Application of substances that increase LSH content or activity in improving heterochromatin stability and enhancing the efficacy of cell therapy.

Abstract

This invention discloses the application of substances that increase LSH content or activity in improving heterochromatin stability and enhancing the efficacy of cell therapy, belonging to the field of biomedical technology. This invention, through overexpression of LSH in IMR90 fibroblasts and T lymphocytes, successfully achieved for the first time complete inhibition of DNA methylation loss in heterochromatin regions during cell expansion. The innovative research results of this invention strongly demonstrate that the insufficient DNA methylation maintenance efficiency caused by the dense chromatin environment in heterochromatin regions is the reason for the continuous loss of DNA methylation during sustained cell expansion. Overexpression of LSH can effectively improve the DNA methylation maintenance efficiency in heterochromatin regions to prevent DNA methylation loss, thereby improving several phenotypic defects caused by sustained cell expansion. This provides a new strategy for effectively maintaining cell function in adoptive cell therapy and has broad application value in enhancing the efficacy of various cell therapies.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of substances that increase LSH content or activity in improving heterochromatin stability and enhancing the efficacy of cell therapy. Background Technology

[0002] Cell therapy is a method of delivering functional or enhanced live cells, either autologous or allogeneic, into a patient's body as therapeutic drugs to repair, replace, or eliminate damaged or abnormal cells, thereby achieving tissue regeneration or lesion clearance. Currently, the most widely used cell therapies in the field of cell therapy include stem cell transplantation therapies (such as hematopoietic stem cell transplantation and mesenchymal stem cell transplantation) and adoptive immune cell therapies (such as CAR-T therapy and TCR-T therapy). As a novel treatment approach, these cell therapy drugs have demonstrated significant application value in the treatment of various complex diseases (such as autoimmune diseases, neurodegenerative diseases, and tumors) compared to traditional small-molecule chemical or large-molecule biological drugs.

[0003] Despite the widespread success of cell therapy, many challenges remain in its practical application. Insufficient cell proliferation capacity and poor post-transplantation sustainability are among the major problems encountered by various cell therapy methods. For example, mesenchymal stem cell transplantation therapy first requires the preparation of a sufficient number of clinical-grade mesenchymal stem cells in vitro. However, the long-term cell passage culture during this process can induce cellular senescence, manifested primarily as decreased cell proliferation capacity, increased chromosomal abnormalities, and elevated inflammatory responses. Similarly, in adoptive immunotherapy (such as CAR-T therapy), it is necessary to first expand a large number of patient-derived T lymphocytes in vitro. However, some patient-derived T lymphocytes have poor in vitro expansion capacity, which is a major limiting factor in obtaining a sufficient number of CAR-T cells for in vivo reinfusion. Furthermore, low-dose CAR-T cells cannot completely and effectively eliminate tumor cells. At the same time, CAR-T therapy still faces bottlenecks in the treatment of solid tumors, with the tumor microenvironment of solid tumors posing a challenge to the sustained maintenance of CAR-T cell proliferation capacity and effector function. After being reinfused into the patient, CAR-T cells often exhibit a depleted or senescent phenotype upon entering the tumor microenvironment, thus failing to effectively and continuously eliminate tumor cells in the long term. Therefore, how to effectively enhance cell proliferation capacity in vitro or in vivo and prevent cell function degeneration during cell therapy is one of the main challenges encountered in the application of various cell therapies to diseases.

[0004] Epigenetic imbalance is a significant factor contributing to cellular functional decline. Loss of DNA methylation in heterochromatin regions caused by excessive cell proliferation is a common epigenetic feature in senescent and tumor cells. This chromatin change is also observed during various cell therapy treatments, such as cellular senescence during in vitro expansion of mesenchymal stem cells and the exhaustion or senescence of CAR-T cells in the tumor microenvironment after infusion. Significantly reduced heterochromatin-specific DNA methylation levels are observed in both cases. DNA methylation, a major mechanism for transcriptional silencing in heterochromatin regions in somatic cells, leads to abnormal activation of numerous repetitive sequences or transposon elements within these regions. This activation triggers intracellular nucleic acid sensing pathways (such as the cGAS-STING pathway), inducing chronic inflammation and impairing normal cellular function. Furthermore, activation of heterochromatin in centromere / paracentromere regions disrupts genomic stability, affecting normal chromosome segregation and resulting in defective cell division.

[0005] Although heterochromatin DNA methylation plays a crucial role in preventing abnormal transposon activation, maintaining genomic stability, and reducing chronic inflammatory responses, the reasons for the continuous loss of heterochromatin DNA methylation during long-term cell proliferation remain unclear, and effective interventions to prevent this loss are lacking. In a previous study, the applicant systematically compared the maintenance rates of DNA methylation in different regions across the entire genome during DNA replication. The results showed that the dense chromatin environment in heterochromatin regions is not conducive to the effective maintenance of DNA methylation, and this insufficient maintenance efficiency may be the reason for the continuous decline in DNA methylation levels in these regions during long-term cell expansion. Furthermore, genetic evidence indicates that the chromatin remodeling factor LSH is specifically crucial for the effective maintenance of heterochromatin DNA methylation. Therefore, we believe that using gene overexpression or other methods to increase LSH levels or activity can effectively inhibit the loss of heterochromatin DNA methylation, thereby improving cell proliferation capacity and preventing cellular functional decline, thus enhancing the persistence and therapeutic efficacy of cell therapy drugs. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention aims to provide the application of substances that increase LSH content or activity (including LSH overexpression recombinant vectors, recombinant viruses, and recombinant cells) in improving heterochromatin stability and enhancing the efficacy of cell therapy. Substances that increase LSH content or activity can effectively prevent the loss of DNA methylation in heterochromatin regions during long-term cell proliferation, thereby reducing the generation of chronic inflammatory responses and significantly improving cell expansion capacity, while promoting the effective maintenance of cell effector functions. Thus, they can be widely used as enhancers in various cell therapies.

[0007] To achieve the above objectives, the present invention employs the following technical solution:

[0008] This invention is the first to reveal the application of substances that increase LSH content / activity in the preparation of drugs / reagents that improve heterochromatin stability.

[0009] Preferably, the substance that increases LSH expression is an LSH overexpression substance that inhibits the loss of DNA methylation in heterochromatin regions.

[0010] More preferably, the heterochromatin region is a chromatin region modified by histone H3K9me3, highly enriched with HP1 protein or Lamin B1 protein, or a late DNA replication region in the S phase of the cell cycle.

[0011] This invention also discloses the application of substances that increase LSH content / activity in the preparation of drugs / reagents that enhance the efficacy of cell therapy.

[0012] Preferably, the drug is a drug that promotes the effective maintenance of cell function during cell therapy.

[0013] further Preferably, the cell therapy includes functional cell transplantation therapy and adoptive immune cell therapy, etc.

[0014] Preferably, the substance that enhances LSH expression level / activity is an LSH overexpression recombinant vector, recombinant virus, or recombinant cell.

[0015] More preferably, the LSH overexpression recombinant vector is obtained by ligating the LSH gene coding sequence to a viral vector.

[0016] More preferably, the LSH gene coding sequence is the original CDS coding sequence of the LSH gene, or the CDS coding sequence of the LSH gene after codon optimization, or the CDS coding sequence of the LSH gene after base sequence modification without changing the biological function of LSH itself.

[0017] More preferably, the viral vector is a lentiviral vector, a retroviral vector, or an AAV viral vector.

[0018] Preferably, the LSH-overexpressing recombinant lentivirus is produced by co-transfecting the LSH-overexpressing recombinant vector and the viral packaging plasmid into human embryonic kidney HEK293T cells.

[0019] More preferably, the viral packaging plasmid is pMDLg / pRRE, pRSV / REV, or pVSVG.

[0020] Preferably, the LSH-overexpressing recombinant cells are obtained by infecting host cells with the LSH-overexpressing recombinant virus.

[0021] More preferably, the host cell is a human embryonic lung IMR90 fibroblast, or a human peripheral blood-derived T lymphocyte, or other cell types used for cell therapy.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention first demonstrated the complete inhibition of DNA methylation loss in heterochromatin regions caused by long-term cell proliferation in a replicative cellular senescence model of human embryonic lung IMR90 fibroblasts by overexpressing LSH. This effectively prevented the abnormal activation of transposon elements, reduced downstream chronic inflammatory responses, significantly improved cell proliferation capacity, and enhanced several cellular senescence-related phenotypes. Simultaneously, this invention also effectively alleviated the decrease in heterochromatin DNA methylation levels during clonal expansion by overexpressing LSH in human peripheral blood-derived T lymphocytes, thereby significantly improving T cell proliferation capacity and cell viability, and promoting the long-term effective maintenance of T cell tumor-killing effects.

[0024] Therefore, the innovative research results of this invention strongly demonstrate that insufficient DNA methylation maintenance efficiency caused by the dense chromatin environment of heterochromatin is the reason for the continuous loss of DNA methylation in this region during long-term proliferation. Overexpression of LSH can specifically improve the methylation maintenance efficiency of heterochromatin to prevent the decrease in methylation level. This provides a new perspective and target for understanding and intervening in the epigenetic information imbalance that occurs after cells undergo massive clonal expansion. At the same time, using LSH overexpression to improve heterochromatin stability can significantly enhance cell proliferation and survival capacity and reduce adverse reactions such as chronic inflammation, thereby promoting the long-term stable maintenance of cell effector functions. This provides a new means to enhance the efficacy of cell therapy drugs and has shown broad application value in various cell adoptive therapies, including CAR-T therapy. Attached Figure Description

[0025] Figure 1a The transcript levels of the HELLS (LSH) gene in IMR90 cells infected with mCherry and hLSH-overexpressing lentiviruses, obtained by RNA-seq sequencing;

[0026] Figure 1b The protein level of LSH in IMR90 cells infected with mCherry and hLSH-overexpressing lentiviruses was determined by Western Blot.

[0027] Figure 1c The overall level of DNA methylation across the entire genome was determined by WGBS in wild-type young IMR90 cells that had not undergone long-term passage (P9) and in replicative senescent IMR90 cells that had undergone long-term, large-scale proliferation (P19) and were overexpressing mCherry / hLSH.

[0028] Figure 1d Visualization of DNA methylation levels at specific locations in the genome in wild-type young IMR90 cells and replicated senescent IMR90 cells overexpressing mCherry / hLSH;

[0029] Figure 1e The heatmap results are obtained by analyzing the differentially methylated regions (DMRs) that show significant changes in DNA methylation between wild-type young IMR90 cells and replicated senescent IMR90 cells overexpressing mCherry / hLSH.

[0030] Figure 1f The DNA methylation levels in early and late replication regions of wild-type young IMR90 cells and replicating senescent IMR90 cells overexpressing mCherry / hLSH;

[0031] Figure 2a The image is a volcano plot, showing the changes in transposon element expression during the replicative senescence process caused by long-term continuous proliferation of IMR90 cells.

[0032] Figure 2b The volcano plot shows the differential expression of transposon elements in replicated senescent IMR90 cells overexpressing mCherry / hLSH;

[0033] Figure 2cA heatmap showing the expression changes of transposon elements that are significantly upregulated during the long-term proliferation of IMR90 cells in IMR90 cells overexpressing mCherry and hLSH. Transposon elements whose activation can be inhibited by LSH overexpression are defined as Cluster 1, while transposon elements that cannot be inhibited by LSH overexpression are defined as Cluster 2.

[0034] Figure 2d This study shows the changes in DNA methylation at transposon elements (Cluster 2) that are upregulated by LSH overexpression (Cluster 1) and those that are not upregulated by LSH overexpression during the long-term proliferation of IMR90 cells.

[0035] Figure 2e The left-hand heatmap shows the expression changes of gene encoding in IMR90 cells overexpressing mCherry and hLSH during long-term continuous proliferation. The right-hand heatmap shows the genes (Cluster 2) that were significantly upregulated during replication and senescence in the left-hand heatmap and were effectively suppressed by LSH overexpression. The results were obtained after biological pathway enrichment analysis (GOterm analysis) using DAVID software. The biological pathways marked with "*" are those related to the inflammatory response.

[0036] Figure 2f A heatmap showing the changes in inflammation-related genes that are significantly upregulated during long-term continuous proliferation, in IMR90 cells overexpressing mCherry and hLSH, during replication and senescence.

[0037] Figure 2g Statistical curves showing the growth of IMR90 cells overexpressing mCherry and hLSH during long-term passage culture;

[0038] Figure 2h The changes in cell size (FSC value) of IMR90 cells overexpressing mCherry and hLSH during long-term passage culture were detected by flow cytometry.

[0039] Figure 3a A schematic diagram of the process of separating and purifying human peripheral blood T cells in vitro, activating antigens, infecting viruses, and culturing them for a long time;

[0040] Figure 3b The changes in LSH protein levels during long-term in vitro expansion of human peripheral blood T cells;

[0041] Figure 3c The results of Western blotting analysis of LSH protein expression in human T cells after infection with mCherry and hLSH lentiviruses;

[0042] Figure 3d The changes in DNA methylation in the heterochromatin region (PMD) of control cells and LSH-overexpressing cells during long-term clonal proliferation of T cells were detected by WGBS technology.

[0043] Figure 3e This is a volcano plot showing the changes in the transcriptome of control T cells and LSH-overexpressing T cells after long-term clonal expansion;

[0044] Figure 3f The GSEA analysis results show the expression changes of T cell effector function-related genes during long-term clonal expansion and after LSH overexpression.

[0045] Figure 3g Statistical curves showing the growth of human T cells overexpressing mCherry and hLSH during long-term in vitro culture;

[0046] Figure 3h The results of flow cytometry analysis of Ki67, a cell proliferation-related marker, on day 11 of in vitro expansion of T lymphocytes in the RFP overexpression control group and the hLSH overexpression experimental group.

[0047] Figure 3i The results are from flow cytometry analysis of the changes in the proportion of dead cells in T cells during the late stage of clonal proliferation using the dead cell fluorescent dye A780.

[0048] Figure 3j The results of flow cytometry analysis of CD27 staining, a co-stimulatory molecule on the cell surface, are presented for T lymphocytes in the RFP overexpression control group and the hLSH overexpression experimental group during in vitro clonal expansion. Detailed Implementation

[0049] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0050] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0052] The present invention will now be described in further detail with reference to the accompanying drawings:

[0053] Example 1

[0054] Overexpression of LSH can delay the loss of heterochromatin DNA methylation and the replicative senescence phenotype that occur during the continuous proliferation of human embryonic lung IMR90 fibroblasts.

[0055] The specific experimental operation steps involved in this embodiment are as follows:

[0056] 1. Obtaining LSH coding sequences and constructing lentiviral recombinant plasmids

[0057] (1) Obtaining the LSH coding sequence

[0058] 1) Cloning of wild-type LSH sequences:

[0059] The wild-type gene sequence encoding human LSH was amplified from a cDNA library of human cervical cancer HeLa-S3 cells using nested PCR. The amplification primers are as follows:

[0060] Nested upstream primer 1: 5′-AGCGGTTGTGAGGAGTTAGC-3′ (shown in SEQ ID NO:1)

[0061] Nested downstream primer 1: 5′-TCTCTCCCCATGAAAAGCCT-3′ (shown in SEQ ID NO:2)

[0062] Nested upstream primer 2: 5′-CATTGCAGGCTCTGAGAGGA-3′ (shown in SEQ ID NO:3)

[0063] Nested downstream primer 2: 5′-ACCTAAAGCCCATGAACTGC-3′ (shown in SEQ ID NO:4)

[0064] After the PCR reaction was completed, the LSH coding sequence fragment of the corresponding size was purified by gel extraction.

[0065] 2) Optimization of LSH encoded sequences:

[0066] The codon composition of the LSH coding sequence was modified using a codon optimization algorithm, and the optimized LSH coding sequence fragment was obtained through gene synthesis and PCR amplification.

[0067] (2) LSH-encoded sequence linked to T vector

[0068] Wild-type or optimized LSH coding sequence fragments were ligated into the pEASY-T5 cloning vector to construct the pEASY-T5-LSH recombinant plasmid. The correctness of the obtained LSH coding sequence was verified by transforming DH5α competent bacteria and performing single-colony Sanger sequencing.

[0069] (3) LSH coding sequence ligated into lentiviral expression vector

[0070] The LSH coding sequence and the pLenti lentiviral expression vector were linearized using PCR, and homologous arms were introduced at both ends of the fragment. Then, the LSH coding sequence was ligated to the pLenti lentiviral expression vector using Gibson Assembly (a molecular biology technique for assembling DNA molecules in vitro) to construct the pLenti-LSH recombinant plasmid. The correctness of the recombinant plasmid was verified by transforming DH5α competent bacteria and performing single-colony Sanger sequencing. At the same time, the control gene mCherry was ligated to the pLenti lentiviral expression vector using the same method to construct the pLenti-mCherry control recombinant plasmid.

[0071] 2. Preparation of recombinant lentivirus packaging for LSH overexpression

[0072] (1) Culture of lentiviral packaging cells 293FT cells

[0073] Human embryonic kidney 293FT cells were cultured in DMEM medium (Gibco, C11995) with the addition of 10% fetal bovine serum (BI) and 1% penicillin-streptomycin (Sangon Biotech). The 293FT cells were cultured in a 37°C cell culture incubator (containing 5% CO2) and, when the cells reached approximately 90% confluence, were trypsinized and passaged at a ratio of 1:4 or 1:6.

[0074] (2) Packaging and preparation of recombinant lentiviruses

[0075] The lentiviral recombinant expression plasmid pLenti-hLSH and lentiviral packaging plasmids (pMDLg / RRE, pVSVG, and pRSV / Rev) were introduced into 293FT cells using the cationic non-liposome transfection reagent VigoFect (Vigras). After transfection, the cell culture medium was replaced with fresh medium 6 hours later for continued culture. After 48 hours and 72 hours of culture, the cell culture medium containing lentivirus was collected. The supernatant was centrifuged and filtered through a 0.45 μm syringe filter to obtain a lentiviral suspension.

[0076] 3. Establishment of IMR90 cell lines with stable LSH overexpression

[0077] (1) Culture of human embryonic fibroblast IMR90 cells

[0078] Human embryonic lung fibroblast IMR90 cells were cultured in MEM medium (Sigma, M4655) supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Sangon Biotech), 1 mM sodium pyruvate (Gibco), 1% non-essential amino acids (Sigma), and 1% penicillin-streptomycin solution (Beyotime Biotech). IMR90 cells were cultured in a 37°C cell culture incubator (containing 5% CO2) and, when reaching approximately 90% confluence, were trypsinized and passaged at a 1:4 ratio.

[0079] (2) Lentiviral infection of IMR90 cells

[0080] IMR90 cells with a low passage number were passaged into 3.5 cm culture dishes. After the cells were fully adhered, the culture medium was aspirated. 1 mL of filtered virus suspension and 1 mL of preheated fresh culture medium were mixed in equal proportions and added to the cells for infection. Simultaneously, 0.5 μL of 10 mg / mL Polybrene was added to improve infection efficiency. After gentle shaking to mix thoroughly, the cells were placed in a 37°C cell culture incubator (containing 5% CO2) for further culture. After 24 hours of infection, the liquid in the culture dish was aspirated, and 2 mL of the equal proportions of virus suspension and cell culture medium were added for a second infection. After 24 hours of the second infection, the liquid in the culture dish was aspirated, and 2 mL of fresh cell culture medium was added for further culture.

[0081] (3) Screening of lentivirus-infected positive cells

[0082] On day 3 or 4 post-lentiviral infection, positive cells are selected using the resistance gene or fluorescent protein carried on the pLenti recombinant plasmid integrated into the host cell genome. Specifically, resistance selection can be performed by adding the corresponding antibiotic (such as Puromycin) to the culture medium of infected cells; cells that survive after one week of continuous selection are considered successfully infected with the lentivirus. Alternatively, flow cytometry can be used to separate cells that stably express the corresponding fluorescent protein; these fluorescently positive cells are considered successfully infected.

[0083] (4) Identification of LSH-stably overexpressing IMR90 cells

[0084] After successfully infecting IMR90 cells with lentivirus, further identification of LSH expression in positive cells is necessary. The results are as follows: Figure 1a This demonstrates that lentiviral infection with LSH overexpression effectively inhibits heterochromatin-specific DNA methylation loss during the replication and senescence of IMR90 fibroblasts. First, at the DNA level, PCR confirmed the insertion of exogenous LSH expression elements into the genome of positive cells. Second, at the RNA level, quantitative reverse transcription PCR (RT-qPCR) or transcriptome sequencing identified LSH overexpression at the transcript level in positive cells (Figure 1a). Finally, at the protein level, Western blotting determined the degree of LSH overexpression at the protein level. Figure 1b (As shown). At both the RNA and protein levels, LSH was significantly overexpressed by several tens of times, indicating the successful establishment of LSH-overexpressing IMR90 cells mediated by lentiviral infection.

[0085] 4. LSH overexpression delays the loss of heterochromatin DNA methylation during long-term proliferation.

[0086] Whole-genome bisulfite sequencing (WGBS) was used to detect changes in DNA methylation levels in heterochromatin regions of IMR90 cells overexpressing mCherry and hLSH during replicative senescence induced by long-term continuous expansion. The specific experimental steps are as follows:

[0087] (1) Establish a long-term cell proliferation-induced replicative senescent cell model

[0088] IMR90 cells with a low passage number (approximately 20 cell divisions, PD20) are continuously passaged in vitro. After prolonged and repeated expansion (approximately PD55-PD65), IMR90 cells will enter the cell cycle arrest phase and exhibit senescence-related phenotypes. This process is known as the replicative senescence of IMR90 cells.

[0089] In young wild-type IMR90 cells that had not undergone long-term expansion culture, mCherry overexpression control group and hLSH overexpression experimental group cells were established using lentiviral infection. The control and experimental groups were then passaged in parallel for a long period. After the cells entered the division arrest phase, the effect of LSH overexpression on the loss of heterochromatin DNA methylation induced by long-term cell proliferation was detected using WGBS technology.

[0090] (2) Extraction of genomic DNA using phenol-chloroform

[0091] 1) IMR90 cells from the control group and the experimental group with lower and higher passage numbers were cultured in 6cm cell culture dishes. When the cells grew to cover about 90% of the bottom area of ​​the culture dish, they were digested with trypsin and collected into a new 1.5mL centrifuge tube. After centrifugation at 900rpm for 3min, the supernatant was discarded and the cells were washed once with PBS.

[0092] 2) Resuspend the cell pellet washed with PBS in 1 mL of cell lysis buffer, and add 30 μL of 10% SDS to each mL of cell lysis buffer and mix thoroughly by pipetting. The solution should become viscous at this point.

[0093] 3) Add 30 μL of 20 mg / mL RNase A and mix well by pipetting. Place in a 37°C water bath for about 3 hours to digest.

[0094] 4) Add 20 μL of 20 mg / mL proteinase K and mix by pipetting. Place in a 65°C water bath overnight.

[0095] 5) The next day, remove the centrifuge tubes from the 65℃ water bath and let them cool to room temperature for 5 minutes.

[0096] 6) Add an equal amount of phenol-chloroform solution to the solution, and vortex at maximum speed for 20 seconds to mix thoroughly.

[0097] 7) Centrifuge at 13.3 krpm for 15 minutes, and transfer the upper aqueous phase containing DNA to a new 1.5 mL centrifuge tube;

[0098] 8) To obtain cleaner DNA, add an equal amount of phenol-chloroform to the supernatant, shake well to mix, centrifuge at 13.3 krpm for 15 minutes, and transfer the upper aqueous phase to a new centrifuge tube.

[0099] 9) Add 1 / 10 volume of 4M NaCl solution to the supernatant, add 2.5 volumes of anhydrous ethanol, add 1 μL of 10 mg / ml glycogen, and mix thoroughly by inverting the container. If the amount of DNA is large enough, filamentous DNA can be observed precipitating out at this time.

[0100] 10) Centrifuge at 13.3 krpm for 2 minutes, discard the supernatant, add 75% ethanol, mix thoroughly by inverting, centrifuge at 13.3 krpm for 2 minutes, and discard the supernatant.

[0101] 11) Add 75% ethanol again and mix by inversion. After centrifuging at 13.3krpm for 2 minutes, discard the supernatant. Gently shake the remaining liquid in the centrifuge to centrifuge to the bottom of the tube. After removing the remaining liquid, let the precipitate dry thoroughly to allow the ethanol to evaporate completely.

[0102] 12) Add 50 μL of 1×TE solution or 10 mM Tris-HCl (pH 8.0) solution to the precipitate to dissolve the DNA. Let it stand at room temperature for several hours or in a 4°C refrigerator overnight. After the DNA is completely dissolved, transfer it to -20°C for storage.

[0103] (3) Genomic DNA ultrasonic fragmentation

[0104] 1) Take 2 μg of genomic DNA into a 200 μL centrifuge tube, add 10 μL of λ-DNA (1 ng / μg) as “Spike-in”, then add an appropriate amount of 1×TE to bring the solution to 125 μL, and transfer it all to a Covaris microTUBE (ultrasonic tube);

[0105] 2) Turn on the Covaris sonicator, place the sonication tube containing the DNA sample into the sonication tank, select the sonication program with a target fragment size of 200bp, and run the program to break the genomic DNA to approximately the target fragment size.

[0106] (4) Genomic DNA fragment size screening

[0107] 1) Take 120 μL of the sonicated DNA solution into a 1.5 mL centrifuge tube, add 72 μL of AMPure magnetic beads (0.6×), mix thoroughly by pipetting, and place at room temperature (23℃) for 10 minutes;

[0108] 2) Place the centrifuge tube on the magnetic rack and let it stand for 5 minutes. At this time, the magnetic beads will gather on one side of the tube wall of the magnetic rack and the solution will become clear.

[0109] 3) Keep the centrifuge tube on the magnetic rack, transfer the supernatant to a new 1.5 mL centrifuge tube, and discard the magnetic bead precipitate;

[0110] 4) Place the new tube containing the supernatant on the magnetic rack for 5 minutes, and then take 185 μL of the supernatant into a new 1.5 mL centrifuge tube to thoroughly remove the magnetic beads from the solution. At this time, DNA fragments larger than 600 bp bound to the magnetic beads will be discarded, while the supernatant will contain DNA fragments smaller than 600 bp.

[0111] 5) Add 105 μL of AMPure magnetic beads to the centrifuge tube, mix thoroughly by pipetting, and place at room temperature (23℃) for 10 minutes. At this time, the total volume of magnetic beads in the solution reaches 1.4 × the DNA solution volume, and the magnetic beads can bind to DNA fragments larger than 150 bp.

[0112] 6) Place the centrifuge tube on the magnetic rack for 5 minutes until the solution becomes clear. Keep the centrifuge tube on the magnetic rack and discard the supernatant (containing non-target DNA fragments smaller than 150 bp).

[0113] 7) Add 500 μL of 80% ethanol to the centrifuge tube, rotate the centrifuge tube quickly 180° on the magnetic rack and repeat 6-10 times. Then let the centrifuge tube stand on the magnetic rack for 1 minute and discard the supernatant. Repeat twice to thoroughly clean the magnetic beads.

[0114] 8) Centrifuge the centrifuge tube at 3,000g for 5 seconds to remove the remaining ethanol to the bottom of the tube. Discard the remaining liquid at the bottom of the tube and keep the centrifuge tube cap open. Let it stand at room temperature for several minutes to allow the remaining ethanol to evaporate completely.

[0115] 9) When the magnetic bead precipitate cracks, add 50 μL of 10 mM Tris-HCl (pH 8.0), blow it up and down more than 15 times, and let it stand at room temperature for 10 minutes.

[0116] 10) Place the centrifuge tube on the magnetic rack for at least 2 minutes to allow the magnetic beads to fully aggregate, and transfer the supernatant to a new 1.5 mL centrifuge tube. Repeat twice to thoroughly remove the magnetic beads from the solution.

[0117] 11) DNA concentration was measured using a Qubit 2.0 fluorometer, and the measurement system is shown in Table 1 below:

[0118] Table 1

[0119] DNA 1μL Qubit dye 1μL Buffer QB 198μL Total 200μL

[0120] 12) If subsequent experimental operations are temporarily interrupted, the genomic DNA after fragment size screening can be stored at -20℃.

[0121] (5) Repair of genome fragment ends

[0122] 1) Take 500 ng of genomic DNA after fragment size screening and perform end-end completion and A-tailing reactions using "KAPA HyperPrep Kits" (DNA library construction kits). The reaction solution preparation system is shown in Table 2 below:

[0123] Table 2

[0124] Genomic DNA fragments 500ng End Repair & A-Tailing Buffer 7μL End Repair&A-Tailing Enzyme Mix 3μL Ultrapure water Add to 60μL Total volume 60μL

[0125] 2) After thoroughly mixing the reaction system, proceed with the reaction on a PCR instrument according to the following procedure:

[0126] 20℃ for 30 minutes

[0127] 65℃ for 30 minutes

[0128] 4℃ ∞

[0129] The temperature of the hot cap should be set to 85℃, instead of the 105℃ set for conventional PCR.

[0130] 3) After the reaction is complete, proceed immediately to the next step of sequencing adapter ligation reaction.

[0131] (6) Methylation sequencing adapter ligation

[0132] 1) Using the “KAPA HyperPrep Kits”, methylated sequencing adapters were ligated to both ends of the genomic DNA fragments after end-filling and A-tailing. The reaction system is shown in Table 3 below, and the preparation method is as follows:

[0133] Table 3

[0134]

[0135]

[0136] 2) After thoroughly mixing the reaction system, incubate it at 20°C for 15 minutes.

[0137] (7) Purification of the ligation reaction products

[0138] 1) Transfer 105 μL of the ligation product from the previous step to a 1.5 mL centrifuge tube, add 126 μL of AMPure magnetic beads (1.2×), mix thoroughly by pipetting, and incubate at room temperature (23℃) for 10 minutes.

[0139] 2) Place the centrifuge tube on the magnetic rack for 5 minutes until the solution becomes clear. Keep the centrifuge tube on the magnetic rack and discard the supernatant.

[0140] 3) Add 500 μL of 80% ethanol to the centrifuge tube, rotate the centrifuge tube quickly 180° on the magnetic rack and repeat 6-10 times. Then let the centrifuge tube stand on the magnetic rack for 1 minute and discard the supernatant. Repeat twice to thoroughly clean the magnetic beads.

[0141] 4) Centrifuge the centrifuge tube at 3,000g for 5 seconds to remove the residual ethanol to the bottom of the tube. Discard the residual liquid at the bottom of the tube and keep the centrifuge tube cap open. Let it stand at room temperature for several minutes to allow the residual ethanol to evaporate completely.

[0142] 5) When the magnetic bead precipitate cracks, add 40 μL of 10 mM Tris-HCl (pH 8.0), blow it up and down more than 15 times, and let it stand at room temperature for 10 minutes.

[0143] 6) Place the centrifuge tube on the magnetic rack for at least 2 minutes to allow the magnetic beads to accumulate fully, and transfer the supernatant to a new 1.5 mL centrifuge tube. Repeat twice to thoroughly remove the magnetic beads from the solution.

[0144] 7) The concentration of purified DNA products was measured using a Qubit 2.0 fluorometer.

[0145] (8) Bisulfite conversion

[0146] 1) The purified ligation product was converted to bisulfite using the "EpiTect Bisulfite Kit" (a kit for bisulfite conversion experiments). First, 800 μL of ultrapure water was added to a tube of "Bisulfite Mix" powder and vortexed thoroughly to dissolve it completely. The solution was then aliquoted into multiple 1.5 mL centrifuge tubes at a volume of 90 μL each and stored at -20°C for long-term storage.

[0147] 2) Prepare the bisulfite reaction system according to the following system, and add each component in the order shown in Table 4 below:

[0148] Table 4

[0149] Genomic DNA fragments 20μL Bisulfite Mix 85μL DNA Protect Buffer 35μL Total volume 140μL

[0150] 3) After thoroughly mixing the reaction system, proceed with the reaction on the PCR instrument according to the following procedure:

[0151]

[0152]

[0153] 4) After the bisulfite conversion reaction is completed, the reaction product is purified by column purification using the "EpiTect Bisulfite Kit", and finally DNA is eluted using 20 μL of elution buffer EB.

[0154] (9) Low cycle number PCR amplification

[0155] 1) High Fidelity HotStart Uracil with ReadyMix (usually abbreviated as HotStart Uracil+ReadyMix, a PCR premixed reagent) was used to amplify bisulfite-converted genomic DNA fragments by PCR. The reaction system is shown in Table 5 below, and the preparation method is as follows:

[0156] Table 5

[0157] DNA after bisulfite conversion 10μL Primers (including i5 index) 3μL Primers (including i7 index) 3μL 2×KAPA Uracil Mix 25μL Ultrapure water 9μL Total volume 50μL

[0158] 2) After thoroughly mixing the reaction system, proceed with the reaction on a PCR instrument according to the following procedure:

[0159]

[0160] Repeat 6 times, 30s-15s-1min.

[0161] 3) After the reaction is complete, transfer the PCR product to a 1.5 mL centrifuge tube and add 50 μL of AMPure magnetic beads (1×) for purification. Finally, elute with 20 μL of 10 mM Tris-HCl (pH 8.0) to obtain the constructed DNA library.

[0162] 4) The concentration of the DNA library was measured using a Qubit 2.0 fluorometer, and an appropriate volume of the library was sent to a next-generation sequencing company for high-throughput sequencing.

[0163] (10) Bioinformatics analysis of WGBS sequencing data

[0164] 1) Use the software "FastQC" to perform quality checks on the raw sequencing files in fastq format;

[0165] 2) Use the software "Trimmomatic" to remove sequencing adapter sequences and low-quality sequences from the original sequencing file;

[0166] 3) Use the software "Bismark Mapper" to align the original sequencing files to the hg19 or hg38 human reference genome;

[0167] 4) Use the software "MethylDackel" to extract DNA methylation level information at all CpG sites on the genome;

[0168] 5) Use the software "Wig / BedGraph-to-bigWig" to convert the bed format file containing all CpG site methylation information into a bigwig file, and then perform visualization analysis in the IGV genome browser.

[0169] (11) Analysis of Experimental Results

[0170] 1) Starting from the genome-wide DNA methylation level, see [reference needed]. Figure 1c It was found that, compared with the IMR90 cells that had not been cultured for a long time at the beginning (left column), the control cells that had been cultured for a long time and overexpressed mCherry showed a significant decrease in the overall DNA methylation level (middle column), while this decrease was very effectively inhibited in the experimental group cells that overexpressed LSH (right column).

[0171] 2) See the visualization results. Figure 1d It can be clearly seen that, compared with the young cells that were not cultured for a long time at the beginning (P9), the control group cells (mCherry P19) showed significant heterochromatin-specific methylation loss during the long-term continuous expansion process, while this loss was effectively inhibited in the experimental group cells with LSH overexpression (LSH P19).

[0172] 3) From the DNA methylation heatmap, see Figure 1e It was observed that, compared with young IMR90 cells that had not proliferated extensively at the beginning, control cells overexpressing mCherry showed a large-scale and significant loss of DNA methylation after long-term passage culture, while LSH overexpression could effectively delay this loss of methylation.

[0173] 4) A detailed analysis was conducted on the changes in DNA methylation in the early and late replication regions during long-term passage, respectively. (See [reference needed]) Figure 1f As shown in the figure, the inhibitory effect of LSH on DNA methylation loss is more pronounced in the late DNA replication region (i.e., the heterochromatin region).

[0174] All of these results demonstrate that LSH overexpression can effectively inhibit the loss of heterochromatin DNA methylation during the long-term continuous proliferation of IMR90 fibroblasts, providing a new and effective perspective and means for intervening in the epigenetic imbalance that occurs during cell therapy.

[0175] 5. LSH overexpression delays transposon activation and inflammatory response during long-term proliferation.

[0176] The differences in gene expression in the mCherry overexpression control group and the hLSH overexpression experimental group before and after long-term passage culture were detected using RNA-seq transcriptome sequencing technology. The specific experimental steps are as follows:

[0177] (1) Total RNA extraction and sequencing from cells:

[0178] 1) Remove IMR90 cells from the incubator, discard the culture medium and add 1 mL TRIzol directly to the culture dish. Use a pipette to repeatedly aspirate and mix, and let stand for about 5 minutes to allow the cells to fully lyse.

[0179] 2) Add 0.2 mL of chloroform to 1 mL of TRIzol lysis buffer, and vortex at maximum speed for 20 s. After mixing thoroughly, let stand and incubate for 2-3 minutes.

[0180] 3) Centrifuge at 12,000×g for 15 minutes at 4℃. The mixture will be separated into a lower organic phase, a middle phase and an upper aqueous phase containing RNA.

[0181] 4) Transfer 0.4 mL of the upper aqueous phase containing RNA to a new 1.5 mL centrifuge tube, add 10 μg of RNase-free Glycogen and 0.5 mL of isopropanol to precipitate the RNA, mix thoroughly, incubate at 4 °C for 10 minutes, and then centrifuge at 12,000 × g for 10 minutes at 4 °C. After centrifugation, a white precipitate can be observed at the bottom of the tube.

[0182] 5) After carefully aspirating the supernatant, wash the RNA precipitate twice with 75% ethanol, centrifuge at 7,500×g for 5 minutes at 4°C, aspirate the supernatant, and let stand for 5-10 minutes to allow the ethanol to evaporate completely.

[0183] 6) Add 20-50 μL of RNase-free ultrapure water to dissolve the RNA precipitate. After complete dissolution, use a Nanodrop micro-spectrophotometer to determine the RNA concentration. At the same time, detect the OD values ​​at wavelengths of 260 nm and 280 nm to assess the RNA purity and quality. Then send it to a next-generation sequencing company for RNA-seq library construction and sequencing.

[0184] (2) Bioinformatics analysis of RNA-seq transcriptome sequencing data

[0185] 1) Use the software "FastQC" to perform quality checks on the raw sequencing files in fastq format;

[0186] 2) Use the software "Trim Galore!" to remove sequencing adapter sequences and low-quality sequences from the original sequencing file;

[0187] 3) Use the software "RNA STAR" to align the raw sequencing files to the hg19 or hg38 human reference genome;

[0188] 4) Use the software "featureCounts" to calculate the number of reads that can be matched on each coding gene or transposon element;

[0189] 5) The DESeq2 software was used to identify genes and transposon elements that were significantly differentially expressed during long-term continuous proliferation;

[0190] 6) Use the software "Volcano Plot" to draw a volcano plot of differentially expressed genes, and use the software "heatmap2" to draw a heatmap of differentially expressed genes;

[0191] 7) Use the online tool "DAVID" to perform GO analysis on differentially expressed genes to identify biological pathways with significant changes.

[0192] (3) Analysis of experimental results

[0193] 1) Differential analysis of transposon element expression in IMR90 cells during long-term passage was performed. (See [reference needed]) Figure 2a As shown, it can be observed that after extensive cell proliferation, there is a large amount of abnormal activation of transposon elements in the genome, and the activation of the vast majority of transposon elements can be significantly inhibited by LSH overexpression. Figure 2b and Figure 2c Furthermore, compared to transposon elements that cannot be inhibited by LSH overexpression, transposon elements that can be effectively inhibited by LSH overexpression exhibit a more dramatic decrease in DNA methylation during long-term sustained amplification, and this loss of DNA methylation can be inhibited by LSH overexpression. Figure 2d This indicates that LSH overexpression can prevent the abnormal activation of various transposon elements in heterochromatin regions by delaying the loss of DNA methylation during long-term proliferation.

[0194] 2) Based on the GO enrichment analysis results of differentially expressed genes, see [link to relevant documentation]. Figure 2eAs shown, a large number of inflammation-related genes were significantly upregulated during the long-term expansion of IMR90 cells, and this inflammatory response was effectively suppressed in the LSH overexpression experimental group compared with the mCherry control group. These inflammation-related genes originated from multiple inflammatory signaling pathways, including the type I interferon pathway (such as IFI6 / 27 / 30 / 44, ISG15, OAS1 / 2 / 3, GBP3, and DDX60L), aging-related secretory phenotypic factors (SASP factors, such as IL-6 and MMP10), the TNF signaling pathway (TRAF1, TNFRSF10A / 11B / 14, and BIRC3), nucleic acid sensing proteins (STING and RIG-I), and innate immune pathways (CD14, C6, PTX3, and NLRP10), etc. Figure 2f These results demonstrate that LSH overexpression can reduce the occurrence of chronic inflammatory responses by inhibiting the activation of transposon elements during long-term continuous proliferation, thereby preventing the activation of various cytoplasmic nucleic acid sensing proteins.

[0195] 6. LSH overexpression improves senescence-related cell phenotypes caused by long-term proliferation.

[0196] (1) LSH overexpression enhances the proliferation ability of senescent cells.

[0197] IMR90 cells were continuously passaged until they entered the replicative senescence stage. At each passage, a small number of cells were counted using a cell counting chamber, and a fixed number of cells were seeded into new cell culture dishes for further passage. Based on the cell count results at each passage, growth curves were plotted for the mCherry overexpression control group and the LSH overexpression experimental group. (See [reference]). Figure 2g It was found that LSH overexpression can significantly delay the cell cycle arrest phenotype during the replication senescence process, enabling IMR90 cells to maintain a strong proliferative capacity after undergoing a large number of cell proliferations.

[0198] (2) LSH overexpression improves the phenotype of increased volume in senescent cells.

[0199] Flow cytometry was used to detect the size of senescent cells after long-term passage culture. Cell diameter was measured by FSC intensity. (See [link to relevant documentation]). Figure 2h It was found that the control group cells exhibited a significant increase in cell volume during the long-term continuous expansion process, while LSH overexpression could significantly improve this cell enlargement phenotype associated with cell senescence.

[0200] These results demonstrate that LSH overexpression can not only mitigate the abnormal activation of transposon elements and the generation of chronic inflammatory responses during long-term proliferation by inhibiting heterochromatin DNA methylation loss, but also further improve various cellular senescence-related phenotypes such as slowed cell proliferation and increased cell volume. This provides a new and effective means to improve the long-term maintenance of cell function during cell therapy and enhance the clinical therapeutic effects of cell drugs.

[0201] Example 2

[0202] Overexpression of LSH can enhance the proliferation and effector function of human T lymphocytes during long-term clonal expansion. The specific experimental steps are as follows:

[0203] 1. Preparation of recombinant lentivirus packaging for LSH overexpression

[0204] (1) Construction of LSH-overexpressing recombinant lentiviral expression vector

[0205] The LSH coding sequence and the pCDH lentiviral vector were linearized using PCR, and homologous arms were introduced at both ends of the fragment. Then, the LSH coding sequence was ligated to the pCDH lentiviral expression vector using Gibson Assembly to construct the pCDH-LSH recombinant plasmid. The correctness of the recombinant plasmid was verified by transforming DH5α competent bacteria and performing single-colony Sanger sequencing. At the same time, the control gene mCherry was ligated to the pCDH lentiviral vector using the same method to construct the pCDH-mCherry control recombinant plasmid.

[0206] (2) Packaging and preparation of LSH-overexpressing recombinant lentivirus

[0207] 1) One day before transfection, passage 293T cells into an appropriate number of 10cm cell culture dishes, with approximately 4 × 10⁻⁶ cells seeded per dish. 6 about;

[0208] 2) When the cell confluence reaches approximately 90%, cell transfection is performed using the calcium phosphate transfection method. The single-plate transfection system and preparation method are shown in Table 6 below:

[0209] Liquid A:

[0210] Table 6

[0211] pCDH lentivirus recombinant plasmid 15μg REV lentivirus packaging plasmid 6μg RRE lentivirus packaging plasmid 7.5μg VSVG lentivirus packaging plasmid 6μg <![CDATA[2.5M CaCl2]]> 50μL Sterile ultrapure water Add to 500μL Total volume 500μL

[0212] Solution B: 500 μL 2×HBS buffer

[0213] 3) After preparing solution A, let it stand at room temperature for 3 minutes. Then add solution A to solution B and quickly mix by pipetting 30-40 times to generate a suitable amount of bubbles. At this time, you can observe that the transfection mixture produces a slight white precipitate (i.e., calcium transfection particles). Add the mixed liquid to 293T cells, shake gently, and then place them in a 37℃ cell culture incubator (containing 5% CO2) for culture.

[0214] 4) 6-8 hours after transfection, aspirate the supernatant containing the transfection reagent and add 15 mL of fresh, preheated cell culture medium to continue culturing.

[0215] 5) After changing the culture medium for 48 hours, collect the cell supernatant containing lentivirus, store it temporarily at 4°C, and add 10 mL of fresh preheated cell culture medium to continue culturing.

[0216] 6) After changing the culture medium for 72 hours, collect the virus-containing supernatant again and combine it with the cell supernatant collected the first time. After centrifugation, take the supernatant and filter it through a 0.45μm syringe filter to obtain the lentivirus stock solution.

[0217] 7) The virus stock solution was concentrated by centrifugation using an ultrafiltration tube with a molecular weight cutoff of 100kD. The concentration factor was 30 times. When the virus reached the corresponding volume, 10mL of human T cell culture medium was added for resuspending, and centrifugation was performed again to the target concentration volume.

[0218] 8) In a biosafety cabinet, the concentrated virus suspension was filtered using a 0.22μm syringe filter and then aliquoted into appropriate volumes and stored at -80℃.

[0219] 2. Lentiviral infection of human T cells to achieve stable LSH overexpression

[0220] After isolating T lymphocytes from human PBMCs, they were activated in vitro using αCD3 / αCD28 antibodies, and the LSH-encoding gene was introduced into the T lymphocytes using lentiviral infection to detect the role of LSH overexpression in the long-term clonal expansion of T lymphocytes (experimental procedure see [link]). Figure 3a The specific experimental steps are as follows:

[0221] (1) Isolation of human peripheral blood mononuclear cells

[0222] 1) Dilute human peripheral blood according to a fixed ratio. Take 50 mL of concentrated human peripheral blood and add 70 mL of blank RPMI 1640. Mix the two thoroughly.

[0223] 2) Add 15 mL of Ficoll-Paque to a 50 mL centrifuge tube. TMLymphocyte separation medium was prepared, and 30 mL of diluted human peripheral blood was slowly added along the wall of the centrifuge tube. Then, the centrifuge tube was gently placed in a horizontal centrifuge, and the centrifuge speed was adjusted to 3 and 1. The tube was centrifuged at 2,200 rpm for 25 min at 20°C.

[0224] 3) After centrifugation, gently remove the centrifuge tube from the centrifuge. You can observe that the liquid in the centrifuge tube separates into four layers from top to bottom: plasma layer, mononuclear cell layer, lymphocyte separation fluid layer, and granulocyte-erythrocyte layer. Slowly aspirate the top layer of plasma, and then slowly aspirate the second layer of mononuclear lymphocytes and transfer it to a 50mL centrifuge tube.

[0225] 4) Add serum-free RPMI 1640 medium to 50 mL of liquid containing monocytes, mix thoroughly, and centrifuge at 1,800 rpm for 10 min at 20 °C, with both the ascending and descending speeds set to 9.

[0226] 5) After centrifugation, cell pellet can be observed at the bottom of the centrifuge tube. Carefully aspirate the supernatant and resuspend the cells in serum-free RPMI 1640 medium.

[0227] 6) Centrifuge at 1,600 rpm for 6 min at 20°C, and resuspend the cell pellet in serum-free RPMI 1640 medium after centrifugation;

[0228] 7) Take a small amount of cells and count them using a cell counting chamber. Add trypan blue to analyze cell viability. Then, aliquot the cells into appropriate quantities and freeze them.

[0229] (2) In vitro activation of T cells in human peripheral blood

[0230] 1) Remove cryopreserved human peripheral blood mononuclear cells (PBMCs) from liquid nitrogen, thaw rapidly in a 37°C water bath, count cells using trypan blue, and adjust the cell density to 1×10⁻⁶. 7 The cells were seeded into 24-well plates and cultured in a 37°C cell culture incubator (containing 5% CO2).

[0231] 2) After culturing the revived PBMCs overnight, cell counting was performed again using trypan blue, and the required volume of αCD3 / αCD28 magnetic beads (Gibco, 40203D) was calculated based on the cell count, with each 4 × 10⁻⁶ bead... 5 Cells require 1 μL of magnetic beads for activation;

[0232] 3) Clean the αCD3 / αCD28 magnetic beads with culture medium. First, add 1 mL of blank RPMI 1640 culture medium to a sterile flow cytometer, then add the corresponding volume of magnetic beads. Gently pipette to mix, then place the sterile flow cytometer on a magnetic rack for about half a minute. After the magnetic beads gather to the side near the wall of the magnetic rack, aspirate the supernatant of the culture medium.

[0233] 4) Add PBMC cell suspension to the magnetic bead precipitate, gently pipette to mix, and incubate on a rotary mixer for 0.5 h. Then transfer the cells to a 24-well plate and incubate overnight in a 37°C cell culture incubator (containing 5% CO2).

[0234] (3) Lentiviral infection of human T cells

[0235] 1) After 24 hours of activation, the T cell density was diluted to 5 × 10⁻⁶. 5 / mL, and add 500μL of cell suspension to a 24-well plate;

[0236] 2) Remove the frozen lentivirus from the -80℃ freezer, thaw it slowly on ice, and then bring it to room temperature. Add 500 μL of virus suspension to each well and mix it with the cell suspension in a 1:1 ratio. At the same time, add Polybrene to promote infection (final concentration of 8 μg / mL) and add 100 U of cytokine IL-2 and mix thoroughly.

[0237] 3) Seal the 24-well plate containing the cultured T cells with plastic wrap and centrifuge at 2,000×g for 90 min. The centrifuge temperature is set to 30℃, the ramp rate is set to 3 and the deceleration rate is set to 1. After centrifugation, remove the plate and place it in a 37℃ cell culture incubator (containing 5% CO2) for incubation and infection.

[0238] 4) After centrifugation for 4-6 hours, add 1 mL of cell culture medium to each well, mix thoroughly, and return to a 37°C cell culture incubator (containing 5% CO2) for further culture.

[0239] 5) The day after lentivirus infection, carefully aspirate 1500 μL of supernatant from the cell culture wells and add 500 μL of lentivirus suspension again. After adding Polybrene and IL-2, perform a second centrifugation infection. After infection, add 1 mL of cell culture medium 4-6 h and place in a 37℃ cell culture incubator (containing 5% CO2) for continued culture.

[0240] 6) On day 3, discard the virus-containing cell supernatant and add 2 mL of preheated fresh T cell culture medium, along with IL-2 (100 U) to continue culturing;

[0241] 7) On day 5, flow cytometry analysis was performed using the fluorescent protein carried by the exogenous expressed gene to determine the lentivirus infection efficiency, and the successfully infected T cells were sorted out by flow cytometer for further culture.

[0242] (4) Western Blot detection of overexpression efficiency

[0243] 1) Cell Collection: Human T cells cultured in vitro were collected into centrifuge tubes and centrifuged at 1,800 rpm for 10 min. After discarding the supernatant, the cells were resuspended in 1 mL of 1×PBS. Cell counting was performed using trypan blue, followed by centrifugation and discarding of the supernatant. Cells were then counted at 1×10⁻⁶ ppm. 6 Cells were resuspended in 100 μL of 1×PBS and 100 μL of 2×SDS loading buffer were added. After thorough mixing, the cells were heated in a 100°C metal bath for 10 min to lyse. The protein lysis buffer was then centrifuged at 13,300 rpm for 5 min. The protein supernatant was transferred to a new centrifuge tube, and cell residue was discarded. Finally, the protein sample was stored at -20°C for long-term preservation.

[0244] 2) SDS-PAGE gel electrophoresis: Take the frozen protein samples out of the -20℃ freezer and heat them in a 100℃ metal bath for 5 min. After the samples have cooled, load them into the SDS-PAGE gel wells in a certain order. Pour an appropriate amount of 1× electrophoresis buffer into the electrophoresis tank and electrophores at a constant current of 20mA for 1.5 h.

[0245] 3) Protein transfer: Cut a PVDF membrane of appropriate size and soak it in methanol to activate it for 1 min. Place a sponge, four layers of filter paper, SDS-PAGE gel, PVDF membrane, four layers of filter paper and sponge in the transfer clamp in the following order. Then place the transfer clamp into the transfer tank, making sure that the side with the gel is facing the negative electrode. Pour in an appropriate amount of 1× transfer buffer. After turning on the power, transfer the membrane at a constant current of 200 mA for 2 h to transfer the protein band from the gel to the PVDF membrane.

[0246] 4) Sealing: After the transfer is completed, remove the PVDF membrane and place it in a sealing solution containing 5% skim milk powder (prepared by TBST). Shake slowly on a shaker at room temperature for 1-2 hours to seal.

[0247] 5) Primary antibody incubation: After blocking, cut out the target band of the corresponding molecular weight, put it into the primary antibody that has been appropriately diluted with blocking solution, and incubate overnight on a shaker at 4°C;

[0248] 6) Washing the membrane: After the primary antibody incubation is complete, immerse the PVDF membrane in TBST and wash it on a shaker at room temperature for 10 minutes each time.

[0249] 7) Secondary antibody incubation: Place the washed PVDF membrane into the secondary antibody that has been appropriately diluted with TBST, and incubate on a shaker at room temperature for 1-2 hours;

[0250] 8) Washing the membrane: After the secondary antibody incubation is completed, immerse the PVDF membrane in TBST and wash it on a shaker at room temperature for 10 minutes each time.

[0251] 9) Protein signal detection: After thoroughly washing the PVDF membrane, dab it dry on absorbent paper and flatten it on a plastic film. Add chemiluminescent HRP substrate (A solution and B solution are mixed in a 1:1 ratio) evenly to the surface of the PVDF membrane. After reacting for an appropriate time, place it in a dark box for pressing. Then, develop it in a darkroom using an automatic X-ray film processor.

[0252] 10) Test results: Figure 3b and Figure 3c The study showed changes in LSH protein expression levels in wild-type and LSH-overexpressing human T lymphocytes. Specifically, by examining the expression of endogenous LSH protein in wild-type T lymphocytes, it was found that LSH expression gradually decreased with prolonged in vitro culture time. Figure 3b In T lymphocytes infected with LSH-overexpressing lentivirus, Western blotting using an antibody targeting the HA tag carried by the exogenous LSH expression element revealed successful expression of the exogenous LSH protein in human T cells, demonstrating that lentiviral infection successfully introduced the exogenous LSH expression element into human T cells. Simultaneously, Western blotting using an antibody targeting the C-terminal sequence of LSH showed significant LSH overexpression in LSH-enhanced human T cells compared to those infected with mCherry control lentivirus. Figure 3c This demonstrates the success of LSH overexpression mediated by lentiviral infection.

[0253] 3. LSH overexpression improves the functional degradation of T cells during long-term clonal expansion.

[0254] (1) LSH overexpression inhibits DNA methylation loss in heterochromatin regions of T cells.

[0255] Changes in DNA methylation levels of human T lymphocytes during long-term in vitro clonal expansion were detected using WGBS technology (see [link]). Figure 3d We were able to find that LSH overexpression can effectively delay the decrease in DNA methylation level in heterochromatin regions during long-term proliferation, indicating that LSH overexpression in human T lymphocytes can indeed significantly improve the maintenance efficiency of DNA methylation in heterochromatin regions, thus preventing the imbalance of epigenetic information in T cells during long-term continuous proliferation.

[0256] (2) LSH overexpression promotes the maintenance of effector function of T cells during long-term expansion.

[0257] Changes in the transcriptome of human T lymphocytes during long-term in vitro clonal expansion were detected by RNA-seq sequencing (see [link]). Figure 3e and 3f This study revealed that LSH overexpression significantly enhances the maintenance of T cell effector function during long-term continuous proliferation. By comparing differentially expressed genes in the control group (4RFP overexpression) and the experimental group (hLSH overexpression) after extensive clonal expansion, we observed a significant increase in the expression of numerous T cell effector function-related genes in LSH-overexpressing cells (see...). Figure 3e This includes effector-related cytokines (such as IL-13, CSF1 / 2, CCL4, and IFNG), cell-killing-related proteins (such as GZMA, GZMB, and FASLG), effector cell surface receptors (such as IL4R and IL2RA), and effector cell differentiation-related transcription factors (such as JAK3-STAT3, BATF, and BHLHE40). Meanwhile, GSEA analysis results showed (e.g.) Figure 3f The expression of genes related to T cell effector function decreases significantly during long-term continuous expansion in vitro, while LSH overexpression can effectively delay the degradation of this T cell effector function. This indicates that LSH overexpression can promote the long-term effective maintenance of T cell effector function by inhibiting the imbalance of epigenetic information during long-term clonal expansion of T lymphocytes.

[0258] (3) LSH overexpression enhances the proliferation capacity of T cells during long-term clonal expansion.

[0259] Human T cells from the mCherry overexpression control group and the LSH overexpression experimental group were passaged in vitro for a long period of time. The number of T cells increased daily during the culture process to plot the growth curves of the control and experimental groups (e.g., ...). Figure 3g The growth curves showed that, during long-term in vitro culture, the LSH-overexpressing experimental group exhibited a significant growth advantage compared to the control group. Simultaneously, flow cytometry was used to detect the level of Ki-67, a proliferation-related marker in T lymphocytes (e.g., ...). Figure 3h The study found that, compared with the control group, the experimental group cells overexpressing LSH had significantly higher Ki-67 levels, and this increase was observed in CD8+ cells. + This effect was more pronounced in T lymphocyte subsets. Therefore, these results indicate that LSH overexpression can enhance T lymphocyte (especially CD8) expression. + The cell proliferation capacity of T lymphocytes during long-term, large-scale expansion.

[0260] (4) LSH overexpression slows down T cell death in the late stage of clonal expansion.

[0261] Given that T cells in the LSH-overexpression experimental group exhibited a significant survival advantage in the late stage of in vitro proliferation, we further examined the survival rate of T cells during this late stage. We used a dead-cell fluorescent dye (A780) and flow cytometry to detect changes in the live / dead cell ratio during the late stage of cell clonal expansion (e.g., Figure 3i We observed that as the culture time increased, the proportion of live cells in the control group decreased significantly, while LSH overexpression could effectively inhibit the gradual increase of this cell death rate, thereby significantly improving the cell survival ability of late-stage clonal expansion T cells.

[0262] (5) LSH overexpression delays the downregulation of CD27 in T cells during long-term clonal expansion.

[0263] Since the downregulation of co-stimulatory molecules CD27 and CD28 is a common cellular phenotype during T cell aging in vivo and long-term in vitro expansion, and CD27 and CD28 co-stimulatory molecules play an important role in T cell's effective response to antigen stimulation and effector function, we further used flow cytometry to detect changes in the expression of CD27 molecules on the surface of T lymphocytes during long-term clonal expansion. The results showed that the proportion of CD27-positive cells decreased significantly during the long-term proliferation of control cells, while LSH overexpression could effectively prevent this downregulation of CD27 molecules. This indicates that LSH overexpression can effectively improve the reduction in the expression level of co-stimulatory molecules in T lymphocytes during long-term clonal expansion and enhance the T cell's ability to respond to antigen stimulation.

[0264] These results indicate that LSH overexpression can effectively delay the loss of DNA methylation in heterochromatin regions during long-term clonal expansion of T lymphocytes and significantly enhance the cell division and survival capabilities of T cells in the late stage of continuous proliferation. At the same time, LSH overexpression can also effectively promote the long-term maintenance of T lymphocyte antigen response capabilities and effector functions, which provides a new optimization strategy for improving the efficacy of CAR-T and other immune cell adoptive therapies.

[0265] Based on the aforementioned experimental results of this invention, LSH overexpression can effectively inhibit DNA methylation loss in heterochromatin regions during the long-term continuous proliferation of human embryonic lung IMR90 fibroblasts, preventing the activation of abnormal transposon elements in this genomic region, thereby reducing chronic inflammatory responses and significantly improving several replication-related senescence phenotypes such as cell cycle arrest and cell volume increase. Simultaneously, LSH overexpression can effectively improve the stability of heterochromatin in human T lymphocytes during long-term clonal expansion, thereby significantly enhancing the proliferative capacity and survival of T cells in the late stage of continuous proliferation and promoting the long-term effective maintenance of T cell effector function.

[0266] The innovative research findings of this invention strongly demonstrate that the insufficient DNA methylation maintenance efficiency caused by the dense chromatin environment in heterochromatin regions is the reason for the continuous loss of DNA methylation during cell proliferation. Overexpression of LSH can effectively improve the DNA methylation maintenance efficiency in heterochromatin regions to prevent DNA methylation loss, thereby improving several phenotypic defects caused by continuous cell proliferation, such as decreased cell proliferation capacity, upregulation of inflammatory response gene expression, and degradation of immune cell effector function. This provides a new perspective for understanding epigenetic imbalances and cellular functional degradation during cell proliferation, and offers a new strategy for effectively maintaining cell function in adoptive cell therapy, with broad application value in enhancing the efficacy of various cell therapies. Therefore, this invention provides a new research perspective and intervention method for effectively improving heterochromatin stability and enhancing the efficacy of cell therapy, and has broad value in the clinical application of various cell drugs.

[0267] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. Application of LSH-overexpressing recombinant vectors, LSH-overexpressing recombinant lentiviruses, or LSH-overexpressing recombinant cells in the preparation of drugs that enhance the efficacy of CAR-T cell immunotherapy.

2. The application as described in claim 1, characterized in that, The drug in question is a cell therapy drug for CAR-T cell therapy.

3. The application as described in claim 1, characterized in that, The LSH overexpression recombinant vector is obtained by ligating the LSH gene coding sequence into a viral vector.

4. The application as described in claim 3, characterized in that, The viral vector is a lentiviral vector, a retroviral vector, or an AAV viral vector.

5. The application as described in claim 3, characterized in that, The LSH-overexpressing recombinant lentivirus was produced by co-transfecting the LSH-overexpressing recombinant vector and the viral packaging plasmid into human embryonic kidney HEK293T cells.

6. The application as described in claim 5, characterized in that, The viral packaging plasmids are pMDLg / pRRE, pRSV / REV, and pVSVG.

7. The application as described in claim 1, characterized in that, The LSH-overexpressing recombinant cells were obtained by infecting host cells with an LSH-overexpressing recombinant virus, and the host cells were human peripheral blood-derived T lymphocytes.

Images