Optogenetic control of the integrated stress response using OPTO-pkr

Abstract

Disclosed herein are compositions and methods for activating a light responsive modified protein kinase R (PKR) and the Integrated Stress Response (ISR) pathway in a cell.

Description

[0001] OPTOGENETIC CONTROL OF THE INTEGRATED

[0002] STRESS RESPONSE USING OPTO-PKR

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application 63 / 552,839, filed February 13, 2024, the disclosure of which is hereby incorporated by reference in its entirety.

[0005] FIELD OF THE INVENTION

[0006] Disclosed herein are compositions and methods for activating a light responsive modified protein kinase R (PKR) and the Integrated Stress Response (ISR) pathway in a. cell.

[0007] INCORPORATION BY REFERENCE

[0008] The contents of the xml file named “11682-011WO1-ST26” which was created on

[0009] February 7 , 2025, and is 117 KB in size, tire hereby incorporated by reference in their entirety.

[0010] BACKGROUND OF THE INVENTION

[0011] Cells continuously map internal and external environments to adjust their functions according to their needs and maintain functional equilibrium — homeostasis. When restoring homeostasis is unachievable, programmed cell death ensues, thereby eliminating cells that cannot function optimally. Both outcomes, preserving functional cells and eliminating those that are not, ensure organism-level homeostasis to protect the organism’s health. Cellular stress responses are the fundamental mechanisms in charge of maintaining homeostasis. As such, they can be thought of as “molecular computers” that cells utilize to constantly collect, transmit, and interpret information about any deviation from functional optimality. Thus, dysfunction or insufficiency is often associated with multiple diseases. A detailed mechanistic understanding of how stress responses allow ceils to compute information and make decisions to restore homeostasis is essential to successfully developing strategies targeting stress responses for therapeutic intervention.

[0012] To better understand the mechanisms of cellular stress responses, there is a. need for new tools to dissect the relationship between perturbations and response mechanisms induced by the Integrated Stress Response (ISR) pathway. Tools are needed to precisely control recurrent stimulation dynamics and enable high-resolution output. Stress-inducing agents such as chemicals and physical stimuli introduce pleiotropic changes in cells due to molecular damage, which is not instantaneously reversed. The cell must undertake repair processes, thereby constraining the ability to investigate how the stress response interprets dynamic inputs. In addition, the combinatorial complexity of the signals turned on by real stressors resulting in the non-linear relationship between stimuli dose and cellular response is difficult to analyze.

[0013] The compositions and methods disclosed herein address the need for a comprehensive strategy to analyze the remodeling of the transcriptome in response to both acute and gradual stressors while assessing the adaptive and terminal outcomes of the ISR. The various aspects discussed herein relate to optogenetic control of the integrated stress response.

[0014] SUMMARY OF THE INVENTION

[0015] The present disclosure provides a synthetic approach that allows circumventing these limitations. The approach consists of chimeric versions of Integrated Stress Response (ISR) kinases that are insensitive to the physiological inputs and, instead, respond to light as virtual stress with no cross-activation of other kinases or stress response pathways. This approach allows dissection of the ISR codes, screening for each branch’s novel modulators, and designing logic gates to modulate cell fate decisions.

[0016] In some aspects, disclosed herein is a method of activating an Integrated Stress Response (ISR) pathway in a cell, the method comprising activating a modified protein kinase R (PKR), wherein PKR has been modified with a light responsive element, wherein the light responsive element comprises VIVID, iLID / SspB, CRY2PHR / CIBN, or a. fragment thereof, whereupon light activation of the light responsive element and PKR activates the ISR pathway.

[0017] In some embodiments, the light responsive element forms an optogenetic PKR (opto- PKR) when fused to PKR. In some embodiments, the opto-PKR forms a. dimer upon light activation.

[0018] In some embodiments, the light-responsive element causes opto-PKR to auto- phosphorylate upon light activation. In some embodiments, the opto-PKR activates the ISR pathway by phosphorylating eukaryotic initiation factor 2α (eIF2α),

[0019] In some embodiments, the light responsive element comprises a functional domain, a chimera, a functional fragment, or combinations thereof. In some embodiments, the light responsive element comprises VIVID, or a fragment thereof.

[0020] In some embodiments, the cell comprises a genetic modification to express the opto- PKR protein. In some embodiments, the genetic modification is a heritable trait. In some embodiments, the cell is in a non-human organism. In some embodiments, the ISR is activated in vitro in the cell.

[0021] In some embodiments, the opto-PKR is introduced into a. cell by a vector. In some embodiments, the vector comprises a bacterial plasmid or a recombinant virus. In some embodiments, the bacterial plasmid comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a fragment thereof. In some embodiments, the recombinant virus is an adeno associated virus (AAV), retrovirus, lentivirus, poxvirus, rabies virus, pseudo-rabies virus, or herpes simplex virus.

[0022] In some embodiments, the ISR pathway is activated using a light source with variable exposure duration, recovery time, intensity, or combinations thereof. In some embodiments, the light source is an optoPlate light delivery device.

[0023] In some embodiments, the method further determines a downstream change in gene expression is measured using western blotting, immunofluorescence, RNAseq, or a combination thereof.

[0024] In some aspects, disclosed herein is a modified protein kinase R (PKR), wherein PKR has been modified with a light responsive element, wherein the light responsive element comprises VIVID, iLID / SspB, CRY2PHR / CIBN, or a fragment thereof.

[0025] In some embodiments, the modified PKR comprises at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the modified PKR comprises at least 80% sequence identity to SEQ ID NO: 4. In some embodiments, the modified PKR comprises at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the modified PKR comprises SEQ ID NO: 4.

[0026] In some embodiments, the modified PKR comprises SEQ ID NO: 6.

[0027] In some embodiments, the light responsive element forms an opto-PKR when fused to PKR. In some embodiments, the opto-PKR comprises SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, or a. variant thereof. In some embodiments, the light-responsive element comprises SEQ ID NO: 2, SEQ ID NO: 13, or a variant thereof. In some embodiments, the light-responsive element forms a dimer upon light activation.

[0028] In some embodiments, the light-responsive element causes the opto-PKR to auto- phosphorylate upon light activation.

[0029] In some embodiments, the light responsive element comprises a functional domain, a chimera, a functional fragment, or combinations thereof. In some embodiments, the light responsive element comprises VIVID, or a fragment thereof. In one aspect, disclosed herein is a nucleic acid construct encoding the modified PKR of any preceding aspect.

[0030] In some embodiments, the construct comprises SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or fragments thereof.

[0031] In some embodiments, the construct comprises a bacterial plasmid or a recombinant virus. In some embodiments, the bacterial plasmid comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a fragment thereof.

[0032] In some embodiments, the recombinant virus comprises an adeno associated virus (AAV), retrovirus, lentivirus, poxvirus, rabies virus, pseudo-rabies virus, or herpes simplex virus.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0035] FIG. 1A depicts the schematic representation of the tool.

[0036] FIG. 1B show's the formation of VIVID*-PKR (169-550aa)-mRuby2 puncta upon illumination in transiently transfected HEK289T cells. Puncta are indicated with a white arrow.

[0037] FIG. 1C shows increased levels of ATF4 upon blue light treatment (Is every 30s for 3hr) in H4 and HEK293T cells transiently transfected with VIVID* -PKR(169-550aa)-mRuby. As a positive control, cells were treated with 300 nM of Thapsigargin (Tg).

[0038] FIG. ID shows H4 cells stability expressing VIVID*-PKR(169-550aa)-mRuby2 with increased levels of ATF4 upon blue light treatment (Is every 30s for Ihr, 2hr, and 6hr). As a positive control, cells were treated with 300nM Thapsigargin (Tg).

[0039] FIGS. 2 A, 213, 2C and 2D depict activation of opto-PKR using VIVID*-mRuby2- PKR(169-550aa) to detect light induced activation of the ISR.

[0040] FIG. 2A shows the scheme of the light-inducible VIVID*-mRuby2-PKR(169-550aa) system.

[0041] FIG. 2B shows H4 cells stably expressing VIVTD*-mRuby2-PKR(169-550aa) demonstrated increased levels of ATF4 upon light treatment (Is every 30s for 3hr or 6hr). As a positive control, cells were treated with 300nM Thapsigargin (Tg).

[0042] FIG. 2C shows that H4 cells stably expressing VIVTD*-mRuby2-PKR(169-550aa) and treated with light (1s every 30s for 3hr or 6hr) have increased transcription levels of downstream stress-induced genes, such as ATF4 (left for each time point) GADD34 (middle for each time pointO and DDIT3(CHOP) (right for each time point). Transcript levels were normalized to levels of housekeeping gene GAPDH and dark condition. As a. positive control, samples were treated with 300 nM Tg.

[0043] FIG. 2D shows H4 cells stably expressing VIVID*-mRuby2-PKR(169-550aa) have more nuclear localization of ATF4 upon light treatment.

[0044] FIGS. 3 A and 3B show a schematic representation of opto-PKR using the iLID- SspBmicro system, attaching truncated PKR to both elements of the iLID-SspBmicro (FIG. 3A), or using an iLID tandem repeat as a scaffold protein to recruit SspBmicro-PKR (FIG. 3B).

[0045] DETAILED DESCRIPTION

[0046] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

[0047] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0048] Terminology

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term ‘ comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and the open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0050] The following definitions are provided for the full understanding of terms used in this specification.

[0051] The terms “about" and “approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0052] As used herein, the terms "may," "optionally," and “may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0053] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0054] The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

[0055] An "increase" can refer to any change that results in a. greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50,

[0056] 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

[0057] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a. condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or

[0058] 100% decrease so long as the decrease is statistically significant.

[0059] A “protein”, "polypeptide", or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids. Thus, for example, the terms kinase, peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like.

[0060] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala. or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, p- alanine, β- Amino-propionic acid, allo-Hydroxylysine acid, 2 -Aminobutyric acid, 3-Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric aacciidd., N -Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N-

[0061] Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0062] The term “variant” means a polypeptide derived from a parent polypeptide by one or more (several) alteration(s), i.e., a substitution, insertion, and / or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid, occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1 or more, such as 1, 2, 3, 4, 5. 6, 7, 8, 9 or 10. preferably 1-3 amino acids immediately adjacent an amino acid occupying a position. In relation to substitutions, "immediately adjacent’ may be to the N-side ("upstream’) or C-side ("downstream’) of the amino acid occupying a position ("the named amino acid’). Therefore, for an ammo acid named / numbered ‘X,’ the insertion may be at position ‘X+1’ (‘downstream’ ) or at position ‘X-1’ (‘upstream’).

[0063] A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a. certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences---a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%', at least 70%, at least 80%, at least 90%, at least 91%, at least 92%', at least 93%, at least 94%, at least 95%, at least 96%, at least

[0064] 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relati ve to a reference polypeptide.

[0065] A variant polypeptide may have substantially the same functional activity as a reference polypeptide. For example, a variant polypeptide may exhibit or more biological activities associated with binding a ligand and / or binding DNA at a. specific binding site.

[0066] As used herein, the term “genetically modified” and other grammatical variations, including but not limited to a “genetic modification” refers to a living cell, tissue, or organism whose genetic material has been altered using genetic engineering techniques. The genetic modification results in an alteration that does not occur naturally by mating and / or natural recombination. Modified genes can be transferred within the same species, across species (creating transgenic organisms), and across kingdoms. New, exogenous genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out.

[0067] A " vector” refers to any vehicle that carries a. polynucleotide into a cell for the expression of the polynucleotide in the cell. The vector may be, for example, a plasmid, a virus, a phage particle, or a nanoparticle. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. In some embodiments, the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation. In other embodiments, the vector is a lipid nanoparticle. Lipid nanoparticles can be used to deliver mRNA to a host cell for expression of the mRNA in the host cell. In some embodiments, the expression vector comprises a plasmid or a virus or viral vector. A plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome. As used herein, the term "integrated" used in reference to an expression vector (e.g., a plasmid or viral vector) means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell. As used herein, a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell. A wide range of viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low toxicity, capability to enter cells). Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others. In some embodiments, the expression vector encoding a chimeric polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.).

[0068] As used herein, the term “probe” refers to a molecule or group of molecules used in molecular biology or chemistry to study the properties of other molecules or structures. If some measurable property of the molecular probe used changes when it interacts with the molecule of interest, the interactions between the probe and the molecule of interest can be studied. This makes it possible to indirectly study the properties of compounds and structures which may be hard to study directly. In some embodiments, a probe comprises the opto-PKR or the modified PKR of the present disclosure.

[0069] A “fluorophore” is a fluorescent chemical compound that can re-emit light upon light excitation. The chemicals are sometimes used alone as a tracer in fluids, as a due for staining certain structures, as an enzyme substrate, or as a probe / indicator. More commonly they are covalently bonded to a macromolecule to serve as a marker for bioactive reagents (i.e.: antibodies, peptides, nucleic acids, etc.) Fluorophores are notably used to stain tissues, ceils, or materials in a variety of analytical methods such as fluorescent imaging and spectroscopy.

[0070] A “biosensor” or “sensor” refers to a cell, protein, nucleic acid, light responding elements, or combinations thereof, which can detect analytes, light, or target molecules. These sensors are applied to both in vitro and in vivo applications. Said sensors can exist as monomers or exist configured as dimers, trimers, or oligomers.

[0071] As used herein, “fused” or “operably fused” refers to two or more compositions or compounds, including but not limited to proteins and light responsive elements, being bound or linked together in such a way the optimizes the intended function. When bound or linked, these compositions or compounds can be linked covalently, electrostatic interaction, through hydrogen bonding, or any combinations thereof.

[0072] A “nucleic acid” is a chemical compound that serves as the primary information- carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5 -carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.

[0073] The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

[0074] The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.

[0075] The term “oligonucleotide” denotes single- or double-stranded nucleotide multimers. Suitable oligonucleotides may be prepared by the phosphoramidite method described by Beaucage and Carruthers, Tetrahedron Lett., 22:1859-1862 (1981), or by the triester method according to Matteucci, et al., J. Am. Chem. Soc., 103:3185 (1981), both incorporated herein by reference, or by other chemical methods using either a commercial automated oligonucleotide synthesizer or VLSIPS™ technology. When oligonucleotides are referred to as “double-stranded,” it is understood by those of skill in the art that a pair of oligonucleotides exist in a hydrogen-bonded, helical array typically associated with, for example, DNA. In addition to the 100% complementary form of double-stranded oligonucleotides, the term “double-stranded,” as used herein is also meant to refer to those forms which include such structural features as bulges and loops, described more fully in such biochemistry texts as Stryer, Biochemistry , Third Ed., (1988), incorporated herein by reference for all purposes.

[0076] The terms “polynucleotide”, “nucleotide sequence”, and “nucleic acid sequence” are used interchangeably herein and refer to a single or double stranded polymer composed of nucleotide monomers.

[0077] The term “recombinant” refers to a human manipulated nucleic acid (e.g. polynucleotide) or a copy or complement of a human manipulated nucleic acid (e.g. polynucleotide), or if in reference to a protein (i.e, a “recombinant protein”), a protein encoded by a recombinant nucleic acid (e.g. polynucleotide). In embodiments, a recombinant expression cassette comprising a promoter operably linked to a second nucleic acid (e.g. polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation (e.g., by methods described in Sambrook et al., Molecular Cloning--™A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). In another example, a recombinant expression cassette may comprise nucleic acids (e.g. polynucleotides) combined in such a way that the nucleic acids (e.g. polynucleotides) are extremely unlikely to be found in nature. For instance, human manipulated restriction sites or plasmid vector sequences may flank or separate the promoter from the second nucleic acid (e.g. polynucleotide). One of skill will recognize that nucleic acids (e.g. polynucleotides) can be manipulated in many ways and are not limited to the examples above.

[0078] Nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, “operably linked” means that the DNA sequences being linked are near each other, and, in the case of a. secretory leader, contiguous and in reading phase. However, operably linked nucleic acids (e.g. enhancers and coding sequences) do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. In embodiments, a promoter is operably linked with a. coding sequence when it is capable of affecting (e.g. modulating relative to the absence of the promoter) the expression of a protein from that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). Methods of Activating the Integrated Stress Response

[0079] The Integrated Stress Response is a complex signaling pathway present primarily in eukaryotic cells, which is activated in response to physiological and pathological stresses, including but not limited to hypoxia, metabolic deprivation (such as, for example glucose deprivation and amino acid deprivation), microbial infections (such as, for example viral infections and bacterial infections), and endoplasmic reticulum (ER) stresses. The initial stress signals can activate protein kinases such as PKR-like ER kinase (PERK), heme-regulated eIF2α kinase (HRI), general control non-depressible 2 (GCN2), and double stranded RNA dependent protein kinase (PKR). Said protein kinases, once activated, can phosphorylate the a subunit of the protein complex, eIF2, further resulting in ATF4 gene activation and alterations in downstream gene expression.

[0080] The present disclosure provides methods of activating an Integrated Stress Response (ISR) pathway using chimeric versions of ISR kinases that respond to light as virtual stress with no cross-activation of other kinases or stress response pathways. This approach allows dissection of the ISR codes, screening for each branch’s novel modulators, and designing logic gates to modulate cell fate decisions.

[0081] In some aspects, disclosed herein is a method of activating an Integrated Stress Response (ISR) pathway in a cell, the method comprising activating a. modified protein kinase R (PKR), wherein PKR has been modified with a light responsive element, wherein the light responsive element comprises VIVID, iLID / SspB, CRY2PHR / CIBN, CRY2PHR, or a fragment thereof, whereupon light activation of the li ght responsive element and PKR activates the ISR pathway.

[0082] As used herein, a “light responsive element” refers to a molecule, peptide, probe, or any construct thereof that alters its physical shape, emits energy in the form of light or heat, alters its functionalities, or a combination thereof in response to exposure to at least one light stimuli.

[0083] In some embodiments, the method of any preceding aspect activates, increases, and / or enhances the ISR pathway by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 100%, or more in a cell.

[0084] In some embodiments, the light responsive element forms an optogenetic PKR (opto- PKR) when fused to PKR.

[0085] In some embodiments, the light responsive element and the PKR are fused to form a chimeric composition. In some embodiments, the chimeric composition comprises the opto- PKR. In some embodiments, the opto-PKR comprises PKR fused to VIVID, or a variant thereof. In some embodiments, the opto-PKR comprises PKR fused to iLID / SspB, or a variant thereof. In some embodiments, the opto-PKR comprises PKR fused to CRY2PHR / CIBN, or a variant thereof. In some embodiments, the opto-PKR comprises PKR fused to CRY2PHR, or a variant thereof.

[0086] In some embodiments, the opto-PKR forms a dimer upon light activation.

[0087] In some embodiments, the light-responsive element causes opto-PKR to auto- phosphorylate upon light activation. In some embodiments, the opto-PKR activates the ISR pathway by phosphorylating eukaryotic initiation factor 2α (eIF2α).

[0088] In some embodiments, the light responsive element comprises a functional domain, a chimera, a functional fragment, or combinations thereof. In some embodiments, the light responsive element comprises a. mutation, including but not limited to a point mutation, a substitution mutation, or a deletion mutation. In some embodiments, the light responsive element comprises VIVID, or a fragment thereof.

[0089] In some embodiments, the VIVID light responsive element comprises a truncated VIVID. In some embodiments, the VIVID is missing the first 36 amino acid residues of a wild- type VIVID sequence.

[0090] In some embodiments, the VIVID light responsive element comprises SEQ ID NO: 1, or a sequence at least 95% identical thereto. In some embodiments, the VIVID light responsi ve element comprises SEQ ID NO: 2, or a sequence at least 95%' identical thereto.

[0091] In some embodiments, the cell comprises a genetic modification to express the opto- PKR protein. In some embodiments, the genetic modification is a heritable trait.

[0092] As used herein, a “heritable trait” refers to traits, characteristics, genotypes, and / or phenotypes that can be passed down from one cellular generation to another cellular generation, or from parent to offspring.

[0093] In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is selected from the group consisting of yeast, insect, avian, fish, worm, amphibian, xenopus, bacteria, algae and mammalian cells. In one embodiment, disclosed herein is a non-human transgenic organism, wherein the organism is an insect, fish, bird, worm, amphibian, xenopus, or non-human mammal. In some embodiments, the cell is in a non-human organism. In some embodiments, the ISR is activated in vitro in the cell.

[0094] In some embodiments, the cell is in a non-human organism.

[0095] In some embodiments, the opto-PKR is introduced into a cell by a vector. In some embodiments, the vector comprises a bacterial plasmid or a recombinant virus. In some embodiments, the vector comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a fragment thereof. In some embodiments, the vector comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a sequence at least 95% identical thereto. In some embodiments, the recombinant virus is an adeno associated virus (AAV), retrovirus, lentivirus, poxvirus, rabies virus, pseudo-rabies virus, or herpes simplex virus.

[0096] In some embodiments, the ISR pathway is activated using a light source with variable exposure duration, recovery time, intensity, or combinations thereof. In some embodiments, the ISR pathway is activated using an LED light source. In some embodiments, the light source simultaneously emits one, two, three, or more wavelengths. In some embodiments, the one, two, three, or more wavelengths range from about 400 (nanometers) nm to about 500nm. In some embodiments, the wavelength is about 450 nm. In some embodiments, one, two, three, or more wavelengths comprising 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415. 416, 417, 418, 419, 420, 421, 422, 423. 424, 425, 426, 427, 428, 429, 430,

[0097] 431, 432, 433, 434, 435, 436, 437. 438, 439, 440, 441, 442, 443, 444, 445. 446, 447, 448. 449.

[0098] 450, 451 . 452, 453. 454, 455, 456, 457, 458, 459. 460, 461. 462, 463, 464, 465, 466, 467, 468,

[0099] 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487,

[0100] 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500nm. In some embodiments, the light source comprises an optoPlate light delivery device. In some embodiments, the light source comprises a light brick.

[0101] In some embodiments, the method further determines a downstream change in gene expression is measured using western blotting, immunofluorescence, real-time PCR, RNAseq, or a combination thereof.

[0102] Expression Vectors

[0103] In one aspect, disclosed herein is a. nucleic acid construct encoding the modified PKR of any preceding aspect.

[0104] In some embodiments, the construct comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9. SEQ ID NO: 12, SEQ ID NO: 14. SEQ ID NO: 16, or fragments thereof.

[0105] In some embodiments, the construct comprises SEQ ID NO: 1 , or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0106] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 1. In some embodiments, the construct comprises SEQ ID NO: 3, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0107] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 3.

[0108] In some embodiments, the construct comprises SEQ ID NO: 5, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0109] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 5.

[0110] In some embodiments, the construct comprises SEQ ID NO: 7, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0111] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 7.

[0112] In some embodiments, the construct comprises SEQ ID NO: 9, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0113] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 9.

[0114] In some embodiments, the construct comprises SEQ ID NO: 12, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0115] 97%, at least 98%, or at least 99%' identical thereto. In some embodiments, construct comprises SEQ ID NO: 12.

[0116] In some embodiments, the construct comprises SEQ ID NO: 14, or a. sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0117] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 14.

[0118] In some embodiments, the construct comprises SEQ ID NO: 16, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0119] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 16.

[0120] In some embodiments, the constnict comprises a bacterial plasmid or a recombinant virus. In some embodiments, the construct comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a fragment thereof.

[0121] In some embodiments, the constnict comprises SEQ ID NO: 17, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the constnict comprises SEQ ID NO: 17.

[0122] In some embodiments, the construct comprises SEQ ID NO: 18, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0123] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the constnict comprises SEQ ID NO: 18.

[0124] In some embodiments, the constnict comprises SEQ ID NO: 19, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%', at least 96%, at least

[0125] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 19.

[0126] In some embodiments, the construct comprises SEQ ID NO: 20, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0127] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 20.

[0128] In some embodiments, the constnict comprises SEQ ID NO: 21 , or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0129] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the construct comprises SEQ ID NO: 21.

[0130] In some embodiments, the construct comprises SEQ ID NO: 22, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0131] 9 !%, at least 98%, or at least 99% identical thereto. In some embodiments, the bac construct comprises SEQ ID NO: 22.

[0132] In some embodiments, the recombinant virus comprises an adeno associated virus (AAV), retrovirus, lentivirus, poxvirus, rabies virus, pseudo-rabies virus, or herpes simplex virus.

[0133] In some embodiments, the recombinant virus comprises a. lentivirus.

[0134] Retroviral Vectors

[0135] In some embodiments, the recombinant virus comprises a. retrovirus. In some embodiments, the retrovirus comprises a Moloney Murine Leukemia Virus (MMLV).

[0136] A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I.M., Retroviral vectors for gene transfer. A retro virus is essentially a package which has packed into it nucleic acid cargo. The nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat. In addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus. Typically a retroviral genome, contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell. Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome. The removal of the gag, pol, and env genes allows for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle. This amount of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.

[0137] Since the replication machinery and packaging proteins in most retroviral vectors have been removed (gag, pol, and env), the vectors are typically generated by placing them into a. packaging cell line. A packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal. When the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.

[0138] Adeno-associated viral vectors

[0139] Another type of viral vector is based on an adeno-associated virus (AAV). In some embodiments, the recombinant virus comprises an adeno-associated viral vector (AAV).

[0140] This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site- specific integration property are preferred. An especially preferred embodiment of this type of vector is the P4.1 C vector produced by A vigen, San Francisco, GA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0141] In another type of AAV virus, the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell-specific expression operably linked to a heterologous gene. Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or B19 parvovirus.

[0142] Typically the AAV and B19 coding regions have been deleted, resulting in a safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer infectivity and site- specific integration, but not cytotoxicity, and the promoter directs cell-specific expression. United states Patent No. 6,261 ,834 is herein incorporated by reference tor material related to the AAV vector.

[0143] Adenoviral Vectors

[0144] In some embodiments, the recombinant virus comprises an adenoviral vector.

[0145] The construction of replication-defective adenoviruses has been described (Berkner et al., J. Virology 61. : 1213-1220 (1987); Massie et al., Mol. Cell. Biol. 6:2872-2883 (1986); Haj- Ahmad et al, J. Virology 57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987); Zhang "Generation and identification of recombinant adenovirus by liposome- mediated transfection and PCR analysis" BioTechniques 15:868-872 (1993)). The benefit of the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but tire unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092 (1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation Research 73: 1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994): Zabner, Cell 75:207-216 (1993); Caillaud, Ear. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen. Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosls, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51:650-655 (1984); Seth, et ah. Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0146] A viral vector can be one based on an adenovirus which has had the E1 gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.

[0147] Large payload viral vectors

[0148] In some embodiments, the recombinant virus comprises a large payload viral vector. In some embodiments, the large payload viral vector comprises herpes simplex virus (HSV) or Epstein-Barr virus (EBV).

[0149] Molecular genetic experiments with large human herpesviruses have provided a. means whereby large heterologous DNA fragments can be cloned, propagated and established in cells permissive for infection with herpesviruses (Sun et al, Nature genetics 8: 33-41, 1994; Cotter and Robertson,. Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the potential to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV recombinants can maintain large pieces of DNA in the infected B-cells as episomal DNA. Individual clones carried human genomic inserts tip to 330 kb appeared genetically stable The maintenance of these episomes requires a specific EBV nuclear protein, EBNA1 , constitutively expressed during infection with EBV. Additionally, these vectors can be used for transfection, where large amounts of protein can be generated transiently in vitro. Herpesvirus amplicon systems are also being used to package pieces of DNA > 220 kb and to infect cells that can stably maintain DNA as episomes.

[0150] Other useful systems include, for example, replicating and host-restricted non- replicating virus vectors.

[0151] Modified protein kinases

[0152] The present disclosure provides chimeric versions of Integrated Stress Response (ISR) kinases that are insensitive to the physiological inputs and, instead, respond to light as virtual stress with no cross-activation of other kinases or stress response pathways. In some aspects, disclosed herein is a modified protein kinase R (PKR), wherein PKR has been modified with a light responsive element, wherein the light responsive element comprises VIVID, iLID / SspB, CRY2PHR / CIBN, CRY2PHR, or a fragment thereof.

[0153] In some embodiments, the modified PKR comprises a full-length PKR. In some embodiments, the modified PKR comprises a truncated PKR. In some embodiments, the truncated PKR has one or more double stranded RNA (dsRNA) binding domains removed.

[0154] In some embodiments, the modified PKR comprises at least 70% sequence identity to SEQ ID NO: 4. In some embodiments, the modified PKR comprises at least 80%' sequence identity to SEQ ID NO: 4. In some embodiments, the modified PKR comprises at least 90% sequence identity to SEQ ID NO: 4. In some embodiments, the bacterial plasmid comprises SEQ ID NO: 4, or a. sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%' identical thereto.

[0155] In some embodiments, the modified PKR comprises SEQ ID NO: 4.

[0156] In some embodiments, the modified PKR comprises SEQ ID NO: 6, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% Identical thereto. In some embodiments, the modified

[0157] PKR comprises SEQ ID NO: 6.

[0158] In some embodiments, the light responsive element forms an opto-PKR when fused to PKR. In some embodiments, the opto-PKR comprises SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, or a variant thereof.

[0159] In some embodiments, the opto-PKR comprises SEQ ID NO: 8, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0160] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the opto-PKR comprises SEQ ID NO: 8.

[0161] In some embodiments, the opto-PKR comprises SEQ ID NO: 10, or a. sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0162] 97%, at least 98%', or at least 99%' identical thereto. In some embodiments, the opto-PKR comprises SEQ ID NO: 10.

[0163] In some embodiments, the opto-PKR comprises SEQ ID NO: 11, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0164] 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the opto-PKR comprises SEQ ID NO: 11.

[0165] In some embodiments, the opto-PKR comprises SEQ ID NO: 15, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the opto-PKR comprises SEQ ID NO: 15.

[0166] In some embodiments, the light-responsive element comprises SEQ ID NO: 2, SEQ ID NO: 13, or a variant thereof.

[0167] In some embodiments, the light-responsive element comprises SEQ ID NO: 2, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the light-responsive element comprises SEQ ID NO: 2.

[0168] In some embodiments, the light-responsive element comprises SEQ ID NO: 13, or a sequence at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the light-responsive element comprises SEQ ID NO: 13.

[0169] In some embodiments, the light-responsive element forms a dimer upon light activation.

[0170] In some embodiments, the light responsive element comprises components, domains, and / or fragments of any preceding aspect. In some embodiments, the light-responsive element forms a dimer upon light activation. In some embodiments, the opto-PKR comprises the components, elements, domains, and / or fragments of any preceding aspect.

[0171] In some embodiments, the light-responsive element causes the opto-PKR to auto- phosphorylate upon light activation.

[0172] In some embodiments, the light responsive element comprises a functional domain, a. chimera, a functional fragment, or combinations thereof. In some embodiments, the light responsive element comprises VIVID, or a fragment thereof.

[0173] In some embodiments, the VIVID light responsive element comprises one or more mutations selected from Y50W, T69L, 174V, Y94E, S99N, N100R, A101H, N133Y, R137K,

[0174] M179I, or a combination thereof, relative to a wild-type VIVID sequence.

[0175] In some embodiments, the VIVID light responsive element comprises a truncated VIVID. In some embodiments, the truncated VIVID is missing amino acid residues 1-36 of a wild-type VIVID sequence. In some embodiments, the VIVID light responsive element comprises SEQ ID NO: 1, or a sequence at least 95% identical thereto. In some embodiments, the VIVID light responsive element comprises SEQ ID NO: 2, or a sequence at least 95% identical thereto.

[0176] In some embodiments, the light responsive element comprises iLID / SspB. In some embodiments, the SspB is operatively fused to the N-terminus of the modified PKR. In some embodiments, the iLID is operatively fused to the N-terminus of the modified PKR. In some embodiments, the SspB is operatively fused to the N-terminus of the modified PKR, and the iLID is arranged as a tandem repeat. In some embodiments, the tandem repeat iLID is a scaffold that recruits the modified PKR fused to the SspB.

[0177] Disclosed are the components to be used to prepare the compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular light responsive composition, such as for example an opto-chimeric peptide, is disclosed and discussed and a number of modifications that can be made to a number of molecules including the peptide and light responsive element discussed, specifically contemplated is each and every combination and permutation of the opto-PKR and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B- F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0178] It is understood that the compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures which can perform the same function which are related to the disclosed structures, and that these structures will ultimately achieve the same result.

[0179] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation: and the number or type of embodiments described in the specification.

[0180] A number of embodiments of tbe disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0181] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0182] EXAMPLES

[0183] The following examples are set forth below to Illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled In the art.

[0184] Example 1: Light-induced activation of the PKR branch of the ISR.

[0185] Protein kinase R (PKR) is a double-stranded RNA (dsRNA) sensor kinase that belongs to the Integrated Stress Response (ISR) signaling network. PKR is activated by dimerization and trans-autophosphorylation following dsRNA binding, and active PKR phosphorylates eIF2α. PKR can also associate into multimeric complexes visualized as discrete dynamic puncta by fluorescence microscopy, which are important for regulating the eIF2α-PKR interaction and thus fine-tune the ISR.

[0186] Previous approaches to develop a synthetic version of PKR replaced PKR' s dsRN A sensor domain with either a mutant version of the small prolyl-isomerase FKBP* (F36V), whose dimerization can be triggered with a small drug-like molecule, or with the photoreceptor protein CRY2olig, which can be Induced to cluster upon illumination with blue light. Both synthetic approaches result in canonical ISR signaling on demand. Each approach has advantages and limitations, but optogenetic procedures are recommended when spatial and temporal resolution are desired. The current disclosure presents an improved version of a light-activatable PKR that better mimics its physiological activation to avoid artifacts resulting from the disordered clustering which is afforded by for example CRY2olig, which may not recapitulate the dimer-on-dimer association that is likely reflective of the natural biophysical properties of PKR.

[0187] The main concern for the previous optogenetic version of PKR using CRY2olig is that CRY2olig is a truncated version (1-498 aa) of the plant photoreceptor CRY2 with a point mutation (E490G) that enhances its oligomerization. CRY2olig clustering - - -as opposed to dimerization — has unknown stoichiometry and solid-like properties; two feattires that do not recapitulate the physiological behavior of PKR activation (i.e., dimer formation, and self- association into liquid-like — not solid — condensates).

[0188] As an alternative to CRY2olig, this example uses photoreceptor proteins that will only dimerize upon illumination or form complexes with known stoichiometry, mimicking the physiological activation of PKR, and thereby allowing the formation and dissolution of dynamic PKR puncta, as observed for the endogenous protein.

[0189] Example 2: Light-induced activation of the PKR branch of the ISR using VIVID.

[0190] A truncated version of PKR (169-550 aa) was tested, missing its two dsRNA binding domains and N-terminal fused to a mutant version of the photoreceptor protein VIVID. VIVID is a fungus protein that forms an antiparallel homodimer in response to blue light and has been engineered to develop heterodimers, change its lifetime photocycle, and improve its thermal stability. This example utilizes a truncated version of VIVID with the first 36 amino acids removed to improve its homodimer affinity (Kd=13uM). Then, one or more mutations are used to generate a new version of VIVID (VVD*) with improved thermal stability (T69L, Y94E, S99N, N100R, A101H, N133Y, R136K, M179I), shorter lifetime photocycle of 730s (I74V), and improved dimer stability (Y50W).

[0191] For all the experiments presented here, the same version of VIVID* was used, having ten mutations (Y50W, T69L, I74V, Y94E, S99N, N100R, A101H, N133Y, R136K, M179I) plus the N-terminus truncation. The first design includes this version of VVD* fused to a truncated version of PKR followed by the fluorescent protein mRuby2 (FIG. 1A). Cloning was done by inserting VVD*-PKR (169-550aa)-mRuby2 into mRuby2-Nl vector, then HEK293T and H4 cells were transiently transfected. Cells treated with light (Is every 30s for 3hr, 5 mW / cmA2, 450 nm) showed an increased level of ATF4, and live cell imaging showed the light-induced formation of VVD*- PKR(169-550aa)-mRuby2 puncta (FIG. 1 B and 1C).

[0192] Upon these positive results, a stable H4 cell line was generated expressing the protein VVD*-PKR(169-550aa)-mRuby2 (CH59). The chimeric protein was cloned in a pLVX vector (Clontech), and standard protocols were followed to make infective lentiviral particles loaded with the design plasmid. Through Fluorescence-activated Cell Sorting (FACS), the expressing population was sorted and tested for ISR activation upon blue light illumination.

[0193] As seen in FIG. ID, H4 cells treated with light for Ih, 3h, and 6h (1s every 30, 5 mW / cmA2, 450 nm ) showed an increased level of ATF4 (FIG. ID).

[0194] In parallel, a different orientation of the protein was tested with mRuby2 fused at the N-terminus of PKR (FIG. 2 A). This chimeric version of the protein was also cloned in a pLVX vector, and H4 cells were infected to generate a stable cell line. FACS was used to select high- expressing cells. Similar to the previous orientation, WD*-mRuby2-PKR exhibited a light- dependent increase of ATF4 (FIG. 2B), and quantitative real-time PCR showed an up- regulation of ATF4-target genes, GADD34 and DDIT3 (also known as CHOP) (FIG. 2C). Light-dependent translation and nuclear localization of ATF4 was also confirmed by immunome fluorescence staining of H4 cells expressing WD*-mRuby2-PKR and treated with light for 4h (1s every 30s, 5 mW / cmA2, 450 nm) (FIG. 2D). As seen in FIG. 2D, the level of nuclear ATF4 correlates with the expression level of opto-PKR.

[0195] Example 3: Light-induced activation of the PKR branch of the ISR using iLID-SspB.

[0196] The iLID-SspB system is a light-inducible heterodimerizing system where the AsLOV2 photoreceptor protein from Avena sativa was engineered to cage the SsrA bacterial peptide at its C-terminus (named iLID). The interaction between the caged SsrA peptide and its partner protein, SspB, is only possible after the light-induced conformational change of iLID, which exposes the C-terminal Ssr.A peptide. The iLID-SspB system has been used to control protein -protein interaction, and mutant versions with different affinity and lifetime photocycle have expanded the use of the system.

[0197] The iLID-SspB system was tested in two different configurations, fusing a. truncated version of PKR to each iLID-SspB component, or using a tandem repeat of iLID as a scaffold protein to recruit a truncated PKR fused to SspB (FIG. 3). For both cases, the SspBmicro, which has a microMolar affinity for its partner iLID, was used. To avoid recombination problems while doing the stable cell lines, one construct was Inserted into a lentivlral plasmid (pLVx), while the other one was inserted into a retroviral plasmid (pMMLV).

[0198] The plasmids (pLVx-EGFP-EFl A-SspBmicro-PKR, pMMLV-CMV-mCh-iLID-PKR, and pMMLV-CMV-mCh-iLID2x) and viruses used to generate the cell lines were ordered from Vector Builder, and the full-length sequence of each plasmid.

[0199] Methods

[0200] Light treatment

[0201] Samples were kept in the dark by covering all the plates with aluminum foil and working with them under red light illumination conditions. To give a light treatment, samples were placed under an opto Plate inside an incubator, and light pulses were provided as described for each case. The optoPlate was built in-house following a design reporter as described previously (Hernandez-Candia, C.N. et al. Advances in optogenetic regulation of gene expression in mammalian cells using cryptochrome 2 (CRY2). Methods. 2019 Jul 15:164- 165:81 -90).

[0202] Fluorescent microscopy

[0203] Confocal and epi fluorescence i mages were acquired with a Ni kon-Crest X-Light V3 and a 40x or 20x objective lens.

[0204] Plasmids

[0205] VVD-PKR-mRuby2 was generated in a two-step cloning strategy. First VVD was PCR amplified using primer F1-5’- AGATCCgctagcGCCACCATGGtgcacactctctacgc-3’ (SEQ ID NO: 33), Rl-5'- aattcgaagcttcgatcccccttcggtttcgcactgaa-3’ (SEQ ID NO: 34) using as a template and IDT gBlock, and then cloned into pmRuby2-N1 vector using Nbel and Hindlll. Then PKR wwaass PCR amplified using primers F2-5’- gatcgaagcttACCTCAGTGAAATCTGACTACC-3’ (SEQ ID NO: 35), R2-5’- ggatcccgggcACATGTGTGTCGTTCATTTTT-3’ (SEQ ID NO: 36) and then cloned Into the previous vector using Hindlll and BamHI.

[0206] Cell line maintenance and. generation

[0207] All the cells were maintained on DMEM / F-12, GlutaMAX (Gibco, cat. 10565018) media, with 10% FBS (Omega Scientific, FB-01, lot 2290025) and 5% antibiotic-antimycotic (Gibco, cat.15240062). Western Blot and Immunofluorescence

[0208] Table 1. Antibodies used. Tables shows antibody concentrations, vendors, and lot numbers. qPCR

[0209] Table 2. Primer sequences

[0210] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. SEQUENCES

[0211] SEO ID NO: 1 VIVID*

[0212] ATGGtgcacactctctacgccccaggcgggtacgatattatgggctggctgatccagatcatgaacaggcccaatccccaggtcg agctgggacccgtggatCTTtcatgtgcactgGTTctgtgcgacctgaagcagaaggatacacctatagtttacgcttcagaagc ctttctgGAAatgacagggtatAATAGACATgaggtgctggggaggaactgtaggttcctccagagtcccgatggtatggt gaaacctaagagtactcgcaaatatgtggatagcaatactattTATaccatgAAGaaagccatcgacagaaacgcagaagttca ggtggaagtggtgaactttaagaagaacggccagcggttcgtgaactttctcacaatgattccagtgcgggacgaaaccggggagta ccggtacagcATTggttttcagtgcgaaaccgaa

[0213] SEP ID NO: 2 VIVID*

[0214] M VHTI ..YAPGG YDIMGWIJQIMN RPNPQ VELGPVDLSC ALVLCDLKQKDTPI V ¥ AS E

[0215] AFLEMTGYNRHEVLGRNCRFLQSPDGMVKPKSTRKYVDSNTIYTMKKAIDRNAEV

[0216] QVEVVNFKKNGQRFVNFLTMIPVRDETGEYRYSIGFQCETE

[0217] SEP ID NO: 3 - FULL PKR

[0218] GCTGGTGATCTTTCAGCAGGTTTCTTCATGGAGGAACTTAATACATACCGTCAGA

[0219] AGCAGGGAGTAGTACTTAAATATCAAGAACTGCCTAATTCAGGACCTCCACATG

[0220] ATAGGAGGTTTACATTTCAAGTTATAATAGATGGAAGAGAATTTCCAGAAGGTG

[0221] AAGGTAGATCAAAGAAGGAAGCAAAAAATGCCGCAGCCAAATTAGCTGTTGAG

[0222] ATACTTAATAAGGAAAAGAAGGCAGTTAGTCCTTTATTATTGACAACAACGAAT

[0223] TCTTCAGAAGGATTATCCATGGGGAATTACATAGGCCTTATCAATAGAATTGCCC

[0224] AGAAGAAAAGACTAACTGTAAATTATGAACAGTGTGCATCGGGGGTGCATGGGC

[0225] CAGAAGGATTTCATTATAAATGCAAAATGGGACAGAAAGAATATAGTATTGGTA

[0226] CAGGTTCTACTAAACAGGAAGCAAAACAATTGGCCGCTAAACTTGCATATCTTC

[0227] AGATATTATCAGAAGAAACCTCAGTGAAATCTGACTACCTGTCCTCTGGTTCTTT

[0228] TGCTACTACGTGTGAGTCCCAAAGCAACTCTTTAGTGACCAGCACACTCGCTTCT

[0229] GAATCATCATCTGAAGGTGACTTCTCAGCAGATACATCAGAGATAAATTCTAAC

[0230] AGTGACAGTTTAAACAGTTCTTCGTTGCTTATGAATGGTCTCAGAAATAATCAAA

[0231] GGAAGGCAAAAAGATCTTTGGCACCCAGATTTGACCTTCCTGACATGAAAGAAA

[0232] CAAAGTATACTGTGGACAAGAGGTTTGGCATGGATTTTAAAGAAATAGAATTAA

[0233] 'ITGGC'rcAGGTGGATTTGGCrAAGTTTTCAAAGCAAAACACAGAATTGACGGAA

[0234] AGACTTACGTTATTAAACGTGTTAAATATAATAACGAGAAGGCGGAGCGTGAAG

[0235] TAAAAGCATTGGCAAAACTTGATCATGTAAATATTGTTCACTACAATGGCTGTTG

[0236] GGATGGATTTGATTATGATCCTGAGACCAGTGATGATTCTCTTGAGAGCAGTGAT

[0237] TATGATCCTGAGAACAGCAAAAATAGTTCAAGGTCAAAGACTAAGTGCCTTTTC

[0238] ATCCAAATGGAATTCTGTGATAAAGGGACCTTGGAACAATGGATTGAAAAAAGA

[0239] actgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaacc accgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaa atactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttacca gtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctga acggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcg ceacgcttcccgaagagagaaaggcggacaggtatecggtaagcggcagggtcggaacaggagagcgcacgagggagcttcca gggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccct gattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagc gaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcc cgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccgg ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctc actaaagggaacaaaagctggagctgcaagctt

[0240] SEO ID NO: 18 - pMMLV<MV-mC.h-iLID-PKR gttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttac ggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagg gactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccct attgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtatt agtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctcca ccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaat gggcggtaggcgtgtacggtgggaggtctatataagcagcgcgttttgcctgtactgagctcaataaaagagcccacaacx:cx:tcact cggcgcgccagtcctccgattgactgagtcgcccgggtacccgtgtatccaataaaccctcttgcagttgcatccgacttgtggtctcg ctgttccttgggagggtctcctctgagtgattgactacccgtcagcgggggtctttcatttgggggctcgtccgggatcgggagacccc tgcccagggaccaccgacccaccaccgggaggtaagctggccagcaacttatctgtgtctgtccgattgtctagtgtctatgactgattt tatgcgcctgcgtcggtactagttagctaactagctctgtatctggcggacccgtggtggaactgacgagttcggaacacccggccgc aaccctgggagacgtcccagggacttcgggggccgtttttgtggcccgacctgagtccaaaaatcccgatcgttttggactctttggtg cacccccctaataggagggatatgtggttctggtaggagacgagaacctaaaacagttcccgcctccgtctgaatttttgctttcggtttg ggaccgaagccgcgccgcgcgtcttgtctgctgcagcatcgttctgtgttgtctctgtctgactgtgtttctgtatttgtctgaaaattaggg ccagactgttaccactcccttaagtttgaccttaggtcactggaaagatgtcgagcggatcgctcacaaccagtcggtagatgtcaaga agagacgttgggttaccttctgctctgcagaatggccaacctttaacgtcggatggccgcgagacggcacctttaaccgagacctcatc acccaggttaagatcaaggtcttttcacctggcccgcatggacacccagaccaggtcccctacatcgtgacctgggaagccttggcttt tgacccccctccctgggtcaagccctttgtacaccctaagcctccgcctcctcttcctccatccgccccgtctctcccccttgaacctcct cgttcgaccccgcctcgatcctccctttatccagccctcactccttctctaggcgcccccatatggccatatgagatcttatatggggcac ccccgccccttgtaaacttccctgaccctgacatgacaagagttactaacagcccctctctccaagctcacttacaggctctctacttagt SEQ ID NO: 32. Hs 28 R

[0241] AAACTCTGGTGGAGGTCCGT

[0242] SEQ ID NO: 33 - VVD Forward

[0243] AGATCCgctagcGCCACCATGGtgcacactctctacgc

[0244] SEQ ID NO: 34 VVD Reverse

[0245] Aattcgaagcttcgatcccccttcggtttcgcactgaa

[0246] SEQ ID NO: 35 - PKR Forward

[0247] GatcgaagcttACCTCAGTGAAATCTGACTACC

[0248] SEO ID NO: 36 PKR Reverse ggatcccgggcACATGTGTGTCGTTCATTTTT

Claims

CLAIMSWhat is claimed is:

1. A method of activating an Integrated Stress Response (ISR) pathway in a cell, the method comprising activating a modified protein kinase R (PKR), wherein PKR has been modified with a light responsive element, wherein the light responsive element comprises VIVID, iLID / SspB, CRY2PHR / CIBN, or a fragment thereof, and whereupon light activation of the light responsive element and PKR activates the ISR pathway.

2. The method of claim 1 , wherein the light responsive element forms an optogenetic PKR (opto-PKR) when fused to PKR.

3. The method of claim 2, wherein the opto-PKR forms a dimer upon light activation.

4. The method of any one of claims 1-3, wherein the light-responsive element causes opto-PKR to auto-phosphorylate upon light activation.

5. The method of any one of claims 1-4, wherein the opto-PKR activates the ISR pathway by phosphorylating eukaryotic initiation factor 2α (eIF2α).

6. The method of any one of claims 1-5, wherein the light responsive element comprises a functional domain, a chimera, a functional fragment, or combinations thereof.

7. The method of any one of claims 1 -6, wherein the light responsive element comprises VIVID, or a fragment thereof.

8. The method of any one of claims 1-7, wherein the cell comprises a genetic modification to express the opto-PKR protein.

9. The method of claim 8, wherein the genetic modification is a heritable trait.

10. The method of any one of claims 1-9, wherein the cell is in a non-human organism.

11. The method of any one of claims 1-10, wherein the ISR pathway is activated in vitro in the cell.

12. The method of any one of claims 2-11 , wherein the opto-PKR is introduced into the cell by a vector.

13. The method of claim 12, wherein the vector comprises a bacterial plasmid or a recombinant virus.

14. The method of claim 13, wherein the bacterial plasmid comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a fragment thereof.

15. The method of claim 13, wherein the recombinant virus comprises an adeno associated virus (AAV), retrovirus, lentivirus, poxvirus, rabies virus, pseudo-rabies virus, or herpes simplex virus.

16. The method of any one of claims 1-15, wherein the ISR pathway is activated using a light source with variable exposure duration, recovery time, intensity, or combinations thereof.

17. The method of claim 16, wherein the light source is an optoPlate light deli very device.

18. The method of any one of ciaims 1-17, wherein a. downstream change in gene expression is measured using western blotting, immunofluorescence, RNAseq, or a combination thereof.

19. A modified protein kinase R (PKR), wherein PKR has been modified with a light responsive element, wherein the light responsive element comprises VIVID, iLID / SspB, CRY2PHR / CIBN, or a fragment thereof.

20. The modified PKR of claim 19, wherein the modified PKR comprises at least 70% sequence identity to SEQ ID NO: 4.

21. The modified PKR of claim 19, wherein the modified PKR comprises at least 80% sequence identity to SEQ ID NO:4.

22. The modified PKR of claim 19, wherein the modified PKR comprises at least 90% sequence identity to SEQ ID NO: 4.

23. The modified PKR of claim 19, wherein the modified PKR comprises SEQ ID NO: 4.

24. The modified PKR of claim 19, wherein the modified PKR comprises SEQ ID NO: 6.

25. The modified PKR of any one of claims 19-24, wherein the light responsive element forms an optogenetic PKR (opto-PKR) when fused to PKR.

26. The modified PKR of claim 25, wherein the opto-PKR comprises SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 15, or a variant thereof.

27. The modified PKR of any one of claims 19-26, wherein the light-responsive element comprises SEQ ID NO: 2, SEQ ID NO: 13, or a variant thereof.

28. The modified PKR of any one of claims 19-27, wherein the light-responsive element forms a dimer upon light activation.

29. The modified PKR of any one of claims 19-28, wherein the light-responsive element causes the opto-PKR to auto-phosphorylate upon light activation.

30. The modified PKR of any one of claims 19-29, wherein the light responsive element comprises a functional domain, a chimera, a functional fragment, or combinations thereof.

31. The modified PKR of any one of claims 19-30, wherein the light responsive element comprises VIVID, or a fragment thereof.

32. A nucleic acid construct encoding the modified PKR of any one of claims 19-31.

33. The nucleic acid construct of claim 32, wherein the construct comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, or fragments thereof.

34. The nucleic acid construct of claim 32, wherein the construct comprises a bacterial plasmid or a recombinant vims.

35. The nucleic acid construct of claim 34, wherein the bacterial plasmid comprises SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, or a fragment thereof.

36. The nucleic acid construct of claim 34, wherein the recombinant virus comprises an adeno associated virus (AAV), retrovirus, lentivirus, poxvirus, rabies virus, pseudo-rabies virus, or herpes simplex virus.

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