阅读说明：本技术 一种诱导死亡的重组细胞及其应用 (Death-inducing recombinant cell and application thereof ) 是由 赖良学 刘洋 邹庆剑 杨洋 李川 陈敏 于 2021-08-27 设计创作，主要内容包括：本发明提供了一种诱导死亡的重组细胞及其应用,所述诱导死亡的重组细胞为经过OCT4基因编辑系统编辑后,OCT4基因中插入了iCASP9基因的多能干细胞。本发明还提供了所述诱导死亡的重组细胞的构建方法。本发明所述重组多能干细胞可以在特定药物诱导下实现自杀,而当干细胞分化为功能性细胞后,不受药物控制,可正常行使功能。多能干细胞用于疾病治疗过程中,通过给药可以清除残留多能干细胞,降低移植细胞的致瘤风险。所述重组细胞具有良好的全能性,可应用于相关的临床干细胞治疗。(The invention provides a death-inducing recombinant cell and application thereof, wherein the death-inducing recombinant cell is a pluripotent stem cell which is edited by an OCT4 gene editing system and has an iCASP9 gene inserted into an OCT4 gene. The invention also provides a construction method of the death-inducing recombinant cell. The recombinant pluripotent stem cell can realize suicide under the induction of a specific medicament, and can normally function without being controlled by the medicament after the stem cell is differentiated into a functional cell. The pluripotent stem cells are used in the disease treatment process, residual pluripotent stem cells can be eliminated through administration, and the tumor risk of transplanted cells is reduced. The recombinant cell has good totipotency and can be applied to relevant clinical stem cell treatment.)

1. An OCT4 gene-targeted sgRNA, wherein the OCT4 gene-targeted sgRNA comprises mgRNA or hgRNA;

wherein, the coding sequence of the mgRNA comprises a nucleotide sequence shown in SEQ ID No.1, and the coding sequence of the hgRNA comprises a nucleotide sequence shown in SEQ ID No. 2.

2. An OCT4 gene editing system, wherein the OCT4 gene editing system comprises the OCT4 gene targeted sgRNA of claim 1 and Cas 9;

preferably, the Cas9 comprises spCas 9;

preferably, the sgRNA targeting the OCT4 gene is linked to Cas9 in the same expression vector;

preferably, the OCT4 gene editing system further comprises a donor plasmid;

preferably, the donor plasmid comprises iCASP9 gene and a selectable marker gene;

preferably, the screening marker gene comprises a fluorescent screening marker gene and/or a drug screening marker gene;

preferably, the fluorescent selectable marker gene comprises a Tdtomato-encoding gene;

preferably, the drug selection marker gene comprises a puromycin resistance gene.

3. A death-inducing recombinant cell comprising the sgRNA of claim 1 targeted to the OCT4 gene;

preferably, the death-inducing recombinant cell comprises the OCT4 gene editing system of claim 2;

preferably, the death-inducing recombinant cell is a pluripotent stem cell with the iCASP9 gene inserted into the OCT4 gene after being edited by the OCT4 gene editing system of claim 2;

preferably, the death-inducing drug comprises AP 1903.

4. A method of constructing a death-inducing recombinant cell according to claim 3, comprising:

constructing an expression vector containing sgRNA of a targeted OCT4 gene and Cas9, constructing a donor plasmid, introducing the expression vector and the donor plasmid into a receptor cell, and screening positive clones to obtain the death-inducing recombinant cell.

5. The method of claim 4, wherein the expression vector is constructed by:

synthesizing a coding sequence of sgRNA of a targeted OCT4 gene, and connecting the coding sequence to a gene editing vector to obtain the expression vector containing the sgRNA of the targeted OCT4 gene and the Cas 9;

preferably, the gene editing vector comprises a PX330 vector.

6. The method of claim 4 or 5, wherein said introducing comprises transfection;

preferably, the recipient cell comprises a pluripotent stem cell.

7. The method for constructing recombinant cell death-inducing cells according to any one of claims 4 to 6, wherein said step of screening positive clones comprises:

performing fluorescence screening and/or drug screening according to the screening marker gene on the donor plasmid;

preferably, the fluorescence screening comprises flow sorting;

preferably, the drug screen comprises a puromycin screen.

8. The method for constructing recombinant cell to induce death according to any one of claims 4 to 7, further comprising the steps of apoptosis detection, pluripotency identification and tumorigenic capacity detection.

9. The method for constructing a recombinant cell that induces death according to any one of claims 4 to 8, wherein the preparation method comprises:

(1) constructing an expression vector containing sgRNA targeting the OCT4 gene and Cas 9:

synthesizing a coding sequence of sgRNA of a targeted OCT4 gene, and connecting the coding sequence to a PX330 vector to obtain the expression vector containing the sgRNA of the targeted OCT4 gene and the Cas 9;

(2) constructing a donor plasmid containing an iCASP9 gene and a screening marker gene;

(3) introducing the expression vector and donor plasmid into a pluripotent stem cell by transfection;

(4) screening positive clones:

carrying out flow sorting and/or puromycin screening according to the screening marker gene on the donor plasmid;

(5) and carrying out apoptosis detection, pluripotency identification and tumorigenic capacity detection on the obtained positive clone to obtain the death-inducing recombinant cell.

10. Use of any one or a combination of at least two of the sgRNA targeting the OCT4 gene of claim 1, the OCT4 gene editing system of claim 2, the death-inducing recombinant cell of claim 3, or the method of constructing a death-inducing recombinant cell of any one of claims 4 to 9 in the preparation of a stem cell therapeutic drug and/or agent.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a death-inducing recombinant cell and application thereof.

Background

Stem Cells (Stem Cells) are a type of pluripotent Cells that have the ability to self-renew and self-replicate. Under certain conditions, it can differentiate into all cells in the body, further form all tissues and organs of the body, and has great application value in the aspects of organ regeneration, repair and disease treatment. At present, retinal pigment epithelial cells, pancreatic progenitor cells and the like from stem cells enter a clinical test stage and have good treatment effect. However, the stem cell product is usually a mixed cell population, and the undifferentiated stem cells with a certain proportion are included in the target therapeutic cells, so that the formation of teratoma after transplantation is easily caused due to the unlimited proliferation and self-differentiation of the undifferentiated stem cells, which is also a safety hazard to be overcome in clinical treatment.

To address this problem, researchers have included methods of delivery by non-integrating viral vectors to increase safety (Takahashi, t., Kawai, t., Ushikoshi, h., Nagano, s., Oshika, h., Inoue, m., Kunisada, t., Takemura, g., Fujiwara, h., and Kosai, K. (2006.) identification and isolation of infectious stem cell-driven target cells by infectious viral infection and isolation, and also directed killing by oncolytic viruses after teratoma formation (Nagano, s., Oshika, h., jiwara, h., s., K., and No. 5. complicated procedures). Suicide gene strategies have also been used for cell killing, with the use of herpes simplex virus thymidine kinase (HSV-TK) gene and its prodrug, ganciclovir, for a wide range of research and applications. HSV-TK initiates the phosphorylation cascade of ganciclovir, and the product produced competes with deoxyguanosine triphosphate and is incorporated into the replicating DNA, leading to cell death. But a potential problem is that the system may be immunogenic to humans.

Therefore, the development of more specific screening tools to target the elimination of tumorigenic undifferentiated cells has become a focus and direction of research. Wu et al improve the safety of transplanted cells by knocking in a suicide Gene at the SOX2 site of a Pluripotent Gene (Wu Y, Chang T, Long Y, et al. Using Gene Editing to inventory a safety System for pluralityot Stem-Cell-Based therapeutics [ J ]. iSience, 2019, 22.). However, SOX2 is not only an important transcriptional regulator of stem cell pluripotency, but is also involved in the differentiation of neural progenitor cells and the formation of foregut endoderm, and is therefore unsuitable for use with cell types expressing SOX2 at high levels, such as neuronal progenitor cells and foregut cells.

Therefore, how to provide a pluripotent stem cell which has low tumorigenicity, good treatment effect and higher safety and can be applied to clinical treatment becomes a problem to be solved urgently.

Disclosure of Invention

Aiming at the defects and practical requirements of the prior art, the invention provides a death-inducing recombinant cell and application thereof, wherein the iCASP9 gene is inserted into the OCT4 gene of the pluripotent stem cell, and the prepared recombinant dry cell can spontaneously induce death under the treatment of a medicament without influencing differentiated cells, so that only the stem cell with the division capability is killed in a targeted manner, the tumorigenicity is lower, and the cell is safer.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an sgRNA targeting an OCT4 gene, the sgRNA targeting an OCT4 gene comprising either mgRNA or hgRNA;

wherein, the coding sequence of the mgRNA comprises a nucleotide sequence shown in SEQ ID No.1, the coding sequence of the hgRNA comprises a nucleotide sequence shown in SEQ ID No.2, and a thickened part is a targeting site.

SEQ ID No.1：

SEQ ID No.2：

In the invention, mgRNA targets the OCT4 gene of a mouse, hgRNA targets the OCT4 gene of a human, and both sgRNAs have good specificity and targeting property, low off-target rate and high editing efficiency.

In a second aspect, the invention provides an OCT4 gene editing system, where the OCT4 gene editing system includes the sgRNA targeting the OCT4 gene and Cas9 of the first aspect.

Preferably, the Cas9 includes spCas 9.

Preferably, the sgRNA targeting the OCT4 gene is linked to Cas9 in the same expression vector.

Preferably, the OCT4 gene editing system further comprises a donor plasmid.

Preferably, the donor plasmid comprises iCASP9 gene and a selectable marker gene.

In the invention, iCASP9 (inducible caspase9, induced caspase9) is a small molecule drug induced dimerization suicide gene system, which has extremely high affinity with a chemical inducer, and the chemical inducer induces rapid apoptosis by activating caspase9 and downstream effect cysteine protease (such as caspase-3). Induction of recipient cell death under drug treatment can be achieved by inserting iCASP9 gene in the donor plasmid; by inserting the screening marker gene, the screening of positive clones is facilitated.

In the invention, through the matching of the OCT4 gene editing system and the donor plasmid, the fixed-point insertion of the iCASP9 gene can be realized in the OCT4 gene of the recipient cell, the non-specific gene editing is avoided, the editing efficiency is high, and the off-target rate is low.

Preferably, the selectable marker gene comprises a fluorescent selectable marker gene and/or a drug selectable marker gene.

Preferably, the fluorescent selectable marker gene comprises a Tdtomato-encoding gene.

Preferably, the drug selection marker gene comprises a puromycin resistance gene.

In a third aspect, the present invention provides a recombinant cell that induces death, the recombinant cell comprising the sgRNA targeting the OCT4 gene of the first aspect.

Preferably, the death-inducing recombinant cell comprises the OCT4 gene editing system of the second aspect.

Preferably, the death-inducing recombinant cell is a pluripotent stem cell in which the iCASP9 gene is inserted into the OCT4 gene after editing by the OCT4 gene editing system described in the second aspect.

In the invention, OCT4 is an important transcription factor in pluripotent stem cells, plays a key role in maintaining the pluripotency of stem cells, is considered to be positioned at the top of a cell pluripotency regulation network, and is the most important transcription factor participating in regulating the self-renewal of the pluripotent stem cells and maintaining the pluripotency of the pluripotent stem cells. High expression of OCT4 exists in both embryonic germ cell tumor and adult cell tumor, and over-expression can cause tumorigenicity and tumor metastasis of different types of cancers and even distant recurrence after chemoradiotherapy.

After stem cells differentiate into therapeutic cells, the suicide gene from OCT4 direct control ensures rapid shut-down of its suicide ability without affecting functional cells due to loss of pluripotency, only targets dry cells for killing, while accurate gene insertion also avoids the risk of insertional mutagenesis, as a direct, rapid safety strategy that can better overcome tumorigenesis in regenerative medicine settings.

Preferably, the death-inducing drug comprises AP 1903.

In a fourth aspect, the present invention provides a method for constructing a death-inducing recombinant cell according to the third aspect, the method comprising:

constructing an expression vector containing sgRNA of a targeted OCT4 gene and Cas9, constructing a donor plasmid, introducing the expression vector and the donor plasmid into a receptor cell, and screening positive clones to obtain the death-inducing recombinant cell.

The construction method is simple to operate, mature in technology, high in success rate and easy to master by technicians in related fields, and promotes popularization and application of related technologies.

Preferably, the method for constructing the expression vector comprises:

synthesizing a coding sequence of sgRNA of the targeted OCT4 gene, and connecting the coding sequence to a gene editing vector to obtain the expression vector containing the sgRNA of the targeted OCT4 gene and the Cas 9.

Preferably, the gene editing vector comprises a PX330 vector.

Preferably, said introducing comprises transfection.

Preferably, the recipient cell comprises a pluripotent stem cell.

In the invention, the pluripotent stem cells are selected as receptor cells, and the edited genes can be transferred to daughter cells in a cell division mode, so that the stability and the inheritability of gene editing are realized.

Preferably, the step of screening for positive clones comprises:

fluorescence screening and/or drug screening were performed based on the selection marker gene on the donor plasmid.

Preferably, the fluorescence screening comprises flow sorting.

Preferably, the drug screen comprises a puromycin screen.

Preferably, the construction method further comprises the steps of apoptosis detection, pluripotency identification and tumorigenic capacity detection.

As a preferred technical scheme, the method for constructing the death-inducing recombinant cell comprises the following steps:

(1) constructing an expression vector containing sgRNA targeting the OCT4 gene and Cas 9:

synthesizing a coding sequence of sgRNA of a targeted OCT4 gene, and connecting the coding sequence to a PX330 vector to obtain the expression vector containing the sgRNA of the targeted OCT4 gene and the Cas 9;

(2) constructing a donor plasmid containing an iCASP9 gene and a screening marker gene;

(3) introducing the expression vector and donor plasmid into a pluripotent stem cell by transfection;

(4) screening positive clones:

carrying out flow sorting and/or puromycin screening according to the screening marker gene on the donor plasmid;

(5) and carrying out apoptosis detection, pluripotency identification and tumorigenic capacity detection on the obtained positive clone to obtain the death-inducing recombinant cell.

In a fifth aspect, the present invention provides a use of any one or a combination of at least two of the sgRNA targeting the OCT4 gene of the first aspect, the OCT4 gene editing system of the second aspect, the death-inducing recombinant cell of the third aspect, or the death-inducing recombinant cell construction method of the fourth aspect, in the preparation of a stem cell therapeutic drug and/or agent.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, the OCT4 site is selected for accurate targeting, compared with the SOX2 site, the therapeutic cells and the tumorigenic cells can be more effectively distinguished, the cells can be differentiated into various cell types such as neural stem cells, and the like, and the method is more applicable to clinical treatment;

(2) compared with an HSV-tk/GCV system, the iCASP9 suicide system is safer and more effective, can effectively reduce immune rejection, has small cytotoxicity, can pass through a blood brain barrier, has no bystander effect, and has wider clinical application;

(3) according to the invention, the CRISPR/Cas9 gene editing technology is selected, so that genome damage can be reduced, and accurate insertion of an exogenous gene is realized, and compared with the defect that an induced suicide gene safety system constructed by using a lentiviral vector and a method for directly and randomly inserting a transgene cannot directionally kill tumorigenic cells and increases the risk of insertional mutagenesis, the method has more advantages;

(4) the invention can directionally kill the undifferentiated stem cells in the stem cell derived cell products without damaging the differentiated cells such as neural stem cells, and can block the formation of teratoma while preserving the activity of the differentiated cells to the maximum extent; the stem cell line can respond to the induced apoptosis of the drug within a few hours in vitro, and the drug is quickly absorbed after administration and has sensitive reaction; the stem cell line can highly express various pluripotent genes, and form teratoma tissues with a three-germ layer structure and expressing three-germ layer marker genes after transplantation, so that the teratoma tissues are proved to have complete pluripotency, and good experimental materials are provided for subsequent directed differentiation and clinical treatment;

(5) according to the invention, the effects of administration at different stages of teratoma formation are analyzed through a mouse experiment, the early administration is found to inhibit the growth of the teratoma, the volume of the early teratoma is reduced through apoptosis, the teratoma formation is blocked by a medicament, the survival time of a tumor-bearing mouse can be obviously prolonged, and the reference is provided for clinical application.

Drawings

FIG. 1 is a schematic diagram of the structure and targeting of a donor plasmid;

fig. 2 is a picture of screening results of positive clones (scale bar 100 μm);

FIG. 3A is a schematic diagram showing the correspondence between primers and sites in PCR identification of MT9 and HT 9;

FIG. 3B is a schematic diagram showing the correspondence between primers and sites in PCR identification of M9P and H9P;

FIG. 4A is a picture showing the results of PCR identification of the 5-terminal and 3-terminal of MT9 (in the figure, DL 5000-standard DNA molecular weight Marker DL5000, 1-11-PCR amplification products of the 5-terminal and 3-terminal of cells numbered 1-11, DL 2000-standard DNA molecular weight Marker DL 2000);

FIG. 4B is a photograph showing the results of PCR identification of 5-terminal and 3-terminal of HT9 (in the figure, DL 2000-Standard DNA molecular weight Marker DL2000, 1-2-PCR amplification products of 5-terminal and 3-terminal of cells numbered 1-2);

FIG. 4C is a photograph showing the results of PCR identification of the 5-and 3-termini of M9P (in the figure, DL 2000-Standard DNA molecular weight Marker DL2000, 1-13-PCR amplification products of the 5-and 3-termini of cells numbered 1-13);

FIG. 4D is a photograph showing the results of PCR identification of the 5-and 3-termini of H9P (in the drawing, DL 2000-Standard DNA molecular weight Marker DL2000, 1-9-PCR amplification products of the 5-and 3-termini of cells numbered 1-9);

FIG. 5A is a graph of the results of the test experiment for optimal concentration of recombinant cell drug treatment;

FIG. 5B is a graph of the results of the recombinant cell drug treatment optimization time test experiment;

fig. 6A is a picture of the results of apoptosis detection after drug treatment of recombinant cells (scale bar 200 μm);

FIG. 6B is a photograph showing the results of flow analysis of staining before and after drug treatment of recombinant cells;

fig. 7 is a photograph showing the results of the apoptosis induction assays of M9P and H9P (scale bar 100 μ M);

fig. 8 is a photograph showing the results of immunofluorescence staining of pluripotency marker genes of M9P and H9P (scale bar 100 μ M);

fig. 9A is a schematic diagram of 3 dosing regimens in a teratoma blocking effect validation experiment;

fig. 9B is a graph of teratoma growth curves injected with M9P and H9P after different administration modes, respectively;

FIG. 9C is a graph of survival curves of mice injected with M9P and H9P after different administration regimens, respectively;

fig. 9D is a graph of statistical results of tumor volumes formed after re-administration induction of mice injected with H9 hES and H9P, 129mES and M9P, respectively;

fig. 10 is a photograph of immunofluorescent staining of teratomas formed with untreated wild type cells, H9P and M9P (scale bar 100 μ M).

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Materials:

t4 ligase was purchased from NEB;

DNA Polymerase was purchased from Takara under the trade name Takara PrimerSTAR Max DNA Polymerase;

DNA purification recovery kit was purchased from magenta;

the recombinant Kit was purchased from Novowed under the trade name Clon express Ultra One Step Cloning Kit;

PX330 vector was purchased from Addgene;

129mES cells were isolated from this laboratory;

h9 hES cells were from ATCC;

NP40, proteinase K and F12 medium were purchased from Thermo Scientific;

PCR amplification reagents were purchased from Novozam Vazyme;

AP1903 from DC Chemicals;

the Annexin V-FITC/PI detection kit is purchased from BD Biosciences;

hoechst33342 purchased from Beyotime, bi yun tian;

DMEM high-glucose medium purchased from Hyclone;

serum was purchased from Gibco;

matrigel was purchased from Corning;

the immunodeficient mice were from the laboratory animal technology Limited of Weitonglihua, Beijing;

nanog antibody Sox2 antibody was purchased from R & D;

OCT4 antibody, a-SMA antibody, AFP antibody, and GFAP antibody were purchased from SANTA CRUZ biotechnoloy;

TRA-1-81 antibody was purchased from Millipore.

Example 1

The present example provides an OCT4 gene editing system that includes an OCT4 gene editing system comprising an sgRNA targeting an OCT4 gene, a spCas9, and a donor plasmid.

The OCT4 gene editing system is prepared by the following method:

(1) constructing an expression vector containing sgRNA targeting the OCT4 gene and Cas 9:

in order to realize the specific expression of iCASP9 at the OCT4 site of the endogenous pluripotency gene and maintain the pluripotency characteristic of stem cells, the normal function of the endogenous gene needs to be interfered as little as possible, so the site-specific insertion is carried out at the position of a gene expression terminator, and the OCT4 and the suicide gene are connected through a self-cleavage polypeptide 2A sequence (self-cleavage 2A peptide, 2A) to achieve the aim of coexpression with the pluripotency gene.

The sgRNA targeting the OCT4 gene comprises mgRNA or hgRNA;

wherein, the coding sequence of the mgRNA comprises a nucleotide sequence shown in SEQ ID No.1, the coding sequence of the hgRNA comprises a nucleotide sequence shown in SEQ ID No.2, and a thickened part is a targeting site.

SEQ ID No.1：

SEQ ID No.2：

Synthesizing a coding sequence of sgRNA of the targeted OCT4 gene, and connecting the sgRNA to a PX330 vector through T4 ligase to obtain expression vectors mgRNA-spCas9 and hgRNA-spCas9 containing the sgRNA of the targeted OCT4 gene and Cas 9.

(2) Constructing a donor plasmid containing an iCASP9 gene and a screening marker gene;

in order to prevent the iCASP9 suicide gene from being excessively expressed in the cells to cause the spontaneous apoptosis of the cells, 2 donor plasmids, 2A-Tdtomato-IRES-iCASP9 (named T9) and 2A-iCASP9-PURO (named 9P), were designed, and the structure and targeting schematic diagram is shown in FIG. 1. T9 reduces the expression level of iCASP9 gene through ribosome entry site (IRES), and Tdtomato is used as positive selection marker to facilitate the subsequent flow sorting. Whereas the 2A peptide structure in 9P is short and the expression balance of upstream and downstream genes is good, so iCASP9 expression is slightly higher in 9P, while 9P is positively selected by puromycin.

The above fragments were amplified using DNA polymerase, recovered on DNA gel using DNA purification recovery kit, and the fragments were recombinantly ligated using recombinant kit.

Through verification, an OCT4 gene editing system is successfully constructed.

Example 2

This example provides a death-inducing recombinant cell, which is a pluripotent stem cell having the iCASP9 gene inserted into the OCT4 gene edited by the OCT4 gene editing system described in example 1.

The recombinant cell is prepared by the following method:

(1) the expression vector and donor plasmid were introduced into pluripotent stem cells by transfection:

transfection experiments were performed as per the instructions for transfection reagents, and the expression vectors and donor plasmids were transfected into mouse embryonic stem cells (129mES) and human embryonic stem cells (H9 hES) at a mass ratio of 1: 3. For 129mES, transfection was performed using the expression vector mgRNA-spCas9 and 2 donor plasmids, respectively; for H9 hES, the expression vector hgRNA-spCas9 and 2 donor plasmids were used for transfection, respectively.

(2) Screening positive clones:

the T9 group was subjected to flow sorting 5 days after transfection to obtain tdTomato-positive cells, and the 9P group was screened for 2 weeks by adding puromycin at a working concentration of 2. mu.g/mL to obtain puromycin-resistant cells. The screening results for positive clones are shown in FIG. 2.

Through identification, 4 positive clones are successfully screened out: MT9 (recipient cell is 129mES cell, donor plasmid is T9), HT9 (recipient cell is H9 hES cell, donor plasmid is T9), M9P recipient cell is 129mES cell, donor plasmid is 9P) and H9P (recipient cell is H9 hES cell, donor plasmid is 9P) can be used in the subsequent experiments.

(3) And (3) PCR identification:

positive clones were picked and cleaved with NP40 lysate (0.45% NP40 and 60. mu.g/mL proteinase K) in a PCR instrument, and the 5-and 3-termini of the insert were PCR-identified using the cleavage products as templates, respectively.

The sizes of the primers and amplification products used for detection are shown in Table 1, and the correspondence between the primer names and the sites is shown in FIGS. 3A and 3B.

TABLE 1

SEQ ID No.3：gttcagccagaccaccatct；

SEQ ID No.4：ggctgttttaacttcctccg；

SEQ ID No.5：tacaagtagatgcggccgccccctctccctcc；

SEQ ID No.6：cagcctggcctacagagttc；

SEQ ID No.7：tgcagaaagaactcgagcaa；

SEQ ID No.8：gcggccgcatctacttgtacagctcgtccat；

SEQ ID No.9：gagccttacaccaagccaaa；

SEQ ID No.10：ttcaagcatcccggtgtagt；

SEQ ID No.11：gcaacctccccttctacgag。

The PCR identification results are shown in FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D. As can be seen, the target fragment was successfully inserted into the genome of the recipient cell. And taking the identified positive clone (namely the recombinant cell) for amplification culture and carrying out subsequent experiments.

Example 3

This example tests the optimal concentration and treatment time required for drug induction of recombinant cells constructed in example 2, as follows:

recombinant cells were induced with drug AP1903 at a concentration gradient of 0-200 nM, and assayed by MTS cytotoxicity assay using wild-type 129mES and H9 hES as controls, with the results shown in FIG. 5A. The optimal drug concentration was determined to be 10nM from the graph. Meanwhile, it can be seen that the 9P group is more sensitive to the reaction than the T9 group under the low concentration treatment of 5nM, and in addition, the overt concentration has obvious hook-back effect in the MT9 and M9P groups, that is, the apoptosis induction efficiency is obviously reduced along with the increase of the concentration, which is probably caused by the saturation of the drug action.

The time required to induce apoptosis of the four recombinant cells was then tested at the optimal treatment concentration of 10nM, and the results are shown in figure 5B. As can be seen from the figure, M9P and H9P can be completely apoptotic within 2H, and the effect is rapid. The expression of MT9 and HT9 is relatively weak, and the induction time is long, wherein the expression time of MT9 is about 72 hours, and the expression time of HT9 is about 6 hours. Meanwhile, for wild 129mES and H9 hES, no obvious toxic effect exists within 72H, and the medicine is further proved to have good selectivity and safety.

Considering that induction of apoptosis as rapidly as possible is required to better block teratoma growth, M9P and H9P were selected for subsequent experiments with treatment concentrations of 10nM and treatment times of 2H.

Example 4

In this example, apoptosis detection is performed on recombinant cells after drug treatment, and the steps are as follows:

AP1903 was added to M9P and H9P at a final concentration of 10nM, treated for 2H, and then assayed for apoptosis by Annexin V-FITC/PI, the results of which are shown in FIG. 6A. Meanwhile, the Annexin V-FITC/PI staining conditions of the cells before and after administration were subjected to flow analysis, and the results are shown in FIG. 6B.

In normal cells, Phosphatidylserine (PS) is distributed only inside the lipid bilayer of the cell membrane, whereas in early apoptosis, PS in the cell membrane is turned outside from inside the lipid bilayer. Annexin V is Ca with molecular weight of 35-36 kD²⁺The phospholipid-dependent binding protein has high affinity for phosphatidylserine, and thus the physiological state of the cell can be determined by the phosphatidylserine exposed outside the cell. Propidium Iodide (PI) is a nucleic acid dye that cannot penetrate the intact cell membrane, and PI, which permeates the cell membrane to red the nucleus of cells in the middle and late stages of apoptosis and dead cells due to increased permeability of the cell membrane. Therefore, by matching Annexin V with PI, cells at different apoptosis stages can be distinguished.

As can be seen in FIG. 6A, almost all cells in M9P and H9P clones broke and shriveled after 2H of administration, while dead cells floated to form a mass with no visible intact cell morphology and no adherent cells visible after 6H of administration. As seen from FIG. 6B, compared with the non-administered group, more than 95% of the cells in the administered group were in the late apoptosis stage with double positive to Annexin V-FITC and PI or in the early apoptosis stage with single positive to Annexin V-FITC and in the dead cell state with single positive to PI, and the effect was rapid and remarkable.

Example 5

In this example, apoptosis-inducing assays were performed on M9P and H9P after induced differentiation, as follows:

when M9P and H9P were cultured in differentiation medium (90% DMEM high-sugar medium and 10% serum), one week later, a heterogeneous cell population was observed, including stem cells in a clumped form (white dotted line) and differentiated cells in a flat-laid form. AP1903 was added at a final concentration of 10nM, treated for 2h, and then stained with PI and Hoechst33342, with the results shown in FIG. 7.

PI and Hoechst33342 can be combined with nuclear DNA (or RNA). However, PI cannot pass through normal cell membranes, so that the cell membranes are destroyed and can be colored red by PI when the cells are in necrosis or late apoptosis. Hoechst is a membrane-permeable fluorescent dye that stains living cells.

As can be seen from FIG. 7, only stem cells in a confluent form were stained with PI in the dosed M9P cells, while the surrounding differentiated cells were stained with Hoechst, while almost all the cells in the non-dosed group were not stained with PI. Similar phenomena were observed with H9P differentiated cells, and the pooled clones floated to form clumps after dosing, consistent with the previous H9P positive clone after dosing.

Taken together, this experiment demonstrates that the M9P and H9P cell lines respond rapidly and specifically to the induction of apoptosis by AP1903, and that AP1903 does not kill differentiated cells.

Example 6

This example verifies the expression of pluripotency marker genes for M9P and H9P by the following steps:

cellular Immunofluorescence (IF) experiments were performed on stem cell pluripotency marker genes Nanog, Sox2, OCT4, TRA-1-81, with wild-type 129mES and H9 hES cells as controls, and the results are shown in FIG. 8.

As can be seen, the expression of the pluripotency gene which is basically consistent with that of the wild type cell can be detected in the recombinant cell, which indicates that the M9P and H9P cell lines have good differentiation potential and can provide good materials for the subsequent stem cell treatment and directed differentiation.

Example 7

This example uses immunodeficient mice to experimentally simulate the formation of teratoma after transplantation in clinical stem cell therapy, while verifying the blocking effect of apoptosis of M9P and H9P on teratoma, as follows:

M9P and H9P were suspended in F12 medium (density 1X 10)⁷pieces/mL), mixed with Matrigel at a volume ratio of 1:1, and placed on ice. 4 weeks of immunodeficient mice were selected, each intraperitoneally administered 200. mu.L of 1.25% Avermectin for anesthesia, shaved on the dorsal side, and then subcutaneously injected 200. mu.L of cell Matrigel cocktail.

Setting a drug administration group: 2H before transplantation, subcutaneous administration at the same time and intraperitoneal administration at different time periods after transplantation, wherein intraperitoneal administration time points of D1, D7, D14, D21 and D28 are set after transplantation for H9P cells, the dosage of AP1903 is 5mg/kg/day, and injection lasts for 3 days; M9P cells were injected at the time point of intraperitoneal administration of D1, D5, D9, D13, D17 and D21 at 10mg/kg/day for 3 days after transplantation. A control group injected with an equal volume of PBS solution was also set. A schematic of the dosing regimen is shown in fig. 9A.

Survival and teratoma growth of transplanted mice were monitored and periodically recorded to create teratoma growth curves and mouse survival curves, with the results shown in fig. 9B and 9C, respectively.

By comparing the growth curves of teratomas (fig. 9B), it can be seen that H9P cells significantly increased the growth rate of teratomas after 10 days, and D14 group was administered when the volume was smaller in the early stage of teratoma formation, and the subsequent tumor volume continued to decrease; in addition, no tumor was generated in the group administered before D14, and the tumor growth was less affected by the administration three weeks later, which was substantially the same as that in the control group. The growth of the teratoma of the M9P cell line is obviously accelerated about one week and progresses earlier than that of H9P, the sizes of the teratomas are reduced after the drug is added to the D9 and D13 groups, the teratoma of the group which is administrated earlier does not grow basically, the influence of the later drug on the sizes of the teratomas is small, and the results are basically consistent with the results of H9P.

It can be seen from the survival curve of the transplanted mice (FIG. 9C) that the survival rate of the mice in the administered group was significantly increased. For H9P, mice in the group of in vitro cell administration 2H before transplantation, subcutaneous administration while transplantation and abdominal cavity administration within two weeks after transplantation survived well within 6 months, which also indicates that the drug has less toxic and side effects on organisms, whereas the PBS control group and the D28 group survived for no more than 35 days and the D21 group can be prolonged to about 60 days. Similar to the results of H9P, the survival rate of mice in the early administration group of M9P cells was greatly improved.

In addition, mice were injected subcutaneously with wild-type H9 hES and H9P, and wild-type 129mES and M9P simultaneously, and administered intraperitoneally for two weeks, and the volume of the tumor was counted, and the results are shown in fig. 9D. It can be seen that AP1903 can specifically kill teratomas formed by H9P and M9P cells, has little influence on wild cells which are not modified and do not carry suicide genes, and simultaneously proves that the induction drug only specifically kills recombinant cells and has little toxic and side effects on other cells. It can also be seen from the pictures that AP1903 had less effect on 129mES WT at individual levels.

In conclusion, the pre-administration (2H in vitro cell administration before transplantation) of H9P and M9P cell lines, subcutaneous administration and early (within two weeks after transplantation) intraperitoneal administration at the same time of transplantation have good blocking effect on teratomas, and the killing effect of drug treatment is reduced when the tumor bodies at the later stage are larger, which also accords with the fact that the differentiation and the expression of pluripotent genes of stem cells are gradually reduced after transplantation. The cells administered in both the pretreatment and the transplantation simultaneously are not tumorigenic, which suggests that the treatment after the directional differentiation using the cell line is preferably carried out before or simultaneously with the cell transplantation.

Example 8

This example performed histological analysis of teratomas formed in example 7, using the following procedure:

after the tumor diameter reached 2cm, the teratoma tissues were frozen and immunofluorescent, and the expression of the trioderm protein and the formation of the trioderm tissues were identified by staining with antibodies a-SMA, AFP and GFAP, respectively, as shown in fig. 10.

alpha-SMA (alpha smooth muscle actin) is a mesodermal marker protein, AFP (alpha fetoprotein) is synthesized by gastrointestinal tissues differentiated from embryo yolk sac, embryo liver cells and other endoderm, and is a marker of liver cancer and germ cell tumor. GFAP (ectoderm) is a glial fibrillary acidic protein, is expressed in astrocytes, ependymal cells, retinal cells, and the like, and is an ectoderm-marker gene.

As can be seen from the figure, the teratoma tissues generated by the H9P and M9P cell lines can detect the expression of three germ layer marker proteins, and have better totipotency.

In conclusion, the iCASP9 suicide system is inserted into the OCT4 gene of the pluripotent stem cell by the CRISPR/Cas9 gene editing technology, and the recombinant cell capable of inducing death is prepared. The recombinant cell can directionally kill undifferentiated stem cells under the treatment of AP1903 without damaging differentiated cells, and has good totipotency; the administration time and the administration effect of different stages of teratoma formation are analyzed through experiments, and the early administration is found to inhibit the growth of the teratoma and reduce the volume of the teratoma, thereby providing reference for clinical application.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

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