(+)-a-(2,3-dimethoxy-phenyl)-l-[2-(4- fluorophenylethyl)]-4-piperidinemethanol (MDL-100907); and the like.

[00184] Suitable ligands for Na⁺ channels include, but are not limited to, lidocaine, novocaine, xylocaine, lignocaine, novocaine, carbocaine, etidocaine, procaine, prontocaine, prilocaine, bupivacaine, cinchocaine, mepivacaine, quinidine, flecainide, procaine, N-[[2’-(aminosulfonyl)biphenyl-4-yl]methyl]- N’-(2,2’-bithien-5-ylmethyl)succinamide (BPBTS), QX-314, saxitoxin, tetrodotoxin, and a type III conotoxin. Suitable ligands for Na⁺ channels also include, but are not limited to, tetrodotoxin, saxitoxin, guanidinium, polyamines (e.g. spermine, cadaverine, putrescine, IJ -conotoxin, and 5-conotoxin.

[00185] Suitable ligands for K⁺ channels include, but are not limited to, quaternary ammonium (e.g., tetraethyl ammonium, tetrabutylammonium, tetrapentylammonium), 4-aminopyridine, sulfonylurea, Glibenclamide; Tolbutamide; Phentolamine, qiunine, qunidine, peptide toxins (e.g., charybdotoxin, agitoxin-2, apamin, dendrotoxin, VSTX1, hanatoxin-1, hanatoxin-2, and Tityus toxin K- a.

[00186] Suitable ligands for CNG and HCN channels include, but are not limited to, 1-cis diltiazem and ZD7288. Suitable ligands for glycine receptors include, but are not limited to, strychnine and picrotoxin.

[00187] Suitable ligands for nicotinic acetylcholine receptors include, but are not limited to, (+)- tubocurarine, Methyllycaconitine, gallamine, Nicotine; Anatoxin A, epibatidine, ABT-94, Lophotoxin, Cytisine, Hexamethonium, Mecamylamine, and Dihydro-P-erythroidine. Suitable ligands for muscarinic acetylcholine receptors include, but are not limited to, a muscarinic acetylcholine receptor antagonist as described in U.S. Patent No. 7,439,255; AF267B (see, e.g., U.S. Patent No. 7,439,251); phenylpropargyloxy- 1,2, 5 -thiadiazole-quinuclidine; carbachol; pirenzapine; migrastatin; a compound as described in U.S. Patent No. 7,232,841; etc.

[00188] Suitable ligands for GABA receptors include, but are not limited to, Muscimol, THIP, Procabide, bicuculine, picrotoxin, gabazine, gabapentin, diazepam, clonazepam, flumazenil, a P- carboline carboxylate ethyl ester, baclofen, faclofen, and a barbiturate.

[00189] In some cases, e.g., where the target ligand-binding polypeptide is an mGluR, the ligand is glutamate. In some cases, where the target ligand-binding polypeptide is an mGluR receptor, the ligand will be a naturally occurring or synthetic ligand of an mGluR receptor (including but not limited to mGluR2, mGluR3, mGluR5 and mGluR6).

[00190] In some cases, where the target ligand-binding polypeptide is an mGluR (e.g., mGluR2), the ligand is glutamate. In some cases, where the target ligand-binding polypeptide is an mGluR (e.g., mGluR2), the ligand is an mGluR2 agonist, e.g., where the agonist is selected from ( l /?,4/?,5.S',6/?)-4- amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268); (IS, 2S,5R, 6S)-2- aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740); (lS,2S,4R,5R,6S)-rel-2-amino-4- methylbicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY395756); (S)-2-amino-2-methyl-4- phosphonobutanoic acid (MAP4); (2S,2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine (DCG IV);

(lS,3R)-l-aminocyclopentane-l,3-dicarboxylic acid ((1S,3R)-ACPD); (2R,4R)-4-aminopyrrolidine-2,4- dicarboxylate ((2R,4R)-APCD); (lS,2S,4R,5R,6S)-2-amino-4-methylbicyclo[3.1.0]hexane-2,6- dicarboxylic acid (LY541850); (lR,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide (LY404039); (lR,4S,5S,6S)-4-((S)-2-amino-4-(methylthio)butanamido)-2- thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide (LY2140023); and a l,2,4-triazolo[4,3- a]pyridine compound as described in US 2019/0047999.

[00191] The structure of LY379268 can be as follows:

[00192] Many suitable ligands will be known to those skilled in the art; and the choice of ligand will depend, in part, on the target (e.g., receptor, ion channel, enzyme, etc.) to which the ligand binds. Fluorophores

[00193] In some cases, a conjugate of the present disclosure comprises a fluorophore. For example, as noted above, Q² can be a label, such as a fluorophore. Examples of fluorophores include, but are not limited to: an Alexa Fluor® dye (e.g., Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 500, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor® 633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, Alexa Fluor® 790, and the like), an ATTO dye (e.g., ATTO 390, ATTO 425, ATTO 465, ATTO 488, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO Rho6G, ATTO 542, ATTO 550, ATTO 565, ATTO Rho3B, ATTO Rhol l, ATTO Rhol2, ATTO Thiol2, ATTO RholOl, ATTO 590, ATTO 594, ATTO Rhol3, ATTO 610, ATTO 620, ATTO Rhol4, ATTO 633, ATTO 647, ATTO 647N, ATTO 655, ATTO Oxal2, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO 740), a DyLight dye, a cyanine dye (e.g., Cy2, Cy3, Cy3.5, Cy3b, Cy5, Cy5.5, Cy7, Cy7.5), a FluoProbes dye, a Sulfo Cy dye, a Seta dye, an IRIS Dye, a SeTau dye, an SRfluor dye, a Square dye, fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), tetramethylrhodamine (TMR), Silicon Rhodamine (SiR), Texas Red, Oregon Green, Pacific Blue, Pacific Green, and Pacific Orange.

Target ligand-binding polypeptides

[00194] As noted above, a conjugate of the present disclosure includes a ligand that binds to a target ligand-binding polypeptide.

[00195] Suitable target ligand-binding proteins include ion and macromolecule permeant channels; receptors (including, but not limited to, ionotropic receptors that bind transmitters; ionotropic receptors that bind hormones; metabotropic receptors and other G protein coupled receptors; receptor tyrosine kinases; growth factor receptors; and other membrane receptors that signal by binding to soluble or membrane-bound or extracellular small molecules or proteins); transporters (including but not limited to ion transporters, organic molecule transporters, peptide transporters, and protein transporters); enzymes (including but not limited to kinases; phosphatases; ubiquitin ligases; acetylases; oxoreductases; lipases; enzymes that add lipid moieties to proteins or remove them; proteases; and enzymes that modify nucleic acids, including but not limited to ligases, helicases, topoisomerases, and telomerases); motor proteins (including kinesins, dyenins and other microtobule-based motors, myosins and other actin-based motors, DNA and RNA polymerases and other motors that track along polynucleotides); scaffolding proteins; adaptor proteins; cytoskeletal proteins; and other proteins that localize or organize protein domains and superstructures within cells.

[00196] In some cases, the target ligand-binding polypeptide is a transcription regulator, an ion channel, a cation channel, a ligand-gated ion channel, a voltage-gated ion channel, a quorum sensor, a pheromone receptor, a neurotransmitter receptor, a G protein-coupled receptor (GPCR), or an enzyme. In some cases, the target ligand-binding polypeptide is a cation channel, e.g., a potassium channel, a sodium channel, or a calcium channel. In some cases, the target ligand-binding polypeptide is a glutamate receptor, a metabotropic glutamate receptor, an ionotropic glutamate receptor, an ionotropic nicotinic acetylcholine receptor, an ionotropic GABA-A receptor, or an ionotropic purinergic P2X receptor. In some cases, the target ligand-binding polypeptide is selected from a glutamate receptor, a metabotropic glutamate receptor (mGluR) an ionotropic glutamate receptor (e.g., a kainate receptor; an AMPA receptor; an NMDA receptor), an ionotropic nicotinic acetylcholine receptor, an ionotropic GABA-A receptor, a metabotropic GABA-B receptor, a metabotropic dopamine receptor, an ionotropic purinergic P2X receptor, a metabotropic purinergic P2Y receptor, a metabotropic serotonin receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, a cation channel, a potassium channel, a calcium channel, a sodium channel, a proton channel, an anion channel and a chloride channel. [00197] In some cases, the target ligand-binding polypeptide is selected from a metabotropic glutamate receptor, where metabotropic glutamate receptors include, e.g., mGluR2, mGluR3, mGluR5, mGluR6, and the like.

Polypeptides that bind target ligand-binding polypeptides

[00198] As noted above, a conjugate of the present disclosure includes an affinity agent that binds: i) a target ligand-binding polypeptide; or ii) a polypeptide that binds a target ligand-binding polypeptide.

[00199] In some cases, a polypeptide that binds a target ligand-binding polypeptide is a fusion polypeptide comprising: i) an antibody that binds the target ligand-binding polypeptide; and ii) a polypeptide that binds the affinity agent present in the conjugate. For example, a polypeptide that binds the affinity agent present in the conjugate can be a SNAP polypeptide (where the affinity agent is benzylguanine), a CLIP polypeptide (where the affinity agent is benzylcytosine), or a HALO polypeptide (where the affinity agent is a chloroalkane).

Exemplary conjugates

[00200] In some cases, a conjugate of the present disclosure is a compound having Formula I: (A)-(X¹)_n-(B)-[(X²)_m-(C)-(X³)p-(D)]_q (Formula I) wherein:

A is an affinity agent;

X¹, when present, is a first linker, wherein n is 0 or 1;

B is a branched linker (e.g., as described herein);

X², when present, is a second linker, wherein m is 0 or 1;

C is a photoisomerizable group;

X³, when present, is a third linker, wherein p is 0 or 1;

D is a ligand; and q is 9.

[00201] Suitable affinity agents are those described in the present disclosure. For example, as described in the present disclosure, the affinity agent can be, but is not limited to, benzylguanine, benzylcytosine, chloroalkane, an antibody, an aptamer, a small molecule or a peptide, and the like. In some cases, the affinity agent is an antibody specific for a target ligand-binding polypeptide. Nonlimiting examples of suitable antibodies include, e.g., a nanobody specific for a target ligand-binding polypeptide (e.g., a nanobody that specifically binds mGluR2); and a scFv antibody specific for a target ligand-binding polypeptide (e.g., a scFv that specifically binds mGluR2). In some instances, the affinity agent is benzylguanine, e.g., O(6)-benzylguanine. [00202] X¹, when present, is a first linker, wherein n is 0 or 1. For instance, when n is 0, then X¹ is not present, and A is connected directly to B. In other instances, n is 1 and X¹ is present.

[00203] Similarly, X², when present, is a second linker, wherein m is 0 or 1. For instance, when m is 0, then X² is not present, and B is connected directly to C (e.g., each arm of the branched linker, B, is connected directly to its respective photoisomerizable group, C). In other instances, m is 1 and X² is present (e.g., each arm of the branched linker, B, includes a second linker, X²).

[00204] Similarly, X³, when present, is a third linker, wherein p is 0 or 1. For instance, when p is 0, then X³ is not present, and C is connected directly to D (e.g., each photoisomerizable group, C, is connected directly to its corresponding ligand, D). In other instances, p is 1 and X³ is present (e.g., each arm of the branched linker, B, includes a third linker, X³).

[00205] In some cases, at least one of X¹, X² and X³ is present. In some cases, only one of X¹, X² and X³ is present. In some cases, X¹ and X² are present and X³ is absent. In some cases, X¹ and X³ are present and X² is absent. In some cases, X² and X³ are present and X¹ is absent. In some cases, X¹, X² and X³ are each present.

[00206] Suitable linkers for X¹, X² and/or X³ include, but are not limited to, a polycarbon chain; poly(ethylene glycol); a peptide; and the like. In some cases, the linker is a C1-C25 alkyl. In some cases, the linker is a substituted C1-C25 alkyl. In some cases, the linker is poly(ethylene glycol) (PEG), where the PEG comprises from 2 to 50 ethylene glycol monomers; e.g., the PEG comprises from 2 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, or from 45 to 50, ethylene glycol units. For example, the linker can be PEG, where the PEG comprises 6 ethylene glycol monomers (i.e., PEG6). In some cases, the PEG linker comprises 12 ethylene glycol monomers (i.e., PEG12) In some cases, the linker is a peptide of from 2 amino acids to 50 amino acids; e.g., from 2 amino acids to 5 amino acids, from 5 amino acids to 10 amino acids, from 10 amino acids to 15 amino acids, from 15 amino acids to 20 amino acids, from 20 amino acids to 25 amino acids, from 25 amino acids to 30 amino acids, or from 30 amino acids to 50 amino acids. In some cases, the linker is a peptide of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length.

[00207] Suitable branched linkers, B, include those described in the present disclosure. For example, suitable branched linkers include branched linkers of Formula (BL), as described herein. Other suitable branched linkers include, but are not limited to dendrimeric structures, such as polyamidoamine (PAMAM) dendrimers, polyethyleneglycol (PEG) dendrimers, and the like.

[00208] Suitable photoisomerizable groups, C, include those described in the present disclosure, such as, but not limited to a moiety selected from an azobenzene, a cyclic azobenzene, an azoheteroarene, a fulgide, a spiropyran, a triphenyl methane, a thioindigo, a diarylethene, and an overcrowded alkene. In some cases, the photoisomerizable group comprises an azobenzene moiety, as described herein.

[00209] Suitable ligands, D, include those described in the present disclosure. In some cases, the ligand is an agonist, an antagonist, an allosteric modulator, or a blocker (e.g., as described herein). In some instances, the ligand, D, comprises glutamate.

[00210] In other embodiments of Formula I, D is a label, a reactive group, or a second affinity agent as described in the present disclosure.

[00211] In some cases, the conjugate has the formula (A)-(X¹)_n-(B)-[(X²)_m-(C)-(D)]_q.

[00212] In some cases, the conjugate has the formula (A)-(X¹)_n-(B)-[(C)-(D)]_q.

[00213] In some cases, the conjugate has the formula (A)-(B)-[(X²)_m-(C)-(X³)p-(D)]_q.

[00214] In some cases, the conjugate has the formula (A)-(B)-[(X²)_m-(C)-(D)]_q.

[00215] In some cases, the conjugate has the formula (A)-(X¹)_n-(B)-[(X²)_m-(C)-(X³)p-(D)]_q.

[00216] Any combination of suitable A, B, C and D moieties, with or without the first linker (X¹), the second linker (X²) or the third linker (X³), may be used in a conjugate according to the present disclosure. Examples of conjugates include, but are not limited to conjugates where:

A is benzylguanine, B is a branched linker of Formula (BL), C is an azobenzene moiety, D is glutamate, and q is 9. For example, in some instances, A is benzylguanine, X¹ is -CH₂NHC(O)(CH₂)₃C(O)NH(PEG₂)CH₂-, B is a branched linker of Formula (BL), X² is -(PEG6)(CH₂)₂C(O)NH(CH₂)₂-, C is azobenzene 460, X³ is -(CH_{2 3}-, D is glutamate, and q is 9. *

[00217] Any combination of suitable A, B, C and D moieties, with or without the first linker (X¹), the second linker (X²) or the third linker (X³), may be used in a conjugate according to the present disclosure. Examples of conjugates include, but are not limited to conjugates where:

A is an antibody specific for a target ligand-binding polypeptide, B is a branched linker of Formula (BL), C is azobenzene 460, D is glutamate, and q is 9;

A is an antibody specific for a metabotropic glutamate receptor (mGluR; e.g., mGluR2, mGluR3, mGluR4, mGluR5, or mGluR6), B is a branched linker of Formula (BL), C is azobenzene 460, D is glutamate, and q is 9;

A is a nanobody specific for mGluR (e.g., mGluR2, mGluR3, mGluR4, mGluR5, or mGluR6), B is a branched linker of Formula (BL), C is azobenzene 460, D is glutamate, and q is 9;

A is a scFv specific for mGluR (e.g., mGluR2, mGluR3, mGluR4, mGluR5, or mGluR6), B is a branched linker of Formula (BL), C is azobenzene 460, D is glutamate, and q is 9; and the like. COMPOSITIONS AND COMBINATION COMPRISING A PHOTOSWITCH CONJUGATE AND A FUSION

POEYPEPTIDE

[00218] In some cases, the present disclosure provides compositions and/or combinations comprising: a) a photoswitch conjugate of the present disclosure; and b) a fusion polypeptide that comprises a polypeptide that binds to the affinity agent present in the photoswitch conjugate, or a polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide. The photoswitch conjugate and the fusion polypeptide or fusion polynucleotide can be a part of the same composition and administered to a subject together. Alternatively, the photoswitch conjugate and the fusion polypeptide or fusion polynucleotide can be a part of a therapeutic approach comprising a combination of agents administered at different times and/or by different means or modes of administration. In some cases, a composition of the present disclosure comprises: a) a photoswitch conjugate of the present disclosure; and b) a fusion polypeptide (or a polynucleotide encoding the fusion polypeptide) comprising: i) a receptor for a ligand (e.g., a ligand present in a photoswitch conjugate of the present disclosure); and ii) a fusion partner, where the fusion partner is a polypeptide that binds the affinity moiety present in the photoswitch conjugate. In some cases, a composition of the present disclosure comprises: a) a photoswitch conjugate of the present disclosure; and b) a first fusion polypeptide (or a polynucleotide encoding it) comprising: i) a receptor for a ligand (e.g., a ligand present in a photoswitch conjugate of the present disclosure); and ii) a fusion partner, where the fusion partner displays an epitope that is bound by an antibody present in a second fusion polypeptide, the second fusion polypeptide comprising: i) an antibody that binds the epitope present in the fusion partner of the first fusion polypeptide; and ii) a polypeptide binds the affinity moiety present in the photoswitch conjugate.

Composition comprising a photoswitch conjugate and a single fusion polypeptide

[00219] In some cases, a composition or combination of the present disclosure comprises: a) a photoswitch conjugate of the present disclosure; and b) a fusion polypeptide (or a polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide) that comprises a polypeptide that binds to the affinity agent present in the photoswitch conjugate. In some cases, the fusion polypeptide comprises: a) a receptor for a ligand (e.g., a ligand present in a photoswitch conjugate of the present disclosure); and b) a fusion partner, where the fusion partner is a SNAP polypeptide, a CLIP polypeptide, or a HALO polypeptide. For example, in some cases, the receptor for the ligand is a glutamate receptor, a metabotropic glutamate receptor (mGluR), an ionotropic glutamate receptor (e.g., a kainate receptor; an AMPA receptor; an NMDA receptor), an ionotropic nicotinic acetylcholine receptor, an ionotropic GABA-A receptor, a metabotropic GABA-B receptor, a metabotropic dopamine receptor, an ionotropic purinergic P2X receptor, a metabotropic purinergic P2Y receptor, a metabotropic serotonin receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, a cation channel, a potassium channel, a calcium channel, a sodium channel, a proton channel, an anion channel, and a chloride channel. [00220] In some cases, a composition or combination of the present disclosure comprises: a) a conjugate of the present disclosure; and b) a nucleic acid comprising a nucleotide sequence encoding the fusion polypeptide. The nucleic acid can be an expression vector. The nucleotide sequence can be operably linked to a promoter. The expression vector can be, e.g., a recombinant viral expression vector. As a non-limiting example, the expression vector can be an adeno-associated virus (AAV) vector, where the recombinant AAV vector comprises a heterologous nucleotide sequence encoding the fusion polypeptide. The recombinant AAV vector can also comprise a nucleotide sequence encoding a variant capsid protein, where the variant capsid protein provides for infection of a retinal cell. In some cases, the recombinant AAV vector will be administered to the eye. In some cases, the recombinant AAV vector will be administered to the eye intravitreally.

[00221] In some cases, a composition or combination of the present disclosure comprises: a) a photoswitch conjugate of the present disclosure, where the affinity agent is a nucleoside base derivative as described above (e.g., benzylguanine; benzylcytosine; chloroalkane; a chloropyrimidine; etc.); and b) a fusion polypeptide that comprises: i) an antibody (e.g., a scFv or a nanobody) specific for a target ligand-binding polypeptide; and ii) a polypeptide (e.g., a SNAP polypeptide; a CLIP polypeptide; a HALO polypeptide; and the like) that binds to the affinity agent present in the conjugate. In some cases, a composition or combination of the present disclosure comprises: a) a photoswitch conjugate of the present disclosure, where the affinity agent is a nucleoside base derivative as described above (e.g., benzylguanine; benzylcytosine; chloroalkane; a chloropyrimidine; etc.); and b) a fusion polypeptide that comprises: i) a nanobody or an scFv specific for an mGluR; and ii) a polypeptide (e.g., a SNAP polypeptide; a CLIP polypeptide; a HALO polypeptide; and the like) that binds to the affinity agent present in the conjugate. In some cases, a composition or combination of the present disclosure comprises: a) a photoswitch conjugate of the present disclosure, where the affinity agent is a nucleoside base derivative as described above (e.g., benzylguanine; benzylcytosine; chloroalkane; a chloropyrimidine; etc.); and b) a fusion polypeptide that comprises: i) a nanobody or an scFv specific for mGluR2, mGluR3, mGluR5, or mGluR5; and ii) a polypeptide (e.g., a SNAP polypeptide; a CLIP polypeptide; a HALO polypeptide; and the like) that binds to the affinity agent present in the conjugate. [00222] In some cases, a fusion polypeptide comprises: a) a SNAP polypeptide; a CLIP polypeptide; a HALO polypeptide; and b) an mGluR2 polypeptide comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

KKVLTLEGDLVLGGLFPVHQKGGPAEDCGPVNEHRGIQRLEAMLFALDRINRDPHLLPGVRLG AHILDSCSKDTHALEQALDFVRASLSRGADGSRHICPDGSYATHGDAPTAITGVIGGSYSDVSIQ VANLLRLFQIPQISYASTSAKLSDKSRYDYFARTVPPDFFQAKAMAEILRFFNWTYVSTVASEGD YGETGIEAFELEARARNICVATSEKVGRAMSRAAFEGVVRALLQKPSARVAVLFTRSEDARELL AASQRLNASFTWVASDGWGALESVVAGSEGAAEGAITIELASYPISDFASYFQSLDPWNNSRNP WFREFWEQRFRCSFRQRDCAAHSLRAVPFEQESKIMFVVNAVYAMAHALHNMHRALCPNTTR LCDAMRPVNGRRLYKDFVLNVKFDAPFRPADTHNEVRFDRFGDGIGRYNIFTYLRAGSGRYRY QKVGYWAEGLTLDTSLIPWASPSAGPLPASRCSEPCLQNEVKSVQPGEVCCWLCIPCQPYEYRL DEFTCADCGLGYWPNASLTGCFELPQEYIRWGDAWAVGPVTIACLGALATLFVLGVFVRHNAT PVVKASGRELCYILLGGVFLCYCMTFIFIAKPSTAVCTLRRLGLGTAFSVCYSALLTKTNRIARIF GGAREGAQRPRFISPASQVAICLALISGQLLIVVAWLVVEAPGTGKETAPERREVVTLRCNHRD ASMLGSLAYNVLLIALCTLYAFKTRKCPENFNEAKFIGFTMYTTCIIWLAFLPIFYVTSSDYRVQT TTMCVSVSLSGSVVLGCLFAPKLHIILFQPQKNVVSHRAPTSRFGSAAARASSSLGQGSGSQFVP TVCNGREVVDSTTSSL (SEQ ID NO:5).

[00223] As one non-limiting example, in some cases, the fusion polypeptide comprises: i) a SNAP polypeptide; and ii) an mGluR2 polypeptide. FIG. 10B and 10D provide amino acid sequences of SNAP-mGluR2 fusion polypeptides. FIG. 10A provides an amino acid sequence of a SNAP-mGluR2 with an mGluR2 signal peptide. FIG. 10C provides an amino acid sequence of a SNAP-mGluR2 with an mGluR5 signal peptide. An expression vector can comprise a nucleotide sequence encoding the amino acid sequence depicted in FIG. 10A or FIG. 10C, such that a SNAP-mGluR2 fusion polypeptide fusion polypeptide comprising a signal peptide is synthesized in a retinal cell. The signal peptide can be cleaved following synthesis, generating a SNAP-mGluR2 fusion polypeptide comprising the amino acid sequence depicted in FIG. 10B or FIG. 10D.

[00224] In some cases, a fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 10B amino acid sequence depicted in FIG. 10B. In some cases, a fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 10D or a functional fragment of the amino acid sequence depicted in FIG. 10D.

[00225] In some cases, an expression vector (e.g., a recombinant AAV vector) comprises a nucleotide sequence encoding a fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 10A. In some cases, an expression vector (e.g., a recombinant AAV vector) comprises a nucleotide sequence encoding a fusion polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, amino acid sequence identity to the amino acid sequence depicted in FIG. 10C. Composition comprising a photoswitch conjugate and two fusion polypeptides

[00226] In some cases, the fusion polypeptide comprises: a) a receptor for a ligand (e.g., a ligand present in a photoswitch conjugate of the present disclosure); and b) a fusion partner, where the fusion partner is an antigen that is bound specifically by an antibody. In some cases, the antibody is a fusion polypeptide comprising: a) the antibody; and b) a fusion partner, where the fusion partner is a SNAP polypeptide, a CLIP polypeptide, or a HALO polypeptide. Other suitable fusion partners include, e.g., an epitope tag (e.g., a hemagglutinin tag, a FLAG tag, a poly(His) tag, and the like). Also suitable for use is a halo-based oligonucleotide binder (HOB) polypeptide. See, e.g., Kossman et al. (2016) Chembiochem. 17:1102. A HOB polypeptide binds chlorohexyl moieties. Also suitable for use is a trimethoprim (TMP) tag, an engineered form of E. coli dihydrofolate reductase (DHFR) that forms a non-covalent high- affinity complex with trimethoprim derivatives. See, e.g., Gallagher et al. (2009) ACS Chem. Biol. 4:547; and Jing and Cornish (2013) ACS Chem. Biol. 8:1704.

SNAP

[00227] In some cases, a SNAP polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPLMQAT AWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYSHLAALAGNPAAT AAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAVKEWLLAHEGHRLGKPGLG (SEQ ID NO:1). A SNAP polypeptide binds O⁶-benzylguanine (BG).

[00228] In some cases, a SNAP polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: DKDCEMKRTTLDSPLGKLELSGCEQGLHEIKLLGKGTSAADAVEVPAPAAVLGGPEPLMQATA WLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISYQQLAALAGNPAAT AAVKTALSGNPVPILIPCHRVVSSSGAVGGYEGGLAVKEWLLAHEGHRLGKPGLG (SEQ ID NO:4).

[00229] In some cases, the SNAP polypeptide or variant thereof binds to benzylguanine.

CLIP

[00230] A CLIP polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence:

MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEVPAPAAVLGGPEPLIQATA WLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKVVKFGEVISESHLAALVGNPAATA AVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLAVKEWLLAHEGHRLGKPGLG (SEQ ID NO:2). A CLIP polypeptide can bind O² -benzylcytosine (BC).

HALO

[00231] A HALO polypeptide can comprise an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%, amino acid sequence identity to the following amino acid sequence: MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAP DLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIA FMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREP FLNPVDREPLWRFPNELPIAGEPANIVALVEEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLA KSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEISG (SEQ ID NOG). A HALO polypeptide binds chloroalkane.

Vectors

[00232] The present disclosure also provides expression vectors, delivery vectors and other vectors comprising the compositions and/or combinations described herein.

[00233] Expression vectors include, but are not limited to, any vector suitable for in vitro or ex vivo delivery of a composition of the disclosure to a cell of the disclosure, by any means. In some embodiments, an expression vector comprises a plasmid. In some cases, the plasmid is electroporated into a cell of the disclosure. Expression vectors of the disclosure may also comprise delivery vectors of the disclosure when used to introduce a composition in vitro or ex vivo.

[00234] Delivery vectors include, but are not limited to, any vector suitable for in vivo delivery of a composition of the disclosure to a cell of the disclosure when in vivo or in situ (in the context of an intact eye). Delivery vectors of the disclosure include, but are not limited, to viral vectors and non-viral vectors. Exemplary viral vectors include, but are not limited to, adeno-associated vectors of any serotype. Exemplary non-viral vectors include, but are not limited to, lipid vectors, polymer vectors and particle vectors. Lipid vectors include, but are not limited to, liposomes, lipid nanoparticles, micelles, lipid polymersomes, and exosomes. Polymer vectors include, but are not limited to, polymersomes, lipid nanoparticles, and nanoparticles. Particle vectors include, but are not limited to, nanoparticles of all geometries and compositions. [00235] In some cases, a delivery vector of the disclosure comprises a composition of the disclosure, including a composition comprising a sequence encoding a promoter operably linked to a polynucleotide encoding a polypeptide of interest. In some embodiments of the delivery vectors of the disclosure, the vector is a viral vector. In some cases, the viral vector is an adeno-associated vector (AAV). In some cases, the AAV is a recombinant AAV (rAAV). In some cases, the rAAV comprises a sequence isolated or derived from an AAV of a first serotype and a sequence isolated or derived from an AAV of a second serotype. In some cases, the rAAV comprises a capsid sequence isolated or derived from an AAV of a first serotype and a capsid insert sequence isolated or derived from an AAV of a second serotype. Exemplary AAV serotypes include, but are not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and any combination thereof. In some cases, an AAV vector of the disclosure comprises a sequence isolated or derived from one or more of AAV2, AAV4, AAV5 and AAV8. In some cases, an AAV vector of the disclosure comprises a wild type sequence from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In some cases, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from one or more of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9. In some cases, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from AAV2 and AAV4. In some cases, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from AAV2 and AAV5. In some cases, an AAV vector of the disclosure comprises a capsid sequence isolated or derived from AAV2 and AAV8. In some cases, an AAV vector of the disclosure comprises a recombinant or chimeric capsid sequence comprising two or more sequences isolated or derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and AAV9.

[00236] In certain specific embodiments of the present disclosure, modified adeno-associated vectors (AAV) are used as described in any of WO 2012/145601, WO 2018/022905, WO 2019/006182, and/or US Application Serial No. 63/032,206, the contents of which are incorporated herein by reference in their entireties. As one non-limiting example, in some cases, a modified AAV comprises a variant AAV capsid protein comprising an insertion of a peptide in the GH loop of the capsid protein, e.g., where the insertion site is within amino acids 570-611 (e.g., between amino acids 587 and 588) of an AAV2 capsid protein, or a corresponding site in another AAV serotype. In some cases, the peptide inserted into the GH loop of the capsid protein comprises the amino acid sequence LGETTRP (SEQ ID NO:6). As another example, in some cases, a peptide inserted into the GH loop of an AAV capsid protein comprises an amino acid sequence selected from the group consisting of LATTSQNKPA (SEQ ID NO:7), LAVDGAQRSA (SEQ ID NO: 8), LAKSDQSKPA (SEQ ID NO: 9) and LAANQPSKPA (SEQ ID NO: 10) as described in WO2018/022905. As another example, in some cases, a peptide inserted into the GH loop of an AAV capsid protein comprises an amino acid sequence selected from the group consisting of LAHQDTTKNS (SEQ ID NO: 11), LAHQDSTKNA (SEQ ID NO: 12), LAHQDATKNA (SEQ ID NO: 13), LALSEATRPA (SEQ ID NO: 14), LAKDETKNSA (SEQ ID NO: 15), LQRGNRQTTTADVNTQ (SEQ ID NO: 16), LQRGNRQATTEDVNTQ (SEQ ID NO: 17), SRTNTPSGTTTQPTLQFSQ (SEQ ID NO: 18) and SKTDTPSGTTTQSRLQFSQ (SEQ ID NO: 19). [00237] In some cases, delivery vectors, including AAV vectors, target a retinal cell type. In some cases, delivery vectors, including AAV vectors, have a tropism for a retinal cell type. In some cases, the retinal cell type is a neuron. In some cases, the retinal cell type is a retinal ganglion cell. In some cases, the retinal cell type is a horizontal cell. In some cases, the retinal cell type is an amacrine cell. In some cases, the retinal cell type is a bipolar cell. In some cases, the retinal cell type is a photoreceptor cell. In some cases, the retinal cell type is not a photoreceptor. Photoreceptor cells include rod cells and cone cells.

[00238] In some cases, the cell is a retinal neuron or a progenitor cell thereof. In some embodiments, the progenitor cell is a neural fold cell, an early retinal progenitor cell (RPC), a late RPC, an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), or a retinal pigmented epithelial (RPE) cell. In some embodiments, ESCs of the disclosure are neither isolated nor derived from a human embryo or human tissue.

[00239] In some cases, a composition of the disclosure may be delivered to a differentiated cell and/or a progenitor cell capable of becoming the differentiated cell type.

Regulatory elements

[00240] In some cases, a nucleotide sequence encoding a polypeptide of the disclosure will be operably linked to one or more transcriptional regulatory elements. For example, in some cases, a nucleotide sequence encoding a gene product of interest is operably linked to a promoter, such as a constitutive promoter. In other cases, a nucleotide sequence encoding a gene product of interest is operably linked to an inducible promoter. In some instances, a nucleotide sequence encoding a gene product of interest is operably linked to a tissue-specific or cell type-specific regulatory element. For example, in some instances, a nucleotide sequence encoding a gene product of interest is operably linked to a retinal cell-specific promoter. For example, in some instances, a nucleotide sequence encoding a gene product of interest is operably linked to a photoreceptor-specific regulatory element (e.g., a photoreceptor-specific promoter), e.g., a regulatory element that confers selective expression of the operably linked gene in a photoreceptor cell. Suitable photoreceptor-specific regulatory elements include, e.g., a rhodopsin promoter; a rhodopsin kinase promoter (Young et al. (2003) Ophthalmol. Vis. Sci. 44:4076); a beta phosphodiesterase gene promoter (Nicoud et al. (2007) J. Gene Med. 9:1015); a retinitis pigmentosa gene promoter (Nicoud et al. (2007) supra)-, an interphotoreceptor retinoid-binding protein (IRBP) gene enhancer (Nicoud et al. (2007) supra)-, an IRBP gene promoter (Yokoyama et al. (1992) Exp Eye Res. 55:225). [00241] Suitable promoters include, but are not limited to, a CAG promoter (Miyazaki et al. (1989) Gene 79:269); a cytomegalovirus (CMV) promoter; a glutamate metabotropic receptor-6 (grm6) promoter (Cronin et al. (2014) EMBO Mol. Med. 6:1175); a Pleiades promoter (Portales-Casamar et al. (2010) Proc. Natl. Acad. Sci. USA 107:16589); a choline acetyltransferase (ChAT) promoter (Misawa et al. (1992) J. Biol. Chem. 267:20392); a vesicular glutamate transporter (V-glut) promoter (Zhang et al. (2011) Brain Res. 1377:1); a glutamic acid decarboxylase (GAD) promoter (Rasmussen et al. (2007) Brain Res. 1144:19; Ritter et al. (2016) J. Gene Med. 18:27); a cholecystokinin (CCK) promoter (Ritter et al. (2016) J. Gene Med. 18:27); a parvalbumin (PV) promoter; a somatostatin (SST) promoter; a neuropeptide Y (NPY) promoter; and a vasoactive intestinal peptide (VIP) promoter. Suitable promoters include, but are not limited to, a red cone opsin promoter, rhodopsin promoter, a rhodopsin kinase promoter, and a GluR promoter (e.g., a GluR6 promoter; also referred to as grm6). Suitable promoters include, but are not limited to, a vitelliform macular dystrophy 2 (VMD2) gene promoter, and an interphotoreceptor retinoid-binding protein (IRBP) gene promoter. Also suitable for use is an L7 promoter (Oberdick et al. (1990) Science 248:223), a thy-1 promoter, a recoverin promoter (Wiechmann and Howard (2003) Carr. Eye Res. 26:25); a calbindin promoter; and a beta-actin promoter. Suitable promoters include synthetic (non-naturally occurring) promoter/enhancer combinations.

[00242] Other suitable promoters useful in accordance with the present disclosure include, for example, a gamma-synuclein (SNCG) promoter (e.g., Chaffiol et al. (2017) Mol. Ther. 25(11) 2546), a CBh promoter (e.g., Grey et al. (2011) Hum. Gene Ther. 22(9): 1143-53), a miniCAG promoter (e.g., Grey et al. (2011) Hum. Gene Ther. 22(9): 1143-53), a neurofilament heavy (NEFH) promoter (Millington- Ward et al. (2020) Sci. Rep. 10:16515), a G protein-coupled receptor kinase 1 (GRK1) promoter (e.g., Khani et al. (2007) Invest. Ophthalmol. Vis. Sci. 48(9):3954-61), a retinaldehyde-binding protein 1 (RLBP1) promoter (e.g., Choi et al. (2015) Mol. Ther. Methods Clin. Dev. 2: 15022; Vogel et al. (2007) Invest. Ophthalmol. Vis. Sci. 48, 3872-3877), a vitelliform muscular dystrophy-2 (VMD2) promoter (e.g., Conlon et al. (2013) Hum. Gene Ther. Clin. Dev. 24, 23-28), a synapsin I (Synl) promoter (e.g., Kugler et al. (2003)), an enhSynl promoter (e.g., Hioki et al. (2007) Gene Ther.l4(l l):872-82), and a neurofilament heavy (NEFH) promoter, or a functional fragment or variant thereof.

[00243] Non-limiting examples of nucleotides sequences of suitable promoters are depicted in FIG. 9A-9I. FIG. 9A presents an example of an SNCG promoter; FIG. 9B presents an example of a CBh (a hybrid promoter comprising a cytomegalovirus (CMV) enhancer and a chicken P-actin (CBA) promoter); FIG. 9C presents an example of a miniCAG promoter; FIG. 9D presents an example of an NEFH promoter; FIG. 9E presents an example of a GRK1 promoter; FIG. 9F presents an example of an RLBP1 promoter; FIG. 9G presents an example of a VMD2 promoter; FIG. 9H presents an example of a synapsin I promoter; and FIG. 91 presents an example of an enhanced synapsin I (enSynl) promoter. [00244] As one non-limiting example, in some cases, a nucleotide sequence encoding a SNAP- mGluR2 fusion polypeptide is operably linked to a human synapsin promoter; e.g., a promoter as depicted in FIG. 9H or FIG. 91.

[00245] In some cases, a suitable promoter comprises a functional fragment or variant of a promoter comprising a nucleotide sequence depicted in any one of FIG. 9A-9I, where the functional fragment or variant retains the ability to promote expression in a retinal cell of an operably linked coding sequence (e.g., a nucleotide sequence encoding a polypeptide of the disclosure). For example, a suitable promoter can comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, nucleotide sequence identity with the nucleotide sequence depicted in any one of FIG. 9A-9I. As an example, a suitable promoter can comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, nucleotide sequence identity with the nucleotide sequence depicted in FIG. 9H. As another example, a suitable promoter can comprise a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99%, nucleotide sequence identity with the nucleotide sequence depicted in FIG. 91.

[00246] In some embodiments of the disclosure, the composition further comprises one or more of a sequence comprising an enhancer, a sequence comprising an intron or any portion thereof, a sequence comprising an exon or any portion thereof, a sequence comprising a Kozak sequence, a sequence comprising a post-transcriptional response element (PRE), a sequence comprising an inverted terminal repeat (ITR) sequence, a sequence comprising a long terminal repeat (LTR) sequence, and a poly-A sequence.

[00247] In some embodiments of the compositions of the disclosure, the composition further comprises a linking element. A linking element of the disclosure may link the sequence encoding the promoter to the sequence encoding the polypeptide of interest. Alternatively, or in addition, a linking element of the disclosure may link, reversible or irreversibly the composition to one or more of a surface, a tag, a label (detectable or sequence barcode), a ligand, an epitope, a capture probe, a selectable marker, or a delivery vehicle of the disclosure.

COMPOSITIONS

[00248] The embodiments further provide compositions comprising a conjugate of the present disclosure. Compositions comprising a conjugate of the present disclosure can include one or more of: a salt, e.g., NaCl, MgCF, KC1, MgSO4, etc.; a buffering agent, e.g., a Tris buffer, N-(2- Hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g., a non-ionic detergent such as Tween-20, Nonidet-P40, etc.; a protease inhibitor; and the like.

PHARMACEUTICAL COMPOSITIONS

[00249] The present disclosure provides pharmaceutical compositions comprising a conjugate of the present disclosure or a composition of the present disclosure. In some cases, the pharmaceutical composition is suitable for administering to an individual in need thereof. In some cases, the pharmaceutical composition is suitable for administering to an individual in need thereof, where the individual is a human.

[00250] A pharmaceutical composition comprising a conjugate or a composition of the present disclosure may be administered to a patient alone, or in combination with other supplementary active agents. The pharmaceutical compositions may be manufactured using any of a variety of processes, including, without limitation, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, and lyophilizing. The pharmaceutical composition can take any of a variety of forms including, without limitation, a sterile solution, suspension, emulsion, lyophilizate, tablet, pill, pellet, capsule, powder, syrup, elixir or any other dosage form suitable for administration. [00251] A pharmaceutical composition comprising a conjugate or a composition of the present disclosure can optionally include a pharmaceutically acceptable carrier(s) that facilitate processing of an active ingredient into pharmaceutically acceptable compositions. As used herein, the term “pharmacologically acceptable carrier” refers to any carrier that has substantially no long-term or permanent detrimental effect when administered and encompasses terms such as “pharmacologically acceptable vehicle, stabilizer, diluent, auxiliary or excipient.” Such a carrier generally is mixed with an active compound, or permitted to dilute or enclose the active compound and can be a solid, semi-solid, or liquid agent. It is understood that the active ingredients can be soluble or can be delivered as a suspension in the desired carrier or diluent. Any of a variety of pharmaceutically acceptable carriers can be used including, without limitation, aqueous media such as, e.g., distilled, deionized water, saline; solvents; dispersion media; coatings; antibacterial and antifungal agents; isotonic and absorption delaying agents; or any other inactive ingredient. Selection of a pharmacologically acceptable carrier can depend on the mode of administration. Except insofar as any pharmacologically acceptable carrier is incompatible with the active ingredient, its use in pharmaceutically acceptable compositions is contemplated. Non-limiting examples of specific uses of such pharmaceutical carriers can be found in “Pharmaceutical Dosage Forms and Drug Delivery Systems” (Howard C. Ansel et al., eds., Lippincott Williams & Wilkins Publishers, 7^th ed. 1999); “Remington: The Science and Practice of Pharmacy” (Alfonso R. Gennaro ed., Lippincott, Williams & Wilkins, 20^th 2000); “Goodman & Gilman’s The Pharmacological Basis of Therapeutics” Joel G. Hardman et al., eds., McGraw-Hill Professional, 10.sup.th ed. 2001); and “Handbook of Pharmaceutical Excipients” (Raymond C. Rowe et al., APhA Publications, 4^th edition 2003).

[00252] A subject pharmaceutical composition can optionally include, without limitation, other pharmaceutically acceptable components, including, without limitation, buffers, preservatives, tonicity adjusters, salts, antioxidants, physiological substances, pharmacological substances, bulking agents, emulsifying agents, wetting agents, sweetening or flavoring agents, and the like. Various buffers and means for adjusting pH can be used to prepare a pharmaceutical composition disclosed in the present specification, provided that the resulting preparation is pharmaceutically acceptable. Such buffers include, without limitation, acetate buffers, citrate buffers, phosphate buffers, neutral buffered saline, phosphate buffered saline and borate buffers. It is understood that acids or bases can be used to adjust the pH of a composition as needed. Pharmaceutically acceptable antioxidants include, without limitation, sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole and butylated hydroxy toluene. Useful preservatives include, without limitation, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric acetate, phenylmercuric nitrate and a stabilized oxy chloro composition, for example, PURITE™. Tonicity adjustors suitable for inclusion in a subject pharmaceutical composition include, without limitation, salts such as, e.g., sodium chloride, potassium chloride, mannitol or glycerin and other pharmaceutically acceptable tonicity adjustor. It is understood that these and other substances known in the art of pharmacology can be included in a subject pharmaceutical composition.

[00253] Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or poly anhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[00254] A conjugate or a composition of the present disclosure can be formulated with one or more pharmaceutically acceptable excipients. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7^th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3^rd ed. Amer. Pharmaceutical Assoc.

[00255] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

[00256] In a method of the present disclosure (described below), a conjugate or a composition of the present disclosure may be administered to the host using any convenient means capable of resulting in the desired reduction in disease condition or symptom. Thus, a conjugate or a composition of the present disclosure can be incorporated into a variety of formulations for therapeutic administration.

More particularly, a conjugate or a composition of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[00257] A conjugate or a composition of the present disclosure can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. Such preparations can be used for oral administration.

[00258] A conjugate or a composition of the present disclosure can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives. Formulations suitable for injection can be administered by an intravitreal, intraocular, intramuscular, subcutaneous, sublingual, or other route of administration, e.g., injection into the gum tissue or other oral tissue. Such formulations are also suitable for topical administration.

[00259] In some cases, a conjugate or a composition of the present disclosure is administered via intravitreal injection. In some cases, a conjugate or a composition of the present disclosure is administered via intraocular administration. In some cases, a conjugate or a composition of the present disclosure is administered via subretinal injection. [00260] A conjugate or a composition of the present disclosure can be utilized in aerosol formulation to be administered via inhalation. A conjugate or a composition of the present disclosure can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[00261] Furthermore, a conjugate or a composition of the present disclosure can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. A conjugate or a composition of the present disclosure can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

[00262] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise a conjugate of the present disclosure in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[00263] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a conjugate of the present disclosure calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for a conjugate of the present disclosure depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

[00264] A conjugate or a composition of the present disclosure can be administered as injectables. Injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.

[00265] A conjugate or a composition of the present disclosure can be formulated in a pharmaceutical composition together with a pharmaceutically acceptable excipient. In some cases, a subject pharmaceutical composition will be suitable for administration to a subject, e.g., will be sterile. For example, in some cases, a subject pharmaceutical composition will be suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and/or other toxins.

[00266] A conjugate or a composition of the present disclosure can be formulated in a pharmaceutical composition that is suitable for administration to the eye of an individual. For example, a conjugate or a composition of the present disclosure can be formulated in a pharmaceutical composition that is suitable for intravitreal administration.

[00267] A conjugate or a composition of the present disclosure can be formulated in a pharmaceutical composition together with a pharmaceutically acceptable excipient that increases the in vivo half life of the conjugate. For example, in some cases, a composition of the present disclosure comprises: i) a conjugate or a composition of the present disclosure; and ii) a cyclodextrin.

[00268] Suitable cyclodextrins include, e.g., a-cyclodextrin, P-cyclodextrin, y-cyclodextrin, 2- hydroxypropyl-P-cyclodextrin, hydroxypropyl-y-cyclodextrin 2,6-di-O-ethyl-P-cyclodextrin, sulfobutylether-P-cyclodextrin, methylated cyclodextrin (e.g., methylated P-cyclodextrin; e.g., P- cyclodextrin with methyl groups on all of the C-2 and C-6 positions, referred to as DIMEB);

CRYSMEB; RAMEB; etc.), heptakis-(2,3,6-tri-O-methyl)-P-cyclodextrin (TRIMEB), maltosyl-P- cyclodextrin, and the like. In some cases, a composition of the present disclosure comprises: i) a conjugate or a composition of the present disclosure; and ii) a cyclodextrin. In some cases, a composition of the present disclosure comprises: i) a conjugate or a composition of the present disclosure; and ii) P- cyclodextrin. In some cases, the present disclosure provides methods for administration of an agent intravitreally to a subject in need of the agent by formulating the agent in combination with a cyclodextrin. As demonstrated herein, superior in vivo efficacy and/or persistence of a therapeutic effect can be achieved following intravitreal administration of a conjugate of the present disclosure formulated in conjunction with a cyclodextrin.

[00269] In some cases, a composition of the present disclosure comprises: i) a conjugate or a composition of the present disclosure; and ii) an alkyl glycoside. In some cases, the alkyl glycoside is selected from the group consisting of undecyl maltoside, dodecyl maltoside, tridecyl maltoside, tetradecyl maltoside, sucrose mono-dodecanoate, sucrose mono-tridecanoate, and sucrose mono- tetradecanoate.

[00270] In some cases, a conjugate or a composition of the present disclosure is delivered by a continuous delivery system. The term “continuous delivery system” is used interchangeably herein with “controlled delivery system” and encompasses continuous (e.g., controlled) delivery devices (e.g., pumps) in combination with catheters, injection devices, and the like, a wide variety of which are known in the art.

[00271] In some cases, a conjugate or a composition of the present disclosure is present in (e.g., encapsulated within) a micelle (e.g., a nanomicelle), a nanoparticle, or a liposome. For example, in some cases, a conjugate of the present disclosure is present in (e.g., encapsulated within) a nanomicelle that comprises a copolymer of polyhydroxyethylaspartamide (PHEA) and pegylated PHEA. As another example, in some cases, a conjugate of the present disclosure is present in (e.g., encapsulated within) a nanomicelle that comprises a poly(ethylene oxide) -poly (propylene oxide)-poly(ethylene oxide) copolymer. As another example, in some cases, a conjugate of the present disclosure is present in (e.g., encapsulated within) a nanoparticle that comprises one or more of albumin, sodium alginate, chitosan, poly(lactide-co-glycolide) (PLGA), poly(lactic acid) (PLA), and polycaprolactone. As another example, in some cases, a conjugate of the present disclosure is present in (e.g., encapsulated within) a liposome that comprises didodecyldimethylammonium bromide, stearylamine, or A-[l-(2,3-dioleoyloxy)propyl]- /V,/V,/V- tri me thy I ammonium chloride. In some cases, a conjugate or a composition of the present disclosure is present in an implant.

METHODS

[00272] A conjugate of the present disclosure finds use in modulating activity of a target ligandbinding polypeptide. A conjugate of the present disclosure finds use in modulating activity of a cell comprising a conjugate of the present disclosure, where the cell comprises a target ligand-binding polypeptide. The present disclosure thus provides a method of modulating activity of a target ligandbinding polypeptide; and a method of modulating activity of a cell comprising a conjugate of the present disclosure, where the cell comprises a target ligand-binding polypeptide. In some cases, a method of the present disclosure comprises exposing the conjugate (or a cell or tissue comprising the conjugate) to appropriate light conditions such that the ligand binds to the ligand-binding site of the target ligandbinding polypeptide. In some cases, a method of the present disclosure comprises exposing the conjugate (or a cell or tissue comprising the conjugate) to appropriate light conditions such that the ligand does not bind to the ligand-binding site of the target ligand-binding polypeptide.

[00273] The present disclosure provides a method of modulating activity of a target ligandbinding polypeptide, the method comprising: a) contacting the target ligand-binding polypeptide with a conjugate of the present disclosure, generating a light-regulatable polypeptide; and b) exposing the light- regulatable polypeptide to light of a wavelength that results in binding of the ligand to the light- regulatable polypeptide, wherein binding of the ligand to the light-regulatable polypeptide modulates activity of the light-regulatable polypeptide. The present disclosure provides a method of modulating activity of a target ligand-binding polypeptide, the method comprising: a) contacting the target ligandbinding polypeptide with a conjugate of the present disclosure, generating a light-regulatable polypeptide; and b) exposing the light-regulatable polypeptide to light of a wavelength that results in release of the ligand from the ligand-binding site of the light-regulatable polypeptide, wherein release of the ligand from the ligand-binding site of the light-regulatable polypeptide modulates activity of the light-regulatable polypeptide.

[00274] “Modulating activity” of a target ligand-binding polypeptide (or a light-regulatable polypeptide) includes increasing an activity of the polypeptide; inhibiting an activity of the polypeptide; sensitizing the polypeptide to another (e.g., non-light) stimulus); reducing the sensitivity of the polypeptide to another stimulus; increasing the efficacy by which another stimulus activates the polypeptide; and decreasing the efficacy by which another stimulus activates the polypeptide. The activity depends on the polypeptide being modulated. For example, in some cases, the ligand is an agonist, and binding of the ligand to the target ligand-binding polypeptide (or light-regulatable polypeptide) results in activation of the target ligand-binding polypeptide (or light-regulatable polypeptide). In other instances, the ligand is an antagonist, and binding of the ligand to the target ligandbinding polypeptide (or light-regulatable polypeptide) results in inhibition, desensitization, or inactivation of the target ligand-binding polypeptide (or light-regulatable polypeptide).

[00275] Target ligand-binding polypeptides include, but are not limited to, a transcription regulator, an ion channel, a cation channel, a ligand-gated ion channel, a voltage-gated ion channel, a quorum sensor, a pheromone receptor, a neurotransmitter receptor, an enzyme, enzyme, a motor protein, a transporter, a membrane transport protein, a G protein-coupled receptor, a G protein, a receptor tyrosine kinase, a scaffolding protein, an adaptor protein, a cytoskeletal protein, an adhesion protein, a membrane-targeting protein, a protein that direct secretion, and a localization or protein interaction domain of a protein. In some cases, the target ligand-binding polypeptide is a cation channel. In some cases, the target ligand-binding polypeptide is an anion channel. In some cases, the target ligand-binding polypeptide is a potassium channel. In some cases, the target ligand-binding polypeptide is a sodium channel. In some cases, the target ligand-binding polypeptide is a calcium channel.

[00276] In some cases, the target ligand-binding polypeptide is in a cell-free composition; i.e., the target ligand-binding polypeptide is not present in a cell.

[00277] In some cases, the target ligand-binding polypeptide is present in a cell in vitro. In some cases, the target ligand-binding polypeptide is present in a cell in vivo.

[00278] Where the target ligand-binding polypeptide is present in a cell, the cell can be any type of cell. For example, the cell can be a mammalian cell, e.g., a human cell, a non-human primate cell, a rodent cell, and the like. The cell can be a retinal cell, a muscle cell, a neuronal cell, a blood cell (e.g., a nucleated blood cell), an epithelial cell, an endothelial cell, a skin cell, a lung cell, etc.

[00279] In some cases, the target ligand-binding polypeptide is present in a cell. In some cases, the cell is a retinal cell. In some cases, the cell is an amacrine cell. In some cases, the cell is a ganglion cell (e.g., a retinal ganglion cell (RGC)). In some cases, the cell is a bipolar cell. In some cases, the cell is a Mueller cell. In some cases, the cell is an ON-bipolar cell (ON-BC). In some cases, the cell is an OFF- bipolar cell.

[00280] The present disclosure provides a method of modulating activity of a target cell, the method comprising exposing the target cell to light, where the target cell comprises a conjugate of the present disclosure and a target ligand-binding polypeptide, where the light is of a wavelength that results in binding of the ligand to the target ligand-binding polypeptide, and where binding of the ligand to the target ligand-binding polypeptide modulates activity of the target cell. The present disclosure provides a method of modulating activity of a target cell, the method comprising exposing the target cell to light, where the target cell comprises a conjugate of the present disclosure and a target ligand-binding polypeptide, where the light is of a wavelength that results in release of the ligand from the target ligandbinding polypeptide, and where release of binding of the ligand from the target ligand-binding polypeptide modulates activity of the target cell. In some cases, the cell is a target cell population. In some cases, the target cell or cell population is present in a tissue.

[00281] The present disclosure provides a method of introducing sensitivity to light into retinal cells that normally are not directly responsive to light or enhancing the light response of already lightsensitive retinal cells, the method comprising exposing the retinal cell to light, wherein the retinal cell comprises a conjugate of the present disclosure and a target ligand-binding polypeptide, where the light is of a wavelength that results in binding of the ligand to the target ligand-binding polypeptide, and where binding of the ligand to the target ligand-binding polypeptide modulates the activity of the retinal cell in response to light. For example, the target polypeptide in the retinal cell may be a metabotropic glutamate receptor, such as mGluR2 or mGluR8 in amacrine cells or mGluR6 or mGluR7 in bipolar cells or mGluR4 in ganglion cells. In these cases, a suitable photo-isomerizable moiety-ligand combination could be azobenzene-glutamate with a D stereoisomer linkage. See, e.g., Broichhagen et al. (2015) ACS Central Science 1, 383-393; and Levitz et al. (2017) Proc. Natl. Acad. Sci. USA 114, E3546-E3554. As other examples, the target polypeptide may be an ionotropic glutamate receptor, such as GluK2, GluK5, GluN2A or GluN2B in bipolar, amacrine or ganglion cells. In these cases, a suitable photo-isomerizable moiety-ligand combination could be azobenzene-glutamate with an L stereoisomer linkage (see, e.g., Volgraf et al. (2006) Nature Chem. Bio. 2:47; Volgraf et al. (2007) J. Am. Chem. Soc. 129:260; and Berlin et al. (2016) Elife 5:el2040), or ATG (see, e.g., Laprell et al. (2015) Nat. Commun. 6:8076. As another example, the target polypeptide may be an ionotropic glutamate receptor, such as GluRAl. In this case, a suitable photo-isomerizable moiety-ligand combination could be ShuBQX-3 (see, e.g., Barber et al. (2017) Chem. Sci. 8:611). As another example, the target polypeptide may be an ionotropic nicotinic acetylcholine receptor in amacrine or ganglion cells and the ligand may be AC-5, MAACh, HoChPE, MG-624 or MAHoCh (see, e.g., Tochitsky et al. (2012) Nat. Chem. 4:105. As another example, the target polypeptide may be an ionotropic GABA-A receptor in amacrine cells or ganglion cells and the ligand may be PAG-2A, PAG-2B, or PAG-3C. As another example, the target polypeptide may be an ionotropic P2X receptor in ganglion cells and the ligand may be MEA-TMA (see, e.g., Lemoine et al. (2013) Proc. Natl. Acad. Sci. USA 110:20813.

[00282] The present disclosure provides method of treating an ocular disorder characterized by reduced responsiveness to light, the method comprising administering a conjugate of the present disclosure, or a composition (e.g., a pharmaceutical composition) comprising a conjugate of the present disclosure, to an eye of an individual having the ocular disorder. In some cases, the conjugate, or a composition (e.g., a pharmaceutical composition) comprising the conjugate, is administered to the individual via intravitreal injection. In some cases, the conjugate, or a composition (e.g., a pharmaceutical composition) comprising the conjugate, is administered to the individual via intraocular administration. In some cases, the conjugate, or a composition (e.g., a pharmaceutical composition) comprising the conjugate, is administered to the individual via subretinal injection.

[00283] Ocular disorders characterized by reduced responsiveness to light include, but are not limited to, inherited retinal degenerative diseases such as retinitis pigmentosa and age-related macular degeneration. Ocular disorders that are suitable for treatment with a method of the present disclosure include, but are not limited to, retinitis pigmentosa, macular degeneration, retinoschisis, and Leber Congenital Amaurosis, and diabetic retinopathy.

METHODS FOR ENHANCING VISUAL FUNCTION

[00284] The present disclosure provides methods for enhancing or restoring visual function in an eye of an individual. The methods comprise administering an effective amount of a conjugate or a composition of the present disclosure to an eye of the individual.

[00285] Administration of a conjugate or a composition of the present disclosure to an eye of an individual can provide for patterned vision and image recognition by the individual. Image recognition can be of a static image and/or of a moving image.

[00286] In some cases, administering an effective amount of a conjugate or a composition of the present disclosure to an eye of an individual provides for image recognition at a light intensity of from about 0.1 pW/cm² to about 10 W/cm². In some cases, administering an effective amount of a conjugate or a composition of the present disclosure to an eye of an individual provides for image recognition at a light intensity of less than 5 W/cm², e.g., less than 3 W/cm², less than 2 W/cm², or less than 1 W/cm². For example, administering an effective amount of a conjugate or a composition of the present disclosure to an eye of an individual provides for image recognition at a light intensity of from about 0.1 pW/cm² to about 0.5 pW/cm², from about 0.5 pW/cm² to about 1.0 pW/cm², or from about 1.0 pW/cm² to about 5 pW/cm². As another example, administering an effective amount of a conjugate or a composition of the present disclosure to an eye of an individual provides for image recognition at a light intensity of from about 10⁵ W/cm² to about 10 ¹ W/cm², from about 10⁴ W/cm² to about 10² W/cm², from about 10⁴ W/cm² to about 1 W/cm², from about 10⁴ W/cm² to about 10 ¹ W/cm², or from about 10⁴ W/cm² to about 5 x 10 ¹ W/cm². In some cases, administering an effective amount of a conjugate or a composition of the present disclosure to an eye of an individual provides for image recognition at a light intensity of from about 10⁵ W/cm² to about 10⁴ W/cm², from about 10⁴ W/cm² to about 10³ W/cm², from about 10³ W/cm² to about 10² W/cm², from about 10² W/cm² to about 10 ¹ W/cm², or from about 10 ¹ W/cm² to about 1 W/cm².

[00287] In some cases, a conjugate of the present disclosure comprises “460 azobenzene” as discussed above. In some cases, a conjugate of the present disclosure that comprises “460 azobenzene” provides for “off’ kinetics that are at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 7,500-fold, at least 10,000-fold, or more than 10,000-fold, faster than the “off’ kinetics conferred on a retinal cell by a conjugate that comprises “azobenzene 380.” “Off’ kinetics refers to the kinetics of turning off the light response upon removal of light (in the dark). For example, in some cases, a conjugate of the present disclosure that comprises “460 azobenzene” provides for “off’ kinetics of less than 10 seconds, less than 5 seconds, less than 1 second, less than 900 milliseconds (ms), less than 800 ms, less than 700 ms, less than 600 ms, less than 500 ms, or less than 400 milliseconds. In some cases, a conjugate of the present disclosure that comprises “460 azobenzene” provides for “off’ kinetics of from about 100 ms to about 200 ms, from about 200 ms to about 300 ms, from about 300 ms to about 400 ms, from about 400 ms to about 500 ms, from about 500 ms to about 600 ms, from about 600 ms to about 700 ms, from about 700 ms to about 800 ms, from about 800 ms to about 900 ms, from about 900 ms to about 1 second, from about 1 second to about 5 seconds, or from about 5 seconds to about 10 seconds.

[00288] In some cases, a conjugate of the present disclosure comprises “460 azobenzene” as discussed above. A conjugate of the present disclosure that comprises “460 azobenzene” provides for excitation by visible light (e.g., blue light). Thus, administration to an eye of a conjugate of the present disclosure that comprises “460 azobenzene” provides for responsiveness of the eye to visible light. [00289] A conjugate or a composition of the present disclosure is administered in an amount effective to increase visual function in an individual by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 2-fold, at least 5-fold, at least 10-fold, or more than 10-fold, compared with the visual function before administration of the conjugate. Tests for visual function are known in the art, and any known test can be applied to assess visual function.

[00290] In some cases, a conjugate of the present disclosure provides for visual detection of a moving image when the image is moving at a speed greater than about 4 centimeter s/second (cm/s), greater than about 5 cm/s, greater than about 6 cm/s, greater than about 7 cm/s, or greater than about 8 cm/s. In some cases, a conjugate of the present disclosure provides for visual detection of a moving image when the image is moving at a speed of greater than 8 cm/s, e.g., from 8 cm/s to about 9 cm/s, from 9 cm/s to 10 cm/s, from 10 cm/s to 12 cm/s, from 12 cm/s to 14 cm/s, or from 14 cm/s to about 16 cm/s. [00291] In some cases, a conjugate of the present disclosure is the conjugate referred to in the Examples (and depicted in FIG. 8) as 9xBGAGi2,46o- The 9xBGAGi2,46o conjugate can provide for enhanced visual function. The 9xBGAGi2,46o conjugate can provide for patterned vision and image recognition. Image recognition can be of a static image and/or of a moving image. In some cases, the 9xBGAGi2,46o conjugate provides for visual detection of a moving image when the image is moving at a speed greater than about 4 centime ters/second (cm/s), greater than about 5 cm/s, greater than about 6 cm/s, greater than about 7 cm/s, or greater than about 8 cm/s. In some cases, the ^9xBGAGi2,46o conjugate provides for visual detection of a moving image when the image is moving at a speed of greater than 8 cm/s, e.g., from 8 cm/s to about 9 cm/s, from 9 cm/s to 10 cm/s, from 10 cm/s to 12 cm/s, from 12 cm/s to 14 cm/s, or from 14 cm/s to about 16 cm/s.

[00292] In some cases, the 9xBGAGi2,46o conjugate provides for from 25- to 250-fold greater light sensitivity than monovalent BGAGi2,46oor 4xBGAGi2,46o- The conjugate 4xBGAGi2,46o has 4 photoisomerizable regulators and a distinct branched linker structure when compared to the conjugates described herein.

[00293] In some cases, the dose of 9xBGAGi2,46o that is required to restore or enhance visual function is at least 25% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 75% less, at least 80% less, at least 85% less, at least 90% less, or at least 95% less, than the dose of 4xBGAGi2,46o required to restore or enhance visual function to the same degree.

[00294] In some cases, the dose of 9xBGAGi2,46o that is administered is a dose that provides for a vitreal concentration of from about 1 pM to about 4 mM. For example, in some cases, the dose of ^9xBGAGi2,46o that is administered is a dose that provides for a vitreal concentration of from about 1 pM to about 10 pM, from about 10 pM to about 25 pM, from about 25 pM to about 50 pM, from about 50 pM to about 100 pM, from about 100 pM to about 250 pM, from about 250 pM to about 500 pM, from about 500 pM to about 750 pM, from about 750 pM to about 1 mM, from about 1 mM to about 2 mM, from about 2 mM to about 3 mM, or from about 3 mM to about 4 mM. In some cases, the dose of 9xBGAGi2, 46o that is administered is a dose that provides for a vitreal concentration of from about 100 pM to about 400 pM. For example, in some cases, the dose of ^9xBGAGi2,46o that is administered is a dose that provides for a vitreal concentration of from about 100 pM to about 150 pM, from about 150 pM to about 200 pM, from about 200 pM to about 250 pM, from about 250 pM to about 300 pM, from about 300 pM to about 350 pM, or from about 350 pM to about 400 pM. In some cases, the dose of 9xBGAGi2, 46o that is administered is a dose that provides for a vitreal concentration of from about 10 pM to about 100 pM. In some cases, the dose of 9xBGAGi2,46o that is administered is a dose that provides for a vitreal concentration of from about 10 pM to about 50 pM. [00295] In some cases, a conjugate of the present disclosure, or a composition (e.g., a pharmaceutical composition) comprising the conjugate, is administered to the individual via intravitreal injection. In some cases, the conjugate, or a composition (e.g., a pharmaceutical composition) comprising the conjugate, is administered to the individual via intraocular administration. In some cases, the conjugate, or a composition (e.g., a pharmaceutical composition) comprising the conjugate, is administered to the individual via subretinal injection.

[00296] In some cases, multiple doses of a conjugate or a composition of the present disclosure are administered to an individual. The frequency of administration can vary depending on any of a variety of factors, e.g., severity of the symptoms, etc. For example, in some cases, a conjugate or a composition of the present disclosure is administered once every 6 weeks, once every 5 weeks, once per month, once every 4 weeks, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week, five times per week, six times per week, every other day (qod), daily (qd), twice a day (qid), or three times a day (tid). [00297] A conjugate or a composition of the present disclosure can be administered to an individual over a period of time of from about 1 day to about 1 year or more than 1 year. For example, a conjugate of the present disclosure can be administered to an individual for a period of time of from 1 week to 2 weeks, from 2 weeks to 1 month, from 1 month to 4 months, from 4 months to 6 months, from 6 months to 1 year, or more than 1 year.

[00298] Individuals suitable for treatment with a method of the present disclosure include individuals having reduced visual function due to loss of rod and cone photoreceptors. In some cases, the individual has an inherited retinal degenerative disease such as retinitis pigmentosa, retinoschisis, or Leber Congenital Amaurosis. In some cases, the individual has an ocular disease (e.g., an inherited ocular disease) selected from retinitis pigmentosa, macular degeneration, age-related macular degeneration, retinoschisis, and Leber Congenital Amaurosis, and diabetic retinopathy. Individuals suitable for treatment with a method of the present disclosure include individuals having a retinal degeneration condition in which the natural light sensitivity is lost and vision is therefore compromised, but where neurons late in the retinal circuit (e.g. bipolar cells or amacrine interneurons or ganglion cells that output to the brain) are spared and can be made directly sensitive to light by introduction of the cone opsin(s).

[00299] Individuals suitable for treatment with a method of the present disclosure include individuals having retinal damage that is traumatic or acute, with no genetic or inherited basis. For example, in some cases, the individual has experienced retinal detachment resulting from blunt trauma, such as a blast injury (e.g., in a military battle), or resulting from an impact to the head, e.g., in the course of an auto accident or other accident resulting in impact to the head. In some instances, the photoreceptors are lost due to traumatic detachment of the retina from the underlying RPE, but the inner retinal neurons are intact. Individuals suitable for treatment with a method of the present disclosure include individuals having photoreceptor loss due to acute light damage, laser exposure, or chemical toxicity.

DEVICES

[00300] The present disclosure provides a device comprising a conjugate or a composition of the present disclosure.

[00301] A device of the present disclosure can comprise: a) a container comprising a composition of the present disclosure (a composition comprising a conjugate of the present disclosure); and b) a means for introducing the composition into the eye of an individual. In some cases, the means for introducing the composition into the eye of an individual comprises a needle. In some cases, the container comprises a syringe. The device will in some cases be sterile. In some cases, the device is implantable.

Examples of Non-Limiting Aspects of the Disclosure

[00302] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

[00303] Aspect 1. A conjugate comprising

[00304] a) an affinity agent that specifically binds:

[00305] i) a target ligand-binding polypeptide; or

[00306] ii) a polypeptide that binds to a target ligand-binding polypeptide;

[00307] b) a branched linker; and

[00308] c) a plurality of photoisomerizable regulators, each independently comprising:

[00309] i) a photoisomerizable group comprising an azobenzene moiety; and

[00310] ii) a ligand that binds to the target ligand-binding polypeptide.

[00311] wherein the branched linker comprises a moiety of Formula (BL):

[00312] -C(O)NH-[C[(CH₂)nC(O)NH]₃]x- (Formula (BL)),

[00313] n is an integer from 1 to 6,

[00314] x is an integer from 1 to 50, and [00315] wherein the branched linker comprises a plurality of arms, each independently comprising a photoisomerizable regulator.

[00316] Aspect 2. The conjugate of aspect 1 , wherein each arm of the branched linker comprises one or more polyethylene glycol (PEG) units.

[00317] Aspect 3. The conjugate of aspect 2, wherein each arm of the branched linker comprises six PEG units.

[00318] Aspect 4. The conjugate of aspect 1 , wherein the photoisomerizable group comprises a structure of Formula 4:

[00319]

[00320] wherein:

[00321] Q² is the ligand;

[00322] w is an integer from 1 to 10;

[00323] R¹ is selected from hydrogen, C_1-10 alkyl, -NR¹⁰Rⁿ, -NR¹²C(O)R¹³, -NR¹²C(O)OR¹³ and

-NR¹²C(O)NR¹²R¹³;

[00324] R² is hydrogen or C_1-10 alkyl;

[00325] R¹⁰ and R¹¹ are independently selected from hydrogen and Ci-io alkyl;

[00326] R¹² is hydrogen or Ci-io alkyl; and

[00327] R¹³ is selected from hydrogen, Ci-io alkyl, C_1-8 alkenyl, Cg io aryl, and substituted Ci-io alkyl,

[00328] or a pharmaceutically acceptable salt thereof.

[00329] Aspect 5. The conjugate of aspect 1, wherein the affinity agent comprises benzylguanine.

[00330] Aspect 6. The conjugate of aspect 1, wherein the affinity agent comprises chloroalkane.

[00331] Aspect 7. The conjugate of aspect 1, wherein the affinity agent comprises benzylcytosine.

[00332] Aspect 8. The conjugate of any one of aspects 1-7, wherein the affinity agent comprises an antibody that specifically binds to the target ligand-binding polypeptide.

[00333] Aspect 9. The conjugate of any one of aspects 1-8, wherein the target ligand-binding polypeptide is selected from a transcription regulator, an ion channel, a cation channel, a ligand-gated ion channel, a voltage-gated ion channel, a quorum sensor, a pheromone receptor, a neurotransmitter receptor, a G-protein-coupled receptor, and an enzyme.

[00334] Aspect 10. The conjugate of aspect 9, wherein the cation channel is a potassium channel, a sodium channel, or a calcium channel.

[00335] Aspect 11. The conjugate of any one of aspects 1-9, wherein the target ligand-binding polypeptide is a glutamate receptor, a metabotropic glutamate receptor, an ionotropic glutamate receptor, an ionotropic nicotinic acetylcholine receptor, an ionotropic GABA-A receptor, a metabotropic GABA-B receptor, a metabotropic dopamine receptor, an ionotropic purinergic P2X receptor, a metabotropic purinergic P2Y receptor, a metabotropic serotonin receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, a cation channel, a potassium channel, a calcium channel, a sodium channel, a proton channel, an anion channel, or a chloride channel.

[00336] Aspect 12. The conjugate of any one of aspects 1-9, wherein the target ligand-binding polypeptide is a glutamate receptor.

[00337] Aspect 13. The conjugate of any one of aspects 1-9, wherein the target ligand-binding polypeptide is a metabotropic glutamate receptor (mGluR).

[00338] Aspect 14. The conjugate of aspect 13, wherein the mGluR is mGluR2, mGluR3, mGluR4, mGluR5, or mGluR6.

[00339] Aspect 15. The conjugate of aspect 14, wherein the target ligand-binding polypeptide is a metabotropic glutamate receptor 2 polypeptide.

[00340] Aspect 16. The conjugate of aspect 15, wherein the ligand comprises an mGluR agonist.

[00341] Aspect 17. The conjugate of any one of aspects 13-15, wherein the ligand comprises glutamate.

[00342] Aspect 18. The conjugate of any one of aspects 13-15, wherein the ligand is selected from (lR,4R,5S,6R)-4-amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268);

(lS,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740); (15,2S,47?,57?,6S)-rel-2- amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY395756); (S)-2-amino-2-methyl-4- phosphonobutanoic acid (MAP4); (2S,2'7?,3'7?)-2-(2',3'-Dicarboxycyclopropyl)glycine (DCG IV);

(15,37?)-l-aminocyclopentane-l,3-dicarboxylic acid ((1S,37?)-ACPD); (27?,47?)-4-aminopyrrolidine-2,4- dicarboxylate ((27?,47?)-APCD); (lS,2S,4R,5R,6S)-2-amino-4-methylbicyclo[3.1.0]hexane-2,6- dicarboxylic acid (LY541850); (lR,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide (LY404039); and (lR,4S,5S,6S)-4-((S)-2-amino-4-(methylthio)butanamido)-2- thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide (LY2140023).

[00343] Aspect 19. The conjugate of any one of aspects 12-18, wherein the affinity agent comprises benzylguanine. [00344] Aspect 20. The conjugate of aspect 8, wherein the antibody is a single-chain Fv (scFv) or a nanobody.

[00345] Aspect 21. The conjugate of aspect 20, wherein the antibody is specific for a metabotropic glutamate receptor (mGluR), optionally wherein the mGluR is mGluR2, mGluR3, mGluR4, mGluR5, or mGluR6.

[00346] Aspect 22. A composition for ocular administration, the composition comprising: [00347] a) a conjugate of any one of aspects 1-21; and

[00348] b) a pharmaceutically acceptable excipient suitable for ocular administration.

[00349] Aspect 23. The composition of aspect 22, wherein the pharmaceutically acceptable excipient comprises a cyclodextrin.

[00350] Aspect 24. The composition of aspect 23, wherein the cyclodextrin is a-cyclodextrin, P- cyclodextrin, y-cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether-P-cyclodextrin, or a derivatized cyclodextrin.

[00351] Aspect 25. The composition of any one of aspects 22-24, wherein the conjugate is encapsulated within a nanoparticle.

[00352] Aspect 26. The composition of aspect 25, wherein the nanoparticle is a nanomicelle, a liposome, a nanosphere, or a nanocapsule.

[00353] Aspect 27. The composition of any one of aspects 22-26, wherein the composition is sterile and free of pyrogens.

[00354] Aspect 28. A composition for ocular administration, the composition comprising: A) a system comprising: a) a conjugate as recited in any one of aspects 1-21; b) a fusion polypeptide comprising: i) a target ligand-binding polypeptide that comprises a binding site for the ligand present in the conjugate; and ii) a heterologous fusion partner that binds the affinity agent; and B) a pharmaceutically acceptable excipient suitable for ocular administration.

[00355] Aspect 29. The composition of aspect 28, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the SNAP polypeptide amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:4.

[00356] Aspect 30. The composition of aspect 28, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the CLIP polypeptide amino acid sequence set forth in SEQ ID NO:2.

[00357] Aspect 31. The composition of aspect 28, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the HALO polypeptide amino acid sequence set forth in SEQ ID NOG. [00358] Aspect 32. A composition for intraocular administration, the composition comprising:

A) a system comprising: a) a conjugate as recited in any one of aspects 1-21; b) a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide comprising: i) a target ligand-binding polypeptide that comprises a binding site for the ligand; and ii) a heterologous fusion partner that binds the affinity agent; and B) a pharmaceutically acceptable excipient suitable for intraocular administration. [00359] Aspect 33. The composition of aspect 32, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the SNAP polypeptide amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:4.

[00360] Aspect 34. The composition of aspect 32, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the HALO polypeptide amino acid sequence set forth in SEQ ID NO:3.

[00361] Aspect 35. The composition of aspect 32, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the CLIP polypeptide amino acid sequence set forth in SEQ ID NO:2.

[00362] Aspect 36. The composition of any one of aspects 30-33, wherein the nucleic acid is present in a recombinant adenovirus-associated virus (AAV) virion.

[00363] Aspect 37. The composition of aspect 36, wherein the AAV virion comprises a variant capsid polypeptide that provides for increased infectivity of a retinal cell by the AAV virion, compared to an AAV virion comprising a corresponding wild- type capsid polypeptide.

[00364] Aspect 38. A composition for intraocular administration, the composition comprising:

A) a system comprising: a) a conjugate as recited in any one of aspects 1-21; b) a first fusion polypeptide comprising: i) a target ligand-binding polypeptide that comprises a binding site for the ligand; and ii) a heterologous polypeptide; and c) a second fusion polypeptide comprising: i) an antibody that binds the heterologous polypeptide; and ii) a heterologous fusion partner that binds the affinity agent; and B) a pharmaceutically acceptable excipient suitable for intraocular administration.

[00365] Aspect 39. The composition of aspect 38, wherein the heterologous fusion partner that binds the affinity agent comprises an amino acid sequence having at least 80% amino acid sequence identity to the SNAP polypeptide amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:4.

[00366] Aspect 40. The composition of aspect 38, wherein the heterologous fusion partner that binds the affinity agent comprises an amino acid sequence having at least 80% amino acid sequence identity to the HALO polypeptide amino acid sequence set forth in SEQ ID NO:3.

[00367] Aspect 41. The composition of aspect 38, wherein the heterologous fusion partner that binds the affinity agent comprises an amino acid sequence having at least 80% amino acid sequence identity to the CLIP polypeptide amino acid sequence set forth in SEQ ID NO:2. [00368] Aspect 42. The composition of any one of aspects 38-41, wherein the antibody is a single-chain Fv or a nanobody.

[00369] Aspect 43. The composition of any one of aspects 38-42, wherein heterologous polypeptide is an epitope tag.

[00370] Aspect 44. A composition for intraocular administration, the composition comprising: A) a system comprising: a) a conjugate as recited in any one of aspects 1-21; and b) a fusion polypeptide comprising: i) an antibody that binds specifically to the target ligand-binding polypeptide; and ii) a polypeptide that binds to the affinity agent, wherein the polypeptide is selected from a SNAP polypeptide, a HALO polypeptide, and a CLIP polypeptide; and B) a pharmaceutically acceptable excipient suitable for intraocular administration.

[00371] Aspect 45. A method of increasing the sensitivity of a retinal cell to light, the method comprising exposing the retinal cell to light, wherein the retinal cell comprises a conjugate as recited in any one of aspects 1-21, the composition of any one of aspects 22-27, or the composition of any one of aspects 28-44, wherein the light is of a wavelength that results in binding of the ligand to the light- regulatable polypeptide, and wherein binding of the ligand to the light-regulatable polypeptide increases the sensitivity of the retinal cell to light.

[00372] Aspect 46. A method of conferring light responsiveness on a retinal cell, the method comprising introducing into the retinal cell a conjugate as recited in any one of aspects 1-21, the composition of any one of aspects 22-27, or the composition of any one of aspects 28-44.

[00373] Aspect 47. A method of treating an ocular disorder characterized by reduced responsiveness to light, the method comprising administering a conjugate as recited in any one of aspects 1-21, the composition of any one of aspects 22-27, or the composition of any one of aspects 28-44, to an eye of an individual having the ocular disorder.

[00374] Aspect 48. The method of aspect 47, wherein the ocular disorder is an inherited retinal degenerative disease.

[00375] Aspect 49. The method of aspect 48, wherein the disease is retinitis pigmentosa or age- related macular degeneration.

[00376] Aspect 50. A medical device comprising: a) a container comprising a conjugate as recited in any one of aspects 1-21, the composition of any one of aspects 22-27, or the composition of any one of aspects 28-44; and b) a means for introducing the composition into the eye of an individual. [00377] Aspect 51. The device of aspect 50, wherein the means for introducing the composition into the eye of an individual comprises a needle.

[00378] Aspect 52. The device of aspect 50 or aspect 51, wherein the container comprises a syringe. [00379] Aspect 53. The device of any one of aspects 50-52, wherein the device is sterile.

EXAMPLES

[00380] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Example 1

[00381] A photoswitch, referred to as “9xBGAGi2,46o”, which bears a light-activated ligand on each of its nine branches, was developed. 9xBGAGi2,46o increased sensitivity by ~10-fold compared to 4xBGAGi2,46o and achieved the same sensitivity when injected at 10% of the concentration. 9xBGAG also supported line pattern recognition at near the acuity limit of wildtype mouse vision. The results suggest that doubling of photoswitched agonist number will reduce the required clinical dose substantially and achieve high acuity vision restoration under room light and using standard computer displays, without the need for goggles (e.g., without the need for light amplification).

[00382] The 9xBGAGi2,46o:SNAP-mGluR system uniquely combines the high sensitivity of GPCRs with five major advantages of BGAG-mGluR: i) robust light response, ii) absence of photobleaching, iii) fast kinetics, iv) no function in wildtype (sighted) mice, suggesting selectivity for RGCs that have lost photoreceptor input (i.e. lack of interference with RGCs that retain input), v) ability to upgrade as new photoswitches are developed, and vi) ability to be discontinued in case of adverse effect.

MATERIALS AND METHODS

Animals, AAVs, and photoswitches

[00383] Mouse experiments were conducted under the express approval of the University of California Animal Care and Use Committee, wt mice (C57BL/6J) and rdl mice (C3H) were purchased from the Jackson Laboratory and housed on a 12-h light/dark cycle with food and water ad libitum. cDNA encoding SNAP-mGluR2 was inserted in an established viral cassette under control of either the human synapsin promoter (hsyn-l) for expression in retinal ganglion cells (RGCs) or a 4-copy concatemer of the mouse grm6 minimal promoter (4xgrm6) for expression in ON-BCs and packaged in the AAV 2/2-4YF capsid. The vector, containing 10¹⁰-10¹² viral genomes was delivered in a 2 □! volume to the vitreous of the rdl mouse eye via microinjection. Recombinant adeno-associated virus (rAAV) injections were at p30-p60 and in vivo and in vitro experiments at p90-pl60. AAVs were produced as previously described. Grieger et al. (2006) Nat. Protocols 1, 1412-1428.

[00384] ChrimsonR was packaged in AAV2 in the same way and under control elements as SNAP-mGluR2 to yield AAV2(4YF):ITR- ChrimsonR-polyA-WPRE-ITR.of the same. The amino acid sequence of the ChrimsonR(K176R) is depicted in FIG. 11B; the nucleotide sequence encoding ChrimsonR(K176R) is depicted in FIG. 11 A.

Photoswitch Preparation

[00385] Photoswitch compounds were synthesized using the protocol described in Broichhagen, et al. (2015) ACS Cent Sci 1, 383-393. Stock solution of 200 mM BGAGn.reo (L-diastereomer) in 100% pharmaceutical grade dimethylsulfoxide (DMSO) (Cryoserv; Bioniche Pharma) was diluted 1:100 in sterile PBS for a final working solution of 1 mM in 1% DMSO. Working solutions were either prepared before administration, prepared in stock stored in the freezer and used as required, or salvaged from the recording bath and stored (either RT or freezer) for reuse. Application of BGAGn.reo or BGAG12 on retinal explants were performed in a volume of 200 pL at a concentration of 50 pM to 50 nM BGAGn.reo (in PBS with >1% DMSO). For in vivo behavioral experiments, a 2-pL volume of ImM or 3.5 pL (final vitreal concentration of 1 pL) of BGAGn.reo solution (in PBS with 1% DMSO) was injected into eyes that treated with AAV >6 wks earlier). For in vivo concentration dependence experiments, the mouse eye was assumed to contain a volume of 5.3 pL and a 2-pL volume of 3.5pM, 1.825pM and 0.1825pM BGAG12, 46o solution (in phosphate buffered saline (PBS) with 1% DMSO) was injected into eyes to obtain a final concentration of IpM, 500nM and 50nM. Reiner et al. (2013) Neuron 79, 209-210. For hydrated slow release 5% pharmaceutical grade beta cyclodextrin (cyclodex) in PBS was mixed with BGAG12.460 for a final concentration of 3 pM and 2 pL were injected bilaterally into the mouse eye. MAGO46O was synthesized and administered in 2 pL at a concentration of 100 pM, identical to the protocol established previously by Kienzler et al. ((2013) J Am Chem Soc 135, 17683-17686) and Gaub et al. ((2014) Proc Natl Acad Sci U S A 111, E5574-5583).

Tissue Preparation and Immunohistochemistry

[00386] Mice >4 wks post-AAV2/2-hsyn-SNAP-mGluR2 treatment were injected with luL of 10 uM of BG-conjugated Alexa Fluor-647 dye into the vitreous. 25 hrs later mice were sacrificed, eyes were fixed in 4% paraformaldehyde (Ted Pella) (30 min), retinas were removed and the tissue incubated in blocking buffer [10% normal goat serum, 1% bovine serum albumin (BSA), 0.5% Triton X-100 in PBS (pH 7.4)] for 2 h at RT. Retinas were washed thoroughly using PBS and flat mounted on slides using Vectashield (Vector Laboratories) medium impregnated with DAPI (cell nuclei stain - blue). Retinas additionally co-injected with AAV2/2-hsyn-LiGluR were exposed to monoclonal antibody against GluK2/K3 (Millipore) (1:500 dilution in blocking buffer overnight at 4 °C) and followed by secondary anti-rabbit Alexa 488 antibody (Invitrogen) was applied (1:1,000 dilution for 2 h at RT) previously described in Gaub., et al. (2014) supra. In vitro sequential labeling of SNAP-mGluR2 with the BG- conjugated Alexa Fluor-647 and antibody staining of the GluK2 subunit recognized in LiGluR was also successfully achieved using minimal fixation (10 min). For retinal sections, whole mounts were embedded in agarose (Sigma) and sectioned transverse using a vibratome (Leica Microsystems) at medium speed, maximum vibration, and 200-pm thickness. Retinal tissues used for immunohistochemistry on retinal cryosections or whole mounts were processed and examined by confocal microscopy (Leica TCS SP5; Leica Microsystems). For cell counting, retina were cryosectioned and stained with DAPI. Z-stack images (24 slices) of 1 pm³ were obtained using the Zeiss LSM-880 NLO Airyscan microscope with 20x objective, increased offset was used to minimize background and differentiate distinct cells, and analysis was performed using the Imaris software to count individual cells in the 3D image.

Multi-electrode array recordings and analysis

[00387] Multi-electrode array (MEA) recordings were performed on retinas from rdl mice at >p90, 6-8wks following AAV injection. Retinas were excised from the eye under dim red light, mounted on 4 pm cell membranes and placed ganglion cell side down in the recording chamber (pMEA 100/30iR- Tpr; Multi Channel Systems, Reutlingen, Germany) of a 60-channel MEA system and perfused with Ames recording media (32 °C). This system has an electrode grid of 6x10, with electrode spacing of 100 lim and electrode size of 30 pm. A Multi Channel Systems harp weight (Scientific Instruments - Slice grids) was placed on the retina to prevent movement and vacuum was applied to the retina using a pump (perforated MEA1060 system with CVP; Multi Channel Systems) to improve electrode-to-tissue contact. [00388] In vitro Illumination was performed by a DG-4 using a 300-W mercury arc lamp (Sutter Instruments, Novato, CA) with a blue (470/60 nm) bandpass filter. See figure legends for details. Relative comparisons with natural light intensities were obtained in various environments using direct light measurement with a power meter (Thorlabs Inc. Newton, NJ). Retinal activity on the MEA was sampled at 25 kHz filtered between 100 and 3,000 Hz. Voltage traces were converted to spike trains offline and the spikes recorded at each electrode were sorted into single units, which we defined as “cells,” via principal component analysis using Offline Sorter (Plexon-64bit) with each electrode commonly identifying 1-3 cells. Single-unit spike clusters were exported to Neuroexplorer and graphed or exported to MATLAB (MathWorks) for graphing with custom software. All firing rates were extracted from traces averaged over 6 light response cycles. Firing rate in the dark was calculated from the average activity during 1-3 s preceding the flash. Responses across cells and across retinas were normalized using the Light Response Index (LRI) adopted from earlier work (LRI = (peak firing rate in the light - average firing rate in dark) / peak firing rate in the light + average firing rate in dark). Kinetic analysis was performed in Graphpad Prism 9. Individual responding cells are displayed in raster plots along with their averages (FIG. 2C) or just as averages (FIG. 3A). rd! retina expressing SNAP-mGluR2 in RGCs received an intravitreal injection of BGAG photoswitches several days before retinal isolation and recording (FIG. 2). Figure legends indicate the number of retinas represented in each raster and average. Behavioral analyses

[00389] The 2-chamber light-dark passive avoidance test was performed as described previously. Lin et al. (2008) Proc. Natl. Acad. Sci. USA 105:16009; Doroudchi et al. (2011) Mol. Ther. 19:1220; van Wyk et al. (2015) PLoS Biol. 13:el002143; and Broichhagen et al. (2015) supra. White light (wavelength range) at -100 pW cm ² or either blue light (460/45 nm) or green light (535/50 nm) at 0.5 - 25 pW cm ² was mounted above the chamber with homogeneously distributed light. Animal movements were tracked using IR sensors on the shuttle box. Time spent in the light and dark chambers was measured and analyzed using Graphic State and Graphic State RT (Coulbourn Instruments).

[00390] Fear conditioning experiments were performed as described previously (van Wyk et al. (2015) supra) using Coulbourn single shock chamber with an LED screen that presented the visual cue mounted to the ceiling of the chamber. Animals were subjected to paired or unpaired light cued fear conditioning consisting of three shock trials at 0.7 mA over a span of 15 min. Freezing behavior in anticipation of the shock was recorded by Coulbourn’ s FreezeFrame software and normalized to movement behavior gathered before the stimulation. Performance was compared between paired and unpaired cohorts in order to determine if a fear response was conditioned to the stimulus transition.

[00391] Modified active avoidance was assayed as previously described (Lin et al. (2008) supra), using the Coulbourn shuttle box (H10-11M-SC), however, now iPad tablet screens were mounted onto the shuttle cage wall, each displaying one of two images that differed in orientation or distance between two lines but were otherwise of equal shape, size, and light intensity. The aversive image side was paired with a foot shock of 0.7 mA. Upon recall the light patterns were reversed to avoid a bias for location and time spent on each side was recorded. Adaptation was tested by dimming or brightening the display to different intensities.

CHEMICAL SYNTHESIS

General

[00392] Solvents for chromatography and reactions were purchased dry over molecular sieves or in HPLC grade. Unless otherwise stated, all other reagents were used without further purification from commercial sources. [00393] Liquid chromatography-mass spectroscopy (LC-MS) was performed on a Shimadzu MS2020 connected to a Nexera UHPLC system equipped with a Waters ACQUITY UPLC BEH Cl 8 (1.7 pm, 50 x 2.1 mm). Buffer A: 0.1% FA in H2O Buffer B: acetonitrile. The typical gradient was from 10% B for 0.5 min gradient to 90% B over 4.5 min

90% B for 0.5 min

gradient to 99% B over 0.5 min with 1 mL/min flow. Retention times (/«) are given in minutes (min).

[00394] High resolution mass spectrometry was performed using a Bruker maXis II ETD hyphenated with a Shimadzu Nexera system. The instruments were controlled via Brukers otofControl 4.1 and Hystar 4.1 SR2 (4.1.31.1) software. The acquisition rate was set to 3 Hz and the following source parameters were used for positive mode electrospray ionization: End plate offset = 500 V; capillary voltage = 3800 V; nebulizer gas pressure = 45 psi; dry gas flow = 10 L/min; dry temperature = 250 °C. Transfer, quadrupole and collision cell settings are mass range dependent and were fine-adjusted with consideration of the respective analyte’s molecular weight. For internal calibration sodium format clusters were used. Samples were desalted via fast liquid chromatography. A Supelco Titan™ Cl 8 UHPLC Column, 1.9 pm, 80 A pore size, 20 x 2.1 mm and a 2 min gradient from 10 to 98% aqueous MeCN with 0.1% FA (H₂O: Carl Roth GmbH + Co. KG ROTISOLV® Ultra LC-MS; MeCN: Merck KGaA LiChrosolv® Acetonitrile hypergrade for LC-MS; FA - Merck KGaA LiChropur® Formic acid 98%- 100% for LC-MS) was used for separation. Sample dilution in 10% aqueous ACN (hyper grade) and injection volumes were chosen dependent of the analyte’s ionization efficiency. Hence, on-column loadings resulted between 0.25-5.0 ng. Automated internal re-calibration and data analysis of the recorded spectra were performed with Bruker’ s DataAnalysis 4.4 SRI software.

[00395] NMR spectra were recorded in deuterated solvents on a Bruker AVANCE III HD 400 equipped with a CryoProbe and calibrated to residual solvent peaks ('H/¹³C in ppm): DMSO-dg (2.50/39.52), acetone-dg (2.05/29.84), CDCL (7.26/77.0), D₂O (4.70). Multiplicities are abbreviated as follows: s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, h = hexet, br = broad, m = multiplet. Coupling constants J are reported in Hz. Spectra are reported based on appearance, not on theoretical multiplicities derived from structural information.

[00396] Preparative RP-HPLC was performed on a Waters e2695 system equipped with a 2998 PDA detector for product collection (at 220, 280, 360 or 460 nm) on a Supelco Ascentis® Cl 8 HPLC Column (5 pm, 250 x 21.2 mm). Buffer A: 0.1% TFA in H₂O Buffer B: acetonitrile. The typical gradient was from 10% B for 5 min gradient to 90% B over 45 min

gradient to 99% B over 5 min with 8 mL/min flow.

[00397] Compound 2, and BG-COOH were previously described in Broichhagen et al. , ACS Cent. Sci. 2015, 1, 383-393 and Levitz et al., PNAS 2017, 114, E3546-E3554, respectively. Abbreviations

[00398] DIPEA: /V./V-diM^pi'opylcthylaminc; DBU: l,8-diazabicyclo[5.4.0]undec-7-ene; DMSO: dimethylsulfoxide; FA: formic acid; HBTU: (2-(177-benzotriazol-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate; TFA: trifluoroacetic acid; THF: tetrahydrofuran;.

Determination of mole numbers

[00399] Mole numbers for reactions were determined by dissolving the parent compound in DMSO or DMF (V) and diluting into DMSO (dilution factor; f = 20 or 50) before measuring absorbance A at a NanoDrop. Calculations were performed according to Eambert-Beer’ s law according to equation 1:

[00400] n = c*V = A*f*V / £ *d (1)

[00401] with:

[00402] d = 0.1 cm

[00403] £ (azobenzene) @ 420 nm: 24,600 M ¹ cm ¹

Synthetic scheme

[00404] A synthetic scheme is depicted in FIG. 7.

[00405] Synthesis of Compound 3 Dimethyl (2S,4S)-2-(4-((4-((E)-(4-((l-amino-21-oxo- 3,6,9,12,15,18-hexaoxa-22-azatetracosan-24-yl)amino)phenyl)diazenyl)phenyl)amino)-4-oxobutyl)-4- ((tert-butoxycarbonyl)amino)pentanedioate (3)

[00406] A round bottom flask was charged with 37 mg (64 pmol, 1.2 equiv.) of FmocPEGgCOOH 1 (Broadpharm, #BP-21630) and 32 mg (54 pmol, 1.0 equiv.) of amine 2 dissolved in 3 mL DMSO and 37 pL DIPEA before 29 mg (75 pmol, 1.4 equiv.) of HBTU was added in one portion. The reaction mixture was stirred o.n. at r.t. before it 150 pL of DBU was added and the reaction was stirred for another 30 min. The solution was quenched by addition of 200 pL HOAc and 100 pL H2O and subjected to RP-HPLC to obtain 33 pmol of the desired compound as a red powder (which turned into a deep red oil upon standing) after lyophilization in 62% yield over 2 steps.

[00407] HRMS (ESI): calc, for C₄5H₇₂N₇Oi4 [M+H]⁺: 934.5132, found: 934.5131.

[00408] Synthesis of Compound 6 4-(l-(9Z7-Fluoren-9-yl)-3-oxo-2,7,10-trioxa-4-azadodecan-12- amido)-4-(2-carboxyethyl)heptanedioic acid (6)

[00409] A round bottom flask was charged with 126 mg (304 pmol, 1.0 equiv.) of amino triester 5 (Frontier Scientific, #NTN1963) and 140 mg (364 pmol, 1.2 equiv.) of FmocPEG2COOH (Broadpharm, #BP-22044) 4 dissolved in 3 mL DMSO and 212 pL DIPEA before 161 mg (426 Dmol, 1.4 equiv.) of HBTU was added. The reaction mixture was stirred o.n. at r.t. before it all volatiles were removed in vacuo, and 1.5 mL of neat TFA was added carefully (exothermic). The resulting mixture was stirred for 3 hours before TFA was removed in vacuo, the residue was taken up in DMF/H2O = 8/2 and the crude was subjected to RP-HPLC to obtain 155 mg (252 pmol) of the desired compound as a white powder after lyophilization in 83% yield.

'H NMR (400 MHz, DMSO-d₆): 6 [ppm] = 11.98 (br s, 3H), 7.88 (d, J = 7.5 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.3 Hz, 2H), 6.88 (s, 1H), 4.29 (d, J = 6.9 Hz, 2H), 4.21 (t, J = 6.9 Hz, 1H), 3.82 (s, 2H), 3.55 (d, J = 4.8 Hz, 4H), 3.44 (t, J = 6.0 Hz, 2H), 3.15 (q, J = 5.9 Hz, 2H), 2.13 (dd, J = 10.2, 6.1 Hz, 6H), 1.87 (dd, J = 10.1, 6.2 Hz, 6H).

¹³C NMR (101 MHz, DMSO-dg) 3 [ppm] = 174.4, 168.8, 156.2, 143.9, 140.8, 127.6, 127.1, 125.2, 120.1, 70.2, 69.8, 69.3, 69.2, 65.4, 56.5, 46.7, 40.1, 29.1, 28.0.

[00410] HRMS (ESI): calc, for C31H3IN2O11 [M+H]⁺: 615.2548, found: 615.2544.

[00411] Synthesis of Compound 7 4,4'-((4-(l-(9Z7-Fluoren-9-yl)-3-oxo-2,7,10-trioxa-4- azadodecan-12-amido)-4-(3-((l,5-dicarboxy-3-(2-carboxyethyl)pentan-3-yl)amino)-3- oxopropyl)heptanedioyl)bis(azanediyl))bis(4-(2-carboxyethyl)heptanedioic acid) (7)

[00412] A round bottom flask was charged with 87.6 mg (146 pmol, 1.0 equiv.) of 6 and 242 mg (584 pmol, 4.0 equiv.) of amino triester 5 dissolved in 5 mL DMSO and 305 pL DIPEA before 276 mg (729 pmol, 1.4 equiv.) of HBTU was added. The reaction mixture was stirred o.n. at r.t. before it all volatiles were removed in vacuo, and 1.5 mL of neat TFA was added carefully (exothermic!). The resulting mixture was stirred for 3 hours before TFA was removed in vacuo, the residue was taken up in DMF/H2O = 8/2 and the crude was subjected to RP-HPLC to obtain 65.0 mg (50 pmol) of the desired compound as a white powder after lyophilization in 34% yield.

'H NMR (400 MHz, DMSO-d₆): <5 [ppm] = 12.02 (br s, 9H), 7.88 (d, J = 7.5 Hz, 2H), 7.69 (d, J = 7.4 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H), 7.32 (t, J = 7.5 Hz, 2H), 7.21 (s, 3H), 7.02 (s, 1H), 4.29 (d, J = 6.9 Hz, 2H), 4.21 (t, J = 6.9 Hz, 1H), 3.81 (s, 2H), 3.67-3.48 (m, 4H), 3.42 (t, J = 6.0 Hz, 2H), 3.15 (q, J = 5.9 Hz, 2H), 2.10 (t, J = 8.2 Hz, 18H), 2.07-1.99 (m, 6H), 1.90-1.71 (m, 24H).

¹³C NMR (101 MHz, DMSO-dg) 3 [ppm] = 174.9, 172.5, 169.0, 156.7, 144.4, 141.2, 128.1, 127.5, 125.7, 120.6, 80.6, 70.6, 69.9, 69.6, 65.8, 57.4, 56.8, 47.2, 40.1 (HSQC), 31.3, 30.6, 29.5, 28.5, 28.2.

[00413] HRMS (ESI): calc, for C6iH₈₅N₅O26 [M+2H]²⁺: 651.7736, found: 651.7729.

[00414] Synthesis of 9xBGAG₄6o (FIG. 8).

[00415] A 1.5 mL Eppendorf tube was charged with 390 nmol (1.0 equiv.) of 7 and 7.8 pmol (20.0 equiv.) of 3 dissolved in 500 pL DMSO and 3.0 pL DIPEA before 3.0 mg (7.8 pmol, 20.0 equiv.) of HBTU was added in one portion. The reaction mixture was vortexed and incubated under shaking (750 rpm) for 4 h before another 3.0 mg of HBTU and 3.0 pL DIPEA were added and the reaction incubated at 50 °C for additional 3 h. Finally, it was quenched by addition of 10 pL HOAc and 200 pL H2O and subjected to RP-HPLC to obtain the desired compound as a red powder after lyophilization, which was used immediately in the next step.

[00416] HRMS (ESI): calc, for C₄66H₇o₉N680i43 [M+5H]⁵⁺: 1910.2085, found: 1910.2093 for FmocHN-PEG₂-(PEG6-AGNHBoc(COOMe)₂)9.

[00417] The Fmoc-protected dendrimer from the previous step was dissolved in MeCN (0.8 mL) and DBU (40 pL) and allowed to incubate at r.t. for 30 min, before it was quenched by addition of 50 pL HOAc and 100 pL H2O and subjected to RP-HPLC to obtain the desired compound as a red powder after lyophilization, which was used immediately in the next step.

[00418] HRMS (ESI): calc, for C451H699N68O141 [M+5H]⁵⁺: 1865.7684, found: 1865.7684 for H₂N-PEG2-(PEG6-AGNHBoc(COOMe)₂)₉.

[00419] The free amine from the previous step was dissolved in DMSO (0.8 mL) and DIPEA (4 pL) and 1 mg BG-COOH and 1.2 mg HBTU were added. The reaction mixture was vortexed and allowed to incubate at 50 °C for 2 h, before it was quenched by addition of 10 pL HOAc and 100 pL H2O and subjected to RP-HPLC to obtain the desired compound as a dark red powder after lyophilization, which was used immediately in the next step.

[00420] HRMS (ESI): calc, for (WIw^Oiu [M+5H]^,+: 1939.0236, found: 1939.0249 for BG-PEG₂-(PEG6-AGNHBoc(COOMe)₂)₉.

[00421] The BG-linked from the previous step was dissolved in MeOH/1 M LiOH (400 pL each) and the resulting suspension was allowed to incubate for 3 h before it was quenched with 50 pL of glacial HOAc, 200 pL of DMF was added and subjected to RP-HPLC to obtain the desired compound as a dark red powder after lyophilization, which was used immediately in the next step.

[00422] HRMS (ESI): calc, for C451H681N74O144 [M+5H]⁵⁺: 1888.5673, found: 1888.5658 for BG-PEG₂-(PEG6-AGNHBOC)₉.

[00423] The free acid from the previous step was put on ice and 1 mL of pre-cooled (4 °C) TFA was added neat. The reaction mixture was vortexed to ensure homogeneity and put back on ice for 1.5 h before all volatiles were removed under a gentle stream of nitrogen. The residue was taken up in DMF/water (9/1) and subjected to RP-HPLC to obtain 20 nmol of ^9xBGAG₄6o as a red powder after lyophilization in 5% yield over 5 steps.

[00424] HRMS (ESI): calc, for C₄06H609N₇₄OI₂6 [M+5H]⁵⁺: 1708.4729, found: 1708.4724 for BG-PEG₂-(PEG6-AG)₉, i.e. ^9xBGAG₄₆o.

RESULTS

9xBGAG endows HEK293 cells with a fast and transient light response

[00425] The original single branched BGAGI_2J46O bears one light-activated azobenzene-glutamate (AG) for each benzylguanine (BG) anchor, with an intervening polyethylene glycol (PEG) linker and where the BG attaches covalently to SNAP fused onto the extracellular N-terminal of mGluR2 (SNAP- mGluR2) (FIG. 1A). Photo-isomerization with blue light of the azobenzene would switch it from the trans configuration, which it assumes in the dark and which obstructs the glutamate so that it cannot bind to the clamshell glutamate binding site of mGluR2, to the cis configuration, which allows glutamate binding and activates the receptor (FIG. 1A). For a single BG anchor, 9XBGAGI_2J46O bears nine PEG branches, each with a light-activated glutamate, for a total of nine photo-activatable agonists (FIG. IB, C). FIG. ID provides a comparison of the chemical structure of ^4XBGAGI_2J46O (left) and ^9XBGAGI_2J46O (right). ^4XBGAGI_2J46O includes four azobenzene-glutamates per SNAP-tag (FIG. ID). The branched photoswitch approach, where each photoswitch bears multiple azobenzene-glutamates, increasing the likelihood of isomerization to the active cis form and, thus, a higher likelihood of receptor agonism, is illustrated schematically in FIG. IE.

[00426] FIG. 1A-1E. Design of multi-branched 9XBGAGI_2J46O for photo-activation of SNAP- mGluR2. (A) BGAGI_2J46O consists of a benzylguanine (BG) that attached covalently to a SNAP domain fused to the N-terminus of mGluR2, a PEG linker with 12 repeats, and an azobenzene-glutamate (AG). Blue light (peak excitation at 460 nm) photo-isomerizes the azobenzene of BGAGI_2J46O from the trans configuration, which obstructs the glutamate (orange ball) and prevents its binding, to the nonobstructing cis configuration, which lets the glutamate bind to the clamshell ligand binding domain of each of the subunits of the mGluR2 dimer. In this way blue light triggers glutamate binding and receptor activation. Azobenzene relaxes back to the trans configuration in the dark to restore the binding incompetent pose of the glutamate and retting state of the receptor. As a result, SNAP-mGluR2 is activated only during blue light exposure. The activation of Gi-coupled mGluR2 leads to Gbg activation of potassium channels, such as the GIRK channel. (B, C) Chemical structure (B) of 9-branched 9xBGAGi2.46o, with one BG attachment site and 9 AG light-controlled glutamates and cartoon (C) depicting one attached to the SNAP domain of each of the two subunits of the mGluR2 dimer. FIG. ID: Chemical structure of ^4xBGAGi2,46o (left) and ^9xBGAGi2,46o (right) with Benzylguanine SNAP-attachment moiety (green), PEG-linkers (black), azobenzene (blue) and L-glutamate ligand (orange) color labeled. FIG. IE: Schematic of SNAP-tagged mGluR2 without (left) and with (middle) covalently tethered ^4xBGAGi2,46o photoswitches. In neurons, photoactivation of mGluR2 (right) leads to binding of Gi/G^y and liberation of G[3y. which binds to, activates and opens the GIRK channel (brown) leading to K⁺ efflux (at physiological low external [K⁺] and negative resting potential) and consequent membrane hyperpolarization.

[00427] To test the function of ^4XBGAGI₂,₄6O and ^9xBG AG 12, 460 in the retina, a viral vector construct was used, with SNAP-mGluR2 under the control of the promoter of the human Synapsin 1 gene, which is preferentially expressed in retinal ganglion cells (RGCs) of mice and humans. The construct, which included inverted terminal repeats (ITRs) and the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (Methods), was packaged into the AAV2(4YF) capsid. Delivery into the eye of >3-month-old rdl mice was via intravitreal injection with a membrane- impermeable BG-dye instead of with the BGAG photoswitch.

9xBGAG restores fast light responses to the isolated rdl retina

[00428] A flash of light to the isolated wildtype retina evokes a diverse set of responses in different retinal ganglion cells (RGCs), as measured on a multi-electrode array (MEA), and these range from transient to sustained bursts of activity during the light and varying degrees of an excitatory response when the light is turned off (FIG. 2A). In rdl mice, which are blind because of degeneration of rods and cones, the light response of RGCs is lost, except in a small number of intrinsically photosensitive RGCs (Berry et al. (2017) Nature Commun. 8:1862; and Berry et al. (2019) Nature Commun. 10:1221). To restore the light response to rdl mice, AAV was used to introduce the gene encoding SNAP-mGluR2 into the RGCs of rdl mice under the human Synapsin promoter (hSyn) (AAV2 4YF: hSyn-SNAP-mGluR2), as done earlier (Berry et al., 2017) supra). Gene delivery was followed 1.5-3 months later with isolation of the retina and MEA recordings on the isolated retina that was incubated for 45 minutes in ImM 9xBGAGi2,46o- Following washout, a relatively uniform inhibitory response to light was observed across the RGCs, which was followed by an excitatory response when the light was turned off (FIG. 2B). The response was similar when the 4-branched 4xBGAGi2,46o or the single branched BGAG12.460 was used (FIG. 2B). [00429] It was found that both 4xBGAGi2,46o and 9xBGAGi2,46o supported a response to very short pulses of light. At a fixed intensity, reduction of the duration of the light pulse decreased the amplitude of both the inhibitory response to light and the after-excitation but reduced the after-excitation more strongly (FIG. 3). The kinetics of the inhibitory response remained fast, so that the disappearance of the slower after-excitation led to a faster reset, suggesting that the approach would support a faster retinal refresh rate when objects are in motion or light is dim.

[00430] FIG. 2A-2B. The function of lx, 4x and 9x branched versions of BGAGi2,46o on SNAP- mGluR2 in RGCs of rdl mouse. A,B) Isolated retina from wildtype (C57) mouse (A) and rdl mouse (B) recording action potential firing from multiple RGCs using a multi-electrode array. Individual units shown below, average displayed above, lx, 4x and 9xBGAGi2,46o on SNAP-mGluR2 responses consist of inhibition during light and rebound excitation when light is turned off.

[00431] Six to ten weeks after intravitreal AAV injection, 2 pl of 1 mM of BGAG12.460, ^4xBGAGi2.46o, or ^9xBGAGi2,46o in PBS was injected into the -5 pl volume mouse eye, to produce a concentration of -290 pM in the vitreous. To assess the light evoked response, SNAP-mGluR2 expressing retinas were removed from rdl mice and mounted on a multi-electrode array (MEA). Due to photoreceptor degeneration, the retinas of untreated rdl adult mice (>12 weeks), and ones expressing SNAP-mGluR2 in RGCs, do not have recordable light-evoked responses at the intensities that were tested. However, rdl mouse retinas expressing SNAP-mGluR2 in RGCs, from animals that had been injected intravitreally with each of the three BGAGi2,46o variants in PBS 2-4 days earlier, showed light- evoked suppression of firing. This was followed by a transient excitatory rebound at the cessation of illumination, a response that resembles the photoreceptor cell driven response of OFF-RGCs in the wildtype retina. The data are shown in FIG. 2C and FIG. 2D.

[00432] With application of progressively shorter light pulses, the amplitude of the rebound excitation declined more than did the inhibition elicited during light exposure, leaving substantial inhibition even in response to a light pulse of only 25 ms (FIG. 3 A) As the duration of the flash (i.e. the number of photons delivered) decreased from 1000 to 25 ms, the rise phase of the inhibitory response maintained the same kinetics and the decay phase slowed by -3.5-fold to a half-time of -140 ms (FIG. 2E-2I). The slowest decay rate was similar to the rate of decay in firing rate at the end of a flash with ChrimsonR in RGCs of the NHP retina. In addition, it was -5 times faster than the deactivation of CoChR-3M current in HEK293 cells, although the OFF after-excitation elicited by longer light flashes to ^4xBGAGi2,46o:SNAP-mGluR2 was -2.5 slower than the deactivation of CoChR-3M current. The onset and decay of inhibition of firing by all three BGAGi2,46o variants was -10-fold and -7-fold faster, respectively, than the rise and fall of firing elicited in RGCs by Opto-mGluR6 expressed in ON-BCs and there was no delay of the kind seen in RGCs with MW cone opsin²³. These comparisons suggest that ^4xBGAGi2,46o:SNAP-mGluR2 in RGCs may have an advantage over CoChR-3M, Opto-mGluR6 and MW cone opsin for motion vision when changes in luminance occur quickly and in dim light.

[00433] FIG. 2C-2D. (C) Light response of RGCs in isolated rdl mouse retina expressing SNAP-mGluR2 in RGCs and labeled with either BGAGi2,46o (black), ^4xBGAGi2,46o (blue) or ^9xBGAG1246o (green). Light triggers a fast suppression of spontaneous firing followed by a rebound excitation when the light is turned off. (Bottom) Raster plot of responses in 65, 93 and 65 RGCs (BGAG12460, ^4xBGAGi246o, ^9xBGAGi246o, respectively) in response to a 3 sec light (blue = 472nm) (each cell shows average response to 5 pulses of light). (Top) Average response of the RGCs shown in the raster (bottom). Activity measured with a 60-channel transparent multielectrode array (ME A). (D) Light response index (LRI) (peak firing rate in light - average firing rate in dark / peak firing rate in light + average firing rate in dark) in rdl mouse retina expressing SNAP-mGluR2 in RGCs and labeled with either BGAG12460 (n = 2), ^4XBGAGI₂46O (n = 4) or ^9xBGAG124₆₀ (n = 4).

[00434] FIG. 2E-2I. Electrophysiological properties of RGC light response in isolated rdl mouse retina expressing SNAP-mGluR2 in RGCs and labeled with 4xBGAGi246o or 9xBGAGi246o- E-H) Change in firing of RGCs of isolated retina in response to light measured on an MEA. FIG. 2E) Average Light response index (LRI) (peak firing rate in light - average firing rate in dark / peak firing rate in light + average firing rate in dark) for inhibitory ON response (during light, blue) and excitatory OFF response (after light, gray) in four retinas from rdl mice expressing SNAP-mGluR2 in RGCs and labeled with either ^4xBGAGi246o (left, in retinas 1-4: n = 97, 25, 17 and 9 cells, respectively) or ^9xBGAGi246o (right, in retinas 1-4: n = 20, 30, 15 and 9 cells, respectively). FIG. 2F) Rise phase of inhibition of firing a start of the light flash (Light ON; upper two sets of superimposed traces) and recovery/rise of rebound excitation following termination of the flash (Light OFF, lower two sets of superimposed traces) from rdl mice expressing SNAP-mGluR2 in RGCs and labeled with either ^4xBGAGi246o or ^9xBGAGi246o- Times of start or end of flash synchronized. Red lines fit to rise and decay phases to estimate rates. Traces from FIG. 3. FIG. 2G) Dependence on flash duration of kinetics measured as slopes of linear fits for traces shown in (F). FIG. 2H and 21) Dependence of normalized integrated firing response on normalized number of photons in RGCs of isolated rdl retina expressing SNAP-mGluR2 in RGCs and labeled with either 4xBGAGi246o (H) or 9xBGAGi246o (I).

[00435] FIG. 3A Dependence of ^4xBGAGi246o:SNAP-mGluR2 (top) and ^9xBGAGi246o:SNAP- mGluR2 (bottom) RGC light response on flash duration in unites shown in (A). Population average firing shows responses down to 25 ms illumination duration. Mean (black) ± SEM (gray). 4xBGAGi246o and 9xBGAGi246o respond to brief pulses of light primarily with inhibition during light and fast on and off kinetics. 4xBGAGi246o:SNAP-mGluR2 and 9xBGAGi246o:SNAP-mGluR2 in RGCs of rdl retina confer similar biphasic light modulation of AP firing as seen in frequency average from multiple trains of pulses in descending duration to example retina of each. Light evokes a rapid inhibition that lasts for the duration of the light pulse and recovers rapidly when the light is turned off and includes a transient afterexcitation phase that is most prominent after long pulses and becomes almost undetectable after short pulses. The on rate of the inhibitory response is fast across fast across light pulse duration. The off excitatory rebound becomes smaller with shorter light pulses.

9xBGAG restores ultra-high-sensitivity light aversion

[00436] Wildtype (C57) mice with intact visual function naturally avoid illuminated spaces, however mice with retinal degeneration, such as rdl, cannot perform this light/dark discrimination. To assess the ability of rdl animals expressing SNAP-mGluR2, which has been derivatized with ^4xBGAGi2.46o, to distinguish light from dark, a 2-chamber shuttle box was employed, with an open doorway between the chambers and an iPad-mini that was installed on the far wall of each chamber, one of which was set to black and the other to white. The illuminated LCD display was set to different white light intensities, ranging from very dim to full brightness of the screen: 0.2, 5, 25 or 88 pW/cm² (i.e. 10^Y photons cm ² s ¹, where y = 11.7, 13.1, 13.8 or 14.3, respectively). Normally sighted wildtype (C57) mice showed maximal photo-aversion at the two dimmer light levels (0.2 and 5 pW/cm²) and spent -600 s of the 900 s observation time in the dark chamber (FIG. 3B, red). Rdl mice spent equal amounts of time (450 s) in the two chambers, even when brightness was increased to 250 pW/cm² using a brighter LCD screen (FIG. 3B, grey and grey dotted line crossing y-axis), indicating that at this age, rdl mice are unable to sense these intensities of light. In contrast, rdl mice expressing SNAP-mGluR2 in RGCs, 5-7 days after injection of 2 pl of 1 mM BGAG12.460, ^4xBGAGi2,46o or ^9xBGAGi2,46o in PBS, spent more time in the dark chamber than in the illuminated chamber. The light levels that produced this photo-aversion differed between the BGAGs. Aversion occurred only at the highest intensity of 1000 «W/cm² with unbranched BGAG12.460, at >5 pW/cm² with ^4xBGAGi2,46o and at >0.2 pW/cm² with ^9xBGAGi2,46o (FIG. 3B- source data 3). Thus, ^4xBGAGi₂,46o:SNAP-mGluR2 and ^9xBGAGi₂,46o:SNAP-mGluR2 in RGCs enable otherwise non-responsive rdl mice to sense light at intensities that are, respectively, only -6% and -0.2% of the maximal brightness of the iPad. The mid-points of the intensity-response relations were -750 pW/cm² for BGAG12.460, -2.5 pW/cm² for ^4xBGAGi2,46o and -0.1 pW/cm² for ^9xBGAGi2,46o (FIG. 3B, dashed black line), i.e. an -300-fold increase in sensitivity due to quadrupling of the number of photo-ligands from 1 to 4 and a further increase of -25-fold increase in sensitivity due to increasing the number of photo-ligands from 4 to 9.

[00437] To contextualize this sensitivity, this performance was compared to rdl mice expressing ChrimsonR(K176R) (ChrimsonR) in the same AAV(4YF) expression vector, using the same Synapsin 1 promoter, ITRs and WPRE. Photo-aversion with ChrimsonR in RGCs had an intermediate sensitivity between that seen with unbranched BGAGi2,46o and ^4xBGAGi2,46o (FIG. 3B). [00438] FIG. 3B. Amount of time spent in the dark compartment of the 2-chamber arena when the illuminated chamber is lit at different intensities for six animal groups (n = 7-11 mice per condition): rdl control (gray); rdl mice expressing SNAP-mGluR2 in RGCs and injected with either BGAGi2,46o (black), ^4xBGAGi2,46o (blue), or ^9xBGAGi2,46o (green); rdl mice expressing ChrimsonR in RGCs (orange); wildtype (C57) mice (red). Values are mean + SEM.

[00439] To determine if 9xBGAGi2,46o could restore vision, rdl mice that received intravitreal injection of AAV2 4YF:hSyn-SNAP-mGluR2 underwent a second intravitreal injection > 6 weeks later with either 4xBGAGi2,46o or 9xBGAGi2,46o in PBS to yield a final concentration in the vitreous of -250 pM. Over the next 3-6 days, vision was tested in several paradigms. The first paradigm tested was whether the animals could distinguish light from dark and, if so, with what sensitivity.

[00440] A 2-chamber shuttle box with an open door in between the chambers was illuminated in one chamber and not in the other (FIG. 4A). Sighted mice are photophobic and spend less time in the illuminated chamber, whereas blind rdl mice spend divide the observation time of 900 s evenly times between the two chambers (FIG. 4B, dashed red line) (Berry et al. (2017) supra). Rdl mice expressing SNAP-mGluR2 in RGCs that were injected intravitreally with 4xBGAGi2,46o in PBS spent more time in the dark chamber than in the illuminated chamber when the illumination intensity was 25 and 88 pW/cm², but dropped off to equal durations in the dark and light chambers when the illumination intensity was reduced to 0.2 and 5 pW/cm² (FIG. 4B), suggesting a threshold sensitivity for light perception between 5 and 25 pW/cm². In contrast, rdl mice expressing SNAP-mGluR2 in RGCs that were injected intravitreally with 9xBGAGi2,46o showed maximal preference for the dark chamber even at 0.2 pW/cm², suggesting an increase in sensitivity by -25-fold or more than the sensitivity endowed by 4xBGAGi2,46o. These results indicate that 9xBGAGi2,46o:SNAP-mGluR2 in RGCs enables previously blind mice to detect very dim light— at least 440-fold dimmer than the maximal brightness of a standard LCD computer display.

[00441] It was asked if the higher sensitivity conferred by 9xBGAGi2,46o compared to 4xBGAGi2,46o means that 9xBGAGi2,46o would be equally effective at a lower dose. To test this, the bias for the dark chamber exhibited by rdl mice who received the same dose of AAV vector for driving expression of SNAP-mGluR2 in RGCs was compared with a similar range of doses of either 4xBGAGi2,46o or 9xBGAGi2,46o- It was found that at 10-fold greater dose, 4xBGAGi2,46o conferred similar or slightly lesser preference for the dark chamber compared to 9xBGAGi2,46o (FIG. 5, compare bars circled in similar colors). Together these results suggest that 9xBGAGi2,46o is equally efficacious at <10% the dose of 4xBGAGi2.46o, roughly consistent with the >25-fold increase in efficacy of the two branched BGAGs when given at the same dose. [00442] FIG. 4. 9XBGAG12.460 confers higher light sensitivity than 4xBGAGi2,46o in restored light avoidance behavior. 4xBGAGi2,46o:SNAP-mGluR2 in the RGCs of rdl mouse retina restores light avoidance when the light chamber has an illumination intensity of 25 or 88 pW/cm² , but not at 0.2 or 5 pW/cm². In contrast, 9xBGAGi2,46o:SNAP-mGluR2 reaches maximal light avoidance at 0.2 pW/cm², suggesting an enhancement in sensitivity of between 25 and 125-fold.

[00443] FIG 5. 9xBGAGi2, 46o ~ 10-fold more potent than xBGAGi2,46o- Similar light avoidance restored by 9xBGAGi2,46o at -10% the concentration compared to 4xBGAGi2,46o-

9xBGAG restores high-acuity line pattern recognition

[00444] Having seen that 9xBGAGi2,46o:SNAP-mGluR2 in RGCs confers high-sensitivity light perception, it was asked whether it would support patterned vision.

[00445] A) Test of discrimination between line patterns in a learned negative association task. 2- chamber arena (bottom), where an aversive mild foot shock can be applied in either chamber. Each chamber has an iPad-mini at far end displaying a pair of parallel vertical lines at one of two spacings (top). Two days of training with mild foot shock paired with one line pattern associated with the foot shock was assigned randomly initially but then kept consistent for that animal throughout the 2 days of training. On the third day, no foot shock was given, and only the displays were shown. The chambers were cleaned thoroughly before and after each training or test session to remove olfactory clues. The room was kept dark to avoid room reference visual cues. In addition, the aversive and non-aversive cues were switched between the end of the second day of training and the test day to avoid location bias. Provided they have sufficient visual acuity to distinguish between the two-line patterns, sighted animals avoid the side with the line pattern that had been previously associated with the foot shock, whereas untreated blind rdl show a location bias that reduces their time on the side that had been aversive during training and on the test day displayed the non-aversive visual cue (Berry et al. (2017) supra-, and Berry et al. (2019) supra).

[00446] Displays of 0.5 cm wide lines separated by either 0.25 or 0.5 cm were compared. In the two-chamber system, given the 18 cm distance from the point of decision to the iPad, the display with 0.5 cm thick lines separated by 0.5 cm is comparable to an optomotor drum spatial frequency of 0.2 cycles per degree (see Methods), and is close to the acuity limit for wildtype mouse (Kretschmer et al. (2017) J. Neurophysiol. 118:300). Mice were trained by pairing either the line pair separate by 0.25 or 0.5 cm with foot shock in untreated rdl mice and rdl mice expressing SNAP-mGluR2 in RGCs following intravitreal injection with either 4xBGAGi2,46o or 9xBGAGi2,46o in PBS to yield a final vitreal concentration of -250 pM.

[00447] Rdl mice expressing SNAP-mGluR2 in RGCs, which were trained and tested within a week after intravitreal injection of BGAG, spent more time on the side of the non-aversive visual display (i.e. the display that was in the chamber that did not have the foot shock during the training period) (FIG. 6B). This preference for the non-aversive chamber was similar to that of wildtype, sighted animals and stood in stark contrast to that of untreated rdl mice whose location bias had them favor the chamber that during the training period displayed the aversive cue, but during the test period displayed the non- aversive cue (FIG. 6B). The strong preference for the chamber with the non-aversive visual cue indicates that both 4xBGAGi2 ,46o and 9xBGAGi2.46o on SblAP-mGluR2 expressed in RGCs restore spatial pattern recognition at an acuity that is near the wildtype limit.

[00448] FIG. 6A-6B. Restoration of high acuity line pattern discrimination. A) Shuttle arena with two chambers, each with a conductive floor that can administer mild foot shock, and each containing an iPad-mini (500 nits) on its far wall, which displays one of line patterns. The black lines on a white background are two 0.5 cm wide black vertical lines separated either by 0.25 or 0.5 cm, as shown above. Animals are trained for two days where foot shock is given in one chamber showing one of the displays. On the third day, the displays are switched and there is no foot shock. Animals able to distinguish between the displays will spend more time in the non-aversive side that shows the display that was not associated during training with the foot shock. B) Sighted wildtype (WT, C57) mice spend approximately twice as much time on the non-aversive side than do untreated rdl mice. Rdl mice expressing SNAP-mGluR2 in RGCs that have received 4xBGAGi2,46o or 9xBGAGi2,46o in PBS 2-4 days earlier show the same preference for the non-aversive side as wild type (WT).

[00449] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A conjugate comprising a) an affinity agent that specifically binds: i) a target ligand-binding polypeptide; or ii) a polypeptide that binds to a target ligand-binding polypeptide; b) a branched linker; and c) a plurality of photoisomerizable regulators, each independently comprising: i) a photoisomerizable group comprising an azobenzene moiety; and ii) a ligand that binds to the target ligand-binding polypeptide. wherein the branched linker comprises a moiety of Formula (BL):

-C(O)NH-[C[(CH₂)nC(O)NH]₃]x- (Formula (BL)), n is an integer from 1 to 6, x is an integer from 1 to 50, and wherein the branched linker comprises a plurality of arms, each independently comprising a photoisomerizable regulator.

2. The conjugate of claim 1, wherein each arm of the branched linker comprises one or more polyethylene glycol (PEG) units.

3. The conjugate of claim 2, wherein each arm of the branched linker comprises six PEG units.

4. The conjugate of claim 1 , wherein the photoisomerizable group comprises a structure of Formula

(Formula 4) wherein:

Q² is the ligand; w is an integer from 1 to 10;

86 R¹ is selected from hydrogen, C_1-10 alkyl, -NR¹⁰Rⁿ, -NR¹²C(O)R¹³, -NR¹²C(O)OR¹³ and - NR¹²C(O)NR¹²R¹³;

R² is hydrogen or C_1-10 alkyl;

R¹⁰ and R¹¹ are independently selected from hydrogen and Ci-io alkyl;

R¹² is hydrogen or Ci-io alkyl; and

R¹³ is selected from hydrogen, Ci-io alkyl, C_1-8 alkenyl, Cg no aryl, and substituted Cino alkyl, or a pharmaceutically acceptable salt thereof.

5. The conjugate of claim 1, wherein the affinity agent comprises benzylguanine.

6. The conjugate of claim 1, wherein the affinity agent comprises chloroalkane.

7. The conjugate of claim 1, wherein the affinity agent comprises benzylcytosine.

8. The conjugate of any one of claims 1-7, wherein the affinity agent comprises an antibody that specifically binds to the target ligand-binding polypeptide.

9. The conjugate of any one of claims 1-8, wherein the target ligand-binding polypeptide is selected from a transcription regulator, an ion channel, a cation channel, a ligand-gated ion channel, a voltagegated ion channel, a quorum sensor, a pheromone receptor, a neurotransmitter receptor, a G-protein- coupled receptor, and an enzyme.

10. The conjugate of claim 9, wherein the cation channel is a potassium channel, a sodium channel, or a calcium channel.

11. The conjugate of any one of claims 1-9, wherein the target ligand-binding polypeptide is a glutamate receptor, a metabotropic glutamate receptor, an ionotropic glutamate receptor, an ionotropic nicotinic acetylcholine receptor, an ionotropic GABA-A receptor, a metabotropic GABA-B receptor, a metabotropic dopamine receptor, an ionotropic purinergic P2X receptor, a metabotropic purinergic P2Y receptor, a metabotropic serotonin receptor, an ionotropic serotonin receptor, an ionotropic glycine receptor, a cation channel, a potassium channel, a calcium channel, a sodium channel, a proton channel, an anion channel, or a chloride channel.

12. The conjugate of any one of claims 1-9, wherein the target ligand-binding polypeptide is a glutamate receptor.

87

13. The conjugate of any one of claims 1-9, wherein the target ligand-binding polypeptide is a metabotropic glutamate receptor (mGluR).

14. The conjugate of claim 13, wherein the mGluR is mGluR2, mGluR3, mGluR4, mGluR5, or mGluR6.

15. The conjugate of claim 14, wherein the target ligand-binding polypeptide is a metabotropic glutamate receptor 2 polypeptide.

16. The conjugate of claim 15, wherein the ligand comprises an mGluR agonist.

17. The conjugate of any one of claims 13-15, wherein the ligand comprises glutamate.

18. The conjugate of any one of claims 13-15, wherein the ligand is selected from (IR^R^S^Rj-d- amino-2-oxabicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268); (lS,2S,5R,6S)-2- aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740); (I .S'.2.S',4/?,5/?.6.Sj-rcl-2-amino-4- methylbicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY395756); (S)-2-amino-2-methyl-4- phosphonobutanoic acid (MAP4); (25',2'R,3'R)-2-(2',3'-Dicarboxycyclopropyl)glycine (DCG IV);

(I .S',3/?)-l -amiiiocyclopciitaiic-l ,3-dicarboxylic acid ((1S,3R)-ACPD); (2R,4R)-4-aminopyrrolidine-2,4- dicarboxylate ((2R,4R)-APCD); (lS,2S,4R,5R,6S)-2-amino-4-methylbicyclo[3.1.0]hexane-2,6- dicarboxylic acid (LY541850); (lR,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide (LY404039); and (lR,4S,5S,6S)-4-((S)-2-amino-4-(methylthio)butanamido)-2- thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide (LY2140023).

19. The conjugate of any one of claims 12-18, wherein the affinity agent comprises benzylguanine.

20. The conjugate of claim 8, wherein the antibody is a single-chain Fv (scFv) or a nanobody.

21. The conjugate of claim 20, wherein the antibody is specific for a metabotropic glutamate receptor (mGluR), optionally wherein the mGluR is mGluR2, mGluR3, mGluR4, mGluR5, or mGluR6.

22. A composition for ocular administration, the composition comprising: a) a conjugate of any one of claims 1-21; and b) a pharmaceutically acceptable excipient suitable for ocular administration.

88

23. The composition of claim 22, wherein the pharmaceutically acceptable excipient comprises a cyclodextrin.

24. The composition of claim 23, wherein the cyclodextrin is a-cyclodextrin, P-cyclodextrin, y- cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether-P-cyclodextrin, or a derivatized cyclodextrin.

25. The composition of any one of claims 22-24, wherein the conjugate is encapsulated within a nanoparticle.

26. The composition of claim 25, wherein the nanoparticle is a nanomicelle, a liposome, a nanosphere, or a nanocapsule.

27. The composition of any one of claims 22-26, wherein the composition is sterile and free of pyrogens.

28. A composition for ocular administration, the composition comprising:

A) a system comprising: a) a conjugate as recited in any one of claims 1-21; b) a fusion polypeptide comprising: i) a target ligand-binding polypeptide that comprises a binding site for the ligand present in the conjugate; and ii) a heterologous fusion partner that binds the affinity agent; and

B) a pharmaceutically acceptable excipient suitable for ocular administration.

29. The composition of claim 28, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the SNAP polypeptide amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:4.

30. The composition of claim 28, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the CLIP polypeptide amino acid sequence set forth in SEQ ID NO:2.

89

31. The composition of claim 28, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the HALO polypeptide amino acid sequence set forth in SEQ ID NO: 3.

32. A composition for intraocular administration, the composition comprising:

A) a system comprising: a) a conjugate as recited in any one of claims 1-21; b) a nucleic acid comprising a nucleotide sequence encoding a fusion polypeptide comprising: i) a target ligand-binding polypeptide that comprises a binding site for the ligand; and ii) a heterologous fusion partner that binds the affinity agent; and

B) a pharmaceutically acceptable excipient suitable for intraocular administration.

33. The composition of claim 32, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the SNAP polypeptide amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:4.

34. The composition of claim 32, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the HALO polypeptide amino acid sequence set forth in SEQ ID NO: 3.

35. The composition of claim 32, wherein the heterologous fusion partner comprises an amino acid sequence having at least 80% amino acid sequence identity to the CLIP polypeptide amino acid sequence set forth in SEQ ID NO:2.

36. The composition of any one of claims 30-33, wherein the nucleic acid is present in a recombinant adenovirus-associated virus (AAV) virion.

37. The composition of claim 36, wherein the AAV virion comprises a variant capsid polypeptide that provides for increased infectivity of a retinal cell by the AAV virion, compared to an AAV virion comprising a corresponding wild-type capsid polypeptide.

38. A composition for intraocular administration, the composition comprising:

A) a system comprising: a) a conjugate as recited in any one of claims 1-21; b) a first fusion polypeptide comprising:

90 i) a target ligand-binding polypeptide that comprises a binding site for the ligand; and ii) a heterologous polypeptide; and c) a second fusion polypeptide comprising: i) an antibody that binds the heterologous polypeptide; and ii) a heterologous fusion partner that binds the affinity agent; and

39. The composition of claim 38, wherein the heterologous fusion partner that binds the affinity agent comprises an amino acid sequence having at least 80% amino acid sequence identity to the SNAP polypeptide amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:4.

40. The composition of claim 38, wherein the heterologous fusion partner that binds the affinity agent comprises an amino acid sequence having at least 80% amino acid sequence identity to the HALO polypeptide amino acid sequence set forth in SEQ ID NO: 3.

41. The composition of claim 38, wherein the heterologous fusion partner that binds the affinity agent comprises an amino acid sequence having at least 80% amino acid sequence identity to the CLIP polypeptide amino acid sequence set forth in SEQ ID NO:2.

42. The composition of any one of claims 38-41, wherein the antibody is a single-chain Fv or a nanobody.

43. The composition of any one of claims 38-42, wherein heterologous polypeptide is an epitope tag.

44. A composition for intraocular administration, the composition comprising:

A) a system comprising: a) a conjugate as recited in any one of claims 1-21; b) a fusion polypeptide comprising: i) an antibody that binds specifically to the target ligand-binding polypeptide; and ii) a polypeptide that binds to the affinity agent, wherein the polypeptide is selected from a SNAP polypeptide, a HALO polypeptide, and a CLIP polypeptide; and

45. A method of increasing the sensitivity of a retinal cell to light, the method comprising:

91 exposing the retinal cell to light, wherein the retinal cell comprises a conjugate as recited in any one of claims 1-21, the composition of any one of claims 22-27, or the composition of any one of claims 28-44, wherein the light is of a wavelength that results in binding of the ligand to the light-regulatable polypeptide, and wherein binding of the ligand to the light-regulatable polypeptide increases the sensitivity of the retinal cell to light.

46. A method of conferring light responsiveness on a retinal cell, the method comprising introducing into the retinal cell a conjugate as recited in any one of claims 1-21, the composition of any one of claims 22-27, or the composition of any one of claims 28-44.

47. A method of treating an ocular disorder characterized by reduced responsiveness to light, the method comprising administering a conjugate as recited in any one of claims 1-21, the composition of any one of claims 22-27, or the composition of any one of claims 28-44, to an eye of an individual having the ocular disorder.

48. The method of claim 47, wherein the ocular disorder is an inherited retinal degenerative disease.

49. The method of claim 48, wherein the disease is retinitis pigmentosa or age-related macular degeneration.

50. A medical device comprising: a) a container comprising a conjugate as recited in any one of claims 1-21, the composition of any one of claims 22-27, or the composition of any one of claims 28-44,; and b) a means for introducing the composition into the eye of an individual.

51. The device of claim 50, wherein the means for introducing the composition into the eye of an individual comprises a needle.

52. The device of claim 50 or claim 51, wherein the container comprises a syringe.

53. The device of any one of claims 50-52, wherein the device is sterile.

92