Optogenetic control of the integrated stress response using OPTO-perk

Abstract

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

Description

[0001] OPTOGENETIC CONTROL OF THE INTEGRATED

[0002] STRESS RESPONSE USING OPTO-PERK

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application 63 / 552,846, 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-like endoplasmic reticulum kinase (PERK) and the Integrated Stress Response (ISR) pathway in a cell.

[0007] INCORPORATION BY REFERENCE

[0008] The contents of the xml file named “11682-014WO1-ST26” which was created on

[0009] February 7, 2025, and is 60.4 KB in size, are 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 the 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 cells 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 one aspect, 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-like endoplasmic reticulum kinase (PERK), wherein PERK has been modified with a light responsive element, whereupon light activation of the light responsive element and PERK activates the ISR pathway.

[0017] In some embodiments, the light responsive element forms an optogenetic PERK (opto- PERK) when fused to PERK. In some embodiments, the opto-PERK is localized in endoplasmic reticulum. In some embodiments, the opto-PERK forms a multimer with another opto-PERK upon light activation.

[0018] In some embodiments, the light-responsive element causes opto-PERK to auto- phosphorylate upon light activation. In some embodiments, the opto-PERK activates the ISR pathway by phosphorylating eukaryotic initiation factor 2a (eIF2a).

[0019] In some embodiments, the light responsive element comprises a functional domain, a chimera, a functional fragment, or a combination thereof. In some embodiments, the light responsive element comprises CRY2olig, CRY2PHR, CIBN / CRY2olig, CIBN / CRY2PHR, iLID / SspB, or a fragment thereof.

[0020] In some embodiments, the cell comprises a genetic modification to express the opto- PERK. In some embodiments, the genetic modification is a heritable trait.

[0021] In some embodiments, the cell is in a non-human organism. In some embodiments, the ISR is activated in vitro in the cell

[0022] In some embodiments, the opto-PERK is introduced into cells by using a vector. In some embodiments, the vector comprises a plasmid or a recombinant virus. In some embodiments, the plasmid comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a fragment thereof.

[0023] In some embodiments, the recombinant virus is an adeno associated virus (AAV), a retrovirus, a lentivirus, a poxvirus, a rabies virus, a pseudo-rabies virus, or a herpes simplex virus.

[0024] In some embodiments, the ISR pathway is activated using a light source with variable exposure duration, recovery time, intensity, or a combination thereof. In some embodiments, the light source is an optoPlate light delivery device.

[0025] In some embodiments, the method further determines a downstream change in gene expression is measured using western blotting, immunofluorescence, RNAseq, or a combination thereof.

[0026] In one aspect, disclosed herein is a modified protein kinase R-like endoplasmic reticulum kinase (PERK), wherein PERK has been modified with a light responsive element.

[0027] In some embodiments, the modified PERK comprises at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the modified PERK comprises at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the modified PERK comprises at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the modified PERK comprises SEQ ID NO: 3.

[0028] In some embodiments, the light responsive element comprises at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the light responsive element comprises at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the light responsive element comprises at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the light responsive element comprises SEQ ID NO: 1.

[0029] In some embodiments, the light responsive element forms an opto-PERK when fused to PERK. In some embodiments, the opto-PERK comprises SEQ ID NO: 1, SEQ ID NO: 3, or variants thereof. In some embodiments, the light-responsive element forms a multimer upon light activation.

[0030] In some embodiments, the light-responsive element causes the opto-PERK to auto- phosphorylate upon fight activation.

[0031] In some embodiments, the fight responsive element comprises a functional domain, a chimera, a functional fragment, or a combination thereof. In some embodiments, the light responsive element comprises CRY2olig, CRY2PHR, CIBN / CRY2olig, CIBN / CRY2PHR, iLID / SspB, or a fragment thereof.

[0032] In one aspect, disclosed herein is a nucleic acid construct encoding the modified PERK of any preceding aspect.

[0033] In some embodiments, the construct comprises SEQ ID NO: 2, SEQ ID NO: 4, or a variant thereof. In some embodiments, the construct comprises a plasmid or a recombinant virus. In some embodiments, the plasmid comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a fragment thereof. In some embodiments, the recombinant virus comprises an adeno associated virus (AAV), a retrovirus, a lentivirus, a poxvirus, a rabies virus, a pseudo-rabies virus, or a herpes simplex virus.

[0034] BRIEF DESCRIPTION OF DRAWINGS

[0035] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0036] FIGS. 1A, IB, and 1C depict photoreceptor protein CRY2PHR and its light-dependent partner CIBN to induce the activation of the chimeric version of PERK.

[0037] FIG. 1A shows the scheme of opto-PERK (PERK-CIBN + CRY2PHR-mCh). Light- dependent interaction of CRY2PHR and CIBN causes the dimerization of PERK and its activation.

[0038] FIG. IB shows that PERK-CIBN localizes to the ER, and CRY2PHR-mCh can get recruited upon illumination.

[0039] FIG. 1C shows stable cell fines expressing PERK-CIBN and CRY2PHR-mCherry exhibit light-dependent phosphorylation of eIF2a and translation of ATF4. DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] Terminology

[0043] 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 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. 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.

[0044] The following definitions are provided for the full understanding of terms used in this specification.

[0045] 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, tire terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0046] 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.

[0047] “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.

[0048] 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.

[0049] 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, 55. 60, 65. 70, 75. 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

[0050] 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, 100%, or more decrease so long as the decrease is statistically significant. 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.

[0051] 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 ( Ile 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 (Vai or V), tryptophan (Tip 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, β-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 acid, N-Methylglycine, sarcosine, 3-Aminoisobutyric acid, N-

[0052] 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.

[0053] The term “variant” means a polypeptide derived from a parent polypeptide by one or more (several) alteradon(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 amino acid named / numbered ‘X,’ the insertion may be at position ‘X+1’ (‘downstream’ ) or at position ‘X-1* (‘upstream’).

[0054] 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). hi 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 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide.

[0055] 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.

[0056] 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.

[0057] 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.).

[0058] 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-PERK or the modified PERK of the present disclosure.

[0059] 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.) Huorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods such as fluorescent imaging and spectroscopy.

[0060] 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, trima's, or oligomers.

[0061] 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.

[0062] 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.

[0063] The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

[0064] The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.

[0065] 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.

[0066] 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.

[0067] 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 add (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.

[0068] 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).

[0069] Methods of Activating the Integrated Stress Response

[0070] 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 eIF2a 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.

[0071] 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.

[0072] In one aspect, 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-like endoplasmic reticulum kinase (PERK), wherein PERK has been modified with a light responsive element, whereupon light activation of the light responsive element and PERK activates the ISR pathway.

[0073] In some embodiments, the modified PERK comprises full-length PERK.

[0074] In some embodiments, the modified PERK comprises truncated PERK.

[0075] 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.

[0076] 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.

[0077] In some embodiments, the light responsive element forms an optogenetic PERK (opto- PERK) when fused to PERK.

[0078] In some embodiments, the light responsive element and the PERK are fused to form a chimeric composition. In some embodiments, the chimeric composition comprises the opto- PERK. In some embodiments, the opto-PERK is localized in endoplasmic reticulum. In some embodiments, the opto-PERK forms a multimer with another opto-PERK upon light activation.

[0079] In some embodiments, the light-responsive element causes opto-PERK to auto- phosphorylate upon light activation. In some embodiments, the opto-PERK activates the ISR pathway by phosphorylating eukaryotic initiation factor 2a (eIF2a).

[0080] In some embodiments, the light responsive element comprises a functional domain, a chimera, a functional fragment, or a combination thereof. In some embodiments, the light responsive element comprises CRY2olig, CRY2PHR, CIBN / CRY2olig, CIBN / CRY2PHR, iLID / SspB, or a fragment 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.

[0081] In some embodiments, the opto-PERK comprises PERK fused to CRY2olig, or a variant thereof. In some embodiments, the opto-PERK comprises PERK fused to CRY2PHR, or a variant thereof. In some embodiments, the opto-PERK comprises PERK fused to CIBN / CRY2olig. or a variant thereof. In some embodiments, the opto-PERK comprises PERK fused to CIBN / CRY2PHR, or a variant thereof. In some embodiments, the opto-PERK comprises PERK fused to iLID / SspB, or a variant thereof.

[0082] In some embodiments, the opto-PERK forms a dimer upon light activation.

[0083] In some embodiments, the cell comprises a genetic modification to express the opto- PERK. In some embodiments, the genetic modification is a heritable trait. 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.

[0084] 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.

[0085] In some embodiments, the opto-PERK is introduced into cells by using a vector. In some embodiments, the vector is a plasmid. In some embodiments, the plasmid comprises SEQ ID NO: 5. SEQ ID NO: 6, or SEQ ID NO: 7.

[0086] In some embodiments, the plasmid 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 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the plasmid comprises SEQ ID NO: 5.

[0087] In some embodiments, the plasmid 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 plasmid comprises SEQ ID NO: 6.

[0088] In some embodiments, the plasmid 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 97%, at least 98%, or at least 99% identical thereto, In some embodiments, the plasmid comprises SEQ ID NO: 7.

[0089] In some embodiments, the vector is a recombinant virus. In some embodiments, the recombinant virus is an adeno associated virus (AAV), a retrovirus, a lentivirus, a poxvirus, a rabies virus, a pseudo-rabies virus, or a herpes simplex virus.

[0090] 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, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453. 454, 455, 456, 457, 458, 459, 460, 461. 462, 463, 464, 465, 466, 467, 468, 469. 470, 471, 472, 473, 474, 475, 476, 477. 478, 479, 480, 481, 482, 483, 484, 485. 486, 487, 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.

[0091] In some embodiments, the method further determines a downstream change in gene expression is measured using western blotting, immunofluorescence, RNAseq, or a combination thereof.

[0092] Expression Vectors

[0093] In one aspect, disclosed herein is a nucleic acid construct encoding the modified PERK of any preceding aspect.

[0094] In some embodiments, the construct comprises SEQ ID NO: 2, SEQ ID NO: 4, or a variant thereof. In some embodiments, the construct comprises SEQ ID NO: 2, SEQ ID NO: 4, or a sequence at least 95% identical thereto.

[0095] In some embodiments, the construct 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 construct comprises SEQ ID NO: 2.

[0096] In some embodiments, the construct 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. In some embodiments, the construct comprises SEQ ID NO: 4.

[0097] In some embodiments, the construct comprises a plasmid or a recombinant virus. In some embodiments, the plasmid comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a fragment thereof. In some embodiments, the plasmid 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 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the bacterial plasmid comprises SEQ ID NO: 5.

[0098] In some embodiments, the plasmid 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 bacterial plasmid comprises SEQ ID NO: 6.

[0099] In some embodiments, the plasmid 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 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the bacterial plasmid comprises SEQ ID NO: 7.

[0100] In some embodiments, the recombinant virus comprises an adeno associated virus (AAV), a retrovirus, a lentivirus, a poxvirus, a rabies virus, a pseudo-rabies virus, or a herpes simplex virus.

[0101] In some embodiments, the recombinant virus comprises a lentivirus.

[0102] Retroviral Vectors

[0103] In some embodiments, the recombinant virus comprises a retrovirus. In some embodiments, the retrovirus comprises a Moloney Murine Leukemia Virus (MMLV).

[0104] 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.

[0105] A retrovirus 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 tire 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.

[0106] 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 tine is a cell tine 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 tines, 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.

[0107] Adeno-associated viral vectors

[0108] 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).

[0109] 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 Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HS V-tk, and / or a marker gene, such as the gene encoding the green fluorescent protein, GFP.

[0110] 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.

[0111] 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 for material related to the AAV vector.

[0112] Adenoviral Vectors

[0113] In some embodiments, the recombinant virus comprises an adenoviral vector.

[0114] 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 51:267-214 (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 are 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, Eur. 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 endocytosis, 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 al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).

[0115] A viral vector can be one based on an adenovirus which has had the El 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. Large payload viral vectors

[0116] 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).

[0117] 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 up 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.

[0118] Other useful systems include, for example, replicating and host-restricted non- replicating virus vectors.

[0119] Modified protein kinases

[0120] 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.

[0121] In one aspect, disclosed herein is a modified protein kinase R-like endoplasmic reticulum kinase (PERK), wherein PERK has been modified with a light responsive element.

[0122] In some embodiments, the modified PERK comprises full-length PERK.

[0123] In some embodiments, the modified PERK comprises truncated PERK.

[0124] In some embodiments, the modified PERK comprises at least 70% sequence identity to SEQ ID NO: 3. In some embodiments, the modified PERK comprises at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the modified PERK comprises at least 90% sequence identity to SEQ ID NO: 3. In some embodiments, the modified PERK 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 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the modified PERK comprises SEQ ID NO: 3.

[0125] In some embodiments, the light responsive element comprises at least 70% sequence identity to SEQ ID NO: 1. In some embodiments, the light responsive element comprises at least 80% sequence identity to SEQ ID NO: 1. In some embodiments, the light responsive element comprises at least 90% sequence identity to SEQ ID NO: 1. In some embodiments, the modified PERK 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 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the light responsive element comprises SEQ ID NO: 1.

[0126] In some embodiments, the light responsive element forms an opto-PERK when fused to PERK. In some embodiments, the opto-PERK comprises SEQ ID NO: 1, SEQ ID NO: 3, or variants thereof.

[0127] In some embodiments, the opto-PERK 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 97%, at least 98%. or at least 99% identical thereto. In some embodiments, the opto-PERK comprises SEQ ID NO: 1.

[0128] In some embodiments, the opto-PERK 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 97%, at least 98%, or at least 99% identical thereto. In some embodiments, the opto-PERK comprises SEQ ID NO: 3.

[0129] 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 multimer upon light activation. In some embodiments, the opto-PERK comprises the components, elements, domains, and / or fragments of any preceding aspect.

[0130] In some embodiments, the light-responsive element causes the opto-PERK to auto- phosphorylate upon light activation.

[0131] In some embodiments, the light responsive element comprises a functional domain, a chimera, a functional fragment, or a combination thereof. In some embodiments, the light responsive element comprises CRY2olig, CRY2PHR, CIBN / CRY2olig, CIBN / CRY2PHR, iLID / SspB, or a fragment thereof.

[0132] In some embodiments, the modified PERK comprises a CIBN fused in between a transmembrane motif and a kinase domain of the modified PERK.

[0133] 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-PERK 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.

[0134] 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.

[0135] 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.

[0136] A number of embodiments of the 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.

[0137] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below.

[0138] EXAMPLES

[0139] 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.

[0140] Example 1: Light-induced activation of the PERK branch of the ISR.

[0141] The pancreatic endoplasmic reticulum kinase (PERK) (also known as EIF2AK3) is a sensor of proteostasis stress and belongs to the Integrated Stress Response (ISR) and Unfolded protein response (UPR) networks. It is localized to the endoplasmic reticulum (ER), with its unfolded protein sensor domain facing the ER lumen and its effector domain, the kinase domain, facing the cytosol where it can bind its downstream target eIF2a. PERK dimerizes upon interaction with unfolded proteins on the ER lumen, and its trans-autophosphorylation of the cytosolic kinase domain leads to its activation.

[0142] Previous strategies to isolate the stress signaling branch of PERK used only its kinase domain fused to a tandem repeat of an inducible dimerizer protein (FKBP(F36V)). This chimeric PERK can trigger eIF2a phosphorylation upon induced dimerization of FKBP and upregulate downstream targets of ATF4. However, this approach dismisses the regulatory effect of PERK ER localization. To gain knowledge on the interactors and modulations of PERK in its physiological context, this study generated a novel chimeric version of PERK that is missing its sensor domain but retains its ER localization and is activatable upon blue-light illumination. To generate a blind-ER-localized PERK version, amino acids corresponding to motifs I, II, and HI that mediate PERK oligomerization were removed.

[0143] The ER signal sequence and region IV were retained to localize the protein and prevent its activation without stress.

[0144] The study used the photoreceptor protein CRY2PHR and its light-dependent partner CIBN to induce the activation of the chimeric version of PERK. CIBN was fused in between the transmembrane motif and the kinase domain of PERK (FIG. 1A), and was co-expressed with CRY2PHR fused to mCherry. Versions of both constructs were generated in the expression plasmid pcDNA3.1+ and were used to transfect COS-7 cells transiently.

[0145] Light fluorescent microscopy of fixed cells treated with light (Is every 30 s for 5 min) or dark conditions showed a light-dependent localization of CRY2PHR-mCherry to the ER (FIG. IB). This shows that the chimeric version of PERK retains its ER localization and can interact with CRY2PHR.

[0146] To test the ISR activation, the PiggyBac system was used to generate a bicistronic plasmid coding for the PERK-CIBN protein and CRY2PHR-mCherry. A COS-7 cell line expressing both proteins was generated. Phosphorylation of eIF2a and ATF4 translation was observed upon light treatment (FIG. 1C).

[0147] Methods

[0148] Light treatment

[0149] 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 a light brick inside an incubator, and light pulses were provided as described for each case. The light brick was built in-house following the design reporter as described previously (HemSndez-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, incorporated by referenced herein).

[0150] Fluorescent microscopy

[0151] To confirm the ER localization of our chimera protein (PERK-CIBN), Cos7 cells were transiently transfected with three different plasmids. Two of them code for our opto-PERK tool (PERK-CIBN and CRY2PHR-mcheny), and the other one was used as an ER marker (mClover-CytERM-N-17; Addgene #56309). Samples were fixed with 4% PFA after being treated with light (Is every 30 seconds for 5 min) or kept in the dark. Confocal images were acquired with a Nikon Wl-SoRa-iLAS, and a 100x objective lens.

[0152] Plasmids pLVx-VVD-PKR-mRuby2 was generated by PCR amplification ofVVD-PKR-mRuby (F- 5*- CGAGACTAGTGCCACCATGGtgcacactctctacgcccca-3’ (SEQ ID NO: 18) and R-5’- GGAAGCGGCCGCttaCTTGTACAGCTCGTCCATCCCACCA-3' (SEQ ID NO: 19)) and insertion into pLVx using Spel and Notl cloning sites. PiggyBac-CRY2PHR-mCh-IRES- PERK(lumen)-CIBN-PERK(kinase) was designed and generated (Vector Builder). pcDNA3.1+-PERK(lumen)-CIBN-PERK(kinase) was designed and generated (Thermofisher, GeneArt). pmCherry-Nl-CRY2PHR-mCh was cloned into pmCherry-Nl using Xhol and Xmal cloning sites. CRY2PHR was PCR amplified from an IDT gBlock using the following primers: Forward 5'-cagatctcgaggccaccatgaagatggacaaaaagact-3' (SEQ ID NO: 20), Reverse 5’- ggtggatcccgggctgctgctccgatcatgat-3* (SEQ ID NO: 21). The PCR product was digested with Xhol and Xmal and then ligated into opened pmCherry-Nl vector.

[0153] Cell line maintenance and generation

[0154] PERK-CIBN and CRY2PHR-mCh were transiently transfected with Lipofectamine 2000. To generate the stable cell lines, PiggyBac-CRY2PHR-mCh-IRES-PERK(lumen)- CIBN-PERK(kinase) and a plasmid coding for the superPiggyBac transposase were transiently transfected using Lipofectamine 2000. Cells were passaged twice, and then using Fluorescence Activation Cell Sorting (FACS), the positive mcherry cells were selected. All the cells were maintained on DMEM / F-12, GlutaMAX (Gibco, cat. 10565018) media with 10% FBS and 5% antibiotic-antimycotic (Gibco, cat.15240062).

[0155] Western Blot and Immunofluorescence

[0156] Table 1. Antibodies used in this study. Table show concentrations, vendors, and lot numbers. qPCR Table 2. Primer sequences

[0157] SEQUENCES

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[0159] 

[0160] 

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-like endoplasmic reticulum kinase (PERK), wherein PERK has been modified with a light responsive element, and whereupon light activation of the light responsive element and PERK activates the ISR pathway.

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

3. The method of claim 2, wherein the opto-PERK is localized in endoplasmic reticulum.

4. The method of claim 2 or 3, wherein the opto-PERK forms a multimer with another opto-PERK upon light activation.

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

6. The method of any one of claims 1-5, wherein the opto-PERK activates the ISR pathway by phosphorylating eukaryotic initiation factor 2a (eIF2a).

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

8. The method of any one of claims 1-7, wherein the light responsive element comprisesCRY2olig, CRY2PHR, CIBN / CRY2olig, CIBN / CRY2PHR, iLID / SspB, or a fragment thereof.

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

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

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

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

13. The method of any one of claims 2-12, wherein the opto-PERK is introduced into cells by using a vector.

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

15. The method of claim 14, wherein the plasmid comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a fragment thereof.

16. The method of claim 14, wherein the recombinant virus comprises an adeno associated virus (AAV), a retrovirus, a lentivirus, a poxvirus, a rabies virus, a pseudo-rabies virus, or a herpes simplex virus.

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

18. The method of claim 17, wherein the light source is an optoPlate light delivery device.

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

20. A modified protein kinase R-like endoplasmic reticulum kinase (PERK), wherein PERK has been modified with a light responsive element.

21. The modified PERK of claim 20. wherein the modified PERK comprises at least 70% sequence identity to SEQ ID NO: 3.

22. The modified PERK of claim 20, wherein the modified PERK comprises at least 80% sequence identity to SEQ ID NO: 3.

23. The modified PERK of claim 20, wherein the modified PERK comprises at least 90% sequence identity to SEQ ID NO: 3.

24. The modified PERK of claim 20, wherein the modified PERK comprises SEQ ID NO:3.

25. The modified PERK of any one of claims 20-24, wherein the light responsive element comprises at least 70% sequence identity to SEQ ID NO: 1.

26. The modified PERK of any one of claims 20-24, wherein the light responsive element comprises at least 80% sequence identity to SEQ ID NO: 1.

27. The modified PERK of any one of claims 20-24, wherein the light responsive element comprises at least 90% sequence identity to SEQ ID NO: 1.

28. The modified PERK of any one of claims 20-24, wherein the light responsive element comprises SEQ ID NO: 1.

29. The modified PERK of any one of claims 20-28, wherein the light responsive element forms an optogenetic PERK (opto-PERK) when fused to PERK.

30. The modified PERK of any one of claims 20-29, wherein the opto-PERK comprises SEQ ID NO: 1, SEQ ID NO: 3, or variants thereof.

31. The modified PERK of any one of claims 20-30, wherein the light-responsive element forms a multimer upon light activation.

32. The modified PERK of any one of claims 20-31, wherein the light-responsive element causes the opto-PERK to auto-phosphorylate upon light activation.

33. The modified PERK of any one of claims 20-32, wherein the light responsive element comprises a functional domain, a chimera, a functional fragment, or a combination thereof.

34. The modified PERK of any one of claims 20-33, wherein the light responsive element comprises CRY2olig, CRY2PHR, CIBN / CRY2olig, CIBN / CRY2PHR, iLID / SspB, or a fragment thereof.

35. A nucleic acid construct encoding the modified PERK of any one of claims 20-34.

36. The construct of claim 35. wherein the construct comprises SEQ ID NO: 2, SEQ IDNO: 4, or a variant thereof.

37. The construct of claim 35 or 36, wherein the construct comprises a plasmid or a recombinant virus.

38. The construct of claim 37, wherein the plasmid comprises SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a fragment thereof.

39. The construct of claim 37, wherein the recombinant virus comprises an adeno associated virus (AAV), a retrovirus, a lentivirus, a poxvirus, a rabies virus, a pseudo-rabies virus, or a herpes simplex virus.

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