Compositions and methods for modulating neuronal excitability

Abstract

The invention features compositions and methods for treating diseases or disorders associated with undesirable neuronal excitability (e.g., de-myelinating disease, neurodegenerative disease, such as Parkinson's disease or Huntington's disease; or chronic pain, or epilepsy).

Description

[0001] COMPOSITIONS AND METHODS FOR MODULATING NEURONAL EXCITABILITY

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims priority to and the benefit of U.S. Application No. 63 / 606,502, filed December 5, 2023, the contents of which is hereby incorporated by reference in its entirety.

[0004] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under grant No. FA9550-22-1-0228 awarded by the Air Force Office of Scientific Research and grant No. DMR2011754 awarded by the National Science Foundation. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] The intrinsic excitability of neurons is governed by two membrane properties: membrane conductivity and capacitance. Electrical, optogenetic, and pharmacological manipulations can transiently change membrane properties through manipulations of ion channels. In particular, conventional optogenetic stimulation harvests optical -driven ion channels (e.g., channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR)), enabling millisecond-timescale, genetically-targeted, all-optical excitation and inhibition of living neurons. In contrast to transiently switching ion channels, naturally occurring neuron development and myelination processes suggest that modulating membrane capacitance is another effective way of manipulating neuron intrinsic excitability during brain development, learning, and aging. Increasing or decreasing the membrane capacitance can decrease or increase the cellular excitability and the velocity of action potential propagation.

[0008] Recent advances in materials science and nanotechnology have shown that the incorporation of miniaturized electrically functional materials and components onto cellular membranes can modulate the membrane capacitance, changing the intrinsic cellular activities in vitro, which can potentially alter the neuron excitability in a long-term manner. However, these techniques do not enable genetically targeted specificity in neuronal circuits, in part due to the difficulty of incorporating prefabricated nanomaterials into biological systems in a cell type- or subcellular-specific manner. In vivo synthesis of functional nanomaterials has recently emerged as a promising alternative strategy for the integration of nanomaterials with living systems, often providing greater control over the location and integration of materials at the cellular level. There remains a need for strategies to increase or decrease membrane excitability with increased specificity while minimizing exposure to toxic side reactions.

[0009] SUMMARY OF THE INVENTION

[0010] The invention features compositions and methods for treating diseases or disorders associated with undesirable neuronal excitability (e.g., autism, de-myelinating diseases (e.g., multiple sclerosis), neurodegenerative disease, such as Parkinson’s disease or Huntington’s disease; or chronic pain, or epilepsy).

[0011] In an aspect, the present disclosure provides a method for modulating neural activity using optogenetic polymerization and assembly of electroactive polymers on specified cellular membranes. The method involves: (a) expressing in a neuronal cell a vector encoding a near-IR excitable photosensitizer dL5** polypeptide in the presence of a malachite green derivative and monomers of 3,3'-diaminobenzidine (DAB), aniline and 7V-phenyl- / ?-phenylenediamine, or 3,4- Ethylenedioxythiophene-thiophene-3,4-Ethylenedi oxythiophene (ETE)-alkyne-sulfonate; and (b) applying near infrared radiation to at least a portion of the neuronal cell to induce singlet oxygen formation to induce polymerization of poly(3, 3 '-diaminobenzidine), polyaniline, or polyETE- alkyne-sulfonate, thereby modulating the neuronal activity.

[0012] In another aspect, the present disclosure provides an adeno-associated viral expression vector (AAV) including a CAG or human synapsin promoter driving expression of a dL5** polypeptide.

[0013] In another aspect, the present disclosure provides a neuronal cell including the AAV of any of the above aspects, or embodiments thereof.

[0014] In another aspect, the present disclosure provides a method for modulating neural activity using optogenetic polymerization and assembly of electroactive polymers on specified cellular membranes. The method involves: (a) contacting a neuronal cell with an adeno-associated viral expression vector (AAV) including a CAG or human synapsin promoter driving expression of a dl5** polypeptide in the presence of a malachite green derivative and monomers of 3,3'- diaminobenzidine, aniline and 7V-phenyl- / ?-phenylenediamine, or ETE-alkyne-sulfonate; and (b) irradiating the neuronal cell to induce polymerization of poly(3,3'-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate, thereby modulating the neuronal activity.

[0015] In another aspect, the present disclosure provides a method for modulating neural activity in a subject having a disorder associated with undesirable neural activity. The method involves: (a) administering to a neuronal cell of the subject an adeno-associated viral expression vector (AAV) including a CAG or human synapsin promoter driving expression of a dl5** polypeptide in the presence of a malachite green derivative and monomers of 3,3'-diaminobenzidine, aniline and A-phenyl- / ?-phenylenediamine, or ETE-alkyne-sulfonate; and (b) irradiating the neuronal cell to induce singlet oxygen production and polymerization of poly(3, 3 '-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate, thereby modulating the neuronal activity of the subject.

[0016] In an aspect, the present disclosure provides a kit for use in the methods of any of the above aspects, or embodiments therof, the kit including a vector including a polynucleotide encoding dl5**, a malachite green derivative, and monomers of 3,3'-diaminobenzidine, aniline and N-phenyl-p-phenylenediamine, and / or ETE-alkyne-sulfonate.

[0017] In any of the above aspects, or embodiments thereof, the method provides for the spatiotemporal control of polymerization.

[0018] In any of the above aspects, or embodiments thereof, the method provides for photopolymerization of poly(3,3'-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate at nanometer-level spatial resolution. In any of the above aspects, or embodiments thereof, spatial control is at the subcellular level.

[0019] In any of the above aspects, or embodiments thereof, the method provides for optical control of polymer assembly on or within the cell membrane.

[0020] In any of the above aspects, or embodiments thereof, the method produces increased levels of singlet oxygen relative to other reactive oxygen species (ROS).

[0021] In any of the above aspects, or embodiments thereof, the method does not reduce neuron viability.

[0022] In any of the above aspects, or embodiments thereof, the method alters neuronal excitability.

[0023] In any of the above aspects, or embodiments thereof, the dl5** polypeptide is expressed under the control of a CAG promoter.

[0024] In any of the above aspects, or embodiments thereof, the vector is a viral vector. In any of the above aspects, or embodiments thereof, the viral vector is an an adeno-associated viral expression vector (AAV) vector.

[0025] In any of the above aspects, or embodiments thereof, the irradiation is near infra-red radiation. In any of the above aspects, or embodiments thereof, the irradiation is carried out using a Cy5 filter. In any of the above aspects, or embodiments thereof, the irradiation is for about 5-8 minutes. In any of the above aspects, or embodiments thereof, the irradiation is for about 9-15 minutes. In any of the above aspects, or embodiments thereof, the neuron is in vitro, ex vivo, or in vivo.

[0026] In any of the above aspects, or embodiments thereof, the neuron is a cell of the central or peripheral nervous system. In any of the above aspects, or embodiments thereof, the neuron is a motor neuron or sensory neuron.

[0027] In any of the above aspects, or embodiments thereof, the dl5** binding to malachite green MG-2I produces increased levels of singlet oxygen relative to other reactive oxygen species (ROS).

[0028] In any of the above aspects, or embodiments thereof, the method treats the disorder or ameliorates at least one symptom of the disorder.

[0029] In any of the above aspects, or embodiments thereof, the disorder is a neurodegenerative disease. In any of the above aspects, or embodiments thereof, the disorder is chronic pain or epilepsy. In any of the above aspects, or embodiments thereof, the disorder is multiple sclerosis.

[0030] In any of the above aspects, or embodiments thereof, the vector is an adeno-associated viral expression vector (AAV).

[0031] In any of the above aspects, or embodiments thereof, the vector comprises a CAG or human synapsin promoter driving expression of the dl5** polypeptide.

[0032] Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

[0033] Definitions

[0034] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

[0035] By “agent” is meant a polypeptide or nucleic acid molecule, or active fragments thereof, or a small molecule chemical compound. In embodiments, the agent is an electroactive polymer whose polymerization is induced using optogenetic polymerization. By “ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. In embodiments, the disease is a neurodegenerative disorder.

[0036] By "alteration" is meant a change (increase or decrease) in the expression levels, structure, or activity of a cell, a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.

[0037] By “aniline” is meant a compound having the chemical formula C6H5NH2 and / or corresponding to CAS Number 62-53-3. Aniline may also be known as Benzenamine. An exemplary chemical structure for aniline may be found below:

[0038] By “3,3’-diaminobenzidine” or “DAB” is meant a compound having the chemical formula (CeH3(NH2)2)2 and / or corresponding to CAS Number 91-95-2. 3,3 ’-diaminobenzidine may also be known as [l,l'-Biphenyl]-3,3',4,4'-tetramine. An exemplary chemical structure for 3,3 ’-diaminobenzidine may be found below:

[0039] In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments. By “decreases” is meant a reduction by at least about 5% relative to a reference level. A decrease may be by 5%, 10%, 15%, 20%, 25% or 50%, or even by as much as 75%, 85%, 95% or more and any intervening percentages.

[0040] “Detect” refers to identifying the presence, absence or amount of the analyte to be detected. In embodiments, the analyte is an electroactive polymer.

[0041] By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

[0042] By “disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. In an embodiment, the disease is a neurodegenerative disease or a disorder characterized by undesirable neuronal activity.

[0043] By “disorder characterized by undesirable neuronal activity” is meant any increase or decrease in neuronal activity in a subject that disrupts the normal electrophysiology of the subject. Examples of such disorders include, but are not limited to, epilepsy, Parkinson’s disease, Huntington’s disease, chronic pain, and other related disorders.

[0044] "Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules, or cells) to become sufficiently proximal to react, interact, affect or physically touch. It should be appreciated, however, that the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents, which can be produced in the reaction mixture. Contacting may include allowing two species to react, interact, or physically touch, wherein the two species may be a recombinant viral particle as described herein and a cell. In embodiments, the two species are an ultrasound contrast agent that is exposed to ultrasound and a cell.

[0045] The word "expression" or "expressed" as used herein in reference to a gene means the transcriptional and / or translational product of that gene. The level of expression of a DNA molecule in a cell may be determined on the basis of either the amount of corresponding mRNA that is present within the cell or the amount of protein encoded by that DNA produced by the cell. The level of expression of non-coding nucleic acid molecules (e.g., siRNA) may be detected by standard PCR or Northern blot methods well known in the art. See, Sambrook et al., 1989 Molecular Cloning: A Laboratory Manual, 18.1-18.88. Expression of a transfected gene can occur transiently or stably in a cell. During "transient expression" the transfected gene is not transferred to the daughter cell during cell division. Since its expression is restricted to the transfected cell, expression of the gene is lost over time. In contrast, stable expression of a transfected gene can occur when the gene is cotransfected with another gene that confers a selection advantage to the transfected cell. Such a selection advantage may be a resistance towards a certain toxin that is presented to the cell. Expression of a transfected gene can further be accomplished by transposon-mediated insertion into to the host genome. During transposon-mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that allow insertion into the host genome as well as subsequent excision. Stable expression of a transfected gene can further be accomplished by infecting a cell with a lentiviral vector, which after infection forms part of (integrates into) the cellular genome thereby resulting in stable expression of the gene.

[0046] The term "exogenous" refers to a molecule or substance (e.g., a compound, nucleic acid or protein) that originates from outside a given cell or organism. For example, an "exogenous promoter" as referred to herein is a promoter that does not originate from the plant it is expressed by. Conversely, the term "endogenous" or "endogenous promoter" refers to a molecule or substance that is native to, or originates within, a given cell or organism.

[0047] The term "gene" means the segment of DNA involved in producing a protein; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons). The leader, the trailer as well as the introns include regulatory elements that are necessary during the transcription and the translation of a gene. Further, a "protein gene product" is a protein expressed from a particular gene.

[0048] By "effective amount" is meant the amount of a vector and / or monomers described herein required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

[0049] By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids. A “host cell” or “cell” is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.

[0050] The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

[0051] By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

[0052] By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0053] By “marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

[0054] By “A-phenyl-p-phenylenediamine” is meant a compound having the chemical formula C6H5NHC6H4NH2 and / or corresponding to CAS Number 101-54-2. N-phenyl- - phenylenediamine may also be known as 4- Aminodiphenylamine. An exemplary chemical structure for A-phenyl-p-phenylenediamine may be found below:

[0055] By “operably linked” refers to a functional linkage between a regulatory sequence and a coding sequence, where a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide. The described components are therefore in a relationship permitting them to function in their intended manner. For example, placing a coding sequence under regulatory control of a promoter means positioning the coding sequence such that the expression of the coding sequence is controlled by the promoter.

[0056] By “poly(3,3’ -diaminobenzidine)” or “PDAB” is meant a polymer comprising monomers of 3,3 ’-diaminobenzidine.

[0057] By “polyaniline” or “PANI” is meant a polymer comprising monomers of aniline.

[0058] By “portion” is meant a fragment of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides.

[0059] By “positioned for expression” is meant that the polynucleotide of the disclosure (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant microRNA molecule described herein).

[0060] The term “promoter” as used herein refers to a sequence of DNA that directs the expression (transcription) of a gene. A promoter may direct the transcription of a prokaryotic or eukaryotic gene. A promoter may be “inducible”, initiating transcription in response to an inducing agent or, in contrast, a promoter may be “constitutive”, whereby an inducing agent does not regulate the rate of transcription. A promoter may be regulated in a tissue-specific or tissuepreferred manner, such that it is only active in transcribing the operable linked coding region in a specific tissue type or types. Exemplary promoters include the CAG promoter and / or the human synapsin promoter, or any other promoter that directs expression in a neuron of interest.

[0061] By “reactive oxygen species” is meant a class of chemically-reactive molecules containing at least one oxygen atom. Reactive oxygen species include oxygen free radicals, such as, for example, superoxide anion radical, hydroxyl radical, hydroperoxyl radical, and singlet oxygen, as well as molecules lacking oxygen free radicals, such as, for example, hydrogen peroxide, ozone, and peroxyni trite.

[0062] The term "recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all. Transgenic cells and plants are those that express a heterologous gene or coding sequence, typically as a result of recombinant methods.

[0063] By “reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%.

[0064] By “reference” is meant a standard or control condition. For example, an untreated cell, tissue, or organ that is used as a reference. In one embodiment, a reference is a cell of normal electrophysiology that is used as the basis for comparison relative to a cell expressing a vector comprising a miniSOG alone or in combination with monomers described herein, which are subsequently irradiated to form polymers.

[0065] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

[0066] By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

[0067] Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3and e'100indicating a closely related sequence.

[0068] By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

[0069] Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0070] By “transformed cell” is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a polynucleotide molecule encoding (as used herein) a polypeptide of the invention.

[0071] The terms "transfection", "transduction", "transfecting" or "transducing" can be used interchangeably and are defined as a process of introducing a nucleic acid molecule or a protein to a cell. Nucleic acids are introduced to a cell using non-viral or viral-based methods. The nucleic acid molecules may be gene sequences encoding complete proteins or functional portions thereof. Non-viral methods of transfection include any appropriate transfection method that does not use viral DNA or viral particles as a delivery system to introduce the nucleic acid molecule into the cell. Exemplary non-viral transfection methods include calcium phosphate transfection, liposomal transfection, nucleofection, sonoporation, transfection through heat shock, magnetifection and electroporation. In some embodiments, the nucleic acid molecules are introduced into a cell using electroporation following standard procedures well known in the art. For viral-based methods of transfection any useful viral vector may be used in the methods described herein. Examples for viral vectors include, but are not limited to retroviral, adenoviral, lentiviral and adeno-associated viral vectors. In some embodiments, the nucleic acid molecules are introduced into a cell using a retroviral vector following standard procedures well known in the art. The terms "transfection" or "transduction" also refer to introducing proteins into a cell from the external environment. Typically, transduction or transfection of a protein relies on attachment of a peptide or protein capable of crossing the cell membrane to the protein of interest. See, e.g., Ford et al. (2001) Gene Therapy 8: 1-4 and Prochiantz (2007) Nat. Methods 4: 119-20.

[0072] As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

[0073] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

[0074] By “vector” is meant a nucleic acid molecule, for example, a plasmid, cosmid, virus, or bacteriophage that is capable of replication in a host cell. In one embodiment, a vector is an expression vector that is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a nucleic acid molecule in a host cell. Typically, expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.

[0075] Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

[0076] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0077] Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0078] BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIGs. 1A-1L Membrane-targeted expression of near-IR, genetically encoded photopolymerase via post-translational modification, a, Membrane localization of dL5**, a genetically encoded fluorogen-activating protein (FAP), in HEK293T cells and rat cortical neurons. The near-infrared genetically encoded photopolymerase becomes active upon binding to the cofactor-targeted and activated photosensitizer (TAP), b, Genetic vector construct for expression of FAP onto the HEK293T cells or neuron membrane. The C-terminal farnesylation (C-Far) motif is fused downstream to FAP gene, allowing the famesyl group to attach to the C- terminal of FAP by post-translational modification, facilitating attachment to the inner side of the cell membrane, c, Schematic illustrating the mechanism of post-translational modification and co-factor binding, d-g, Confocal fluorescence images displaying representative expression patterns of FAP (red) and mCherry (yellow) in HEK293T cells (d-e) and neurons (f-g). h-i, Fluorescence intensity profile plots along the purple lines marked on (e) and (g), respectively.

[0080] FIGs. 2A-2G. Near-IR optogenetic polymerization of electrically functional materials in cells, a, Schematic illustrating the polymerization mechanism of DAB and aniline / PPD. b-c, Relative brightness change before and after 10 minutes of irradiation in the presence of 1 mM DAB to form PDAB or 0.75 mM aniline + 0.75 mM PPD polymerization to form PANI. Values represent means ± SEM; unpaired, two-tailed t-tests. ****p < 0.0001. d-g, Merged confocal fluorescence (left) of fixed HEK293T cells and neurons, and TL (right) images after polymerization.

[0081] FIGs. 3A-3F. Neuron intrinsic excitability modulation by near-IR optogenetic polymerization, a, Schematic illustrating the patch-clamp characterization for cultured rat cortical neurons. The neurons are maintained in the Tyrode’s solution during monomer perfusion of 1 mM DAB or 0.5 mM aniline + 0.5 mM PPD, photopolymerization and wash away of the monomers. Whole-cell recordings are performed before monomer perfusion and after washing away of the monomers. Neurons are maintained at the whole-cell patch state during the entire experiment process, b-c, Comparison of membrane capacitance (Cm) before and after PDAB (b) or PANI (c) polymerization (n = 5). d, representative traces of stepwise tonic current injection (20 pA per step) before and after PDAB and PANI polymerization at rheobase + 40 pA. e-f, Comparison of spike numbers evoked by stepwise tonic current injection with different current values (n = 5). Bar graphs represent means ± SEM; dots with the same color within each group indicate the same neuron. Paired two-tailed t-test. *p < 0.05 and **p < 0.01; t-tests with nonsignificant p values are not shown for clarity.

[0082] FIGs. 4A and 4B. Molecular engineering for polymerization of biocompatible conductive polymers in cells, a, Design of polyETE-alkyne-sulfonate, a 3,4- Ethylenedi oxy thiophene (EDOT)-based, biocompatible conductive polymer for optogenetic polymerization, b, Synthetic scheme and polymerization process of EDOT-thiophene-EDOT (ETE)-alkyne-sulfonate monomer.

[0083] FIGs. 5A-5E. Modulation of neuronal excitability in intact brain circuits, a-b, Schematic illustration of the workflow for modulating neuronal excitability in brain circuits, a, Stereotaxic injection of adeno-associated virus (AAV) into the mouse brain, b, Following robust FAP expression in the CAI region, the mouse brain is harvested, sectioned into acute slices, and analyzed to assess neuromodulation before and after polymerization, c, Fluorescence confocal imaging of mouse brain slices after AAV vector injection into the CAI region. Red: FAP; Blue: Hoechst 33342. d, Representative current-clamp recordings showing action potential traces induced by current injection, e, Quantification of CAI neuronal capacitance changes before and after polymerization.

[0084] DETAILED DESCRIPTION OF THE INVENTION

[0085] The invention features compositions and methods for modulating neuronal excitability and for treating diseases or disorders associated with undesirable neuronal excitability (e.g., autism, de-myelinating diseases (MS), neurodegenerative disease, such as Parkinson’s disease or Huntington’s disease; or chronic pain, or epilepsy).

[0086] The disclosure is based, at least in part, on the discovery that a near-IR excitable photosensitizer, dL5**, which is one of the fluorogen-activating proteins (FAPs) that binds to various malachite green (MG) derivatives as the targeted and activated photosensitizer (TAPs), can be used for the optogenetic polymerization of conductive and insulating polymers (e.g., polyaniline (PANI) and poly(3, 3 '-diaminobenzidine) (PDAB)) in the cell, to alter excitability of the cell. As detailed below, the inventors demonstrated the membrane-targeted expression of dL5** in living cells for enhanced specificity; and showed that dL5**-enabled optogenetic polymerization and assembly of electrically functional polymers can achieve increases or decreases of both the membrane capacitance and intrinsic excitability of neurons by using both in vitro whole-cell patch-clamp and ex vivo brain slice patch-clamp recordings. In addition, through side-chain engineering, the inventors developed 3, 4-Ethylenedi oxythiophene (EDOT)- thiophene-EDOT (ETE) derivatives as a conductive polymer monomer for optogenetic polymerization with improved biocompatibility.

[0087] Compositions and Methods for Modifying Neuronal Excitability

[0088] Cell-type-specific neuronal intrinsic excitability changes are associated with many neurological disorders. Ionic conductivity and membrane capacitance are two foundational parameters that govern neuron excitability. Conventional optogenetic stimulation has proven to be a powerful tool to temporarily manipulate membrane ionic conductivity from genetically targeted components in intact biological systems. However, no analogous method exists for precisely manipulating cell membrane capacitance to enable long-lasting modulation of neuronal excitability with genetically targeted specificity. Genetically targetable polymerization and assembly of conductive and insulating polymers inside the cell can modulate cell membrane capacitance, but further development of this technique has been hindered by poor spatiotemporal control of the polymer deposition and cytotoxicity from the widely diffused reactive oxygen species. The present disclosure provides compositions and methods to address these issues by harnessing genetically targetable photosensitizer proteins to polymerize and assemble electrically functional polymers in neurons with precise spatiotemporal control enabled by light activation. Using whole-cell patch-clamp recordings, this optogenetic polymerization and assembly of electrically functional polymers was demonstrated, which can achieve stepwise increases or decreases of both the membrane capacitance and intrinsic excitability of neurons. Furthermore, cytotoxicity can be limited by controlling light exposure, demonstrating a promising new, optogenetically controlled, biosynthetic method for precisely modulating cell excitability.

[0089] As such, an engineered peroxidase was modified (Liu et al., Science. 80, (2020)) to be expressed in genetically specified neurons in brain tissues. The peroxidase can catalyze the oxidative polymerization of small molecule precursors into electrically functional (conductive or insulating) polymers at the plasma membrane in the presence of H2O2. Whole-cell patch-clamp showed that the in situ synthesized conductive / insulating polymers increased / decreased the membrane capacitance and reduced / elevated the excitability of polymer-coated neurons, respectively. This method shows promise to change the excitability of specific types of neurons in intact neural circuits. However, this peroxidase / ^Ch-driven polymerization has the following limitations: (i) diffusion of H2O2 introduces acute toxicity to the neural systems and (ii) the H2O2-triggered polymerization cannot control the location and extent of in situ polymerization in neurons. These limitations prevent the further application of this technique to living cell membrane modulation with cellular and subcellular spatiotemporal resolution.

[0090] Here, these issues are addressed by developing an optically controlled, genetically targeted (optogenetic) polymerization and assembly of conductive and / or insulating polymers on the neuronal plasma membrane, which, akin to conventional optogenetic stimulations, not only precisely modulates the membrane capacitance in a light-controlled and stepwise manner, but also achieves cell-type-specific control over neuron excitability.

[0091] Whole-cell patch-clamp was used to characterize the electrophysiological properties of the neurons before and after the optogenetic polymerization, showing that the in situ synthesized conductive or insulating materials can increase or decrease the neuronal membrane capacitance and decrease or increase the intrinsic cellular excitability, respectively. Finally, it was shown that optogenetic polymerization can precisely control the location and density of polymers in cells by controlling the light intensity and exposure, thus enabling an iterative, stepwise increase / decrease of membrane capacitance.

[0092] Expression of Recombinant dL5**, a fluorogen-activating protein (FAP)

[0093] In an embodiment, a cell of interest (e.g., neuron, such as a motor neuron, sensory neuron, neuron of the central nervous system, or neuronal cell lines) is engineered to express a polynucleotide encoding FAP. Near-infrared stimulation of such cells induces optogenetic polymerization and assembly of electrically functional polymers for modulation of neuronal excitability. dL5** is a tandem dimer of a double-mutant (E52D, L91S) of the parent L5-MG FAP, a 25 kDa binder for malachite green derivatives (MG) with thousands-fold fluorescence activation, low pM dissociation constant, and robust function in various compartments of living cells. dL5** is discussed in more detail in, for example, He et al., Nat Methods. 2016 Jan 25; 13(3):263-268, and Szent-Gyorgyi et al., J. Mol. Biol., 2013 Aug 23; 425(22):4595-4613, the entireties of each which is hereby incorporated by reference. dL5** may be constitutively expressed or its expression may be regulated by an inducible promoter or other control mechanism where conditions necessitate highly controlled regulation or timing of the expression of a dL5**. This can be accomplished by homologous recombination or by viral integration into the host cell genome. The desired gene sequence can also be incorporated into a cell, particularly into its nucleus, using a plasmid expression vector and a nuclear localization sequence. Methods for directing polynucleotides to the nucleus have been described in the art. The genetic material can be introduced using promoters that will allow for the gene of interest to be positively or negatively induced using certain chemicals / drugs, to be eliminated following administration of a given drug / chemical, or can be tagged to allow induction by chemicals, or expression in specific cell compartments.

[0094] A variety of vectors can be used to introduce a dL5** polynucleotide to a cell of interest. In one embodiment, transducing viral (e.g., retroviral, adenoviral, lentiviral and adeno-associated viral) vectors can be used to introduce dL5** to a cell, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94: 10319, 1997). For example, a polynucleotide encoding a dL5** protein, can be cloned into a retroviral vector and expression can be driven from a neuronal promoter, a CAG promoter, synapsin promoter, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275- 1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1 :55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No.5,399,346).

[0095] Calcium phosphate transfection can be used to introduce plasmid DNA containing a dL5** polynucleotide into cells and is a standard method of DNA transfer to those of skill in the art. DEAE-dextran transfection, which is also known to those of skill in the art, may be preferred over calcium phosphate transfection where transient transfection is desired, as it is often more efficient. Since the cells of the present invention are isolated cells, microinjection can be particularly effective for transferring genetic material into the cells. This method is advantageous because it provides delivery of the desired genetic material directly to the nucleus, avoiding both cytoplasmic and lysosomal degradation of the injected polynucleotide. Cells can also be genetically modified using electroporation. Liposomal delivery of DNA or RNA to genetically modify the cells can be performed using cationic liposomes, which form a stable complex with the polynucleotide. For stabilization of the liposome complex, dioleoyl phosphatidylethanolamine (DOPE) or dioleoyl phosphatidylcholine (DOPQ) can be added. Commercially available reagents for liposomal transfer include Lipofectin (Life Technologies). Lipofectin, for example, is a mixture of the cationic lipid N-[l-(2, 3-dioleyloxy)propyl]-N-N-N- trimethyl ammonia chloride and DOPE. Liposomes can carry larger pieces of DNA, can generally protect the polynucleotide from degradation, and can be targeted to specific cells or tissues. Cationic lipid- mediated gene transfer efficiency can be enhanced by incorporating purified viral or cellular envelope components, such as the purified G glycoprotein of the vesicular stomatitis virus envelope (VSV-G). Gene transfer techniques which have been shown effective for delivery of DNA into primary and established mammalian cell lines using lipopolyamine-coated DNA can be used to introduce target DNA into the de-differentiated cells or reprogrammed cells described herein.

[0096] Naked plasmid DNA can be injected directly into a tissue comprising cells of interest. Microprojectile gene transfer can also be used to transfer genes into cells either in vitro or in vivo. The basic procedure for microprojectile gene transfer was described by J. Wolff in Gene Therapeutics (1994), page 195. Similarly, microparticle injection techniques have been described previously, and methods are known to those of skill in the art. Signal peptides can be also attached to plasmid DNA to direct the DNA to the nucleus for more efficient expression.

[0097] Viral vectors are used to genetically alter cells of the present invention and their progeny. Viral vectors are used, as are the physical methods previously described, to deliver one or more polynucleotide sequences encoding dL5**, for example, into the cells. Viral vectors and methods for using them to deliver DNA to cells are well known to those of skill in the art. Examples of viral vectors that can be used to genetically alter the cells of the present invention include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors (including lentiviral vectors), alphaviral vectors (e. g., Sindbis vectors), and herpes virus vectors.

[0098] Adeno-Associated Virus (AAV)

[0099] In embodiments, the dL5** polynucleotide is delivered by an AAV. AAV is a small (25 nm), nonenveloped virus that contains a linear single-stranded DNA genome packaged into the viral capsid. It belongs to the family Parvoviridae and is of the genus Dependovirus. because productive infection by AAV occurs only in the presence of either an adenovirus or herpesvirus helper virus. In the absence of helper virus, AAV (serotype 2) can establish latency after transduction into a cell by specific but rare integration into chromosome 19ql 3.4. Accordingly, AAV is the only mammalian DNA virus known to be capable of site-specific integration. (Daya, S. and Berns, K.I., 2008, Clin. Microbiol. Rev., 21(4):583-593).

[0100] There are two stages to the AAV life cycle after successful infection: a lytic stage and a lysogenic stage. In the presence of adenovirus or herpesvirus helper virus, the lytic stage persists. During this period, AAV undergoes productive infection characterized by genome replication, viral gene expression, and virion production. The adenoviral genes that provide helper functions for AAV gene expression include Ela, Elb, E2a, E4, and VA RNA. While adenovirus and herpesvirus provide different sets of genes for helper function, they both regulate cellular gene expression and provide a permissive intracellular milieu for a productive AAV infection. Herpesvirus aids in AAV gene expression by providing viral DNA polymerase and helicase as well as the early functions necessary for HSV transcription. rAAV as a vector for gene delivery and therapeutic treatment

[0101] AAVs are well suited for use as vectors and vehicles for gene transfer to the nervous system. AAVs provide safe, long-term expression in the nervous system. Most of the foregoing applications rely on local AAV injections into the adult brain to bypass the blood-brain barrier (BBB) and to temporally and spatially restrict transgene expression.

[0102] AAV vectors have been highly successful in fulfilling all of the features desired for a delivery vehicle, such as the ability to attach to and enter the target cell, successful transfer to the nucleus, the ability to be expressed in the nucleus for a sustained period of time, and a general lack of pathogenicity and toxicity. Recombinant AAV (rAAV) is advantageous as a delivery vector, particularly for delivery to neurons in brain tissue, as it is focally injectable; it exhibits stable expression over time; and it is both non-pathogenic and non-integrative into the genome of the cell into which it is transduced. Twelve human serotypes of AAV (AAV serotype 1 (AAV- 1) to AAV-12) and more than 100 serotypes from nonhuman primates have been reported to date. (Daya, S. and Berns, K.I., 2008, Clin. Microbiol. Rev., 21 (4): 583-593). In addition, rAAV has been approved by the FDA for use as a vector in at least 38 protocols for a number of different human clinical trials. AAV’s lack of pathogenicity, persistence and its many available serotypes have increased the potential of the virus as a delivery vehicle for a gene therapy application in accordance with the described compositions and methods. rAAV vectors have been constructed that do not encode the replication (Rep) proteins and that lack the cv.s-active, 38 base pair integration efficiency element (IEE), which is required for frequent site-specific integration. The inverted terminal repeats (ITRs) are retained because they are the cis signals required for packaging. Thus, current recombinant AAV (rAAV) vectors persist primarily as extrachromosomal elements.

[0103] Recombinant AAV (rAAV) vectors for gene therapy have been based mostly on the AAV-2 serotype. AAV-2-based rAAV vectors can transduce muscle, liver, brain, retina, and lungs, requiring several weeks for optimal expression. The efficiency of rAAV transduction is dependent on the efficiency at each step of AAV infection, i.e., virus binding, entry, trafficking, nuclear entry, uncoating, and second-strand synthesis.

[0104] Several novel AAV vector technologies have been developed to either increase the genome capacity for AAV or enhance gene expression. 7ra / / .s-splicing AAV vectors have been used to increase the capacity of the vector for harboring heterologous polynucleotides by taking advantage of AAV's ability to form head-to-tail concatemers via recombination in the ITRs. In this approach, the transgene cassette is split between two rAAV vectors containing adequately placed splice donor and acceptor sites. Transcription from recombined AAV molecules, followed by the correct splicing of the mRNA transcript, results in a functional gene product. While somewhat less efficient than rAAV vectors, / ra / z.s-spl icing AAV vectors permit delivery of therapeutic genes up to 9 kb in size and have been successfully used for gene expression in the retina, lung and muscle. rAAV polynucleotides may include additional elements, for example, a sequence encoding a reporter or a detectable marker, such as a fluorescent protein, or an element such as a Woodchuck Hepatitis Virus Posttrascriptional Regulatory Element (WPRE), which may increase RNA stability and protein yield. An rAAV polynucleotide may also comprise a promoter to drive transcription of one or more polynucleotides (genes) which are inserted between inverted terminal repeats (ITRs). A polyadenylation signal, such as bovine growth hormone polyadenylation signal and / or SV40 polyomavirus simian virus 40 polyadenylation signal, may be included as elements in the rAAV polynucleotide. The rAAV polynucleotide can comprise a minimal promoter, e.g., a human beta-globin minimal promoter (phPg) and a chimeric intron sequence (Hermeming et al., 2004, J Virol Methods, 122(l):73-77). Without wishing to be bound by theory, ITRs may aid in concatamer formation in the nucleus after the single-stranded, AAV vector DNA is converted into double stranded (ds) DNA by host cell DNA polymerase complexes. Thus, the administration of the described rAAVs may form episomal concatemers in the nucleus of neuronal cells into which they are transduced. In non-dividing cells, concatemers may remain intact in these cells for the lifetime of the neurons. Advantageously, integration of rAAV polynucleotides into host chromosomes is likely to be negligible or absent and will not alter or affect the expression or regulation of any other human gene. Recombinant AAV vectors can be made using standard and practiced techniques in the art and employing commercially available reagents. It will be appreciated by the skilled practitioner that rAAV vectors that been used in several clinical trials that have yielded promising results. By way of example, rAAV based therapy received marketing approval by the European Union in 2012, as reported by Kotterman, M.A. et al., 2014, Nat. Rev. Genet., 15:445- 451. In some embodiments, plasmid vectors may encode all or some of the well-known replication (rep), capsid (cap) and adeno-helper components. The rep component comprises four overlapping genes encoding Rep proteins required for the AAV life cycle (e.g., Rep78, Rep68, Rep52 and Rep40). The cap component comprises overlapping nucleotide sequences of capsid proteins VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. A second plasmid that encodes helper components and provides helper function for the AAV vector may also be co-transfected into cells. The helper components comprise the adenoviral genes E2A, E4orf6, and VA RNAs for viral replication.

[0105] In an embodiment, a method of making rAAVs for the products, compositions, and uses described herein involves culturing cells that comprise an rAAV polynucleotide expression vector as described; culturing the cells to allow for expression of the polynucleotides to produce the rAAVs within the cell, and separating or isolating the rAAVs from cells in the cell culture and / or from the cell culture medium. Such methods are known and practiced by those having skill in the art. The rAAVs can be purified from the cells and cell culture medium to any desired degree of purity using conventional techniques.

[0106] Cell-specific AAV capsids

[0107] The rational design of AAV vectors that display selective tissue / organ targeting has broadened the applications of AAV as vector / vehicle for gene therapy. Both direct and indirect targeting approaches have been used to enhance AAV vector cell targeting specificity and retargeting. By way of example, in direct targeting, AAV vector targeting to certain cell types is mediated by small peptides or ligands that have been directly inserted into the viral capsid sequence. This approach has been successfully employed to target endothelial cells. Direct targeting requires detailed knowledge of the capsid structure such that peptides or ligands are positioned at sites that are exposed to the capsid surface; the insertion does not significantly affect capsid structure and assembly; and the native tropism is ablated to maximize targeting to a specific cell type. In indirect targeting, AAV vector targeting is mediated by an associating molecule that interacts with both the viral surface and the specific cell surface receptor. Such associating molecules for AAV vectors may include bispecific antibodies and biotin. The advantages of indirect targeting are that different adaptors can be coupled to the capsid without resulting in significant changes in the capsid structure, and the native tropism can be easily ablated. A disadvantage of using adaptors for targeting involves a potential for decreased stability of the capsid-adaptor complex in vivo.

[0108] In addition, AAV vectors may be produced that comprise capsids that allow for the increased transduction of cells and gene transfer to the central nervous system and the brain via the vasculature. (Chan, K.Y. et al., 2017, Nat. Neurosci., 20(8): 1172-1179). Such vectors facilitate robust transduction of neuronal. When used with enhancers and cell-type specific promoters, such AAVs provide targeted gene expression in neuronal cells of the nervous system.

[0109] For applications that do not require high expression levels per cell, the amount of virus used, i.e., the viral dose, could be lowered. Lowering the viral load used for systemic gene delivery can reduce cost and production burden and minimize a potential risk for adverse reactions to viral components.

[0110] To achieve enhanced therapy or treatment, the dose of AAV vector that is required for a therapeutic response may be reduced, e.g., by using certain AAV serotypes. Alternatively, the surface of the AAV vector capsid may be altered to include specific ligands for attachment to target tissues and cells as described above. Another approach takes into consideration the trafficking of the virus particle from the endocytoplasmic vesicle to the nucleus. (Zhao, W. et al., 2007, Gene Ther., 14:545-550; Daya, S. and Berns, K.I., 2008, Clin. Microbiol. Rev., 21(4):583- 593). Typically, the virus particle-to-infectivity ratio of AAV vector preparations ranges from 10:1 to 100: 1. The high ratios reflect incomplete or empty vector particles, as well as trafficking from the endocytoplasmic vesicle to the nucleus. During trafficking, the vector particle may become ubiquitinated and directed to a proteasome for degradation, rather than to the nucleus where the transgene may be expressed. It was found that ubiquitination and direction to the proteasome require phosphorylation of tyrosine residues on the surface of the AAV vector capsid. When the seven tyrosine residues on the surface of the AAV-2 capsid were replaced phenylalanine residues, the multiplicity of infection (MOI) required for the detection of transgene expression was greatly reduced both in cell culture and in several mouse models of transduction of cells in the liver and eye. Consequently, the ability to increase transgene expression to therapeutic levels in the treatment of diseases may be enhanced.

[0111] One or more treatment approaches to gain control over seizures are embraced by the therapeutic products, compositions and methods described herein involving state-of-the-art gene therapy or pharmaco-genetic approaches. Such approaches may likely lead to the development of a clinically relevant therapies to alleviate the seizure symptoms of epilepsy. For direct delivery to the brain, AAV vectors may be administered by open neurosurgical procedure or by focal injection in order to bypass the blood-brain barrier, to temporally and spatially restrict transgene expression, and to target specific areas of the brain.

[0112] Systemic AAV delivery (by intravenous injection) provides a non-invasive alternative for broad gene delivery to the nervous system. Several groups have developed AAV capsids that enhance gene transfer to the CNS and certain tissues and cell populations after intravenous delivery. By way of example, AAV-AS capsidl8 utilizes a polyalanine N-terminal extension to the AAV9.4719 VP2 capsid protein to provide higher neuronal transduction, particularly in the striatum. The AAV-BR1 capsid20, based on AAV2, may be useful for more efficient and selective transduction of brain endothelial cells. Another AAV capsid, AAV-PHP.eB, comprises a capsid that transduces the majority of neurons and astrocytes across many regions of the adult mouse brain and spinal cord after intravenous injection.

[0113] Other modes of AAV vector administration may include lipid-mediated vector delivery, hydrodynamic delivery, and a gene gun.

[0114] Promoters

[0115] Promoters useful in expressing a dL5** polynucleotide in a cell (e.g., neuronal cell) are known in the art and are described herein. In one embodiment, the promoter is a CAG promoter constructed from the (C) Cytomegalovirus (CMV) early enhancer element; (A) the promoter, the first exon and the first intron of chicken beta- Actin gene, and (G) the splice acceptor of the rabbit beta-Globin gene. The CAG promoter is known in the art and described, for example, by Miyazaki et al., Gene. 79 (2): 269-77, 1989 and Niwa et al., Gene. 108 (2): 193-9, 1991, each of which is incorporated herein by reference in their entirety.

[0116] In another embodiment, the promoter is a human synapsin promoter. The human synapsin 1 gene promoter confers highly specific long-term expression in the brain. Synapsin promoters are described, for example, by Kugler et al., Gene Therapy 10:337-347, 2003, by Jackson et al., Front. Mol. Neurosci., 04 November 2016, Sec. Methods and Model Organisms.

[0117] Other promoters expressed in neurons are known in the art.

[0118] Treatment of Disorders Characterized by Undesirable Activity

[0119] The virus vectors comprising dL5** polynucleotide and compositions thereof (comprising FAP and TAP to photopolymerize polyaniline (PANI) and poly(3,3'- diaminobenzidine) (PDAB)) as described herein may be used in the treatment of neurological and neurodegenerative diseases and disorders, particularly, for the treatment of epilepsy. A characteristic that distinguishes categories of seizures is whether the seizure activity is partial (e.g., focal) or generalized. In an embodiment, virus vectors and compositions thereof as described herein are used to treat partial and / or generalized seizures. Partial seizures are typically considered to be those in which the seizure activity is restricted to discrete areas of the cerebral cortex. As will be appreciated by the skilled practitioner, a seizure is characterized as a simple-partial seizure if consciousness is fully preserved during the course of the seizure. If consciousness is impaired, then the seizure is characterized as a complex-partial seizure. Complex-partial seizures also include those that initiate as partial seizures and subsequently extend through the cortex; as such, these types of seizures are considered to be partial seizures with secondary generalization.

[0120] Generalized seizures encompass distant regions of the brain simultaneously in a bilaterally symmetric manner and can include sudden, brief lapses of consciousness, such as in the case of absence or petit mal seizures, without loss of postural control. Atypical absence seizures usually include a longer period of lapse of consciousness and more gradual onset and termination. Generalized tonic-clonic or grand mal seizures, considered as the main type of generalized seizures, typically have an abrupt onset without warning. The initial phase of the seizure usually involves tonic contraction of muscles, impaired respiration, a marked enhancement of sympathetic tone leading to increased heart rate, blood pressure and pupil size. After approximately 10-20 seconds, the tonic phase of the seizure typically evolves into a clonic phase, which is produced by periods of muscle relaxation superimposed on the tonic muscle contraction. The periods of relaxation progressively increase until the end of the ictal phase, which usually lasts no more than one minute. The postictal phase is characterized by unresponsiveness, muscular flaccidity, and excessive salivation that can cause stridorous breathing and partial airway obstruction.

[0121] Atonic seizures are characterized by sudden loss of postural muscle tone lasting approximately 1-2 seconds. While consciousness is briefly impaired, there is usually no postictal confusion. Myoclonic seizures are characterized by a sudden and brief muscle contraction that may involve one part of the body or the entire body. Without limitation, the rAAV products, compositions and methods of use thereof as described herein embrace the prophylactic and / or therapeutic treatment of the above-described seizures, including those associated with epilepsy. In an embodiment, the rAAV products, compositions and methods of use thereof as described herein are used for the prophylactic and / or therapeutic treatment of epilepsy. Targeted Cell Types dL5** can be expressed in virtually any eukaryotic or prokaryotic cell of interest. In one embodiment, the cell is a neuronal cell type that requires modulation of its excitability. In another embodiment, the cell is a human neuron (e.g., motor neuron, sensory neuron, neuron of the central nervous system, and neuronal cell line).

[0122] Methods Of Stimulating A Neural Cell

[0123] The methods provided herein are, inter alia, useful for the stimulation of the neuronal cells. In particular, irradiation and / or light stimulation induces photopolymerization of polyaniline (PANI) and / or poly(3,3'-diaminobenzidine) (PDAB) as conductive and insulating polymers. Expression of dL5** in a cell, which is contacted with MG-2I and subsequent photostimulation with near infrared induces polymerization of PANI and / or PDAB, which alters the excitability of the cell.

[0124] The term “neural cell” as provided herein refers to a cell of the brain or nervous system. Non-limiting examples of neural cells include neurons, glia cells, astrocytes, oligodendrocytes and microglia cells. Where a neural cell is stimulated, a function or activity (e.g., excitability) of the neural cell is modulated by modulating, for example, the expression or activity of a given gene or protein (e.g., a dL5** polynucleotide) within said neural cell. The change in expression or activity may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a control (e.g., unstimulated cell). In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or activity in the absence of stimulation. In certain instances, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression or activity in the absence of stimulation. The neural cell may be stimulated by irradiating the neural cell.

[0125] The term “applying” as provided herein is used in accordance with its plain ordinary meaning and includes the meaning of the terms contacting, introducing and exposing. In embodiments, the neural cell forms part of an organism. In embodiments, the organism is a bacterial cell or mammalian cell (e.g., human, murine, bovine, feline, canine).

[0126] Stimulation is achieved by exciting the cell using energy of various wavelengths. In particular embodiments, light is used. In one embodiment, blue light irradiation is used. In other embodiments, light of other colors or of multiple colors is used. In particular embodiments, a neuron is irradiated with 300, 350, 375, 400, 425, 450, 475, 500, 525, 550 nm. In embodiments, the irradiation continues for 1-20 minutes (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 minutes). Methods Of Treatment

[0127] In another aspect, a method of treating a disease or disorder characterized by undesirable neuronal activity (e.g., neurodegenerative disease, such as Parkinson’s disease or Huntington’s disease; or chronic pain, or epilepsy) in a subject in need thereof is provided. The method includes (i) administering to a subject a therapeutically effective amount of a vector comprising recombinant nucleic acid encoding a dL5**, as well as DAB and / or polyaniline. In step (ii) light stimulation (irradiation) is applied to a cell of the subject, resulting in optically controlled, genetically targeted (optogenetic) polymerization and assembly of conductive and / or insulating polymers on the neuronal plasma membrane. This assembly provides for a change in neuronal excitability. In one embodiment, the methods described herein treat a neurological disease by altering neural activity in the subject. In embodiments, the disease is a neurodegenerative disease, such as Parkinson’s disease or Huntington’s disease; or chronic pain, epilepsy, or another disease associated with an undesirable alteration in neuronal excitability.

[0128] Kits

[0129] The invention provides kits for preventing or treating diseases and disorders characterized by undesirable neuronal activity, including neurodegenerative disorders, such as Parkinson’s disease or Huntington’s disease, as well as chronic pain, seizures and / or epilepsy. In one embodiment, the kit provides a therapeutic or prophylactic composition containing an effective amount of a rAAV vector or viral particle as described herein, which comprises a promoter (e.g., CAG) or neuronal specific promoter (e.g., human synapsis) that drives expression of DL5** . In addition to the vector, the kit further comprises polyaniline (PANI) and poly(3,3'- diaminobenzidine) (PDAB) that are photopolymerized to form conductive and insulating polymers throughout the cell, respectively. In some embodiments, the kit comprises a sterile container which contains the therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. The containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0130] A composition comprising an rAAV vector comprising at least a DL5** polynucleotide sequence as described herein is provided together with instructions for administering the composition to a subject having or at risk of developing neurodegenerative disorders, such as Parkinson’s disease or Huntington’s disease, as well as chronic pain, seizures and / or epilepsy. The instructions will generally include information about the use of the composition for the treatment or prevention of the disease or disorder. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent (rAAV comprising DL5** polynucleotide sequence, PDAB, PANI etc.); dosage schedule and administration for treatment or prevention of the disease or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0131] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0132] 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 assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0133] EXAMPLES

[0134] Example 1: Expanding optogenetic polymerization technology. dL5**, a fluorogen-activating protein (FAP), was selected due to its capability of binding to various derivatives of malachite green (MG) as the targeted and activated photosensitizer (TAP). He, J. et al. A genetically targetable near-infrared photosensitizer. Nature Methods 13, 263-268 (2016), which is incorporated herein by reference in its entirety for all purposes. Methods of making dl5** are described, for example, by Saunders et al., Bioconjug Chem. 2013 May 15; 24(5): 803-810. By employing the iodine-substituted MG (MG-2I) analog with near-IR absorbance as a co-factor (FIG. 1A), the efficiency of inter-system crossing could be significantly increased to form a near-IR excitable photosensitizer with high singlet oxygen (xAgO2) generation yield (He, J. et al. A genetically targetable near-infrared photosensitizer. Nature Methods 13, 263-268 (2016)). Since singlet oxygen has a short lifetime and diffusion radius at the microsecond and nanometer scale, respectively, the electron transfer and polymerization process occur within a small submicrometer space around the photo-catalysis center. Hence, it was hypothesized that localizing the FAP-TAPs to the cell membrane would enhance the integration of electrically functional polymers with the cell membrane for membrane modulation. To achieve this goal, a C-terminal famesylation sequence (C-Far) was fused to the C-terminal of FAP, enabling successful famesylation of FAP proteins through post-translational modification (FIG. 1B-C). Additionally, an mCherry fluorescence reporter was inserted upstream of the FAP-C-Far sequence, separated by a self-cleaving peptide T2A. The expression of the designed gene vector in HEK293T cells driven by the CAG promoter and in cultured rat cortical neurons driven by hSyn promoter exhibited distinct membrane-localization expression patterns, while mCherry expression remained within the cytosol (FIG. 1D-I).

[0135] Then, the capability of FAP-TAPs to polymerize electrically functional polymers was evaluated by introducing membrane-localized FAPs into HEK293T cells and rat cortical neurons using the CAG promoter and hSyn promoters, respectively (FIG. 2A). Following incubation of the HEK293T cells and neurons in 1 mM DAB or 0.75 mM aniline + 0.75 mM PPD solutions, the cells and neurons were irradiated with a Cy5 filter set for 10 minutes at an intensity of approximately 70 mW / mm. In TL modes, dark precipitates were visible, leading to a substantial reduction in the relative brightness of individual cells after the polymerization of DAB and aniline / PPD (FIG. 2B-C). Notably, PDAB exhibited a distinctive pattern of membrane localization, with depositions primarily occurring along the cell boundaries. Conversely, nonspecific polymerization and aggregation of PANI was observed within the cells, possibly attributed to the relatively extended lifetime of aniline / PPD cations, resulting in the diffusion of aggregates (Fig. 2D-G)

[0136] Next, the potential modulation of membrane capacitance and intrinsic excitability through the integration of PDAB and PANI onto the cell membrane was explored. To assess the electrical and electrophysiological properties of neurons, the whole-cell patch-clamp technique was used (FIG. 3A). Rat cortical neurons were transfected with AAV carrying membrane- localized FAPs and subsequently incubated in Tyrode’s solution containing 2 pM of MG-2I for 10 minutes, allowing the formation of photo-activatable FAP-TAPs. The neurons were then patched and polymerized using 1 mM DAB or 0.5 mM aniline + 0.5 mM PPD under a Cy5 filter set at an intensity of approximately 3 mW / mm2for 7 minutes. Following polymerization, the monomers were washed away with Tyrode’s solution to minimize any potential side effects. Consequently, a significant decrease or increase in membrane capacitance was observed upon the polymerization of DAB or PANI, respectively (FIG. 3B-C). Furthermore, neurons exhibited increased or decreased firing rates at different current injection levels after PDAB or PANI polymerization, respectively (FIG. 3D-f).

[0137] Further molecular engineering of monomers and polymers to enhance their biocompatibility is contemplated. For instance, incubation with the aniline monomer resulted in the suppression of neuron activity and potential cytotoxicity (Sessler et al., Science Advances 8, eadel 136 (2022)).

[0138] Consequently, engineering the monomers for conductive polymers to improve their biocompatibility (FIG. 4) was performed. For example, EDOT-thiophene-EDOT (ETE) trimer, which has demonstrated biocompatibility in various biological models, and the resulting PEDOT are known for their high conductivity. However, the high ratio of rigid and hydrophobic aromatic structures in ETE limits its solubility in biological fluids. Modification of the sulfonate group can enhance solubility, but the water solubility of the final PEDOT polymers prevents their deposition onto targeted cell structures (Strakosas et al., Science 379, 795-802 (2023)). To address this, side-chain engineering was used to finely control the aggregation state of the ETE monomer / polymer in biological fluids. Specifically, a hydrophilic moiety of the sulfonate group was attached to the center thiophen group of ETE, while alkyl chains were attached to the EDOT structure on the side. The solubility can be precisely adjusted by varying the length of the side chains to achieve the optimal structure. Furthermore, this design serves as a platform for incorporating arbitrary moieties into the side chains, potentially increasing the functionality of the polymers that can be integrated to the cell membrane. This approach significantly enhances the biocompatibility of the polymers, ultimately expanding the toolbox for optogenetic polymerization technology.

[0139] Example 2: Modulation of neuronal excitability in intact brain circuits dL5** FAP-TAP was tested for its ability to modulate neuronal excitability in intact brain circuits. Mice brains were injected with AAV vector encoding FAP-TAPs and the resulting mice were harvested and the brains sectioned into acute slices (FIG. 5A). Confocal imaging demonstrated the integration FAP-TAPs into the cell membranes of neurons in CAI (FIG. 5C). The slices were irradiated with near-infrared light in the presence of MG-2I and DAB (FIG. 5B). Current-clamp recordings of the brain slices demonstrated differences in firing after irradiation (FIG. 5D). Quantification of CAI neuronal capacitance further demonstrated that FAP-TAPs were capable of modulating neuronal excitability in intact brain circuits (FIG. 5E).

[0140] Other Embodiments

[0141] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0142] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0143] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference. The present disclosure may be related to the disclosure provided at U.S. App. No. 63 / 430,617, filed December 6, 2022, which is incorporated herein by reference in its entirety for all purposes.

Claims

What is claimed is:

1. A method for modulating neural activity using optogenetic polymerization and assembly of electroactive polymers on specified cellular membranes, the method comprising(a) expressing in a neuronal cell a vector encoding a near-IR excitable photosensitizer dL5** polypeptide in the presence of a malachite green derivative and monomers of 3,3'- diaminobenzidine (DAB), aniline and A-phenyl- / ?-phenylenediamine, or 3,4- Ethylenedioxythiophene-thiophene-3,4-Ethylenedi oxythiophene (ETE)-alkyne-sulfonate; and(b) applying near infrared radiation to at least a portion of the neuronal cell to induce singlet oxygen formation to induce polymerization of poly(3,3'-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate, thereby modulating the neuronal activity.

2. The method of claim 1, wherein the method provides for the spatiotemporal control of polymerization.

3. The method of claim 1, wherein the method provides for photopolymerization of poly(3,3'-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate at nanometer-level spatial resolution.

4. The method of claim 2, wherein spatial control is at the subcellular level.

5. The method of claim 4, wherein the method provides for optical control of polymer assembly on or within the cell membrane.

6. The method of claim 1, wherein the method produces increased levels of singlet oxygen relative to other reactive oxygen species (ROS).

7. The method of claim 1, wherein the method does not reduce neuron viability.

8. The method of claim 1, wherein the method alters neuronal excitability.

9. The method of claim 1, wherein the dl5** polypeptide is expressed under the control of aCAG promoter.

10. The method of claim 1, wherein the vector is a viral vector.

11. The method of claim 10, wherein the viral vector is an an adeno-associated viral expression vector (AAV) vector.

12. The method of claim 1, wherein the irradiation is near infra-red radiation.

13. The method of claim 1, wherein the irradiation is carried out using a Cy5 filter.

14. The method of claim 1, wherein the irradiation is for about 5-8 minutes.

15. The method of claim 1, wherein the irradiation is for about 9-15 minutes.

16. The method of claim 1, wherein the neuron is in vitro, ex vivo, or in vivo.

17. An adeno-associated viral expression vector (AAV) comprising a CAG or human synapsin promoter driving expression of a dL5** polypeptide.

18. A neuronal cell comprising the AAV of claim 17.

19. The neuronal cell of claim 18, wherein the neuron is a cell of the central or peripheral nervous system.

20. The neuronal cell of claim 18, wherein the neuron is a motor neuron or sensory neuron.

21. A method for modulating neural activity using optogenetic polymerization and assembly of electroactive polymers on specified cellular membranes, the method comprising(a) contacting a neuronal cell with an adeno-associated viral expression vector (AAV) comprising a CAG or human synapsin promoter driving expression of a dl5** polypeptide in the presence of a malachite green derivative and monomers of 3,3'-diaminobenzidine, aniline and N- phenyl- / ?-phenylenediamine, or ETE-alkyne-sulfonate; and(b) irradiating the neuronal cell to induce polymerization of poly(3,3'-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate, thereby modulating the neuronal activity.

22. The method of claim 21, wherein the method provides for the spatiotemporal control of polymerization.

23. The method of claim 21, wherein the method provides for photopolymerization of DAB at nanometer-level spatial resolution.

24. The method of claim 23, wherein spatial control is at the subcellular level.

25. The method of claim 24, wherein the method provides for optical control of polymer assembly on or within the cell membrane.

26. The method of claim 21, wherein the dl5** binding to malachite green MG-2I produces increased levels of singlet oxygen relative to other reactive oxygen species (ROS).

27. The method of claim 26, wherein the method alters neuronal excitability.

28. A method for modulating neural activity in a subject having a disorder associated with undesirable neural activity, the method comprising(a) administering to a neuronal cell of the subject an adeno-associated viral expression vector (AAV) comprising a CAG or human synapsin promoter driving expression of a dl5** polypeptide in the presence of a malachite green derivative and monomers of 3,3'- diaminobenzidine, aniline and 7V-phenyl- / ?-phenylenediamine, or ETE-alkyne-sulfonate; and(b) irradiating the neuronal cell to induce singlet oxygen production and polymerization of poly(3,3'-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate, thereby modulating the neuronal activity of the subject.

29. The method of claim 28, wherein the method treats the disorder or ameliorates at least one symptom of the disorder.

30. The method of claim 28, wherein the method provides for the spatiotemporal control of polymerization.

31. The method of claim 30, wherein the method provides for photopolymerization of poly(3,3'-diaminobenzidine), polyaniline, or polyETE-alkyne-sulfonate at nanometer-level spatial resolution.

32. The method of claim 28, wherein the disorder is a neurodegenerative disease.

33. The method of claim 28, wherein the disorder is chronic pain or epilepsy.

34. The method of claim 28, wherein the disorder is multiple sclerosis.

35. A kit for use in any of the above methods, the kit comprising a vector comprising a polynucleotide encoding dl5**, a malachite green derivative, and monomers of 3,3'- diaminobenzidine, aniline and N-phenyl-p-phenylenediamine, and / or ETE-alkyne-sulfonate.

36. The kit of claim 35, wherein the vector is an adeno-associated viral expression vector (AAV).

37. The kit of claim 35, wherein the vector comprises a CAG or human synapsin promoter driving expression of the dl5** polypeptide.

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