System for directly inducing intracellular huntingtin protein aggregation and use thereof

Abstract

The present inventors confirmed that HTT aggregation can be reproducibly, synchronously, and quantitatively induced through an optogenetic method using a huntingtin protein fused with Cry2PHR, and that aggregation toxicity and neuronal vulnerability appear differently depending on polyglutamine length. In addition, the association between HTT aggregation levels and neuronal viability can be quantitatively evaluated using the system, and it was confirmed that an aggregation inhibitor exhibits a neuronal protective effect. Accordingly, the present invention can be advantageously used for quantitatively analyzing Huntington's disease-related HTT aggregation and neuronal vulnerability, and for screening candidate substances for preventing or treating Huntington's disease.

Description

Intracellular Huntington Protein Aggregation Direct-Induction System and Its Uses

[0001] One example of the present invention relates to a method for preparing an in vitro Huntington's disease model, an in vitro Huntington's disease model prepared by the said method, and a screening method for a drug for the prevention or treatment of Huntington's disease.

[0002]

[0003] Huntington's disease (HD) is caused by the extension of CAG repeats in the huntingtin (HTT) gene, resulting in the formation of an extended polyglutamine (polyQ) region exceeding approximately 35 repeats in the HTT protein, which leads to toxic gain-of-function that induces progressive neurodegeneration. Since CAG repeat length is known to be a major determinant of disease development and influences the age of onset, it suggests the need to develop a model that can controllably and quantitatively reproduce the early pathogenesis.

[0004] Neuropathologically, the corticostriatal circuitry is primarily affected in Huntington's disease, and medium spiny neurons (MSNs) in the striatum are reported to be selectively vulnerable in the early stages. MSNs are GABAergic projection neurons that constitute approximately 90–95% of striatal neurons and can generally be classified into direct pathway groups of D1 receptor expression and indirect pathway groups of D2 receptor expression. In postmortem human studies, the accumulation of HTT in intranuclear inclusions and dystrophic neurites in brain regions affected by Huntington's disease lesions has been observed, indicating that protein aggregation is a major pathological feature of Huntington's disease. However, the mechanistic relationship between aggregate formation and MSN dysfunction or loss has not yet been fully elucidated, as the aggregation phenotype in human cell models is slow, heterogeneous, and difficult to standardize. N-terminal fragments of HTT, particularly those containing exon 1, are known to have a high tendency to aggregate and have been reported as species involved in pathogenesis, so they can be utilized in aggregation-based modeling.

[0005] Huntington's disease modeling using human pluripotent stem cells (hPSCs) has been actively developed, including patient-derived induced pluripotent stem cell (iPSC) models and methods for induced differentiation into the striatal lineage. However, in the context of human neurons, particularly in MSNs, reproducibly and quantitatively inducing aggregation of mutant HTT (mtHTT) species without long-term culture, without artifacts caused by overexpression, and without variability that undermines the reliability of quantitative evaluation remains a challenging task. As a complementary approach to this, an approach that induces aggregation in an externally controllable manner can be proposed. This enables synchronization of aggregation induction and improves the signal-to-noise ratio in imaging-based quantitative analysis, thereby enabling reliable quantitative analysis.

[0006] Optogenetics provides a modular framework for spatiotemporal control of protein behavior using light-responsive domains. The cryptochrome-derived Cry2 photolyase homology region (Cry2PHR) undergoes blue light-dependent oligomerization and has been utilized to induce the controllable clustering of fused proteins.

[0007]

[0008] As a result of the inventors' diligent research,

[0009] A method for preparing an in vitro Huntington's disease model, an in vitro Huntington's disease model prepared by the said method, and a screening method for drugs for the prevention or treatment of Huntington's disease were designed.

[0010]

[0011] Accordingly, the present invention

[0012] (a) introducing a nucleic acid encoding a fusion protein in which Cry2PHR (cryptochrome 2 photolyase homology region) and huntingtin (HTT) protein are fused into cells in vitro; and

[0013] The purpose is to provide a method for preparing an in vitro Huntington disease model, comprising the step of (b) irradiating the cells with blue light to induce aggregation of huntingtin protein within the cells.

[0014]

[0015] In addition, the present invention

[0016] The purpose is to provide an in vitro Huntington's disease model prepared by the above-described manufacturing method.

[0017]

[0018] In addition, the present invention

[0019] (a) a step of treating an in vitro Huntington's disease model prepared by the method according to claim 1 with a candidate drug for the prevention or treatment of Huntington's disease;

[0020] (b) a step of calculating the H-CAS value according to the following [Mathematical Formula 1]; and

[0021] (c) a step of determining the candidate drug as a drug for the prevention or treatment of Huntington's disease if the calculated H-CAS value is reduced compared to the drug-untreated group; the present invention aims to provide a screening method for a drug for the prevention or treatment of Huntington's disease, comprising: (c) a step of determining the candidate drug as a drug for the prevention or treatment of Huntington's disease if the calculated H-CAS value is reduced compared to the drug-untreated group.

[0022] [Mathematical Formula 1]

[0023]

[0024] Here,

[0025] A k aggis the area of ​​individual (k) huntingtin protein aggregates in the drug-treated group, and

[0026] I k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-treated group, and

[0027] C total is the total number of cells in the drug-treated group, and

[0028] BA k agg is the individual (k) huntingtin protein aggregate area of ​​the drug-untreated group, and

[0029] BI k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-untreated group, and

[0030] BC total represents the total number of cells in the drug-untreated group.

[0031]

[0032] In order to achieve the above objective,

[0033] The present invention

[0034] (a) introducing a nucleic acid encoding a fusion protein in which Cry2PHR (cryptochrome 2 photolyase homology region) and huntingtin (HTT) protein are fused into cells in vitro; and

[0035] (b) a step of inducing intracellular huntingtin protein aggregation by irradiating the cells with blue light; the present invention provides a method for preparing an in vitro Huntington disease model, comprising: (b) a step of inducing intracellular huntingtin protein aggregation by irradiating the cells with blue light.

[0036] In one embodiment of the present invention, the fusion protein may be composed of an amino acid sequence represented by SEQ ID NO. 1, but is not limited thereto.

[0037] In another embodiment of the present invention, the huntingtin protein may comprise a polyglutamine (polyQ) composed of 35 or more glutamines, but is not limited thereto.

[0038] In another embodiment of the present invention, the huntingtin protein may comprise an HTT exon 1 fragment represented by SEQ ID NO. 2, but is not limited thereto.

[0039] In another embodiment of the present invention, the nucleic acid may be inserted into an AAVS1 safe harbor locus within the cell, but is not limited thereto.

[0040] In another embodiment of the present invention, the cell may be an SH-SY5Y cell, a human embryonic stem cell (hESC), or a neuron differentiated therefrom, but is not limited thereto.

[0041] In another embodiment of the present invention, the neuron may be a medium spiny neuron (MSN), but is not limited thereto.

[0042] In another embodiment of the present invention, the manufacturing method may further include a step of inducing differentiation into intermediate spinous neurons when the cell is a human embryonic stem cell, but is not limited thereto.

[0043] In another embodiment of the present invention, the intermediate spindle cell may express one or more markers selected from the group consisting of DARPP32, CTIP2, GABA and MAP2, but is not limited thereto.

[0044] In another embodiment of the present invention, the blue light may be light with a wavelength of 450 to 490 nm, but is not limited thereto.

[0045] In another embodiment of the present invention, the aggregation of the huntingtin protein may be dependent on the presence of endogenous huntingtin protein, but is not limited thereto.

[0046]

[0047] In addition, the present invention

[0048] An in vitro Huntington's disease model prepared by the above-described manufacturing method is provided.

[0049] In one embodiment of the present invention, the disease model may be capable of quantitatively evaluating the aggregation of huntingtin protein, but is not limited thereto.

[0050] In another embodiment of the present invention, the huntingtin protein may comprise a polyglutamine composed of 35 or more glutamines, but is not limited thereto.

[0051] In another embodiment of the present invention, the huntingtin protein may comprise an HTT exon 1 fragment represented by SEQ ID NO. 2, but is not limited thereto.

[0052] In another embodiment of the present invention, the aggregation of the huntingtin protein may be dependent on the presence of endogenous huntingtin protein, but is not limited thereto.

[0053]

[0054] In addition, the present invention

[0055] (a) a step of treating an in vitro Huntington's disease model prepared by the method according to claim 1 with a candidate drug for the prevention or treatment of Huntington's disease;

[0056] (b) a step of calculating the H-CAS value according to the following [Mathematical Formula 1]; and

[0057] (c) a step of determining the candidate drug as a drug for the prevention or treatment of Huntington's disease if the calculated H-CAS value is reduced compared to the drug-untreated group; comprising a screening method for a drug for the prevention or treatment of Huntington's disease.

[0058] [Mathematical Formula 1]

[0059]

[0060] Here,

[0061] A k agg is the area of ​​individual (k) huntingtin protein aggregates in the drug-treated group, and

[0062] I k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-treated group, and

[0063] C total is the total number of cells in the drug-treated group, and

[0064] BA k agg is the individual (k) huntingtin protein aggregate area of ​​the drug-untreated group, and

[0065] BI k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-untreated group, and

[0066] BC total represents the total number of cells in the drug-untreated group.

[0067]

[0068] In one embodiment of the present invention, the huntingtin protein may comprise a polyglutamine composed of 35 or more glutamines, but is not limited thereto.

[0069] In another embodiment of the present invention, the huntingtin protein may comprise an HTT exon 1 fragment represented by SEQ ID NO. 2, but is not limited thereto.

[0070] In another embodiment of the present invention, the aggregation of the huntingtin protein may be dependent on the presence of endogenous huntingtin protein, but is not limited thereto.

[0071]

[0072] The inventors confirmed that HTT aggregation can be reproducibly, synchronized, and quantified through an optogenetic method using a Cry2PHR-fused huntingtin protein, and that the toxicity of aggregation and neuronal fragility vary depending on the polyglutamine length. Furthermore, they confirmed that the link between HTT aggregation levels and neuronal viability can be quantitatively evaluated using the above system, and that aggregation inhibitors exhibit neuroprotective effects. Therefore, the present invention can be usefully applied to quantitatively analyze Huntington's disease-related HTT aggregation and neuronal fragility, and to screen candidate substances for the prevention or treatment of Huntington's disease.

[0073]

[0074] The effects of the present invention are not limited to those mentioned above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0075]

[0076] FIGS. 1A to 1E relate to optogenetics-assisted HTT aggregation induction in SH-SY5Y cells using a transient expression system. Specifically, FIG. 1A is a schematic diagram of an optogenetics-assisted HTT aggregation induction system (hereinafter referred to as “OHAIS”) (HTTex1 represents the HTT exon 1 fragment), FIG. 1B is an immunofluorescence image of HTT aggregates in SH-SY5Y cells transiently transfected with the indicated expression vector under dark or blue light conditions (HA immunostaining (red) detects the expressed HTT fusion protein, and DAPI (blue) labels the nucleus), and FIG. 1C is an aggregation-positive (aggregate) SH-SY5Y cells transfected with the indicated expression vector under dark or blue light conditions. + Figure 1D shows the quantification of the proportion of cells (n=3), and is an immunofluorescence image of SH-SY5Y cells transfected with expression vectors encoding HA-Cry2PHR, HTT30Q-HA, or mtHTT81Q-HA under dark or blue light conditions (HA immunostaining (green) detects HA tag proteins, and DAPI (blue) labels the nucleus). Figure 1E shows the quantification of the proportion of agglutination-positive cells among SH-SY5Y cells transfected with the indicated expression vectors under dark or blue light conditions (n=3). Blue light conditions: 38.4 μW / mm² at 470 nm. 2 , continuous investigation for 1 hour. The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ns indicates insignificance, ***p< 0.001, ****p< 0.0001.

[0077] FIGS. 2A through 2F relate to blue light-induced HTT aggregation in the AAVS1KI SH-SY5Y cell line. Specifically, FIGS. 2A and 2D are schematic diagrams of AAVS1 locus-targeted knock-in (KI) using CRISPR-Cas9-mediated homologous recombination (SA stands for splice acceptor), and FIG. 2B shows AAVS1::HTT under dark or blue light conditions. 30Q -HA-Cry2PHR(opto-HTT 30Q ) or AAVS1::mtHTT 81Q -HA-Cry2PHR(opto-mtHTT 81Q This is an immunofluorescence image of HTT aggregation in the KI SH-SY5Y cell line expressing ) (HA immunostaining (green) detects the expressed HTT fusion protein, and DAPI (blue) labels the nucleus), and Fig. 2C is Opto-HTT 30Q and opto-mtHTT 81Q Figure 2E shows the quantification of the proportion of agglutination-positive cells in the KI SH-SY5Y cell line under dark or blue light conditions (n=3), and AAVS1::HA-Cry2PHR(opto-mock), AAVS1::HTT under dark or blue light conditions. 30Q -HA(HTT 30Q ) or AAVS1::mtHTT 81Q -HA(mtHTT 81Q This is an immunofluorescence image of the HA tag protein in the KI SH-SY5Y cell line expressing ) (HA immunostaining (green) detects the HA tag protein, and DAPI (blue) labels the nucleus), and Fig. 2F quantifies the proportion of agglutination-positive cells in the KI SH-SY5Y cell line under dark or blue light conditions (n=3). Blue light condition: 38.4 μW / mm² at 470 nm. 2, continuous investigation for 1 hour. The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ns indicates insignificance, and ****p < 0.0001.

[0078] Figures 3A and 3B illustrate the absence of cytotoxicity associated with blue light-induced HTT aggregation in SH-SY5Y cells. Specifically, Figure 3A evaluates CCK-8-based cell viability under dark or blue light conditions in transiently transduced SH-SY5Y cells (n=4), and Figure 3B evaluates CCK-8-based cell viability under dark or blue light conditions in the KI SH-SY5Y cell line (n=4). Blue light conditions: 38.4 μW / mm² at 470 nm. 2 , 1-hour continuous survey. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ns indicates insignificance.

[0079] FIGS. 4A to 4E relate to the necessity of endogenous HTT for efficient HTT aggregation in SH-SY5Y cells. Specifically, FIG. 4A shows endogenous HTT knockout (HTT) in SH-SY5Y cells. - / - ) Strategy schematic and verification results, and HTT - / - AAVS1::opto-HTT in the background 30Q and AAVS1::opto-mtHTT 81Q This represents the construction of the KI cell line (gRNA targets HTT exon 1 downstream of the start codon (ATG), and F and R indicate the primer binding sites. HTT + / + The cell line exhibits the wild-type (WT) sequence, and HTT - / -The cell line includes a 2 bp deletion (p.[Glu12AspfsTer72];[Glu12AspfsTer72]) that induces frameshift. Red inverted triangles indicate expected Cas9 cleavage sites, and PAM sequences are shown in blue. Figure 4B shows HTT under dark conditions. + / + and HTT - / - This is an HTT immunofluorescence image of the SH-SY5Y cell line (HTT via HTT immunostaining (red)). - / - Loss of endogenous HTT can be confirmed in the cell line, and DAPI (blue) labels the nucleus), Fig. 4C is HTT + / + and HTT - / - Figure 4D shows the HTT Western blot results of the SH-SY5Y cell line (HSP90 is used as a loading control), and KI HTT under blue light conditions. + / + or HTT - / - Immunofluorescence images of HTT agglutination in SH-SY5Y cell lines (HA immunostaining (green) detects expressed HTT fusion proteins, and DAPI labels the nucleus), and Fig. 4E is KI HTT under blue light conditions + / + or HTT - / - This quantifies the proportion of agglutination-positive cells in the SH-SY5Y cell line (n=5). Blue light conditions: 38.4 μW / mm² at 470 nm. 2 , continuous examination for 1 hour. The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ****p< 0.0001.

[0080] FIGS. 5A to 5G relate to the fabrication and validation of Opto-HTTKI and HTTKO hESC cell lines. Specifically, FIG. 5A is a schematic diagram of KI targeting the AAVS1 locus using CRISPR-Cas9-mediated homologous recombination in hESCs (SA stands for splice acceptor). 30Q is AAVS1::HTT 30Q-HA, mtHTT 81Q is AAVS1::mtHTT 81Q -HA, opto-HTT 30Q is AAVS1::HTT 30Q -HA-Cry2PHR, opto-mtHTT 81Q Figure 5B is an immunofluorescence image of the expression of pluripotency markers (OCT3 / 4, NANOG, TRA-1-81) in parental cell line (H9) and KI hESC cell line (DAPI labels the nucleus), Figure 5C is an immunofluorescence image of HTT aggregation in hESCs under dark or blue light conditions (showing HTT (green) and HA (red) signals, with DAPI (blue) labeling the nucleus), and Figure 5D is an endogenous HTT knockout (HTT) in hESCs (H9). - / - ) Schematic diagram of the strategy and validation results (gRNA targets HTT exon 1 downstream of the start codon (ATG), and F and R indicate primer binding sites. Sanger sequencing results, HTT + / + The cell line exhibits the wild-type (WT) sequence, and HTT - / - Cell lines include a 1 bp deletion (p.[Glu12GlyfsTer91];[Glu12GlyfsTer91]) that induces a frameshift. Red inverted triangles indicate expected Cas9 cleavage sites, and PAM sequences are shown in blue. HTT - / - opto-HTT using cell lines 30Q and opto-mtHTT 81Q KI hESC cell lines were constructed), and Figure 5E shows HTT under dark conditions. + / + and HTT - / - This is an HTT immunofluorescence image of the hESC cell line (HTT via HTT immunostaining (red)). - / - Loss of endogenous HTT can be confirmed in cell lines, and DAPI labels the nucleus), Fig. 5F is HTT + / + and HTT - / -These are the HTT Western blot results of the hESC cell line (HSP90 is used as a loading control), and Fig. 5G is HTT under blue light conditions. + / + or HTT - / - opto-HTT in the background 30Q or opto-mtHTT 81Q Immunofluorescence image of HTT aggregation in KI hESC cell line (HA immunostaining (green) detects expressed HTT fusion protein, and DAPI labels the nucleus). Blue light conditions: 38.4 μW / mm² at 470 nm. 2 , continuous irradiation for 1 hour. The scale bars are 100 μm (yellow) and 20 μm (white).

[0081] Figures 6A to 6H show the differentiation of hESCs into MSNs and the progression of differentiation over time. Specifically, Fig. 6A is a schematic diagram of the culture conditions and schedule for differentiating hESCs into MSNs (NIM refers to neural induction medium, NPM to neural proliferation medium, and NDM to neural differentiation medium. XAV represents 4 μM XAV939, SAG to 100 nM SAG, LDN to 100 nM LDN193189, SB to 10 μM SB431544, BF to 20 ng / mL BDNF, GF to 10 ng / mL GDNF, AA to 0.2 mM ascorbic acid, DT to 1 μM DAPT, and TGFβ to 1 ng / mL TGFβ3. Phase contrast images at each time point are shown at the bottom), Fig. 6B is an immunofluorescence image showing the co-expression of NES and FOXG1 or FOXP2 in LGE-like striatal progenitor cells at DIV 15 (DAPI labels the nucleus), and Fig. Figure 6C quantifies the proportion of FOXG1-positive, NES-positive, and FOXP2-positive cells among DAPI-positive cells in DIV 15 (n=3); Figure 6D is an immunofluorescence image showing the co-staining of DARPP32, MAP2, GABA, and CTIP2 in MSNs in DIV 30; Figure 6E quantifies the proportion of DARPP32-positive, CTIP2-positive, and GABA-positive cells among DAPI-positive cells in DIV 30 (n=3); Figure 6F shows the RT-qPCR analysis results for stepwise markers (FOXG1, GAD1, DARPP32, CTIP2), normalized relative to hESC (n=3); and Figure 6G is an immunofluorescence image showing the temporal expression of LGE markers and neuronal markers in DIV 14 (LGE-like progenitor cells) and DIV 21 (early MSNs) (FOXG1 and NES represent progenitor cells, MAP2 and TUJ1 indicate neuronal differentiation.DAPI labels the nucleus), and Figure 6H quantifies the proportion of FOXG1-positive, NES-positive, TUJ1-positive, and MAP2-positive cells among DAPI-positive cells at each time point (n=5). Scale bars are 100 μm (black) and 20 μm (white). Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ns indicates insignificance, and *p< 0.05, ***p< 0.001, and ****p< 0.0001.

[0082] Figures 7A to 7G show similar differentiation efficiencies of parent and KI hESC cell lines into LGE-like progenitor cells and MSNs under cancer conditions. Specifically, Fig. 7A is a schematic diagram of the schedule for differentiating hESCs into LGE-like progenitor cells and MSNs under cancer conditions; Fig. 7B shows the results of RT-qPCR analysis of LGE-related genes (DLX2, DLX5, FOXG1, GSX2, NES, PAX6) for parental cell lines (H9) and KI hESC cell lines differentiated at DIV 15 (n=3); Fig. 7C is an immunofluorescence image showing the expression of FOXG1, NES, and FOXP2 in hESC cell lines differentiated at DIV 15 (DAPI labels the nucleus); Fig. 7D quantifies the ratio of FOXG1-positive, NES-positive, and FOXP2-positive cells among DAPI-positive cells at DIV 15 (n=3); and Fig. 7E shows MSN-related genes (DRD1, DRD2, GAD1, DARPP32, CTIP2, Figure 7F shows the results of RT-qPCR analysis of MAP2 (n=3), and Figure 7G shows the immunofluorescence images of the expression of DARPP32, MAP2, GABA, and CTIP2 in hESC cell lines differentiated from DIV 30. Figure 7G quantifies the proportion of DARPP32-positive, CTIP2-positive, and GABA-positive cells among DAPI-positive cells in DIV 30 (n=3). The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons following two-way ANOVA. ns indicates insignificance.

[0083] Figures 8A to 8H show opto-mtHTT following optogenetic-based HTT aggregation induction. 81QThis relates to the selective loss of hESC-derived MSNs. Specifically, Fig. 8A is a schematic diagram of differentiation and blue light-induced conditions in Opto-HTTKI hESC-derived MSNs, and Fig. 8B is an immunofluorescence image of HTT aggregation in Opto-HTTKI hESC-derived MSNs under blue light conditions at DIV 30 (HA immunostaining (green) detects HTT fusion proteins, MAP2 (red) labels neurons, and DAPI (blue) labels nuclei. Opto-HTT 30Q is AAVS1::HTT 30Q -HA-Cry2PHR, opto-mtHTT 81Q is AAVS1::mtHTT 81Q -HA-Cry2PHR), Figures 8C to 8E are immunofluorescence images showing the expression of DARPP32 / MAP2 (C), CTIP2 / MAP2 (D), and GABA / MAP2 (E) in Opto-HTTKI hESC-derived MSNs under dark or blue light conditions at DIV 30, and Figures 8F to 8H quantify the proportion of DARPP32-positive, CTIP2-positive, and GABA-positive cells among DAPI-positive cells in Opto-HTTKI hESC-derived MSNs according to dark or blue light conditions (n=5). Blue light condition: 26 μW / mm² at 470 nm. 2 , 0.5 second pulse (0.15 Hz), investigated for 8 days. The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ns indicates insignificance, and ****p < 0.0001.

[0084] Figures 9A to 9E relate to the effect of blue light irradiation on the differentiation status of parent and KI hESC-derived MSNs. Specifically, Figure 9A is a schematic diagram of the differentiation process and blue light irradiation conditions in parent cell line (H9) and KI hESC-derived MSNs, Figure 9B is an immunofluorescence image showing the expression of DARPP32 and MAP2 in parent and KI hESC-derived MSNs under dark or blue light conditions in DIV 30, Figure 9C is an immunofluorescence image showing the expression of CTIP2 and MAP2 in DIV 30, Figure 9D is an immunofluorescence image showing the expression of GABA and MAP2 in DIV 30, and Figure 9E is a quantification of the ratio of DARPP32-positive, CTIP2-positive, and GABA-positive cells among DAPI-positive cells under dark or blue light conditions in DIV 30 (n=5). Blue light conditions: 26 μW / mm at 470 nm 2 , 0.5 second pulse (0.15 Hz), investigated for 8 days. The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ns indicates insignificance.

[0085] Figures 10A to 10D relate to the effects of blue light irradiation on the viability and autophagy of parent and KI hESC-derived MSNs. Specifically, Figure 10A evaluates CCK-8-based cell viability in Opto-HTTKI hESC-derived MSNs (DIV 30) under dark or blue light conditions (n=5); Figure 10B evaluates CCK-8-based cell viability in parent cell line (H9) and KI hESC-derived MSNs (DIV 30) under dark or blue light conditions (n=5); and Figure 10C shows cell density (cells / mm²) under dark or blue light conditions under the same initial seeding density conditions. 2) was quantified (n=9), and Fig. 10D shows the Western blot results for LC3A / B (LC3-I and LC3-II) of parental and KI hESC-derived MSNs at DIV 30 (β-actin was used as a loading control). Blue light conditions: 26 μW / mm² at 470 nm. 2 , 0.5 second pulse (0.15 Hz), investigated for 8 days. Error bars represent the standard deviation (SD). Statistical significance was evaluated using Sidak multiple comparisons after two-way ANOVA. ns indicates insignificance, and ****p < 0.0001.

[0086] FIGS. 11A to 11J relate to an HCI analysis method and quantitative indicators for optogenetic-based HTT aggregation. Specifically, FIG. 11A is an Opto-mtHTT 81Q This is a schematic diagram of a high-content imaging (HCI)-based analysis method for quantifying optogenetically induced HTT aggregation in KI SH-SY5Y cells, and Fig. 11B is the definition of the HTT Comprehensive Aggregation Score (H-CAS) (under each condition, H-CAS is the area of ​​aggregates (A) for all detected aggregates k). k agg ) and fluorescence intensity (I k agg Sum the values ​​multiplied by ) and calculate the number of DAPI-positive cells (C total Calculated by normalizing to ) and then further normalizing to the blue light-induced positive control value), Fig. 11C shows opto-mtHTT under dark or blue light conditions. 81Q HCI images showing HTT immunostaining and DAPI signals in KI SH-SY5Y cells, and Fig. 11D is opto-mtHTT under dark or blue light conditions. 81Q The H-CAS values ​​of KI SH-SY5Y cells were quantified (n = 8), and Fig. 11E is Opto-mtHTT 81QThis is a schematic diagram of an HCI-based assay method for evaluating the inhibition of optogenetic HTT aggregation by AEE in KI SH-SY5Y cells, and Fig. 11F shows opto-mtHTT treated with different concentrations of AEE under blue light conditions. 81Q HCI image of HTT immunostaining of KI SH-SY5Y cells (DAPI labels the nucleus), Fig. 11G quantifies H-CAS-based HTT aggregation according to AEE concentration under blue light conditions (n=8), and Fig. 11H shows opto-mtHTT treated with Vehicle (dark or blue light conditions) or 10 mM AEE (blue light conditions). 81Q These are HCI images showing HTT immunostaining and DAPI signals in KI SH-SY5Y cells; Fig. 11I quantifies H-CAS values ​​under Vehicle or 10 mM AEE treatment conditions (n=8), and Fig. 11J evaluates CCK-8-based cell viability according to AEE concentration under dark conditions (n=6). Blue light conditions: 38.4 μW / mm² at 470 nm. 2 , 1-hour continuous examination. The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated using the two-group t-test (D) or Tukey multiple comparison (I, J) after one-way ANOVA. ****p< 0.0001.

[0087] FIGS. 12A to 12C are Opto-mtHTT 81Q This evaluates the AEE effect in MSNs derived from KI hESC. Specifically, Fig. 12A shows the Opto-mtHTT 81Q Figure 12B is a schematic diagram of the AEE pretreatment and cotreatment conditions during blue light induction in KI hESC-derived MSNs, and shows opto-mtHTT treated with vehicle, 1 mM AEE, or 10 mM AEE under dark or blue light conditions. 81QHTT immunostaining of KI hESC-derived MSNs and immunofluorescence images showing the expression of MSN markers (GABA, CTIP2, DARPP32, MAP2) (DAPI labels the nucleus), and Fig. 12C is opto-mtHTT under Vehicle or AEE treatment conditions (dark or blue light conditions). 81Q This quantifies the proportion of DARPP32-positive, CTIP2-positive, and GABA-positive cells among DAPI-positive cells from KI hESC-derived MSNs (n=4). Blue light conditions: 26 μW / mm² at 470 nm. 2 , 0.5 second pulse (0.15 Hz), investigated for 7 days. The scale bar is 20 μm. Error bars represent the standard deviation (SD). Statistical significance was evaluated by Sidak multiple comparisons following two-way ANOVA. *p< 0.05, ***p< 0.001, ****p< 0.0001.

[0088]

[0089] The present invention will be described in detail below.

[0090] The present invention

[0091] (a) introducing a nucleic acid encoding a fusion protein in which Cry2PHR (cryptochrome 2 photolyase homology region) and huntingtin (HTT) protein are fused into cells in vitro; and

[0092] (b) a step of inducing intracellular huntingtin protein aggregation by irradiating the cells with blue light; the present invention provides a method for preparing an in vitro Huntington disease model, comprising: (b) a step of inducing intracellular huntingtin protein aggregation by irradiating the cells with blue light.

[0093] The term "Cry2PHR (cryptochrome 2 photolyase homology region)" as used in this specification refers to a photosensitive protein domain that induces protein-protein interactions or oligomerization in response to blue light, as a photolyase homology region of plant-derived cryptochrome 2. The Cry2PHR is known to have the characteristic of inducing protein clustering or aggregation by causing structural changes in response to light of a specific wavelength.

[0094] The above-mentioned Cry2PHR is fused with the huntingtin protein in the present invention and is used as an optogenetic means to artificially induce the oligomerization and aggregation of the huntingtin protein upon blue light irradiation. Through this, it serves to realize temporally and spatially controllable huntingtin protein aggregation within the cell.

[0095] As used herein, the term "huntingtin protein (HTT)" refers to a protein expressed by the HTT gene, which is associated with Huntington's disease, and which is involved in various physiological functions within a cell. The huntingtin protein plays an important role, particularly in nerve cells, and is characterized by containing polyglutamine (polyQ) repeat sequences.

[0096] In the present invention, the huntingtin protein is provided in a form fused with Cry2PHR and functions as a target protein that is aggregated by blue light. In particular, it is used to implement a Huntington's disease model by utilizing the fact that aggregation characteristics and cytotoxicity vary depending on the polyglutamine repeat length.

[0097] As used herein, the term "blue light" refers to light having a wavelength of about 450 to 490 nm in the visible light region and includes light stimulation used to activate specific photosensitive proteins. It is known that blue light is utilized as a means to induce protein activation or interaction in various optogenetics systems.

[0098] In the present invention, the blue light is used as a stimulus to induce oligomerization and aggregation of huntingtin protein by activating the Cry2PHR domain. Accordingly, huntingtin protein aggregation can be controlled temporally by adjusting the irradiation conditions of the blue light.

[0099] As used herein, the term "Huntington's disease (HD)" refers to a neurodegenerative disease that is an autosomal dominant genetic disorder caused by polyglutamine repeat expansion of the huntingtin protein. The disease is characterized by the selective degeneration of neurons, particularly intermediate spinous neurons, primarily in the striatum region, and the accumulation of huntingtin protein aggregates.

[0100] In order to mimic the pathological characteristics of Huntington's disease, the present invention provides a system that optogenetically induces the aggregation of huntingtin protein, which is utilized to evaluate Huntington's disease-related neuronal fragility and drug effects.

[0101]

[0102] In one embodiment of the present invention, the fusion protein may be composed of an amino acid sequence represented by SEQ ID NO. 1, but is not limited thereto.

[0103]

[0104] In another embodiment of the present invention, the huntingtin protein may comprise a polyglutamine (polyQ) composed of 35 or more glutamines, but is not limited thereto.

[0105] As used herein, the term "polyglutamine (polyQ)" refers to an amino acid sequence in which glutamine residues are repeatedly linked, and includes a domain that can induce structural and functional changes depending on the repeat length within a specific protein. In particular, polyglutamine repeats exceeding a certain length can induce abnormal folding and aggregation of the protein.

[0106] In the present invention, the polyglutamine repeat length acts as a major factor determining the aggregation characteristics and cytotoxicity of the huntingtin protein. Accordingly, huntingtin proteins with different repeat lengths can be used to distinguish between non-toxic and toxic aggregation and to evaluate neuronal fragility.

[0107]

[0108] In another embodiment of the present invention, the huntingtin protein may comprise an HTT exon 1 fragment represented by SEQ ID NO. 2, but is not limited thereto.

[0109]

[0110] In another embodiment of the present invention, the nucleic acid may be inserted into an AAVS1 safe harbor locus within the cell, but is not limited thereto.

[0111] As used herein, the term "AAVS1 safe harbor locus" refers to a specific location on the human genome known to be a safe gene insertion site where the insertion of a foreign gene does not have a significant impact on cell survival or function, and includes regions primarily used as target loci to ensure the stability and reproducibility of gene expression.

[0112]

[0113] In another embodiment of the present invention, the cell may be an SH-SY5Y cell, a human embryonic stem cell (hESC), or a neuron differentiated therefrom, but is not limited thereto.

[0114] As used herein, the term "SH-SY5Y cell" refers to an immortalized cell line derived from human neuroblastoma, which exhibits morphological and physiological characteristics similar to neurons and possesses the ability to differentiate into neurons under experimental conditions.

[0115] As used herein, the term "human embryonic stem cell (hESC)" refers to a stem cell derived from the early developmental stage of a human embryo that possesses the ability to self-replicate and pluripotency, which allows it to differentiate into various cell types.

[0116]

[0117] In another embodiment of the present invention, the neuron may be a medium spiny neuron (MSN), but is not limited thereto.

[0118] As used herein, the term "medium spiny neuron (MSN)" refers to a major type of neuron present in the striatum that exhibits a characteristic morphology with spinous processes and plays an important role in motor control and neural circuit functions. The medium spiny neuron is known to be a cell that is selectively damaged in Huntington's disease.

[0119] In this invention, intermediate spinous neurons are differentiated from human embryonic stem cells and used as a Huntington's disease model, and are utilized as primary target cells to evaluate cell loss and functional changes resulting from huntingtin protein aggregation.

[0120]

[0121] In another embodiment of the present invention, the manufacturing method may further include a step of inducing differentiation into intermediate spinous neurons when the cell is a human embryonic stem cell, but is not limited thereto.

[0122]

[0123] In another embodiment of the present invention, the intermediate spindle cell may express one or more markers selected from the group consisting of DARPP32, CTIP2, GABA and MAP2, but is not limited thereto.

[0124] The term “DARPP32 (Dopamine- and cAMP-regulated phosphoprotein of 32 kDa; DARPP32)” as used in this specification refers to a phosphorylated protein of about 32 kDa that is regulated by dopamine and cAMP signals, is mainly expressed in neurons, and is involved in nerve signal transmission and the regulation of cell function.

[0125] The term “CTIP2 (COU-TF interacting protein 2; CTIP2)” as used in this specification refers to a protein involved in transcriptional regulation, which performs the function of a transcription factor cofactor that regulates specific gene expression and is involved in neural development and cell differentiation processes.

[0126] The term "GABA (gamma-aminobutyric acid; GABA)" as used in this specification refers to an amino acid derivative that acts as a major inhibitory neurotransmitter in the central nervous system and regulates nerve excitation by inhibiting signal transmission between nerve cells.

[0127] The term “MAP2 (Microtubule-associated protein 2; MAP2)” as used in this specification refers to a protein that binds to microtubules of neurons, stabilizes the cytoskeletal structure, and is involved in the formation and maintenance of neurites.

[0128]

[0129] In another embodiment of the present invention, the blue light may be light with a wavelength of 450 to 490 nm, but is not limited thereto.

[0130]

[0131] In another embodiment of the present invention, the aggregation of the huntingtin protein may be dependent on the presence of endogenous huntingtin protein, but is not limited thereto.

[0132]

[0133] In addition, the present invention

[0134] An in vitro Huntington's disease model prepared by the above-described manufacturing method is provided.

[0135] The terms used in this in vitro Huntington's disease model are consistent with the terms described in the method for preparing the in vitro Huntington's disease model.

[0136]

[0137] In one embodiment of the present invention, the disease model may be capable of quantitatively evaluating the aggregation of huntingtin protein, but is not limited thereto.

[0138]

[0139] In another embodiment of the present invention, the huntingtin protein may comprise a polyglutamine composed of 35 or more glutamines, but is not limited thereto.

[0140]

[0141] In another embodiment of the present invention, the huntingtin protein may comprise an HTT exon 1 fragment represented by SEQ ID NO. 2, but is not limited thereto.

[0142]

[0143] In another embodiment of the present invention, the aggregation of the huntingtin protein may be dependent on the presence of endogenous huntingtin protein, but is not limited thereto.

[0144]

[0145] In addition, the present invention

[0146] (a) a step of treating an in vitro Huntington's disease model prepared by the method according to claim 1 with a candidate drug for the prevention or treatment of Huntington's disease;

[0147] (b) a step of calculating the H-CAS value according to the following [Mathematical Formula 1]; and

[0148] (c) a step of determining the candidate drug as a drug for the prevention or treatment of Huntington's disease if the calculated H-CAS value is reduced compared to the drug-untreated group; comprising a screening method for a drug for the prevention or treatment of Huntington's disease.

[0149] [Mathematical Formula 1]

[0150]

[0151] Here,

[0152] Ak agg is the area of ​​individual (k) huntingtin protein aggregates in the drug-treated group, and

[0153] I k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-treated group, and

[0154] C total is the total number of cells in the drug-treated group, and

[0155] BA k agg is the individual (k) huntingtin protein aggregate area of ​​the drug-untreated group, and

[0156] BI k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-untreated group, and

[0157] BC total represents the total number of cells in the drug-untreated group.

[0158] The terms used in the screening method for drugs for the prevention or treatment of Huntington's disease are consistent with the terms described in the method for preparing the in vitro Huntington's disease model and the in vitro Huntington's disease model.

[0159]

[0160] In one embodiment of the present invention, the huntingtin protein may comprise a polyglutamine composed of 35 or more glutamines, but is not limited thereto.

[0161]

[0162] In another embodiment of the present invention, the huntingtin protein may comprise an HTT exon 1 fragment represented by SEQ ID NO. 2, but is not limited thereto.

[0163]

[0164] In another embodiment of the present invention, the aggregation of the huntingtin protein may be dependent on the presence of endogenous huntingtin protein, but is not limited thereto.

[0165]

[0166] To aid in understanding the present invention, it will be explained in more detail below through examples. However, the following examples are merely illustrative for explaining the content of the present invention, and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the technical field to which the invention belongs.

[0167]

[0168] [Example]

[0169] Example 1. Experimental Materials and Methods

[0170] 1-1. Cell Culture

[0171] SH-SY5Y cells (ATCC, CRL-2266) contain AAVS1::HA-Cry2PHR, AAVS1::HTT 30Q -HA, AAVS1::mtHTT 81Q -HA, AAVS1::HTT 30Q -HA-Cry2PHR and AAVS1::mtHTT 81Q -HA-Cry2PHR knock-in (KI) cell lines were cultured in SH medium supplemented with 15% heat-inactivated fetal bovine serum (FBS; Gibco, 12483020) and 2 mM GlutaMAX (Gibco, 35050079) in DMEM / F12 (Gibco, 11330032). Cells were cultured under humid conditions maintained at 37°C and 5% CO2, and the medium was changed every 2 days. For transient transduction, plasmids were introduced into SH-SY5Y cells using jetOPTIMUS (Polyplus, 101000025) according to the manufacturer's instructions.

[0172] Undifferentiated H9 human embryonic stem cells (hESCs; WiCell, WA09) are AAVS1::HTT 30Q -HA, AAVS1::mtHTT 81Q-HA, AAVS1::HTT 30Q -HA-Cry2PHR and AAVS1::mtHTT 81Q Cells, including the -HA-Cry2PHR KI cell line, were maintained on a feeder layer of mouse embryonic fibroblasts (MEF; Applied StemCell, ASF-1215) with inhibited division. Cells were cultured in hESC medium supplemented with DMEM / F12 containing 20% ​​KnockOut serum replacement (KSR; Gibco, 10828028), 0.1 mM MEM non-essential amino acids (MEM-NEAA; Gibco, 11140050), 2 mM GlutaMAX, 55 μM β-mercaptoethanol (Gibco, 21985023), and 10 ng / mL FGF2 (R&D Systems, 4114-TC-01M). Cells were cultured in a humid environment at 37 °C and 5% CO2, and the medium was changed daily. All hESC-related experiments were confirmed to be exempt from review by the Institutional Review Board of Hanyang University (HYUIRB-202512-002). hESCs with passage numbers ranging from 30 to 55 were used in the experiments.

[0173]

[0174] 1-2. MSN Differentiation

[0175] hESCs were differentiated into medium spiny neurons (MSNs) using a protocol partially modified from a previously reported method (Wu, M., Zhang, D., Bi, C., Mi, T., Zhu, W., Xia, L., Teng, Z., Hu, B., and Wu, Y. (2018). A chemical recipe for generation of clinical-grade striatal neurons from hESCs. Stem Cell Reports 11, 635-650). The neural induction medium (NIM) was prepared by mixing DMEM / F12 and Neurobasal medium (Gibco, 21103049) in a 1:1 ratio and adding 2 mM GlutaMAX, 1× B-27 Supplement (Gibco, 12587010), and 1× N-2 Supplement (Gibco, 17502048). Neural proliferation medium (NPM) was prepared by adding 2 mM GlutaMAX, 0.5× B-27 Supplement, and 0.5× N-2 Supplement to the same basic medium (DMEM / F12:Neurobasal = 1:1). Neural differentiation medium (NDM) was prepared by adding 2 mM GlutaMAX and 1× B-27 Supplement to Neurobasal medium. For differentiation, hESCs were isolated using Dispase (Gibco, 17105041) and cultured as embryoid bodies (EB) on low-attachment plates. DIV 0 through DIV 4 were treated with NIM supplemented with 100 nM LDN193189, 10 μM SB431542, 4 μM XAV939, and 100 nM SAG. During the early stages of EB formation (DIV 0), 10 μM Y-27632 was added only for the first 24 hours to ensure cell survival.In DIV 4, EBs were transferred to plates coated with poly-L-ornithine / laminin / fibronectin (PLF) and cultured in NPM containing the same small molecules. Subsequently, in DIV 5 and DIV 7, the medium was replaced with NPM containing only 100 nM LDN193189 and 10 μM SB431542. From DIV 9 onwards, cells were cultured in NPM from which the small molecules had been removed. In DIV 11, cells were separated into single cells using Accutase (Sigma-Aldrich, A6964) and then transferred to PLF-coated plates at a volume of 4 × 10⁶. 5 cells / cm 2 Resown at a density of [value]. Subsequently, cultured in NDM supplemented with 20 ng / mL BDNF (PeproTech, 450-02), 10 ng / mL GDNF (PeproTech, 450-10), 0.2 mM ascorbic acid (AA; Sigma-Aldrich, A8960), and 1 μM DAPT (TCI, D4257), with 10 μM Y-27632 added only during the initial 24 hours. From DIV 12 to DIV 16, the medium was replaced daily using the same composition excluding Y-27632. From DIV 17 to DIV 30, cultured in NDM containing 1 ng / mL TGFβ3 (R&D Systems, 243-B3), with the medium replaced every other day.

[0176]

[0177] 1-3. Plasmid Production

[0178] HTT fused with Cry2PHR 30Q or mtHTT 81QPlasmids expressing were constructed using a standard restriction enzyme-based cloning method. PfuUltra II Fusion HS DNA Polymerase (Agilent, 600672) was used for PCR amplification, and the ligation reaction was performed using a DNA Ligation Kit.<Mighty Mix> The procedure was performed using (Takara, 6023). All restriction enzymes used were purchased from New England Biolabs. HTTexon 1 fragments containing 30 or 81 glutamine repeats, respectively, were obtained from pJOP-HTT-HR30Q (Addgene, plasmid #92247; provided by Dr. Mahmoud Pouladi) and pJOP-HTT-HR81Q (Addgene, plasmid #92250; provided by Dr. Mahmoud Pouladi). The coding sequence for Cry2PHR was obtained from the previously reported pHR3-AAVS1-opto-TGFβRs (Lee, J., Lee, G., and Oh, Y. (2026). Optogenetic activation of TGFbeta signaling drives ligand-free chondrogenesis in hESC-derived MSCs. Stem Cells44.). Each fragment was subcloned into the pRK5-HA vector (Stratagene) to form pRK5-HTT 30Q -HA-Cry2PHR and pRK5-mtHTT 81Q -HA-Cry2PHR was constructed. Simultaneously, pRK5-HTT, a control plasmid in which Cry2PHR is not bound, was prepared. 30Q -HA and pRK5-mtHTT 81Q pRK5-HA-Cry2PHR, which expresses only -HA and Cry2PHR, was also constructed. A pRK5-based plasmid was used for transient expression experiments, and the donor vector described below was used for genome insertion experiments.

[0179] For KI at the AAVS1 locus, donor vectors were constructed by inserting gene cassettes into the AAV-CAGGS-EGFP backbone (Addgene, plasmid #22212; provided by Rudolf Jaenisch). Each donor construct consisted of a splice acceptor (SA) and a puromycin resistance gene linked by a T2A peptide (Puro R ), and transgene cassettes (HTT) expressed by the CAG promoter 30Q -HA, mtHTT 81Q -HA, HTT 30Q -HA-Cry2PHR, mtHTT 81QIt was configured to include -HA-Cry2PHR or HA-Cry2PHR). The cassette was surrounded by homology arms designed to target the AAVS1 safe insertion locus located in intron 1 of the PPP1R12C gene, with the lengths of the left and right homology arms being 804 bp and 837 bp, respectively. The guide RNA (gRNA) plasmid used to target the AAVS1 locus was the previously reported PX458-AAVS1 (Kim, MS, Ra, EA, Kweon, SH, Seo, BA, Ko, HS, Oh, Y., and Lee, G. (2023). Advanced human iPSC-based preclinical model for Parkinson's disease with optogenetic alpha-synuclein aggregation. Cell Stem Cell30, 973-986 e911.). To construct the plasmid for HTTKO, a gRNA sequence targeting the downstream of the start codon of HTT gene exon 1 was prepared using CRISPR Design It was designed using the Tool(IDT). Candidate sequences were selected based on on-target efficiency and off-target potential. The finally selected gRNA was cloned into PX458 (pSpCas9(BB)-2A-GFP; Addgene, plasmid #48138; provided by Feng Zhang) and used as PX458-HTT. The oligonucleotides used were forward 5′-CACCGAAGGACTTGAGGGACTCGA-3′ and reverse 5′-AAACTCGAGTCCCTCAAGTCCTTC-3′.

[0180] The sequence accuracy and in-frame fusion status of all plasmids were verified through Sanger sequencing (COSMO Genetech).

[0181]

[0182] 1-4. Preparation of KI Cell Line

[0183] SH-SY5Y cells and hESCs were isolated as single cells using TrypLE Express (Gibco, 12604013) or Accutase, respectively. Total 1 × 10⁶ 6 After suspending dogs' cells in Opti-MEM Reduced Serum Medium (Gibco, 31985062), 2.5 μg of PX458-AAVS1 plasmid and the corresponding dsDNA donor plasmid (HTT 30Q -HA, mtHTT 81Q -HA, HTT 30Q -HA-Cry2PHR, mtHTT 81Q-HA-Cry2PHR or HA-Cry2PHR (refer to Section 1-3. Plasmid Construction) was mixed to include 7.5 μg. Subsequently, electroporation was performed using a NEPA21 Super Electroporator (Nepa Gene) at 125 V and 5 ms according to the manufacturer's instructions. For SH-SY5Y cells, cells were seeded into 100 mm dishes containing SH medium after electroporation, and homologous recombination cells were selected by adding 2 μg / mL puromycin (InvivoGen, ant-pr-1) 24 hours after electroporation and performing selection for 7 days. For hESCs, electroporated cells were seeded onto an irradiated MEF feeder layer and cultured in hESC medium containing 10 μM Y-27632. Seven days after electroporation, selection was performed for four days using 0.5 μg / mL puromycin in MEF-conditioned medium prepared using hESC medium from which FGF2 had been removed, with the addition of 10 ng / mL FGF2 and 10 μM Y-27632. After selection, the presence of KI was confirmed by PCR analysis of genomic DNA in puromycin-resistant colonies. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen, 69504) according to the manufacturer's instructions.The following primers were used for PCR: AAVS15′ junction PCR (forward 5′-GGAACTCTGCCCTCTAACGC-3′, reverse 5′-ACGTCACCGCATGTTAGAAGA-3′), AAVS13′ junction PCR (forward 5′-CAATGCCCTGGCTCACA-3′, reverse 5′-GGCTCCATCGTAAGCAAACC-3′), and AAVS1 integration PCR (forward 5′-TTCGGGTCACCTCTCACTCC-3′, reverse 5′-GGCTCCATCGTAAGCAAACC-3′).

[0184]

[0185] 1-5. Preparation of HTT KO cell line

[0186] SH-SY5Y cells and hESCs were isolated as single cells using TrypLE Express or Accutase, respectively. Total 1 × 10⁶ 6After suspending cells in Opti-MEM Reduced Serum Medium containing 10 μg of PX458-HTT plasmid (see Plasmid Construction section), electroporation was performed using a NEPA21 Super Electroporator at 125 V and 5 ms according to the manufacturer's instructions. After electroporation, SH-SY5Y cells were cultured in SH medium, while hESC cells were cultured in hESC medium containing 10 μM Y-27632 and on an irradiated MEF feeder layer. After 30 hours of electroporation, cells were separated into single cells using TrypLE Express or Accutase and 40 μg / mL DNase I (Roche, 11284932001), followed by fluorescence-activated cell sorting (FACS). In the sorting process, gating was established based on eGFP fluorescence intensity relative to the non-transgenic control group to select the top 20% of eGFP-positive cells, which were then isolated using FACSAria III (BD Biosciences). The selected cells were subsequently cultured to form monoclonal colonies. For each colony, KO status was confirmed by PCR analysis of genomic DNA. Genomic DNA was extracted using QuickExtract DNA Extraction Solution (Lucigen, QE09050). The HTT genomic region containing the gRNA target site was amplified using the following primers: forward 5′-GTTCTGCTTTTACCTGCGGC-3′ and reverse 5′-CTGCTGGAAGGACTTGAGGG-3′. The PCR products were purified using a Gel Extraction Kit (COSMO Genetech, CMG0111), and the formation of indels at the target site was confirmed via Sanger sequencing (COSMO Genetech).

[0187]

[0188] 1-6. Blue Light Irradiation and Culture Conditions

[0189] Blue light stimulation was performed using a custom TouchBright W-96 LED excitation system (TB-X6-Z004; Live Cell Instrument) installed inside a cell culture incubator. All irradiation conditions, including light intensity, frequency, and irradiation time, were controlled via software provided by the manufacturer. The wavelength of the light source used was 470 nm. To ensure consistency of conditions between experiments, the light intensity at the culture surface was measured periodically using a power meter (LaserCheck; Coherent). For the SH-SY5Y cell line, the value was 38.4 μW / mm². 2 Blue light was applied using a continuous irradiation method at an intensity of 26 μW / mm² for 1 hour. For hESC-derived MSNs, 26 μW / mm² 2 Blue light was applied using a pulsed irradiation method consisting of 0.5-second pulses repeated at 0.15 Hz with a specific intensity. For the phenotypic analysis following aggregation induction, blue light stimulation was administered for a total of 8 days from DIV 22 to DIV 30. For the aggregation control experiment using arginine ethyl ester (AEE), blue light stimulation was applied for 7 days from DIV 30 to DIV 37 under the same irradiation conditions. The control wells were wrapped in aluminum foil to completely block blue light. To prevent unintentional optogenetic activation, all cell culture and sample handling processes were performed under strict control of light conditions. Cells were maintained under conditions free of blue light, and all manipulation procedures were conducted in a red light environment unless otherwise noted.

[0190]

[0191]

[0192] 1-7. Evaluation of High Content Analysis and Screening Applicability for HTT Aggregation

[0193] AAVS1::mtHTT to quantitatively evaluate HTT aggregation 81Q A high-content-based screening suitability analysis system was constructed using -HA-Cry2PHR KI SH-SY5Y cells. To enable treatment with compounds under various conditions, the cells were placed in a 96-well plate at 0.03 × 10⁻⁶ per well. 6 Cells were seeded at a density of [number]. Each plate included a positive control (cells exposed to blue light without compound treatment) and a negative control (cells maintained under dark conditions) to establish the dynamic range of the analysis. To evaluate the applicability of this system to aggregation control conditions, the test compound was diluted in SH medium and treated to the cells, while the control group was treated with an equal volume of a vehicle (e.g., DMSO). Treatment was performed a total of three times at 24-hour intervals. After the final treatment, cells were exposed to blue light at a wavelength of 470 nm (38.4 μW / mm²) to induce HTT aggregation. 2 The cells were irradiated continuously with ) for 1 hour. The negative control wells were shielded with aluminum foil to completely block exposure to blue light. After blue light irradiation, the cells were fixed with 4% paraformaldehyde (PFA; FUJIFILM Wako Pure Chemical, 163-20145), and immunocytochemical analysis using anti-HTT antibodies was performed to detect HTT aggregates (see Section 1-13, Immunocytochemical Analysis).

[0194]

[0195] 1-8. Automated Image Acquisition and Analysis

[0196] Fluorescence images were acquired using a Lionheart FX automated microscope (BioTek) equipped with a laser-based autofocus system. To ensure unbiased quantitative analysis between experiments, all image acquisition conditions were fixed and applied identically. For SH-SY5Y cells, nuclei were imaged using the DAPI channel (illumination = 5, integration time = 5, gain = 10), and HTT aggregates were detected using the Cy5 channel (illumination = 1, integration time = 50, gain = 24). For each well, a total of nine fields of view were captured in a 3 × 3 array centered on the well center; the size of each field of view was 403 × 297 μm, and the distance between adjacent fields of view was set to 200 μm (edge-to-edge).

[0197] Image analysis was performed using the Cellular Analysis module of Gen5 software (BioTek). DAPI-positive nuclei (DAPI + ) and HTT-positive aggregates (HTT + ) was detected based on predefined user-independent criteria that apply equally to all conditions. DAPI +The nucleus detection conditions are as follows: threshold = 1000; advanced detection options_background flattening_rolling ball diameter = 10; advanced detection options_image smoothing strength = 1; advanced detection options_evaluate background on = 5; object selection_Min. object size = 5 μm; object selection_Max. object size = 20 μm; subpopulation_integral <6000000>0; subpopulation_circularity > 0.1. HTT + The aggregate detection conditions are as follows: threshold = 3000; advanced detection options_background flattening_rolling ball diameter = 1; advanced detection options_image smoothing strength = 0; advanced detection options_evaluate background on = 5; object selection_Min. object size = 0.2 μm; object selection_Max. object size = 5 μm; subpopulation_integral <2000000>; subpopulation_circularity > 0.1; subpopulation_peak < 65000.

[0198]

[0199] 1-9. HTT Aggregate Quantification (H-CAS)

[0200] HTT aggregation was quantified using a composite index defined as the HTT Comprehensive Aggregation Score (H-CAS). H-CAS is an index designed to reflect the total aggregation burden by simultaneously considering variations in aggregate size, fluorescence intensity, and cell number. Since existing aggregation evaluation methods—such as aggregate count or total fluorescence intensity alone—are unable to adequately reflect changes in aggregate size and intensity, H-CAS integrates these two factors to produce a single index. H-CAS was calculated as follows:

[0201]

[0202] For individual aggregates (k) detected under each condition, the aggregate area (A k agg ) and fluorescence intensity (I k agg After calculating the product of ), it was summed for all aggregates. The value obtained in this way was used to correct for differences in the total number of cells, specifically the number of DAPI-positive cells (C total It was normalized by dividing by ). To enable comparison between experiments, the value of the positive control (conditions where aggregation was induced by blue light, no compound treatment) calculated in the same way—i.e., aggregate area (BA k agg ), aggregate fluorescence intensity (BI k agg ), total cell count (BC total Further normalization was performed using )- as the reference value. Through this, a dimensionless relative index was derived. This normalization process reduces inter-experimental variability and ensures comparability between conditions by correcting for differences in aggregation signals and cell numbers.

[0203]

[0204] 1-10. Cell Viability Analysis

[0205] To evaluate cell viability following optogenetically induced HTT aggregation, analysis was performed using the Cell Counting Kit-8 (CCK-8; ABBKINE, KTA1020) according to the manufacturer's instructions. SH-SY5Y cells were seeded in a 96-well plate and subjected to blue light stimulation according to established conditions. For hESC-derived MSNs, analysis was performed at a DIV of 30 following differentiation and photostimulation. CCK-8 solution was added to each well to a final concentration of 10% (v / v) and incubated at 37°C for 2 hours. Subsequently, absorbance was measured at 450 nm using a microplate reader (SpectraMax iD3; Molecular Devices), with 650 nm used as the reference wavelength.

[0206]

[0207] 1-11. Reverse Transcription Quantitative PCR Analysis (RT-qPCR)

[0208] Total RNA was isolated from cells using TRIzol reagent (Invitrogen, 15596026). cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, FSQ-301) with 500 ng of isolated RNA as a template. Quantitative PCR (qPCR) was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad) and THUNDERBIRD Next SYBR qPCR Mix (Toyobo, QPX-201). The expression levels of each gene were quantified via RT-qPCR and normalized based on GAPDH expression levels. The primer sequences used and additional information are presented in Table 1 below.

[0209] GenesForwardReverseCTIP25'-ATCCTCAGCCCCTTTTGTTT-3'5'-GCCGTTGTTCCTGAATTGTT-3'DARPP325'-CCTGAAGGTCATCAGGCAGT-3'5'-GGTCTTCCACTTGGTCCTCA-3'DLX25'-ACCAGACCTCGGGATCCGCC-3'5'-CTGCGGGGTCTGAGTGGGGT-3'DLX55'-TTCAGAAGACTCAGTACCTCGC-3'5'-GAGTTACACGCCATTGGGTC-3'DRD15'-AGGGACTTCTCTGTTCGTATCC-3'5'-GGAACCTGATAACGGCAGCA-3'DRD25'-CTGAGGGCTCCACTAAAGGAG-3'5'-CATTCTTCTCTGGTTTGGCG-3'FOXG15'-TGGCCCATGTCGCCCTTCCT-3'5'-GCCGACGTGGTGCCGTTGTA-3'GAD15'-AGGCAATCCTCCAAGAACC-3'5'-TGAAAGTCCAGCACCTTGG-3'GAPDH5'-AGGGCTGCTTTTAACTCTGGT-3'5'-CCCCACTTGATTTTGGAGGGA-3'GSX25'-TATGTCGACTCGCTCATCATC-3'5'-CAAGCGGGATGAAGAAATCC-3'MAP25'-AAACTGCTCTTCCGCTCAGACACC-3'5'-GTTCACTTGGGCAGGTCTCCACAA-3'NES5'-CAACAGCGACGGAGGTCTC-3'5'-GCCTCTACGCTCTCTTCTTTGA-3'PAX65'-GCCCTCACAAACACCTACAG-3'5'-TCATAACTCCGCCCATTCAC-3'

[0210]

[0211] 1-12. 웨스턴 블롯 분석

[0212] Cells were washed twice with PBS and then lysed in a lysis buffer containing 1× Cell Lysis Buffer (Cell Signaling Technology, #9803), 1% Triton X-100 (Sigma-Aldrich, X100), 10% glycerol (Sigma-Aldrich, G5516), 1× Protease / Phosphatase Inhibitor Cocktail (Cell Signaling Technology, #5872), 20 mM N-ethylmaleimide (NEM; Sigma-Aldrich, E3876), and 1 mM PMSF (Cell Signaling Technology, #8553). Protein concentration was measured using the Quick Start Bradford Protein Assay Kit (Bio-Rad, 5000204). The sample was denatured in 1× Laemmli sample buffer (Sigma-Aldrich, S3401) at 98 °C for 2 minutes, separated by SDS-PAGE, and transferred to a nitrocellulose membrane (GE Healthcare, 10600001). The membrane was blocked for 1 hour at room temperature in a solution of TBST (Cell Signaling Technology, 9997) containing 5% skim milk (BD Difco, 232100). Subsequently, the membrane was incubated overnight at 4 °C using a primary antibody diluted in TBST containing 3% skim milk. After washing three times with TBST, the membrane was incubated for 2 hours at room temperature using an HRP-conjugated secondary antibody diluted in TBST containing 3% skim milk. After the reaction, the sample was washed three times with TBST, protein bands were detected using an ECL substrate (PerkinElmer, NEL103001EA), and images were acquired using an Amersham Imager 680 (GE Healthcare). The antibody information used is presented in Table 2 below.

[0213] Primary antibodiesanti-CompanyCat. No.IsotypeApplicationDilutionβ-actinSigma-AldrichA5441Mouse IgG1Western Blot1:5,000CTIP2abcamab18465Rat IgG 2a Immunocytochemistry1:1,000DARPP32abcamab40801Rabbit IgGImmunocytochemistry1:1,000FOXG1abcamab196868Rabbit IgGImmunocytochemistry1:1,000FOXP2abcamab16046Rabbit IgGImmunocytochemistry1:1,000GABASigma-AldrichA2052Rabbit IgGImmunocytochemistry1:1,000HAabcamab9110Rabbit IgGImmunocytochemistry1:1,000HABioLegend901502Mouse IgG1Immunocytochemistry1:1,000HSP90abcamab203126Rabbit IgGWestern Blot1:10,000HTT (2B7)Coriell InstituteCH03023Mouse IgG1Immunocytochemistry1:1,000Western Blot1:1,000LC3A / BCell Signaling Technology#12741Rabbit IgGWestern Blot1:1,000MAP2Sigma-AldrichM1406Mouse IgG1Immunocytochemistry1:1,000NANOGCell Signaling Technology#3580Rabbit IgGImmunocytochemistry1:200NESBioLegend656802Mouse IgG1Immunocytochemistry1:500OCT3 / 4Santa Cruz Biotechnologysc-5279Mouse IgG 2bImmunocytochemistry1:100TRA-1-81Cell Signaling Technology#4745Mouse IgMImmunocytochemistry1:200TUJ1BioLegend801201Mouse IgG 2a Immunocytochemistry1:1,000

[0214]

[0215] 1-13. Immunocytochemical Analysis

[0216] After fixing the cells with 4% PFA at room temperature for 10 minutes, Ca 2+ / Mg 2+ It was washed with PBS (Corning, 21-030-CV) containing [substance]. Subsequently, it was washed with PBS (Ca [substance] containing 0.5% BSA (GenDEPOT, A0100-010) and 0.1% Triton X-100. 2+ / Mg 2+ Permeation and blocking were performed simultaneously in the (including) solution at room temperature for 20 minutes. The primary antibody was diluted in the same blocking solution and incubated overnight at 4 °C. After washing three times with PBS, the samples were incubated at room temperature for 2 hours using a species-specific Alexa Fluor-conjugated secondary antibody. Nuclear staining was performed for 5 minutes using 1 μg / mL DAPI (Invitrogen, D1306). After additional washing, images were acquired using a TCS SP5 confocal microscope (Leica) or a Lionheart FX automated microscope (BioTek). Immunocytochemical analysis was performed using the antibodies presented in Table 2.

[0217]

[0218] 1-14. Statistical Analysis

[0219] All data were analyzed using Prism v10.6 (GraphPad). Results were expressed as mean ± standard deviation (SD) obtained from at least three repeated biological experiments. Comparisons between two groups were performed using the unpaired Student's test (two-tailed). For comparisons between three or more groups, Tukey's multiple comparison test after one-way ANOVA or Sidak's multiple comparison test after two-way ANOVA was applied as specified in the figure description. Statistical significance was determined based on p < 0.05.

[0220]

[0221] Example 2. Experimental Results

[0222] 2-1. Optogenetics-based HTT aggregation enables rapid and reproducible aggregate induction in SH-SY5Y cells

[0223] In order to construct an adjustable HTT aggregation system, HTT 30Q or mtHTT 81Q We fused it to the Cry2PHR domain and evaluated blue light-induced aggregation in SH-SY5Y cells (see Fig. 1A). Upon blue light illumination, HTT 30Q -HA-Cry2PHR and mtHTT 81Q Both -HA and Cry2PHR formed distinct intracellular aggregates in most transfected cells, while almost no aggregates were observed under dark conditions (see Figures 1B and 1C). The expression vector lacking Cry2PHR did not exhibit blue light-dependent aggregation, and mtHTT 81Q Aggregates were observed in only a few cells with single overexpression. This suggests that aggregation in this system is primarily driven by Cry2PHR-mediated oligomerization rather than non-specific overexpression (see Figs. 1D and 1E).

[0224] For stable and reproducible expression, HTT 30Q -HA-Cry2PHR (hereinafter “opto-HTT 30Q (referred to as ”) or mtHTT 81Q -HA-Cry2PHR (hereinafter “opto-mtHTT 81Q An AAVS1-targeted KI SH-SY5Y cell line expressing (referred to as “)" was constructed, and OHAIS was established in SH-SY5Y cells (see Fig. 2A). AAVS1::opto-HTT upon blue light irradiation 30Q and AAVS1::opto-mtHTT 81Q KI cells exhibited reproducible aggregate formation, while no aggregates were observed under dark conditions (see Figs. 2B and 2C). In contrast, KI cell lines lacking Cry2PHR or KI cell lines expressing Cry2PHR alone did not form aggregates even upon blue light irradiation (see Figs. 2D to 2F), confirming that Cry2PHR is required for blue light-dependent aggregation induction. Under these conditions, blue light exposure did not affect cell viability as assessed by CCK-8 analysis (see Figs. 3A and 3B).

[0225]

[0226] 2-2. Endogenous HTT is required for optogenetically induced HTT aggregation

[0227] To determine whether endogenous HTT is required for optogenetic aggregation induction, HTTKO SH-SY5Y cells were constructed using CRISPR-Cas9 targeting exon 1 of the HTT gene (see Figs. 4A to 4C). Subsequently, opto-HTT was placed on the AAVS1 locus of the HTT KO cells. 30Q and opto-mtHTT 81Q ...was introduced. Under blue light irradiation conditions, aggregate formation was significantly reduced in HTTKO cells compared to the control group (see Figures 4D and 4E). That is, opto-HTT 30Q and opto-mtHTT 81QBlue light-induced aggregation was significantly attenuated in HTT-KO cells (aggregation-positive cells about 1-4%), which implies that endogenous HTT is required for reproducible aggregate formation in OHAIS. These results suggest that aggregation operates efficiently in an intracellular environment where endogenous HTT is present, rather than being driven by the opto-HTT fusion protein alone.

[0228]

[0229] 2-3. Construction of Opto-HTT KI hESC cell line and differentiation into MSNs

[0230] To establish a human MSN-based system, opto-HTT 30Q or opto-mtHTT 81Q AAVS1-targeted KI hESC cell lines expressing [variable] were constructed, along with corresponding control cell lines lacking Cry2PHR (see Fig. 5A). All KI cell lines maintained pluripotency marker expression similar to that of the parental cell line H9 (see Fig. 5B). opto-HTT upon blue light irradiation 30Q and opto-mtHTT 81Q The KI hESC cell line formed aggregates, but no aggregates were observed in the control cell line (see Fig. 5C). Consistent with the OHAIS SH-SY5Y cells, aggregation was significantly reduced in the HTTKO hESC background (see Figs. 5D to 5G).

[0231] Subsequently, a differentiation protocol for generating MSNs from hESCs was established (see Fig. 6A). At day 15 of differentiation (dayin vitro 15; DIV 15), most cells exhibited lateral ganglionic eminence (LGE)-like progenitor cell characteristics expressing FOXG1, NES, and FOXP2 (see Figs. 6B and 6C). At DIV 30, the differentiated cells expressed MSN markers such as DARPP32, CTIP2, GABA, and MAP2 (see Figs. 6D and 6E), and progressive neuronal maturation was confirmed by gene expression and immunocytochemical analysis over time (see Figs. 6F to 6H).

[0232] Furthermore, under dark conditions, no significant difference in differentiation efficiency was observed between the parent cell line and the KI cell line at both the LGE-like progenitor cell stage and the MSN stage (see Fig. 7). This is HTT 30Q or mtHTT 81Q This means that the introduction of the AAVS1 target (regardless of the presence or absence of Cry2PHR) does not affect basic lineage differentiation and MSN maturation.

[0233]

[0234] 2-4. Optogenetic-induced mtHTT aggregation causes MSN fragility

[0235] To evaluate aggregation-related phenotypes in a Huntington's disease-associated human MSN environment, KI hESC cell lines were differentiated into MSNs and pulsed blue light stimulation was applied from DIV 22 to DIV 30 (see Fig. 8A). Under the above conditions, opto-HTT 30Q and opto-mtHTT 81Q Intracellular aggregates were formed in all MSNs (see Fig. 8B). Opto-mtHTT 81QIn MSN, the proportion of DARPP32, CTIP2, and GABA-positive cells was significantly reduced compared to the dark-condition control group (see Figs. 8C–8H, 9A–9E), indicating the loss of the MSN population. Considering that differentiation efficiency was similar among cell lines under dark conditions (see Fig. 7), the decrease in MSN marker-positive cells reflects cell loss due to aggregation rather than reduced differentiation. On the other hand, opto-HTT 30Q Although MSNs formed aggregates, no significant changes were observed in MSN marker-positive cells. This indicates that the aggregation of polyQ-extended mtHTT selectively induces MSN loss, and that the aggregation of non-pathological HTT alone is insufficient to induce toxicity. Furthermore, opto-mtHTT after long-term blue light exposure 81Q The overall survival rate of MSN was significantly reduced, but opto-HTT 30Q No significant changes were observed in MSN and control cell lines (see Figs. 10A to 10C). This indicates polyQ length-dependent neuronal fragility associated with mtHTT aggregation. In addition, opto-mtHTT 81Q MSN's LC3-II levels decreased after aggregation induction (see Fig. 10D), suggesting that the autophagy process may be modified in response to polyQ extended mtHTT aggregation.

[0236]

[0237] 2-5. Development of a Quantitative High-Content Imaging Analysis Method for HTT Aggregation

[0238] To quantitatively analyze HTT agglutination, opto-mtHTT 81QA high-content imaging (HCI)-based analysis method using KI SH-SY5Y cells was established (see Fig. 11A). HTT aggregates were visualized by immunocytochemistry and quantified using an automated HCI pipeline with predefined, user-independent criteria, thereby minimizing operator-dependent bias. For each detected aggregate k, the product of area and fluorescence intensity (A k agg ×I k agg A raw aggregation index was defined by calculating the value, summed for all aggregates, and normalized by the total number of DAPI-positive cells. The value represents a normalized aggregation measurement per cell that integrates aggregate size and signal intensity while correcting for differences in cell numbers. To enable direct comparison between experimental conditions, the raw value was further normalized by the blue light-induced positive control value, thereby introducing the dimensionless composite index, the HTT Comprehensive Aggregation Score (H-CAS) (see Fig. 11B). This normalization improves inter-experimental comparability and dynamic range by providing a consistent reference point for blue light-induced HTT aggregation. Blue light-induced aggregation by OHAIS significantly increases the H-CAS value compared to dark conditions, thereby ensuring a dynamic range between aggregation and non-aggregation states (see Figs. 11C and 11D). Accordingly, HCI-based analysis methods and H-CAS can be utilized as quantitative analysis tools for evaluating the high content of HTT aggregation.

[0239]

[0240] 2-6. Reduction in HTT Cohesion Mitigates MSN Vulnerability in Human OHAIS MSN

[0241] To evaluate the utility of the platform, arginine ethyl ester (AEE), for which HTT aggregation reduction has been reported, was tested. Opto-mtHTT 81Q In KI SH-SY5Y cells, AEE reduced aggregation in a concentration-dependent manner, which was reflected in a reduction in H-CAS (see Figs. 11E to 11I). At a concentration of 10 mM, continuous blue light irradiation (38.4 μW / mm²) was applied after 3 days of AEE pretreatment. 2 Aggregation was reduced without affecting cell viability even under conditions of , 470 nm, 1 hour (see Fig. 11J). This indicates that no cytotoxicity was observed in SH-SY5Y cells under the above conditions.

[0242] Subsequently, AEE [resources] hESC-derived opto-mtHTT 81Q We evaluated whether it modulates aggregation-related phenotypes in MSNs (see Fig. 12A). AEE treatment was initiated 2 days prior to light exposure and maintained for a 7-day stimulation period (pulsed blue light: 26 μW / mm²). 2 , 470 nm, 0.5 second pulse, 0.15 Hz). opto-mtHTT treated with AEE under the above conditions 81QIn MSNs, HTT aggregation was reduced, and DARPP32, CTIP2, and GABA-positive cell populations recovered stepwise; partial recovery was observed at 1 mM, and more pronounced recovery at 10 mM (see Figures 12B and 12C). This indicates that the reduction in HTT aggregation induced by AEE is associated with the recovery of MSN loss due to aggregation. Furthermore, while no apoptosis was observed in OHAIS SH-SY5Y cells despite efficient aggregation induction under short-term high-intensity irradiation conditions, pronounced MSN loss was induced in OHAIS MSNs by prolonged aggregation induction under different irradiation conditions. Despite these differences in conditions and responses, AEE consistently reduced HTT aggregation in both systems and induced the recovery of MSN loss due to aggregation in human OHAIS MSNs. This indicates that the aggregation-regulating effect identified in HCI-based analyses is also confirmed in the human MSN model and is associated with neuronal fragility.

[0243]

[0244] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

(a) introducing a nucleic acid encoding a fusion protein in which Cry2PHR (cryptochrome 2 photolyase homology region) and huntingtin (HTT) protein are fused into cells in vitro; and (b) a step of inducing intracellular huntingtin protein aggregation by irradiating the cells with blue light; comprising a method for preparing an in vitro Huntington disease model. In claim 1, A method for preparing an in vitro Huntington's disease model, characterized in that the fusion protein is composed of an amino acid sequence represented by SEQ ID NO.

1. In claim 1, A method for preparing an in vitro Huntington's disease model, characterized in that the above-mentioned huntingtin protein comprises a polyglutamine (polyQ) composed of 35 or more glutamines. In claim 1, A method for preparing an in vitro Huntington's disease model, characterized in that the above-mentioned huntingtin protein comprises an HTT exon 1 fragment represented by SEQ ID NO.

2. In claim 1, A method for preparing an in vitro Huntington's disease model, characterized in that the above nucleic acid is inserted into the AAVS1 safe harbor locus within the cell. In claim 1, A method for preparing an in vitro Huntington's disease model, characterized in that the above-mentioned cells are SH-SY5Y cells, human embryonic stem cells (hESCs), or neurons differentiated therefrom. In claim 6, A method for preparing an in vitro Huntington's disease model, characterized in that the nerve cell is a medium spiny neuron (MSN). In claim 6, The above manufacturing method is characterized by further including a step of inducing differentiation into intermediate spinous neurons when the cells are human embryonic stem cells. In claim 7, A method for preparing an in vitro Huntington's disease model, characterized in that the above-mentioned intermediate auricular neurons express one or more markers selected from the group consisting of DARPP32, CTIP2, GABA, and MAP2. In claim 1, A method for preparing an in vitro Huntington's disease model, characterized in that the blue light is light with a wavelength of 450 to 490 nm. In claim 1, A method for preparing an in vitro Huntington's disease model, characterized in that the aggregation of the above-mentioned huntingtin protein is dependent on the presence of endogenous huntingtin protein. An in vitro Huntington's disease model prepared by the method according to claim 1. In claim 12, The above disease model is an in vitro Huntington's disease model characterized by being able to quantitatively evaluate the aggregation of huntingtin protein. In claim 13, An in vitro Huntington's disease model characterized in that the above-mentioned huntingtin protein comprises a polyglutamine composed of 35 or more glutamines. In claim 13, An in vitro Huntington's disease model characterized in that the above-mentioned huntingtin protein comprises an HTT exon 1 fragment represented by SEQ ID NO.

2. In claim 13, An in vitro Huntington's disease model characterized in that the aggregation of the above-mentioned huntingtin protein is dependent on the presence of endogenous huntingtin protein. (a) a step of treating an in vitro Huntington's disease model prepared by the method according to claim 1 with a candidate drug for the prevention or treatment of Huntington's disease; (b) a step of calculating the H-CAS value according to the following [Mathematical Formula 1]; and (c) a step of determining the candidate drug as a drug for the prevention or treatment of Huntington's disease if the calculated H-CAS value is reduced compared to the drug-untreated group; comprising a screening method for a drug for the prevention or treatment of Huntington's disease. [Mathematical Formula 1] Here, A k agg is the area of ​​individual (k) huntingtin protein aggregates in the drug-treated group, and I k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-treated group, and C total is the total number of cells in the drug-treated group, and BA k agg is the individual (k) huntingtin protein aggregate area of ​​the drug-untreated group, and BI k agg is the fluorescence intensity of individual (k) huntingtin protein aggregates in the drug-untreated group, and BC total represents the total number of cells in the drug-untreated group. In claim 17, A screening method for a drug for the prevention or treatment of Huntington's disease, characterized in that the above-mentioned huntingtin protein comprises a polyglutamine composed of 35 or more glutamines. In claim 17, A screening method for a drug for the prevention or treatment of Huntington's disease, characterized in that the above-mentioned huntingtin protein comprises an HTT exon 1 fragment represented by SEQ ID NO.

2. In claim 17, A screening method for drugs for the prevention or treatment of Huntington's disease, characterized in that the aggregation of the above-mentioned huntingtin protein is dependent on the presence of endogenous huntingtin protein.

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