WO2025071367A1 - Calcium-modulating fusion protein that can be expressed by adeno-associated virus - Google Patents

Abstract

A calcium-modulating fusion protein that can be expressed by an adeno-associated virus, according to one aspect of the present invention, has monSTIM1 of which the coding sequence size is reduced compared to before so as to fit the packaging capacity of AAV, thereby establishing an AAV-based system that can be expressed in neurons and glial cells of a mouse brain. The monSTIM1 variant expressed by AAV according to one aspect can minimize problems such as tissue damage, glial scar formation, inflammation, and tissue heating, caused by an optical fiber inserted for a long period and has an effect of modulating neural activity in a non-invasive way, and thus is a promising tool that enables studying the spatiotemporal roles of calcium-mediated cellular activities in various brain functions and also has potentials to be applied in treating brain diseases related to abnormal calcium signaling.

Description

Calcium-regulated fusion protein expressible by adeno-associated virus

It relates to a calcium-regulated fusion protein that can be expressed by adeno-associated virus.

Calcium ion (Ca2 ⁺ ) is a ubiquitous second messenger that plays a critical role in regulating a wide range of brain functions, including cognition, emotion, movement, learning, and memory. Intracellular Ca2 ⁺ signals are tightly regulated in space and time, and abnormal regulation in the central nervous system has been associated with various neurological disorders, such as epilepsy, chronic pain, psychiatric diseases, and neurodegeneration. Brain cells possess molecular machinery, such as channels and pumps located in the plasma membrane and membranes of cell organelles, that regulate Ca2 ⁺ signals. Ca2 ⁺ signals regulate neuronal functions, such as synaptic plasticity, neurotransmitter release, and gene expression, and their functions vary depending on various parameters, such as signal amplitude, duration, and location. In addition, Ca2 ⁺ signals also play a role in regulating important processes, such as neurotransmitter release, homeostasis, and immune responses, in glial cells.

In the optogenetic technology field, various technologies are being actively studied to directly investigate the causal relationship between Ca ²⁺ signals and cellular functions in space and time by controlling intracellular Ca ²⁺ levels through light stimulation. In particular, CRAC (Ca ²⁺ -release-activated Ca ²⁺ ) channels with high selectivity for Ca ²⁺ are attracting attention as a major target. In these technologies, STIM1 (Stromal interaction molecule 1), a key regulator of the store-operated Ca ²⁺ entry (SOCE) process, plays a key role, and two types of optogenetic actuators based on different photoreceptors have been developed to activate CRAC channels.

The first type of optogenetic actuator utilizes the light-oxygen-voltage-sensing domain 2 (LOV2) domain of the phototropin isolated from oat ( Avena Sativa) . Conjugation of the LOV2 domain to an active STIM1 fragment causes steric hindrance, which inhibits STIM1 activity in the absence of light. Blue light stimulation induces a conformational change within the LOV2 domain that dissociates it from the C-terminal Jα helix. This dissociation relieves STIM1 inhibition, allowing activation of the CRAC channel and increasing intracellular Ca2 ⁺ levels.

The second type of optogenetic actuator utilizes cryptochrome 2 (CRY2) isolated from Arabidopsis thaliana . Taking advantage of the light-mediated homo-interaction property of CRY2, the cytoplasmic fragment of STIM1 binds to CRY2 and undergoes homo-oligomerization upon light stimulation. Homo-oligomerized STIM1 translocates to the cell membrane and activates CRAC channels, mimicking the natural action mechanism of STIM1 (Fig. 1a). This second type of cryptochrome 2 (CRY2)-based optogenetic actuator is named OptoSTIM1.

The OptoSTIM1 has been demonstrated to be effective in activating Ca2 ⁺ influx in various cell types and successfully controls targeted brain functions (Nat Biotechnol. 2015;33:1092-6). Although LOV2 domain-based actuators have the advantage of faster kinetics for (de)activation and smaller size compared to OptoSTIM1, OptoSTIM1 was found to induce larger fold changes in regulating intracellular Ca2 ⁺ levels (Cell Calcium. 2017;64:36-46).

Despite the design advantage of the LOV2 domain, further studies were conducted to improve the design of CRY2 of OptoSTIM1 because OptoSTIM1 showed a superior performance in regulating calcium influx. In particular, during the process of improving the CRY2 design, it was found that the CRY2(E281A) mutation decreased the basal activity of OptoSTIM1 in the dark and increased its sensitivity to blue light. In addition, it was confirmed that combining CRY2(E281A) and A9 conjugate significantly increased light-dependent oligomerization. Based on this, the second-generation OptoSTIM1, monSTIM1, was developed, which showed approximately 55-fold higher sensitivity to blue light than the previous one. These improvements enabled the activation of calcium signals in the mouse brain noninvasively without optical fiber insertion.

Meanwhile, adeno-associated virus (AAV) is widely used in neuroscience research and gene therapy due to its high gene transfer efficiency, low host genome integration probability, and low immunogenicity. Moreover, recent advances in AAV capsid engineering have led to the development of systemic delivery technologies that enable targeted expression in specific tissues and cells. However, when combining AAV cassette components, including the coding sequence of monSTIM1 and inverted terminal repeats (ITRs), woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and human growth hormone (hGH) polyadenylation signal, the total sequence size excluding the promoter is ~ 5.1 kb, which exceeds the packaging capacity of adeno-associated virus (AAV) (~ 5.0 kb) (Fig. 1b).

Previous studies have used lentiviruses to express optogenetic ^{Ca2+ modulators} in mouse brain due to the size constraints of monSTIM1. Lentiviruses can carry larger genes, but they have the property of integrating into the host genome, which may raise safety issues for gene therapy, and their limited diffusion within brain tissue reduces the number of transduced cells.

Herein, in order to expand the diversity of monSTIM1 by utilizing the potential advantages of AAV, the present inventors developed AAV-compatible monSTIM1 variants (monSTIM1 variants) by shortening the monSTIM1 coding sequence through several strategies to fit the packaging capacity of AAV. The monSTIM1 variants of the present invention efficiently increase intracellular Ca2 ⁺ levels in various brain cells such as neurons, astrocytes, and microglia, and maintain the ultra-high light sensitivity property of the existing monSTIM1, so that they can effectively induce Ca2 ⁺ -mediated gene expression in neurons and astrocytes of the mouse brain in response to non-invasive light illumination.

One aspect is to provide a fusion protein comprising a Tag protein; CRY2 (cryptochrome 2) protein or a variant thereof; and STIMI1 (Stromal interaction molecule 1) protein.

Another aspect is to provide a polynucleotide encoding the above fusion protein.

Another aspect is to provide a vector comprising a polynucleotide encoding the fusion protein.

Another aspect is to provide a Ca2+ modulator comprising STIMI1 (Stromal interaction molecule 1) protein to which CIB1 (cryptochromeinteracting basic-helix-loop-helix 1) protein or a fragment thereof is attached; and CRY2 (cryptochrome 2) protein or a variant thereof.

Another aspect provides a Ca2 ⁺ modulator comprising a STIMI1 protein to which a tag protein is attached; and a CRY2 protein or a variant thereof to which a nanobody antibody capable of binding to the tag protein is attached.

Another aspect provides an expression system comprising a first vector comprising a polynucleotide encoding a STIMI1 protein or a fragment thereof; and a second vector comprising a polynucleotide encoding a CRY2 protein or a variant thereof.

One aspect provides a fusion protein comprising a Tag protein; CRY2 (cryptochrome 2) protein or a variant thereof; and STIMI1 protein.

The term "tag protein" in this specification refers to a protein that is attached to a specific protein and used to track or purify the location of the protein. The tag is connected to the C-terminus or N-terminus of the protein, and allows the expression level of the protein to be monitored or easily detected.

In one specific example, the tag protein can be a fluorescent protein. For example, the fluorescent protein can be green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), orange fluorescent protein (OFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), far-red fluorescent protein, or a tetracysteine motif. Here, the green fluorescent protein is EGFP (enhanced green fluorescent protein), Emerald (Tsien, Annu. Rev. Biochem., 67: 509-544, 1998), Superfolder (Pedelacq et al., Nat. Biotech., 24: 79-88, 2006), and GFP (Prendergast et al., Biochem., 17(17): 3448-3453, 1978), Azami Green (Karasawa, et al., J. Biol. Chem., 278: 34167-34171, 2003), TagGFP (Evrogen, Russia), TurboGFP (Shagin et al., Mol. Biol. Evol., 21(5): 841-850, 2004), ZsGreen (Matz et al., Nat. Biotechnol., 17: 969-973, 1999) or T-Sapphire (Zapata-Hommer et al., BMC Biotechnol., 3:5, 2003). In one specific example, the green fluorescent protein may be composed of an amino acid sequence of SEQ ID NO: 41.

In one specific example, the tag protein may be an epitope tag. The epitope tag is a short amino acid sequence that is added to a foreign protein and acts as a label that can be recognized by a specific antibody, and may be, for example, Myc, V5, T7, AU1/AU5, VSV-G (Vesicular Stomatitis Virus Glycoprotein), and preferably, FLAG or HA, but is not limited thereto. Specifically, the FLAG is an epitope tag composed of an eight amino acid sequence (DYKDDDDK), and HA is an abbreviation for Hemagglutinin epitope tag, and is an epitope tag composed of a nine amino acid sequence (YPYDVPDYA) derived from the hemagglutinin protein of influenza A virus.

The term CRY2 (cryptochrome 2) protein used in this specification refers to a protein that mainly detects blue light and regulates circadian rhythm, and is found in plants, fungi, and animals. In one specific example, the CRY2 (cryptochrome 2) protein may be extracted from Arabidopsis thaliana , but is not limited thereto.

The term fragment, as used herein, is intended to mean a polypeptide consisting only of a portion of the complete polypeptide sequence and structure, wherein a C-terminal deletion or an N-terminal deletion of the variant may be present. For example, the CRY2 fragment means a functional fragment of a CRY2 polypeptide having CRY2 activity.

The term variant, as used herein, may mean a change in the basic structure or sequence of a gene or protein.

In one specific example, the CRY2 variant may refer to a CRY2(E281A) mutation. The CRY2(E281A) mutation reduces the basal activity of OptoSTIM1 in the dark and increases sensitivity to blue light.

In one specific example, the CRY2 variant may be a combination of CRY2(E281A) and A9 conjugate (CRY2(E281A, A9)). The CRY2(E281A, A9) has significantly increased light-dependent oligomerization and sensitivity to blue light.

In one specific example, the CRY2 (E281A, A9) protein may be composed of an amino acid sequence of SEQ ID NO: 31.

The term STIMI1 (Stromal interaction molecule 1) protein in this specification is a transmembrane protein present in the endoplasmic reticulum (ER) membrane of cells, and is a key regulator of the SOCE (store-operated Ca2+ entry) process. It plays a role in sensing the Ca2 ⁺ level in the lumen of the ER, and when ER Ca2 ⁺ is depleted, STIM1 oligomerizes and undergoes a conformational change in the C-terminal domain. It then translocates to the plasma membrane, binds to and activates the CRAC (Ca2+-release-activated Ca2+) channel, thereby increasing the intracellular Ca2 ⁺ level.

In one specific example, the STIMI1 protein may be a cytosolic STIM1 fragment. The cytosolic STIM1 fragment may refer to the cytosolic carboxyl terminus (STIM1ct) of the STIM1 protein, and specifically, may refer to a portion of the amino acid sequence 238-685 of the STIM1 protein. For example, the STIM1ct may consist of an amino acid sequence of SEQ ID NO: 30.

In one specific example, the cytoplasmic STIM1 fragment may include a STIM1 CRAC activation domain (CRAC-activation domain, CAD; 342-448), which plays a role in binding to and opening a Ca2+ channel. For example, the STIM1 CRAC activation domain may be a portion of 342-448 of the STIM1 protein and may consist of an amino acid sequence of SEQ ID NO: 34.

For example, the cytoplasmic STIM1 fragment may be, but is not limited to, a portion of the amino acid sequence 238-685 (SEQ ID NO: 30), 238-448 (SEQ ID NO: 35), 248-448 (SEQ ID NO: 36), 336-448 (SEQ ID NO: 37), 318-463 (SEQ ID NO: 38), 318-450 (SEQ ID NO: 39), or 342-483 (SEQ ID NO: 40) of the STIMI1 protein.

Another aspect provides a polynucleotide encoding the fusion protein.

Another aspect provides a vector comprising the polynucleotide. The vector may be a recombinant expression vector capable of expressing the fusion protein.

In one specific example, the vector may be an adeno-associated virus (AAV) vector.

The term monSTIM1 in this specification may mean a structure comprising a fusion of GFP (Green Fluorescent Protein), cryptochrome 2 (CRY2), and STIM1 protein. Specifically, the STIM1 protein may mean a STIM1 fragment, and the CRY2 may be a mutant (CRY2 (E281A, A9)) combining CRY2 (E281A) and A9.

In one specific example, the CRY2 variant (E281A, A9) may be composed of the amino acid sequence of SEQ ID NO: 31.

The term monSTIM1 variants in this specification means a variant of the structure of monSTIM1.

In one specific example, the monSTIM1 variant may mean that a small tag protein other than GFP is replaced in the monSTIM1.

In one specific example, the tag protein may refer to FLAG or HA. Specifically, the tag protein may be composed of an amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33.

In one specific example, the monSTIM1 variant may mean a STIM1 protein that is truncated from a portion of the STIM1 protein in the monSTIM1. For example, the truncated STIM1 protein may be a cytoplasmic STIM1 fragment, and the cytoplasmic STIM1 fragment may be a portion of the amino acid sequence 238-685 (SEQ ID NO: 30), 238-448 (SEQ ID NO: 35), 248-448 (SEQ ID NO: 36), 336-448 (SEQ ID NO: 37), 318-463 (SEQ ID NO: 38), 318-450 (SEQ ID NO: 39), or 342-483 (SEQ ID NO: 40) of the STIMI1 protein, but is not limited thereto.

In one specific example, the monSTIM1 variant may mean that the monSTIM1 is separated into two structures. For example, the two structures may include a first vector comprising a polynucleotide encoding a STIMI1 protein or a fragment thereof; and a second vector comprising a polynucleotide encoding a CRY2 protein or a variant thereof.

Another aspect provides a Ca2+ modulator comprising a STIMI1 protein to which a CIB1 (cryptochromeinteracting basic-helix-loop-helix 1) protein or a fragment thereof is attached; and a CRY2 protein or a variant thereof.

The term CIB1 (cryptochromeinteracting basic-helix-loop-helix 1) protein in this specification refers to a bHLH (basic-helix-loop-helix) transcription factor found mainly in plants, which interacts with CRY2 (Cryptochrome 2) and is activated by blue light.

In one specific example, the fragment of the CIB1 protein may mean an N-terminal fragment of the CIB1 protein (CIBN).

In one specific example, the fragment of the CIB1 protein may consist of an amino acid sequence of SEQ ID NO: 42.

The term Ca2+ modulator used in this specification refers to a substance that regulates cell function by controlling the concentration, influx, and release of calcium, and mainly regulates calcium channels or calcium pumps to influx or release calcium into cells and regulates cell activity through signal transduction processes.

The definitions of the above “tag protein”, “CRY2 protein”, “STIMI1 protein”, “fragment”, “variant”, etc. are as described above.

Another aspect provides a Ca2+ modulator comprising a STIMI1 protein to which a tagged protein is attached; and a CRY2 protein or a variant thereof to which a nanobody antibody capable of binding to the tagged protein is attached.

The term nanobody antibody as used herein means a type of single-domain antibody (sdAb) derived from a heavy chain antibody of camelids or cartilaginous fish, which has no light chain and contains only the VHH domain of the heavy chain.

In one specific example, the nanobody is meant to be capable of specifically binding to the tag protein. For example, a GFP nanobody can specifically bind to GFP with high affinity (Kd = 0.23 nM), thereby efficiently delivering the homologous interaction of CRY2 required for STIM1 oligomerization through GFP-GFP nanobody (vhhGFP) interaction.

In one specific example, the GFP nanobody (vhhGFP) may be composed of the amino acid sequence of SEQ ID NO: 43.

In one specific example, the nanobody antibody capable of binding to the tag protein can be attached to the N-terminus or C-terminus of the CRY2 protein or a variant thereof.

In one specific embodiment, one or more copies of the nanobody antibody capable of binding to the tag protein can be attached to the N-terminus or the C-terminus of the CRY2 protein or a variant thereof. For example, but not limited to, one, two or three copies of the nanobody antibody capable of binding to the tag protein can be attached to the N-terminus or the C-terminus of the CRY2 protein or a variant thereof. Specifically, one, two or three copies of a GFP nanobody (vhhGFP) can be fused to the N- or C-terminus of the CRY2 protein or a variant thereof.

Another aspect provides an expression system comprising a first vector comprising a polynucleotide encoding a STIMI1 protein or a fragment thereof; and a second vector comprising a polynucleotide encoding a CRY2 protein or a variant thereof.

In one specific embodiment, the first vector may additionally comprise a polynucleotide encoding a CIB1 protein or a fragment thereof; or a tag protein.

In one specific embodiment, the second vector may comprise a polynucleotide encoding a nanobody antibody capable of binding to a tag protein.

The definitions of the above “tag protein”, “CRY2 protein”, “STIMI1 protein”, “CIB1 protein”, “fragment”, “variant”, etc. are as described above.

The terms and methods described for the above inventions are equally applicable to each invention.

According to one aspect of the present invention, a calcium-regulatory fusion protein expressible by adeno-associated virus was constructed by reducing the size of the existing monSTIM1 coding sequence to fit the AAV packaging capacity, thereby establishing an AAV-based system that can be expressed in neurons and glial cells of the mouse brain. The monSTIM1 variant expressed by AAV according to one aspect can minimize problems such as tissue damage, glial scar formation, inflammation, and tissue heating caused by long-term optical fiber insertion, and has the effect of regulating neural activity in a noninvasive manner.

Figure 1 illustrates the optogenetic activation pattern of monSTIM1 mutants or OptoCRAC in one aspect:

FIG. 1a is a schematic diagram showing the mechanism of action of GFP-monSTIM1; FIG. 1b is a structural diagram showing the coding sequence of monSTIM1 and the sizes of components of the AAV cassette; FIG. 1c is a photograph showing a fluorescent image of a cell co-expressing R-GECO1 and one aspect of monSTIM1 variants or OptoCRAC; FIG. 1d is a graph showing the change in normalized intensity of R-GECO1 over time according to the transient activation of one aspect of monSTIM1 variants and OptoCRAC; FIG. 1e is a graph showing the maximum fold change (△F/F0) of R-GECO1 intensity for one aspect of monSTIM1 variants and OptoCRAC upon blue light stimulation; FIG. 1f is a graph quantifying the activation kinetics of one aspect of monSTIM1 variants; FIG. 1g is a graph quantifying the deactivation kinetics of one aspect of monSTIM1 variants; Figure 1h is a graph showing the light sensitivity of each optogenetic module (EGFP-monSTIM1, FLAG-monSTIM1, HA-monSTIM1, OptoCRAC).

Figure 2 shows the structures of six fusion proteins of CRY2-fused STIM1 fragments containing the STIM1 CRAC activation domain (CAD; 342-448) and the correlation between the expression levels of these proteins and basal R-GECO1 intensity:

Figure 2a is a schematic diagram showing the structure of CRY2 fused STIM1 fragments; Figure 2b is a graph showing the expression levels of six CRY2 fused STIM1 fragments (monSTIM1 variants) containing the STIM1 CRAC activation domain (CRAC-activation domain, CAD; 342-448); Figure 2c is a photograph showing the result of fluorescence images of R-GECO1 in HeLa cells co-expressing each CRY2 fused STIM1 fragment (monSTIM1 variant sets 1-6) under blue light illumination; Figure 2d is a graph showing the maximum fold change ((△F/F0) of R-GECO1 intensity upon activation of each CRY2-fused STIM1 fragment (monSTIM1 mutant set 1-6); Figure 2e is a graph showing the fluorescence intensity of R-GECO1 in the absence of blue light, and Figure 2f is a graph showing the correlation between the expression level of CRY2-fused STIM1 fragments and the basal R-GECO1 intensity; Figure 2g is a schematic diagram showing the structure of CRY2-fused STIM1 fragments (set 1-7) whose expression is induced by IRES2; Figure 2h is a photograph showing the fluorescence image of R-GECO1 in HeLa cells co-expressing each IRES2-induced CRY2-fused STIM1 fragment (set 1-7) when illuminated with blue light; Figure 2i is a graph showing the fluorescence image of R-GECO1 upon activation of each IRES2-induced CRY2-fused STIM1 fragment (set 1-7). Graph showing the maximum fold change in R-GECO1 intensity ((△F/F0); Fig. 2j is a graph showing the fluorescence intensity of R-GECO1 in CRY2 fused STIM1 fragments (set 1-7) whose expression is induced by each IRES2 in the absence of blue light.

Figure 3 shows the system that separates the monSTIM1 protein into two components and the correlation between the expression levels of these proteins and the basal R-GECO1 intensity:

Figure 3a is a schematic diagram showing the working mechanism of the two-component system of monSTIM1 protein; Figure 3b is a schematic diagram showing the fusion structure of the system in which monSTIM1 protein is separated into two components; Figure 3c is a photograph showing the fluorescence image of R-GECO1 in HeLa cells co-expressing each protein pair (set 1-5) when illuminated with blue light; Figure 3d is a graph showing the maximum fold change ((△F/F0)) of R-GECO1 intensity upon activation of monSTIM1 mutants; and Figure 3e is a graph showing the fluorescence intensity of R-GECO1 in the absence of blue light.

Figure 4 is a graph showing the relative Ca2+ levels measured by Fura-2 imaging in cells expressing each monSTIM1 mutant (EGFP-monSTIM1; FLAG-monSTIM1; GFP-CRY2-STIM1(318-450); GFP-IRES2-CRY2-STIM1(238-448); GFP-STIM1+vhhGFP-CRY2; CIBN-STIM1+CRY2) (left: Fura-2 ratio measured under dark conditions (emission 340 nm/380 nm); right: Fura-2 ratio measured after blue light irradiation).

Figure 5 shows the Ca2 ⁺ increase induced by activation of one monSTIM1 mutant in cultured hippocampal neurons:

Figure 5a is a schematic diagram illustrating the light illumination protocol; Figure 5b is a photograph (top) showing fluorescence images of R-GECO1 illuminated with blue light in neurons expressing each monSTIM1 variant (EGFP-monSTIM1; FLAG-monSTIM1; GFP-CRY2-STIM1(318-450); GFP-IRES2-CRY2-STIM1(238-448); GFP-STIM1+vhhGFP-CRY2; CIBN-STIM1+CRY2) and a graph (bottom) showing the relative change in R-GECO1 intensity over time upon repetitive light illumination; Figure 5c is a graph showing the maximum fold change ((△F/F0)) in R-GECO1 intensity upon activation of monSTIM1 variants.

Figure 6 shows that activation of one monSTIM1 mutant in cultured astrocytes and microglia increases Ca2 ⁺ :

Figure 6a is a photograph showing R-GECO1 fluorescence images in astrocytes (left) illuminated with blue light and microglia (BV2 cells, right) co-expressing each monSTIM1 mutant (EGFP-monSTIM1; FLAG-monSTIM1; GFP-CRY2-STIM1(318-450); GFP-IRES2-CRY2-STIM1(238-448); GFP-STIM1+vhhGFP-CRY2; CIBN-STIM1+CRY2); Figure 6b is a schematic illustrating the illumination protocol; Figure 6c is a graph showing the relative change in R-GECO1 intensity over time upon illumination; Figure 6d is a graph showing the maximum fold change ((△F/F0) of R-GECO1 intensity upon activation of the monSTIM1 mutant; Figure 6e is a photograph showing a fluorescence image of R-GECO1 in astrocytes when light was shined on the subcellular region indicated by the circle with the white line; Figure 6f is a kymograph of R-GECO1 corresponding to lines a-b and c-d of Figure 6e.

Figure 7 shows the results of application of one aspect of monSTIM1 mutants in neurons:

Figure 7a is a schematic representation of the experiment in which one AAV-compatible monSTIM1 variant was expressed under the control of the CaMKIIα promoter targeting the CA1 region of the hippocampus (top) and mice were irradiated with blue LED light via a custom transcranial light illumination system, followed by retention for immunohistochemistry and analysis (bottom); Figure 7b is a schematic representation indicating the region used for quantification of activated neurons expressing cFos and monSTIM1 variants in the CA1 region (two AAV-compatible monSTIM1 variants were used: AAV-CaMKIIα-FLAG-monSTIM1 and AAV-CaMKIIα-EGFP-STIM1(318-450). Blue, DAPI; green, FLAG; pink, NeuN. Scale bar, 50 μm); Figure 7c is a representative image showing cFos-positive cells expressing each monSTIM1 mutant, with or without non-invasive light delivery and when expressing EGFP (control) (scale bar, 50 μm); Figure 7d is a graph quantifying cFos-positive cells expressing FLAG-monSTIM1, and Figure 7e is a graph quantifying cFos-positive cells expressing GFP-CRY2-STIM1(318-450).

Figure 8 shows the results of applying one type of monSTIM1 mutant to astrocytes:

Figure 8a is a photograph showing FLAG-monSTIM1 expressed under the control of the GfaABC1D promoter targeting the CA1 region of the hippocampus (green, FLAG; red, c-Fos; blue, DAPI. Scale bar, 100 μm); Figure 8b is a photograph showing cFos-positive cells expressing FLAG-monSTIM1 with or without noninvasive light delivery (green, FLAG; blue, DAPI; pink, GFAP; red, c-Fos. Scale bar, 50 μm); Figure 8c is a graph showing the quantification of cFos-positive cells expressing FLAG-monSTIM1 in CA1 astrocytes.

Figure 9 is a photograph showing an image showing SST-positive cells expressing FLAG-monSTIM1 in the SST-Cre mouse line (blue is DAPI, green is FLAG, and red is tdTomato).

The following experimental examples will be described in more detail. However, these experimental examples are intended to exemplify one or more specific examples, and the scope of the present invention is not limited to these experimental examples.

Example 1. Preparation of plasmids

The expression plasmid for R-GECO1 (Addgene plasmid #32444) was obtained from Addgene. To construct expression plasmids of HA-monSTIM1 and FLAG-monSTIM1, the monSTIM1 sequence of GFP-monSTIM1 was amplified by polymerase chain reaction (PCR) using HA-F, FLAG-F, and HA-R primers. The amplified sequence was ligated to GFP-monSTIM1 using NheI and SalI restriction sites after excising EGFP.

The LOV2 (404-546) sequence of OptoCRAC was PCR amplified using LOV2-F and LOV2-R primers and ligated into the EGFP-C1 vector at the BsrGI and HindIII sites to generate the EGFP-LOV2 vector.

The STIM1(336-486) sequence of GFP-monSTIM1 was PCR amplified using STIM1(336-486)-F and STIM1(336-486)-R primers, and then ligated into the EGFP-LOV2 vector at the HindIII and BamHI sites to generate the OptoCRAC expression plasmid.

For the construction of GFP-CRY2-STIM1(238-448), GFP-CRY2-STIM1(248-448), GFP-CRY2-STIM1(336-448), and GFP-CRY2-STIM1(342-448) expression plasmids, sequences encoding STIM1(238-448), STIM1(248-448) were generated, and the STIM1(336-448) and STIM1(342-448) fragments were PCR amplified using STIM1(238-448)-F, STIM1(248-448)-F, STIM1(336-448)-F, STIM1(342-448)-F, and STIM1(238-448)-R primers and ligated into monSTIM1 using BspEI and BamHI restriction enzyme sites. Combined.

To construct GFP-CRY2-STIM1(318-463) and GFP-CRY2-STIM1(318-450) expression plasmids, sequences encoding STIM1(318-463) and STIM1(318-450) fragments were PCR amplified using STIM1(318-463)-F primers. The sequences were amplified using STIM1(318-463)-R and STIM1(318-450)-R primers and ligated into monSTIM1 at the BspEI and BamHI sites.

To generate the IRES2-CRY2-STIM1(238-448) expression plasmid, the IRES2 sequence was PCR amplified using the IRES2-F and IRES2-R primers, excised EGFP, and ligated into GFP-CRY2-STIM1(238-448) at the NheI and BsrGI sites. Then, the sequence encoding EGFP was inserted into the NheI and NotI sites to generate the EGFP-IRES2-CRY2-STIM1(238-448) expression plasmid. The STIM1(238-448) fragment was replaced with other STIM1 mutants at the BspEI and BamHI sites.

The CRY2 (E281A, A9) sequence of monSTIM1 was PCR amplified using CRY2-F and CRY2-R primers, excised EGFP to generate the CRY2-N1 vector, and then ligated into EGFP-N1 at AgeI and BsrGI sites.

The vhhGFP sequence of mCherry-CRY2-vhhGFP was PCR amplified using vhhGFP-F1 and vhhGFP-R1 primers and ligated into CRY2-N1 at NheI and AgeI sites to generate the vhhGFP-CRY2 expression plasmid. The CRY2 (E281A, A9) sequence of CRY2-N1 was digested with AgeI and BsrGI, EGFP was excised, and ligated into EGFP-C1 (Clontech) to generate the CRY2-C1 vector.

The vhhGFP sequence of mCherry-CRY2-vhhGFP was PCR amplified using primers vhhGFP-F2 and vhhGFP-R2 and ligated to CRY2-C1 at the BsrGI and XhoI sites to generate the CRY2-vhhGFP vector.

To construct the viral vector for pAAV-CamKIIa-GFP-monSTIM1, an exchange enzyme site was designed using NheI-BamHI oligomers and inserted into pAAV-CamKIIa-EGFP (addgene #50469). GFP-monSTIM1 was ligated to pAAV-CamKIIa-EGFP at the NheI and BamHI sites after excising EGFP.

The W3SL sequence of pAAV-CW3SL-EGFP (Addgene plasmid #61463) was PCR amplified using W3SL-F and W3SL-R primers, then ligated into pAAV-CamKIIa-GFP-monSTIM1 vector at BamHI and RsrII sites after excising WPRE and hGH poly(A) signals, generating pAAV-CamKIIa-GFP-monSTIM1-W3SL vector.

To generate viral vectors of pAAV-CamKIIa-FLAG-monSTIM1-W3SL and pAAV-CamKIIa-GFP-STIM1(318-450)-W3SL, GFP-monSTIM1 was excised, and FLAG-monSTIM1 or GFP-STIM1(318-450) was inserted into the pAAV-CamKIIa-GFP-monSTIM1-W3SL vector using NheI and BamHI sites.

The GfaABC1D promoter sequence was PCR amplified using primers GfaABC1D-F and GfaABC1D-R, and the CaMKIIα promoter sequence was excised, followed by ligation into pAAV-CaMKIIα-FLAG-monSTIM1 at the MluI and XbaI sites to generate pAAV-GfaABC1D-FLAG-monSTIM1. The sequences of the primers used for plasmid construction are shown in Table 1.

Primer order Sequence number HA-F 5'-GACTGCTAGCGCCACCATGGGATACCCATACGACGTGCCTGACTACGCCCCACCGGTCATGAAGATGGAC-3' 1 HA-R 5'-GCATCCACGAGTGGGTACC-3' 2 FLAG-R 5'-GACTGCTAGCGCCACCATGGGAGACTACAAGGATGACGACGATAAGCCACCGGTCATGAAGATG-3' 3 LOV2-F 5'-GACTTGTACAAGGGCAGCCTGGCCACCACTCTAGAGCG-3' 4 LOV2-R 5'-GACTAAGCTTCAGCTCCTTGGCGGCCTC-3' 5 STIM1(336-486)-F 5'-GACTAAGCTTGAATCTCACAGCTCATGGTATGCTC-3' 6 STIM1(336-486)-R 5'-GACTGGATCCTTAAGACACAATCTCCTCATCCATGTCATC-3' 7 STIM1(238-448)-F 5'-GGAGGCTCCGGACTCAGATC-3' 8 STIM1(238-448)-R 5'-GACTGGATCCCTAGTGGATGCCAGGGTTTGTTG-3' 9 STIM1(248-448)-F 5'-GACTTCCGGATTGGAGGGGTTACACCGAGC-3' 10 STIM1(336-448)-F 5'-GACTTCCGGAGAATCTCACAGCTCATGGTATGC-3' 11 STIM1(342-448)-F 5'-GACTTCCGGATATGCTCCAGAGGCCC-3' 12 STIM1(318-463)-F 5 -GACTTCCGGAGAGGAGGAGTTGG-3' 13 STIM1(318-463)-R 5'-GACTGGATCCCTAACTGCCCATCCA-3' 14 STIM1(318-450)-R 5'-GACTGGATCCCTACAGTGAGTGGATGC-3' 15 IRES2-F 5'-GATCGCTAGCGATCGATCGCGGCCGCATCCGCCCCTCTCCCTCC-3' 16 IRES2-R 5'-GATCTGTACACCATGGTTGTGGCCATATTATCATC-3' 17 CRY2-F 5'-GACTACCGGTCGCCACCATGAAGATGGACAAAAAGACCATCG-3' 18 CRY2-R 5'-GACTTGTACAATTCGTTGTCGAGGTCGGG-3' 19 vhhGFP-F1 5'-GACTGCTAGCGCCACCATGGTCCAACTGGTGGAGTCTGG-3' 20 vhhGFP-R1 5'-GACTACCGGTGGGCTTCCGCCGCTGGAGACGGTGACCTG-3' 21 vhhGFP-F2 5'-GACTTGTACAGCCACCATGGTCCAACTGGTGGAGTCTGG-3' 22 vhhGFP-R2 5'-GACTCTCGAGGGGCTTCCGCCGCTGGAGACGGTGACCTG-3' 23 Agel-Nhel-BamHⅠ-HindⅢ oligomer-F 5'-CCGGGCTAGCGGTTCATCAGGTTCATCAGGATCC-3' 24 Agel-Nhel-BamHⅠ-HindⅢ oligomer-R 5-AGCTGGATCCTGATGAACCTGATGAACCGCTAGC-3' 25 W3SL-F 5'-TGGATGGGCAGTTAGGGATCCCTCGAGATAATCAACCTCTGGATTACAAAATTTG-3' 26 W3SL-R 5'-TCCTGCGGCCGCTCGGTCCGTCCTGCGGCCGCTTTTAAAAAAC-3' 27 GfaABC1D-F 5'-ATCGACGCGTAACATATCCTGGTGTGGAGTAGGGG-3' 28 GfaABC1D-R 5'-ATCGTCTAGAGCGAGCAGCGGA-3' 29

Example 2. Cell culture and infection

HeLa cells (ATCC) and BV2 cells (ATCC) were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, Cat# 11965092, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Cat# 16,000-044) at 37°C in a humidified 5% CO2 atmosphere.

Hippocampal neurons were prepared from embryonic day 15–16 mice, and the collected embryonic hippocampi were dissected in Hank's balanced salt solution (HBSS; Gibco, Cat# 14175-095). The collected hippocampi were dissociated by incubation with 0.05% trypsin for 5 min at 37 °C, filtered through a 0.4-μm filter, and seeded onto coated 24-well polymer-coverslip-bottom plates (ibiTreat; ibidi, Cat# 82426, Gräfelfing, Germany) coated with 50 μg/mL poly-D-lysine (Millipore, Cat# A003-E, MA, USA).

Neurons were cultured in Neurobasal medium (Cat# 21103-049) supplemented with 2% B-27, 2% N-2 supplement, 2 mM GlutaMAX (Gibco, Cat# 35050061), 1000 units/mL penicillin-streptomycin and maintained at 37°C in a humidified 5% CO2 atmosphere. Astrocytes were dissected from P0-P1 C57BL/6 mouse pups by removing attached meninges and dissociated into single cell suspensions by osmotic pressure through a Pasteur pipette. Dissociated cells were plated on 60 mm dishes coated with 50 μg/mL poly-D-lysine. Cells were cultured in high-glucose DMEM (Gibco) containing L-glutamine and supplemented with 10% horse serum, 10% FBS, and 1000 units/mL penicillin-streptomycin and maintained at 37°C in a humidified atmosphere with 5% CO2.

Cells were transfected using Lipofectamine LTX (Invitrogen, Cat# 15338-100, CA, USA) according to the manufacturer's instructions.

Example 3. Confirmation of live-cell imaging

For live cell imaging, a Nikon A1R confocal microscope (Nikon Instruments) mounted on a Nikon Eclipse Ti body and equipped with a CFI Plan Apochromat VC objective (Х60/1.4 numerical aperture (NA)) and digital zoom Nikon imaging software (NIS Element AR 64-bit version 3.21; Laboratory Imaging) was used.

Cells were maintained at 5% CO ₂ and 37°C during imaging with a Chamlide TC system (Live Cell Instruments, Inc., Korea) mounted on the microscope. Ca ²⁺ influx in cells co-expressing R-GECO1 was measured using a red Ca ²⁺ indicator genetically encoded with each monSTIM1 mutant, exciting cells at 30 s intervals using 488 and 561 nm laser sources. Captured images were analyzed using NIS-Elements AR microscope imaging software (NIS-element AR 64-bit version 3.21, Nikon). Time-lapse images of Ca ²⁺ influx were analyzed in defined regions of interest (ROIs) of the cells, and changes in R-GECO1 intensity were quantified.

Example 4. Modification and optimization for monSTIM1 size reduction

Example 4-1. Reduction of monSTIM1 size by replacing GFP with a small tag

To address the issue of exceeding the packaging capacity of AAV of the existing monSTIM1 (Figure 1b), which consists of three components: green fluorescent protein (GFP), CRY2 (E281A, A9) (SEQ ID NO: 31), and cytoplasmic STIM1 fragment (aa 238-685) (SEQ ID NO: 30), GFP of monSTIM1 was replaced with a smaller tagging component (HA (SEQ ID NO: 33) or FLAG tag (SEQ ID NO: 32)) by the method of Examples 1 and 2, ^and each mutant was expressed in HeLa cells together with R-GECO1, a red fluorescent Ca ²⁺ indicator.

To confirm the Ca2 ⁺ influx promoting efficiency of FLAG-monSTIM1 and HA-monSTIM1, live-cell imaging was confirmed and analyzed using the method of Example 3, and the results are shown in Figures 1c to 1h.

Figure 1c is a fluorescence image of cells co-expressing R-GECO1 and a monSTIM1 mutant or OptoCRAC (LOV2-STIM1). Blue light was delivered for 2 min at 30-min intervals, and changes in intracellular Ca2 ⁺ levels were monitored by imaging R-GECO1 (left, pink (magenta) image), while expression of monSTIM1 mutant and OptoCRAC is shown in green images (right, green image).

Figure 1d is a graph showing the change in normalized intensity of R-GECO1 over time upon transient activation of one aspect of monSTIM1 mutants and OptoCRAC; Figure 1e is a graph showing the maximum fold change (△F/F0) in R-GECO1 intensity for one aspect of monSTIM1 mutants and OptoCRAC upon blue light stimulation (EGFP-monSTIM1: n=18; FLAG-monSTIM1: n=23; HA-monSTIM1: n=24; OptoCRAC: n=31; FLAG-monSTIM1(CRY2 ^D387A ): n=38 cells).

As shown in Figures 1d to 1e, upon blue light stimulation, FLAG-monSTIM1 promoted Ca2 ⁺ influx with an efficiency similar to that of the original GFP-monSTIM1, whereas HA-monSTIM1 showed a decreased increase in Ca2 ⁺ compared to the original.

Meanwhile, the activation and deactivation kinetics and sensitivity to blue light of monSTIM1 mutants of different types were measured.

Figure 1f is a graph quantifying the activation kinetics of one aspect of monSTIM1 mutants; Figure 1g is a graph quantifying the deactivation kinetics of one aspect of monSTIM1 mutants (EGFP-monSTIM1: n=30-38; FLAG-monSTIM1: n=42-50; HA-monSTIM1: n=45-47 cells); Figure 1h is a graph showing the light sensitivity of each optogenetic module (EGFP-monSTIM1, FLAG-monSTIM1, HA-monSTIM1, OptoCRAC) (Data are presented as mean ± SEM. (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Student two-tailed t test); ns, not significant (p>0.05)).

As shown in Figures 1f and 1g, HA-tagged monSTIM1 exhibited slower activation and faster deactivation kinetics than GFP or FLAG-tagged monSTIM1, suggesting that the properties of monSTIM1 may be influenced to some extent by the labeling components.

In addition, FLAG-monSTIM1 coupled with the light-insensitive CRY2(D387A) mutant, a negative control construct, did not induce an increase in Ca2+ levels. Both FLAG- and HA-monSTIM1 mutants coupled with CRY2 showed similar sensitivity to blue light (Fig. 1h), indicating that the light sensitivity is mainly determined by the CRY2 photoreceptor. In contrast, the LOV2-based method, OptoCRAC, confirmed that the light sensitivity was reduced and induced a significantly smaller increase in Ca2+ compared to the monSTIM1 mutant (Figs. 1c to 1e and 1h).

These results indicate that replacing GFP with a small tag can significantly reduce the size of monSTIM1 while maintaining the original monSTIM1 function.

Example 4-2. Size reduction of monSTIM1 through STIM1 truncation

To test whether truncation of STIM1 could further reduce the size of monSTIM1 while maintaining the efficiency of calcium influx and basal calcium levels in the dark, a series of fusion proteins were generated using the method of Example 1.

Specifically, among the STIM1 fragments fused to CRY2, six fusion proteins (monSTIM1 mutants) containing the STIM1 CRAC-activation domain (CAD; 342-448), which plays a role in binding to and opening Ca2 ⁺ channels, were designed (Fig. 2a, Table 2).

protein Amino acid sequence Sequence number STIM1(342-448) (CRAC-activation domain, CAD) YAPEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIH 34 STIM1(238-448) KEHMKKMMKDLEGLHRAEQSLHDLQERLHKAQEEHRTVEVEKVHLEKKLRDEINLAKQEAQRLKELREGTENERSRQKYAEEELEQVREALRKAEKEKELESHSSWY APEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIH 35 STIM1(248-448) LEGLHRAEQSLHDLQERLHKAQEEHRTVEVEKVHLEKKLRDEINLAKQEAQRLKELREGTENERSRQKYAEEELEQVREALRKAEKELESHSSWYAPEAL QKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIH 36 STIM1(336-448) ESHSSWYAPEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIH 37 STIM1(318-463) EEELEQVREALRKAEKELESHSSWYAPEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIHSLVAALNIDPSWMGS 38 STIM1(318-450) EEELEQVREALRKAEKELESHSSWYAPEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIHSL 39 STIM1(342-483) YAPEALQKWLQLTHEVEVQYYNIKKQNAEKQLLVAKEGAEKIKKKRNTLFGTFHVAHSSSLDDVDHKILTAKQALSEVTAALRERLHRWQQIEILCGFQIVNNPGIHSLVAALNIDPSWMGSTRPNPAHFIMTDDVDDMDEE 40

Each truncated STIM1 construct is surrounded by different domain regions responsible for CAD autoinhibition. The expression levels of the six CRY2-fused STIM1 fragments (monSTIM1 mutants) were measured, and the results are shown in Fig. 2b.

Figure 2b is a graph showing the expression levels of six CRY2-fused STIM1 fragments (monSTIM1 mutants) containing the STIM1 CRAC-activation domain (CAD; 342-448) (expressed as mean ± SEM (one way ANOVA followed by multiple comparison test); ns, not significant (p>0.05)).

As shown in Fig. 2b, we found that there was no significant difference in the average expression levels of the expressed CRY2 fusion STIM1 protein fragments.

Meanwhile, in order to confirm the Ca2 ⁺ influx promoting efficiency of the above six CRY2 fused STIM1 fragments (monSTIM1 mutant set 1-6), live-cell imaging was confirmed and analyzed using the method of Example 3, and the results are shown in Figs. 2c to 2e.

Figure 2c is a photograph showing the result of fluorescence imaging of R-GECO1 in HeLa cells co-expressing each CRY2-fused STIM1 fragment (monSTIM1 mutant set 1-6) under blue light illumination; Figure 2d is a graph showing the maximum fold change ((△F/F0)) of R-GECO1 intensity upon activation of each CRY2-fused STIM1 fragment (monSTIM1 mutant set 1-6); Figure 2e is a graph showing the fluorescence intensity of R-GECO1 in the absence of blue light, and Figure 2f is a graph showing the correlation between the expression level of CRY2-fused STIM1 fragment and the basal R-GECO1 intensity.

As shown in Figures 2c to 2e, among the six CRY2-fused STIM1 fragment constructs, the STIM1 construct of set 5 (aa 318-450 fused EGFP-CRY2) was confirmed to exhibit similar levels of Ca2 ⁺ influx upon light stimulation and basal R-GECO1 intensity in the absence of light stimulation (dark) to monSTIM1.

Although sets 1 to 4 induced Ca ²⁺ influx, the intensity of R-GECO1 fluorescence was increased in the dark state, indicating an increase in basal Ca ²⁺ levels, which may be due to differences in the level of autoinhibition within the STIM1 domain. Notably, the basal R-GECO1 intensity in each cell was positively correlated with the level of EGFP-CRY2-STIM1 expression, suggesting that a subpopulation of CRY2-STIM1 molecules is activated even in the absence of blue light (Fig. 2f).

Meanwhile, to determine whether the basal strength of R-GECO1 could be reduced by lowering the expression level of the CRY2-STIM1 mutant, protein expression was induced using internal ribosome entry sequence 2 (IRES2) (SEQ ID NO: 44).

Figure 2g is a schematic diagram showing the structures of CRY2 fusion STIM1 fragments (sets 1-7) whose expression is induced by IRES2; Figure 2h is a photograph showing a fluorescence image of R-GECO1 in HeLa cells co-expressing each IRES2-induced CRY2 fusion STIM1 fragment (sets 1-7) when illuminated with blue light; Figure 2i is a graph showing the maximum fold change ((△F/F0) of R-GECO1 intensity upon activation of CRY2 fusion STIM1 fragments (Sets 1-7) whose expression is induced by each IRES2; Figure 2j is a graph showing the fluorescence intensity of R-GECO1 in CRY2 fusion STIM1 fragments (Sets 1-7) whose expression is induced by each IRES2 in the absence of blue light (monSTIM1: n=42; Set 1: n=30; Set 2: n=32; Set 3: n=43; Set 4: n=27; Set 5: n=41; Set 6: n=26; Set 7: n=81 cells. Data are presented as means±SEM (*p<0.05, ***p<0.001, ****p<0.0001; Student two-tailed t test); ns, not significant (p>0.05)).

As shown in Figs. 2g to 2j, the method using internal ribosome entry sequence 2 (IRES2) resulted in a marked decrease in basal R-GECO1 fluorescence, and in particular, the set 1 construct showed a much lower level of basal R-GECO1 fluorescence than monSTIM1. However, set 7 showed a much higher basal R-GECO1 intensity because it contains the constitutively activated domain of STIM1. In particular, unlike Fig. 2d, the set 5 construct expressed by IRES2 did not efficiently induce Ca2 ⁺ influx under light stimulation, and only the set 1 construct expressed by IRES2 induced a Ca2 ⁺ increase at a level similar to monSTIM1.

This suggests that the expression level of CRY2-fused STIM1 protein can affect both the basal Ca2 ⁺ level in the dark and the maximal Ca2 ⁺ level under light illumination.

Example 4-3. Size reduction through splitting of monSTIM1

To reduce the size of the original monSTIM1 coding sequence to fit AAV packaging capacity, we tested two approaches to split the monSTIM1 protein into two components.

Specifically, in the first approach (set 1), STIM1 (238-685) (SEQ ID NO: 30) was conjugated to CIBN (SEQ ID NO: 42), an N-terminal fragment of the cryptochrome-interacting basic-helix-loop-helix 1 (CIB1) protein that binds to oligomerized CRY2 in a light-dependent manner.

In the second approach (sets 2–5), GFP-tagged STIM1 and GFP nanobody (vhhGFP)-conjugated CRY2 were utilized. To determine the optimal composition of the fusion protein and the copy number of vhhGFP (SEQ ID NO: 43), one or two copies of vhhGFP were fused to the N- or C-terminus of CRY2 (E281A, A9) (Fig. 3B). GFP nanobodies are single-domain antibodies derived from the variable domain of heavy-chain antibodies and can specifically bind GFP with high affinity (Kd = 0.23 nM). Therefore, the GFP-vhhGFP interaction efficiently transmits the homotypic interaction of CRY2 required for STIM1 oligomerization.

The system in which the above monSTIM1 protein was separated into two components and the correlation between the expression levels of these proteins and the basal R-GECO1 intensity are shown in Figure 3.

Figure 3a is a schematic diagram showing the working mechanism of the two-component system of monSTIM1 protein; Figure 3b is a schematic diagram showing the fusion structure of the system in which monSTIM1 protein is separated into two components; Figure 3c is a photograph showing the fluorescence image of R-GECO1 in HeLa cells co-expressing each protein pair (set 1-5) when illuminated with blue light; Figure 3d is a graph showing the maximum fold change ((△F/F0) of R-GECO1 intensity upon activation of monSTIM1 mutant; Figure 3e is a graph showing the fluorescence intensity of R-GECO1 in the absence of blue light (monSTIM1: n=52; Set 1: n=27; Set 2: n=24; Set 3: n=14; Set 4: n=21; Set 5: n=51 cells. Data are presented as means±SEM (*p<0.05, **p<0.01, ****p<0.0001; Student two-tailed t test); ns, not significant (p>0.05)).

As shown in Figure 3, when cells co-expressing each protein pair with R-GECO1 were stimulated with blue light, the fluorescence of R-GECO1 increased the most in Set 1 (Figures 3c and 3d).

In addition, proteins fused to the N-terminus of CRY2 with vhhGFP (sets 4 and 5) effectively induced Ca ²⁺ influx, indicating that the N-terminal fusion of vhhGFP maintained the GFP-binding properties and the light-dependent oligomerization of CRY2. In contrast, the C-terminal fusion proteins (sets 2 and 3) showed significantly lower levels of Ca ²⁺ elevation, suggesting that the C-terminal fusion interfered with GFP binding and the light-dependent oligomerization of CRY2. Notably, all sets did not show an increase in basal R-GECO1 fluorescence in the dark, indicating that the STIM1 fragment (aa 238-685) sufficiently inhibited CAD activity in the absence of light stimulation (Fig. 3e).

Based on the results of Examples 4-1 to 4-3 above, five monSTIM1 variants that are potentially compatible with AAV were selected. The five monSTIM1 variants that are potentially compatible with AAV are as follows:

FLAG-CRY2-STIM1(238-685) (FLAG-monSTIM1) (SEQ ID NO: 45); GFP-CRY2-STIM1(318-450)(SEQ ID NO: 46); GFP-IRES2-CRY2-STIM1(238-448); CIBN-STIM1(238-685)(SEQ ID NO: 47) + CRY2 (SEQ ID NO: 31) and GFP-STIM1(238-685)(SEQ ID NO: 48) + vhhGFP-CRY2 (SEQ ID NO: 49).

Information on the size of the coding sequence for each of the above monSTIM1 variants is shown in Table 3.

Size (bp) Expression vector of the stray Single configuration GFP-CRY2-STIM1(238-685) 3555 HA-CRY2-STIM1(238-685) 2817 FLAG-CRY2-STIM1(238-685) 2868 GFP- CRY2-STIM1(318-450) 2610 GFP-IRES2-CRY2-STIM1(238-448) 3432 Two configurations VhhGFP-CRY2 1920 GFP-STIM1(238-685) 2061 CIBN-STIM1(238-685) 1854 CRY2 1494 AAV expression vector Insert size (~4.8 kb) AAV-CaMKIIα-FLAG-STIM1(238-685) 4147 AAV-CaMKIIα-EGFP-CRY2-STIM1(318-450) 3864 AAV-GfaABC1D-FLAG-STIM1(238-685) 4467

Experimental Example 1. Measurement of relative Ca2+ levels using Fura-2 imaging

R-GECO1 signals were utilized as surrogate measurements of relative Ca2+ levels. To more precisely characterize Ca2 ⁺ levels under both dark and light conditions, Fura-2 imaging was performed on cells expressing each monSTIM1 mutant (EGFP-monSTIM1; FLAG-monSTIM1; GFP-CRY2-STIM1(318-450); GFP-IRES2-CRY2-STIM1(238-448); GFP-STIM1+vhhGFP-CRY2; CIBN-STIM1+CRY2).

Specifically, HeLa cells expressing each of the above monSTIM1 mutants (EGFP-monSTIM1; FLAG-monSTIM1; GFP-CRY2-STIM1(318-450); GFP-IRES2-CRY2-STIM1(238-448); GFP-STIM1+vhhGFP-CRY2; CIBN-STIM1+CRY2) were loaded with Fura-2 AM (Invitrogen, Cat# F6774) diluted to 2 μM in DMEM and incubated at room temperature for 30 min, after which the cells were washed three times for 5 min each. Fura-2 imaging was performed using a LAMBDA DG-4 lamp (Sutter Instrument Company) and a × 40/0.75 NA CFI Plan Fluor objective with intermittent excitation using 340 and 380 nm filtered fluorescent lamps. The emitted light was collected with a Nikon DS-Qi1 black-and-white digital camera after passing through a 510 nm emission filter, and the results are shown in Fig. 4.

Figure 4 is a graph showing the relative Ca2+ levels measured by Fura-2 imaging in cells expressing each monSTIM1 mutant (EGFP-monSTIM1; FLAG-monSTIM1; GFP-CRY2-STIM1(318-450); GFP-IRES2-CRY2-STIM1(238-448); GFP-STIM1+vhhGFP-CRY2; CIBN-STIM1+CRY2) (Left: Fura-2 ratio measured under dark condition (emission 340 nm/380 nm); Right: Fura-2 ratio measured after blue light irradiation (EGFP-monSTIM1: n=120; FLAG-monSTIM1: n=150; EGFP-CRY2-STIM1(318-450): n=201; EGFP-IRES2-CRY2-STIM1(238-448): n=146; EGFP-STIM1+vhhGFP-CRY2: n=152; CIBN-STIM1+CRY2: n=144 cells. Data are presented as mean ± SEM (****p<0.0001; Student two-tailed t test)).

As shown in Fig. 4, we confirmed that all monSTIM1 mutants significantly increased Ca2 ⁺ concentration in response to light stimulation. In the absence of blue light, all mutants except GFP-CRY2-STIM1(318-450) showed basal Ca2 ⁺ levels similar to those observed in untransfected cells, which is consistent with the trend observed using R-GECO1. Meanwhile, the slightly elevated Ca2 ⁺ level observed in GFP-CRY2-STIM1(318-450) implies that the N-terminal autoinhibitory domain of STIM1 is insufficient to completely inhibit the activity of CAD.

Experimental Example 2. Confirmation of light-induced Ca2+ influx in neurons, astrocytes, and microglia

To evaluate the efficacy of monSTIM1 mutants in modulating Ca2 ⁺ signals in the brain, the proteins (EGFP-monSTIM1; FLAG-monSTIM1; EGFP-CRY2-STIM1(318-450); EGFP-IRES2-CRY2-STIM1(238-448); EGFP-STIM1+vhhGFP-CRY2; CIBN-STIM1+CRY2) were introduced into cultured neurons, astrocytes, and microglia using the method of Example 2. Neurons expressing the above monSTIM1 mutant were treated with ACSF for 30 minutes, and then stimulated with blue light eight times for 0.5 seconds at 2-minute intervals. Astrocytes and microglia were given repetitive light stimulation (five times for 2 minutes at 30-second intervals), and changes in Ca2 ⁺ levels were confirmed using the method of Example 3.

2.1 Confirmation of light-induced Ca2+ influx in neurons

Results related to changes in Ca2 ⁺ levels through activation of monSTIM1 mutants in cultured hippocampal neurons are shown in Figure 5.

Figure 5a is a schematic diagram illustrating the light illumination protocol; Figure 5b is a photograph (top) showing fluorescence images of R-GECO1 illuminated with blue light in neurons expressing each monSTIM1 mutant and a graph (bottom) showing relative changes in R-GECO1 intensity over time upon repeated light illumination; Figure 3c is a graph showing the maximum fold change ((△F/F0) of R-GECO1 intensity upon activation of monSTIM1 mutants (EGFP-monSTIM1: n=6; FLAG-monSTIM1: n=5; EGFP-CRY2-STIM1(318-450): n=7; EGFP-IRES2-CRY2-STIM1(238-448): n=7; EGFP-STIM1+vhhGFP-CRY2: n=5; CIBN-STIM1+CRY2: n=6 cells. Data are expressed as mean ± SEM (one way ANOVA followed by multiple comparison test); ns, not significant (p>0.05)).

As shown in Figures 5a to 5c, we observed a rapid and sustained increase in intracellular Ca2 ⁺ levels following repeated light exposure (five times at 2-minute intervals) in neurons expressing monSTIM1 mutants (Figure 5b). Meanwhile, there was no significant difference in the level of Ca2 ⁺ increase in monSTIM1 mutants (Figure 5c).

These results suggest that monSTIM1 mutants can effectively regulate intracellular Ca2 ⁺ levels in neurons.

2.2 Confirmation of light-induced Ca2+ influx in astrocytes and microglia

The results of changes in Ca2 ⁺ levels through activation of monSTIM1 mutants according to daily pattern in cultured astrocytes and microglia are shown in Figure 6.

Figure 6 confirms that activation of monSTIM1 mutants increases Ca2+ in cultured astrocytes and microglia: Figure 6a is a photograph showing R-GECO1 fluorescence images in astrocytes (left) illuminated with blue light and microglia (BV2 cells, right) co-expressing each monSTIM1 mutant; Figure 6b is a schematic illustrating the illumination protocol; Figure 6c is a graph showing the relative change in R-GECO1 intensity over time upon illumination; Figure 6d is a graph showing the maximum fold change ((△F/F0)) of R-GECO1 intensity upon activation of monSTIM1 mutants.

As shown in Figures 6a to 6d, although there were differences in the maximum fold changes among the mutants, all mutants significantly increased Ca ²⁺ levels in astrocytes and microglia upon light stimulation. Notably, the magnitude of the increase in Ca ²⁺ signals induced in glial cells by the monSTIM1 mutants exceeded that observed in neurons, which may be due to differences in the expression levels or densities of endogenous Ca ²⁺ channels in different cell types.

Meanwhile, to confirm the local regulation of Ca2 ⁺ levels by monSTIM1, FLAG-monSTIM1 was expressed in astrocytes and light was irradiated to specific subcellular regions for 1 s, and the results are shown in Fig. 6e and Fig. 6f.

Figure 6e is a photograph showing a fluorescence image of R-GECO1 in astrocytes when light is shone on the subcellular region indicated by a circle with a white line, and Figure 6f is a kymograph of R-GECO1 corresponding to lines a-b and c-d of Figure 6e.

As shown in Figures 6e and 6f, the local stimulation induced a reversible, local increase in Ca2 ⁺ levels within the light-irradiated region, while no detectable increase in Ca2 ⁺ signals was observed in the contralateral, non-irradiated subcellular region.

These results imply that monSTIM1 mutants can effectively regulate intracellular Ca2 ⁺ levels in neurons and glial cells in a temporal and spatial manner.

Experimental Example 3. In vivo application of monSTIM1 mutants in the mouse brain

3.1. Application of monSTIM1 mutants in neurons

We investigated whether monSTIM1 mutants can modulate Ca2 ⁺ signals in the mouse brain in vivo .

The mice used in the experiment were male C57BL/6 mice aged 12 to 15 weeks. Male C57BL/6 mice aged 12 to 15 weeks were group-housed on a 12-hour light/dark cycle and had free access to food and water. The experimental protocol was approved by the Institutional Animal Care and Use Committee of IBS (Daejeon, Republic of Korea). Mice were randomly assigned to experimental groups.

Surgical procedures for mice were performed according to the IBS IACUC guidelines. Mice were anaesthetized with 5% isoflurane and maintained with 1–2% isoflurane during stereotaxic surgery. After fixation of the skull in a stereotaxic device (RWD), the skin was shaved, the scalp was sterilized with povidone iodine, and a small craniotomy was performed. The following coordinates were used for microinjections into CA1: AP, 2.0 mm; ML, ± 1.5 mm; DV, -1.4 mm. All microinjections were performed using a glass capillary Picospritzer (Parker), and after each injection, the capillaries were held in place for an additional 10 min to allow for diffusion and then slowly withdrawn. In vivo photostimulation experiments were performed 3 weeks after injection using 473 nm light delivered through a solid-state LED excitation system (Live Cell Instrument), with an illumination time of 30 min.

AAV vectors were generated using the method of Example 1 using two single-component mutants (FLAG-monSTIM1 and GFP-CRY2-STIM1(318-450)). To increase the space available for packaging the coding sequence, WPRE and the hGH polyadenylation signal in the AAV cassette were replaced with a smaller component, W3SL, to enhance gene expression. In addition, monSTIM1 mutants were selectively expressed in excitatory neurons using the small CaMKIIα promoter. Three weeks after AAV injection into the mouse hippocampal CA1 region, immunohistochemical staining was performed to confirm this.

Immunohistochemical staining was performed as follows. Mice were anesthetized with a mixture of alfaxan (40 mg/kg) and xylazine (10 mg/kg) for 90 min under dim or bright light conditions, and perfused transcardially first with phosphate-buffered saline (PBS) and then with PBS containing 4% paraformaldehyde (PFA). The brains were removed, fixed in 4% PFA overnight at 4°C, and then sectioned at 30 μm thickness using a vibratome (Leica). For immunostaining, free-floating sections were washed with PBS, incubated in blocking solution (PBS containing 5% normal goat serum and 0.3% Triton X-100) for 1 h, and then primary antibodies were added to the blocking solution and incubated overnight at 4°C. Sections were then washed with PBS and incubated with secondary antibodies in PBS for 2 h at room temperature. Finally, brain tissue sections were washed with PBS and slide-mounted with Vectashield antifade mounting medium containing DAPI (4',6-diamidino-2-phenylindole) (Vector Laboratories, Cat# H-1200, CA, USA). For CA1 imaging, fluorescence images were acquired using a Leica Stellaris 8 confocal microscope equipped with ×20, ×40, and ×60 objectives. For all brain regions, fluorescence images were acquired using a Zeiss Axio scan Z1 equipped with ×10 objective, and images were analyzed using ImageJ (NIH). For each mouse sample, at least three coronal brain sections were analyzed to quantify cFos+ and FLAG+ cells.

Figure 7 shows the results of application of one aspect of monSTIM1 mutants in neurons:

Figure 7a is a schematic representation of the experiment in which AAV-compatible monSTIM1 variants were expressed under the control of the CaMKIIα promoter targeting the CA1 region of the hippocampus (top) and mice were irradiated with blue LED light via a custom transcranial light illumination system, followed by retention for immunohistochemistry and analysis (bottom); Figure 7b is a schematic representation indicating the region used for quantification of activated neurons expressing cFos and monSTIM1 variants in the CA1 region (two AAV-compatible monSTIM1 variants were used: AAV-CaMKIIα-FLAG-monSTIM1 and AAV-CaMKIIα-EGFP-STIM1(318-450). Blue, DAPI; green, FLAG; pink, NeuN. Scale bar, 50 μm); Figure 7c is a representative image showing cFos-positive cells expressing each monSTIM1 mutant, with or without non-invasive light delivery and when expressing EGFP (control) (scale bar, 50 μm); Figure 7d is a graph quantifying cFos-positive cells expressing FLAG-monSTIM1, and Figure 7e is a graph quantifying cFos-positive cells expressing GFP-CRY2-STIM1(318-450).

As shown in Figures 7a and 7b, 3 weeks after AVV injection into the hippocampal CA1 region, the monSTIM1 mutant showed broad and selective expression in neurons (NeuN-positive cells).

Additionally, as shown in Figures 7c to 7e, activation of the monSTIM1 mutant by blue light led to significant induction of cFos, ^{a Ca 2+ -responsive} immediate-early gene, and this response was observed in approximately 10% of STIM1-positive neurons.

In contrast, no significant increase in cFos expression was observed in mice under dark conditions and in mice expressing only GFP under light stimulation conditions, suggesting that cFos expression was induced by the activity of the monSTIM1 mutant and not by the light stimulus itself.

These results suggest that gene expression was successfully induced by noninvasive light stimulation, and that the two mutants (FLAG-monSTIM1 and GFP-CRY2-STIM1(318-450)) still possess ultrahigh light sensitivity similar to that of native monSTIM1.

3.2. Application of monSTIM1 mutants in astrocytes

To evaluate the ability of monSTIM1 to regulate Ca2+ signals in astrocytes, we examined whether FLAG-monSTIM1 expression driven by the GfaABC1D promoter was expressed in CA1 astrocytes and whether selective expression occurred in GFAP-positive cells, and the results are shown in Fig. 8.

Figure 8 shows the results of applying one aspect of monSTIM1 mutants to astrocytes: Figure 8a is a photograph showing FLAG-monSTIM1 expressed under the control of the GfaABC1D promoter targeting the hippocampal CA1 region (green, FLAG; red, c-Fos; blue, DAPI. Scale bar, 100 μm); Figure 8b is a photograph showing cFos-positive cells expressing FLAG-monSTIM1 with or without noninvasive light delivery (green, FLAG; blue, DAPI; pink, GFAP; red, c-Fos. Scale bar, 50 μm); Figure 8c is a graph quantifying cFos-positive cells expressing FLAG-monSTIM1 in CA1 astrocytes (FLAG-monSTIM1 (-Light): n=5; FLAG-monSTIM1 (+Light): n=6 mice. Data are presented as mean ± SEM (*p<0.05, **p<0.01; Unpaired student's t test); ns, not significant (p>0.05)).

As shown in Fig. 8a and Fig. 8b, we confirmed that FLAG-monSTIM1, driven by the GfaABC1D promoter, was expressed in CA1 astrocytes and selectively expressed in GFAP-positive cells. In addition, when light stimulated, cFos expression was strongly increased in about 50% of monSTIM1-positive astrocytes, whereas this response was not observed in monSTIM1-negative astrocytes (Fig. 8b and Fig. 8c).

These results imply that noninvasive light stimulation of the investigated monSTIM1 mutant effectively and selectively activates Ca2 ⁺ signaling in neurons and glial cells.

Experimental Example 4. Cre-dependent FLAG-monSTIM1 expression control in the SST-Cre mouse strain

To determine whether Cre-dependent FLAG-monSTIM1 expression control exists in the SST-Cre mouse strain, The virus (AAV-nEF1α-DIO-FLAG-CRY2(EA9)-STIM1(238-685)) was injected into the right anterior cingulate cortex (ACC) and CA1 region of the hippocampus of the brain of mice (SST-Cre; Ai-14), which were crossbred with Ai14 mice (Rosa26 locusCAG(promoter)-loxp-Stop-loxp-tdTomato) in which Cre recombinase is expressed in somatostatin (SST) interneurons, and immunohistochemical staining was performed using the method of Experimental Example 3.

Figure 9 is a photograph showing the results of applying Cre-dependent FLAG-monSTIM1 expression control in the SST-Cre mouse strain, showing images of SST-positive cells expressing FLAG-monSTIM1 (blue is DAPI, green is FLAG, and red is tdTomato).

As shown in Fig. 9, it was confirmed that green fluorescent protein (FLAG) was specifically expressed in SST interneurons identified by red fluorescent protein (tdTomato). This means that FLAG-monSTIM1 is selectively expressed in SST interneurons, implying that FLAG-monSTIM1 is a platform that can be specifically expressed in cells expressing Cre recombinase as well as excitatory neurons and astrocytes.

Claims (11)

A fusion protein comprising a Tag protein; CRY2 (cryptochrome 2) protein or a mutant thereof; and STIMI1 (Stromal interaction molecule 1) protein. A fusion protein according to claim 1, wherein the tag protein is a FLAG protein or an HA protein. A fusion protein according to claim 1, wherein the STIMI1 protein is a cytosolic STIM1 fragment. A polynucleotide encoding a fusion protein according to any one of claims 1 to 3. A vector comprising the polynucleotide of claim 4. A vector according to claim 5, wherein the vector is an adeno-associated virus (AAV) vector. A Ca2 ⁺ modulator comprising STIMI1 (Stromal interaction molecule 1) protein to which CIB1 (cryptochromeinteracting basic-helix-loop-helix 1) protein or a fragment thereof is attached; and CRY2 (cryptochrome 2) protein or a variant thereof. A Ca2 ⁺ modulator comprising a STIMI1 protein to which a tag protein is attached; and a CRY2 protein or a variant thereof to which a nanobody antibody capable of binding to the tag protein is attached. A first vector comprising a polynucleotide encoding a STIMI1 protein or a fragment thereof; and An expression system comprising a second vector comprising a polynucleotide encoding a CRY2 protein or a variant thereof. An expression system according to claim 9, wherein the first vector additionally comprises a polynucleotide encoding a CIB1 protein or a fragment thereof; or a tag protein. An expression system according to claim 9, wherein the second vector comprises a polynucleotide encoding a nanobody antibody having binding ability to a tag protein.

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