CN114457031A - Pluripotent stem cell expressing B7-H5 blocking substance or derivative thereof and application - Google Patents
Pluripotent stem cell expressing B7-H5 blocking substance or derivative thereof and application Download PDFInfo
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Abstract
The invention discloses a pluripotent stem cell expressing a B7-H5 blocker or a derivative thereof and application thereof, wherein the genome of the pluripotent stem cell or the derivative thereof is introduced with an expression sequence of a B7-H5 blocker, and the B7-H5 blocker is an anti-B7-H5 antibody. The pluripotent stem cells or derivatives thereof expressing the B7-H5 blocker provided by the invention can be used for inducing iPSCs (induced pluripotent stem cells) or differentiating the iPSCs into low-immunogenicity cells such as MSCs (mesenchymal stem cells) for application, can continuously express the B7-H5 blocker in vivo, and are used for treating B7-H5 high-expression tumors and related diseases.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a pluripotent stem cell expressing a B7-H5 blocker or a derivative thereof and application thereof.
Background
B7-H5 (also known as VISTA, Gi24, PD-1H or Dies 1): is a type I membrane protein with 55-65 KD, belonging to the immunoglobulin super family member. Consisting of an IgV domain, a stalk of about 30 amino acids, a transmembrane domain, and a cytoplasmic domain of 95 amino acids, and uniquely having 4 additional cysteine residues, 3 in the IgV domain and one in the stalk region. The cytoplasmic tail does not have the classical ITIM/ITAM motif, but has the SH2 domain in the middle of the cytoplasmic tail. B7-H5 was expressed predominantly on the surface of mature myeloid APC cells (antigen presenting cells) including macrophages, mature mouse bone marrow-derived dendritic cells (BMDCs), neutrophils and CD11c DC cells, and activated regulatory T cells (tregs). Both APC and T cell surface expressed B7-H5 inhibit T cell growth and thus arrest cell cycle and down regulate cytokine production. There are few reports of studies on B7-H5 expression in human cancers. B7-H5 is expressed in diseased tissues of human colon cancer and lung cancer, and it is found that membranous B7-H5 protein is expressed in normal duct epithelium in pancreas in pancreatic cancer and is down-regulated in pancreatic cancer. In gastric cancer, myofibroblasts overexpress B7-H5 in the Tumor Microenvironment (TME). Therefore, B7-H5 has become a new target for treating tumors, and a B7-H5 blocker (anti-B7-H5 antibody) can be used as a tumor treatment drug. However, the anti-B7-H5 antibody has a short duration of action, requires long-term injection, and is costly for patients.
Stem cells are "seed" cells with self-renewal ability and differentiation ability into specific functional somatic cells, have the potential to regenerate into various tissues, organs and human bodies, and play a central and irreplaceable role in immune response, aging, tumorigenesis and other important biological activities. Stem cells are mainly classified into: totipotent stem cells (Totipotent stem cells), Pluripotent Stem Cells (PSCs), and adult stem cells (adult stem cells). The typical PSCs mainly include Embryonic Stem Cells (ESCs), Embryonic Germ Cells (EGCs), Embryonic Carcinoma Cells (ECCs), Induced Pluripotent Stem Cells (iPSCs), and the like, and such cells have a very deep and wide application prospect due to their powerful functions and can be restricted to some extent by ethics.
Therefore, it is of great significance to develop a pluripotent stem cell or a derivative thereof that can express the B7-H5 blocker in humans.
However, the conception or establishment of the autologous iPSCs cell bank or the immune matched PSCs cell bank requires great expenditure of money, material resources and manpower. The molecular immunological basis for allogeneic recipient organ, tissue or cell transplantation is based primarily on the matching of the classical major histocompatibility complexes MHC-I and MHC-II (human HLA-I, HLA-II). By 6 months 2019, over 20000 HLA system alleles have been identified and named, and only 5000 allele factors of classical HLA-A, B, C are respectively exceeded, and various possible random combinations of these classical HLA-I/II alleles will be astronomical numbers, and as the number of combinations found for new alleles increases, there is a great obstacle to tissue matching and donor selection before organ, tissue and cell transplantation, and also great difficulty in constructing a PSCs cell library covering the immune match of the human population.
Thus, the construction of allogeneic immune-compatible, universal PSCs is imminent. In recent years, a plurality of reports have been provided that the deletion expression of genes on the cell surfaces of HLA-I and HLA-II or the genes thereof is realized by knocking out genes such as B2M, CIITA and the like, so that the cells have immune tolerance or escape T/B cell specific immune response, and universal PSCs with immune compatibility are generated, thereby laying an important foundation for the application of wider universal PSCs source cells, tissues and organs. Also, cells have been reported to overexpress CTLA4-Ig, PD-L1 and thereby inhibit allogeneic immune rejection. Recently, it has been reported that when B2M and CIITA are knocked out, CD47 is knocked in, so that cells obtain escape specific immune response, and have immune tolerance or escape natural immune response of cells such as NK cells, so that the cells have more comprehensive and stronger immune compatibility characteristics. However, these approaches are either not fully immune compatible, and still allow for immunological rejection of the allogens by other routes; or completely eliminate the allogeneic immune rejection response, but simultaneously make the cells of the donor-derived transplant lose the antigen presenting capability, which brings great risk of diseases such as tumorigenicity and virus infection to the recipient.
Therefore, it is also reported that, when the B2M is not directly knocked out, the HLA-A, HLA-B is knocked out or the CIITA is knocked out together, the HLA-C is kept, 12 HLA-C immune matching antigens covering more than 90% of people are constructed, so that the transplanted cells still have a certain degree of antigen presenting function, and the inherent immune response of NK cells can be inhibited through the HLA-C. However, in the cells, the antigen type presented by HLA-I antigen is reduced by more than two thirds, the integrity of the presented antigen is reduced irreversibly, the presenting of various tumor, virus and other disease antigens has great bias, the risk of diseases such as tumor and virus infection is still kept to a certain extent, and the pathogenic risk is higher under the condition that CIITA is knocked out simultaneously; secondly, 12 high-frequency immune match HLA-C antigen species are very different, and the part of the area can only account for 70 percent by verification and calculation, while the HLA data of large sample size which is not authoritative currently in China, Indian and other big countries is displayed, so that the prepared general PSCs are still subjected to huge match vacancy tests; thirdly, the method can go through repeated gene editing for a plurality of times, at least two rounds of single cell isolation culture meters are needed according to each gene editing, the whole process needs at least more than six rounds of single cell isolation culture, and the processes are inevitable and cause various unpredictable mutations of cells due to multiple times of gene editing off-target or unstable chromatin or due to passage proliferation of a large number of single cells, thereby further inducing various problems of carcinogenesis, metabolic diseases and the like. It follows that such immuno-compatible schemes are also a matter of convenience in the "transition period", and many problems remain that are not better solved.
In addition, inducing killing of the suicide gene after donor tissue and cell disease has been induced, which results in serious tissue necrosis, cytokine storm and other unpredictable disease risk problems, and it is a big problem that proper donor cells, tissues and organs do not exist after the cell death of the design.
Disclosure of Invention
In order to overcome the defects of the prior art, the first aspect of the invention aims to provide a pluripotent stem cell expressing a B7-H5 blocker or a derivative thereof, and the pluripotent stem cell comprises at least one of a non-immune compatible pluripotent stem cell expressing a B7-H5 blocker or a derivative thereof, an immune compatible pluripotent stem cell expressing a B7-H5 blocker or a derivative thereof, and an immune compatible reversible pluripotent stem cell expressing a B7-H5 blocker or a derivative thereof; wherein, the immune compatible pluripotent stem cell or the derivative thereof expressing the B7-H5 blocker can be realized by the following scheme: knocking out B2M and/or CIITA gene in the genome of the pluripotent stem cell or the derivative thereof and/or introducing an expression sequence of an immune compatible molecule into the genome of the pluripotent stem cell or the derivative thereof; the immune compatible reversible pluripotent stem cell or the derivative thereof expressing the B7-H5 blocker is realized by the following scheme: introducing immune compatible molecules and an inducible gene expression system into the genome of the pluripotent stem cells or the derivatives thereof, wherein the expression of the immune compatible molecules introduced into the genome of the pluripotent stem cells or the derivatives thereof is regulated by the inducible gene expression system, and the on and off of the inducible gene expression system are regulated by an exogenous inducer; when the immune compatible molecule is normally expressed, the expression of genes related to immune response in the pluripotent stem cell or the derivative thereof is inhibited or overexpressed, so that the allogeneic immune rejection response between the donor cell and the recipient can be eliminated or reduced; when the donor cell is diseased, the expression of the immune compatible molecules can be switched off through the induction of an exogenous inducer, and the antigen presenting capability of the donor cell is recovered, so that the diseased donor cell can be eliminated by a receptor.
The second aspect of the invention aims to provide the application of the pluripotent stem cells or the derivatives thereof in preparing medicines for treating B7-H5 high-expression tumors.
In a third aspect, the present invention provides a preparation comprising the pluripotent stem cells or derivatives thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
in the first aspect of the invention, the pluripotent stem cell or the derivative thereof expressing the B7-H5 blocker is provided, the genome of the pluripotent stem cell or the derivative thereof is introduced with an expression sequence of the B7-H5 blocker, and the B7-H5 blocker is an anti-B7-H5 antibody.
The heavy chain sequence of the anti-B7-H5 antibody is shown as SEQ ID NO.1, and the light chain sequence is shown as SEQ ID NO. 2.
The anti-B7-H5 antibody is preferably a secreted antibody.
The introduction site of the expression sequence of the B7-H5 blocker is a genome safety site of the pluripotent stem cell or the derivative thereof.
The genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.
As another technical scheme of the invention: the B2M and/or CIITA gene of the pluripotent stem cell or the derivative thereof is knocked out, so that an immune compatible pluripotent stem cell expressing a B7-H5 blocking substance or the derivative thereof is obtained.
As another technical scheme of the invention: the genome of the pluripotent stem cell or the derivative thereof is further introduced with one or more immune compatible molecule expression sequences for regulating the expression of genes associated with immune response (allogeneic immune rejection) in the pluripotent stem cell or the derivative thereof, thereby obtaining an immune compatible pluripotent stem cell or the derivative thereof expressing the B7-H5 blocker.
The genes associated with the immune response include:
(1) major histocompatibility complex genes including at least one of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB 1;
(2) major histocompatibility complex-associated genes including at least one of B2M and CIITA.
The introduction site of the expression sequence of the immune compatible molecule is a genome safety site of the pluripotent stem cell or the derivative thereof.
The genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.
The immune-compatible molecule includes any one or more of:
(1) immune tolerance related genes including CD47 or HLA-G;
(2) HLA-C molecules, including HLA-C multiple alleles of which the proportion in the population is over 90 percent in total, or fusion protein genes consisting of the HLA-C multiple alleles of which the proportion is over 90 percent and B2M;
(3) shRNA and/or shRNA-miR of major histocompatibility complex genes including at least one of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB 1;
(4) shRNA and/or shRNA-miR of a major histocompatibility complex-associated gene that includes at least one of B2M and CIITA.
The target sequence of the shRNA and/or shRNA-miR of B2M is at least one of SEQ ID NO. 3-SEQ ID NO. 5;
the target sequence of the shRNA and/or shRNA-miR of the CIITA is at least one of SEQ ID NO. 6-SEQ ID NO. 15;
the target sequence of the shRNA and/or shRNA-miR of the HLA-A is at least one of SEQ ID NO. 16-SEQ ID NO. 18;
the target sequence of the shRNA and/or shRNA-miR of the HLA-B is at least one of SEQ ID NO. 19-SEQ ID NO. 24;
the target sequence of the shRNA and/or shRNA-miR of the HLA-C is at least one of SEQ ID NO. 25-SEQ ID NO. 30;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRA is at least one of SEQ ID NO. 31-SEQ ID NO. 40;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB1 is at least one of SEQ ID NO. 41-SEQ ID NO. 45;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB3 is at least one of SEQ ID NO. 46-SEQ ID NO. 47;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB4 is at least one of SEQ ID NO. 48-SEQ ID NO. 57;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB5 is at least one of SEQ ID NO. 58-SEQ ID NO. 66;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DQA1 is at least one of SEQ ID NO. 67-SEQ ID NO. 73;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DQB1 is at least one of SEQ ID NO. 74-SEQ ID NO. 83;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DPA1 is at least one of SEQ ID NO. 84-SEQ ID NO. 93;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DPB1 is at least one of SEQ ID NO. 94-SEQ ID NO. 103.
shRNA and/or miRNA processing complex related genes and/or anti-interferon effector molecules are also introduced into the genome of the pluripotent stem cell or the derivative thereof.
The shRNA and/or miRNA processing complex related gene comprises at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA) and DGCR 8; the anti-interferon effector molecule is shRNA and/or shRNA-miR of at least one of PKR, 2-5As, IRF-3 and IRF-7.
The introduction site of the shRNA and/or miRNA processing complex related gene and/or anti-interferon effector molecule is a genome safety site of the pluripotent stem cell or the derivative thereof.
The genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.
The target sequence of the shRNA and/or shRNA-miR of the PKR is at least one of SEQ ID NO. 104-SEQ ID NO. 113;
the target sequence of the shRNA and/or shRNA-miR of the 2-5As is at least one of SEQ ID NO. 114-SEQ ID NO. 143;
the target sequence of the shRNA and/or shRNA-miR of the IRF-3 is at least one of SEQ ID NO. 144-SEQ ID NO. 153;
the target sequence of the shRNA and/or shRNA-miR of the IRF-7 is at least one of SEQ ID NO. 154-SEQ ID NO. 163.
The expression frameworks of the major histocompatibility complex gene, the major histocompatibility complex related gene, PKR, 2-5As, the shRNA and/or shRNA-miR of IRF-3 or IRF-7 are As follows:
(1) shRNA expression framework: sequentially comprising an shRNA target sequence, a stem-loop sequence, a reverse complementary sequence of the shRNA target sequence and Poly T from 5 'to 3'; the two reverse complementary target sequences are separated by a middle stem-loop sequence to form a hairpin structure, and finally Poly T is connected to be used as a transcription terminator of RNA polymerase III;
(2) shRNA-miR expression framework: replacing a target sequence in the microRNA-30 or the microRNA-155 with the shRNA-miR target sequence of the major histocompatibility complex gene, the major histocompatibility complex related gene, PKR, 2-5As, IRF-3 or IRF-7 to obtain the gene.
The length of a stem-loop sequence in the shRNA expression frame is 3-9 bases; the length of the Poly T is 5-6 bases.
The expression frame can be added with a constitutive promoter or an inducible promoter, such as a U6 promoter and an H1 promoter, and matched promoter regulatory elements at the 5' end according to requirements.
As another technical scheme of the invention: an inducible gene expression system is also introduced into the genome of the pluripotent stem cell or the derivative thereof and used for regulating the expression of the immune compatible molecules, so that the pluripotent stem cell expressing the B7-H5 blocking substance or the derivative thereof which is immune compatible and reversible is obtained.
The inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system.
The introduction site of the inducible gene expression system is a genome safety site of the pluripotent stem cell or the derivative thereof.
The genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.
The introduction of the B7-H5 blocker expression sequence, the expression sequence of immune compatible molecules, shRNA and/or miRNA processing complex related genes, anti-interferon effector molecules and inducible gene expression system adopts a method of viral vector interference, non-viral vector transfection or gene editing.
The method of gene editing comprises gene knock-in.
The pluripotent stem cells comprise embryonic stem cells, embryonic germ cells, embryonic cancer cells or induced pluripotent stem cells; the pluripotent stem cell derivative includes an adult stem cell, each germ layer cell or tissue into which the pluripotent stem cell is differentiated.
The adult stem cells comprise mesenchymal stem cells and neural stem cells.
In a second aspect of the invention, the application of the pluripotent stem cell or the derivative thereof in preparing a B7-H5 high-expression tumor treatment drug is provided.
In a third aspect of the invention, there is provided a formulation comprising the pluripotent stem cells or derivatives thereof.
The formulation further comprises a pharmaceutically acceptable carrier, diluent or excipient.
The invention has the beneficial effects that:
the pluripotent stem cells or derivatives thereof expressing the B7-H5 blocker provided by the invention can be used for inducing iPSCs (induced pluripotent stem cells) or differentiating the iPSCs into low-immunogenicity cells such as MSCs (mesenchymal stem cells) for application, can continuously express the B7-H5 blocker in vivo, and are used for treating B7-H5 high-expression tumors and related diseases.
The B2M and CIITA genes in the pluripotent stem cells or the derivatives thereof are knocked out, or an immune compatible molecule expression sequence is introduced into the genome of the pluripotent stem cells or the derivatives thereof, so that the pluripotent stem cells or the derivatives thereof have low immunogenicity, and when the pluripotent stem cells or the derivatives thereof are transplanted into a recipient, the problem of allogeneic immune rejection between donor cells and the recipient can be overcome, so that the donor cells can continuously express the B7-H5 blocker in the recipient for a long time.
The genome of the immune compatible reversible pluripotent stem cell or the derivative thereof for expressing the B7-H5 blocker is introduced with an inducible gene expression system and an immune compatible molecule expression sequence. The inducible gene expression system is controlled by an exogenous inducer, and the opening and closing of the inducible gene expression system are controlled by adjusting the addition amount, the continuous action time and the type of the exogenous inducer, so that the expression quantity of the epidemic compatible molecular expression sequence is controlled. While the immune-compatible molecule may regulate the expression of genes associated with an immune response in the pluripotent stem cell or derivative thereof. When the immune compatible molecule is normally expressed, the expression of genes related to the immune response in the pluripotent stem cell or the derivative thereof is suppressed or overexpressed, and the allogeneic immune rejection response between the donor cell and the recipient can be eliminated or reduced, so that the donor cell can continuously express the B7-H5 blocker in the recipient for a long time. When the donor cell is diseased, the expression of the immune compatible molecules can be closed by induction of an exogenous inducer, so that the HLA class I molecules can be reversibly re-expressed on the surface of the donor cell, the antigen presenting capability of the donor cell is recovered, and the diseased cell can be eliminated by a receptor, thereby improving the clinical safety of the general pluripotent stem cell or the derivative thereof, and greatly expanding the value of the general pluripotent stem cell in clinical application.
In addition, the addition amount and the lasting action time of the exogenous inducer can be adjusted to ensure that the transplant gradually expresses low-concentration HLA molecules to stimulate the receptor, so that the receptor gradually generates tolerance on the transplant, and finally stable tolerance is achieved. At the moment, even if the HLA class I molecules with unmatched HLA class I molecule expression on the surface of the transplanted cells can be compatible with the recipient immune system, so that after the expression of the immune compatible molecules in the transplanted cells is induced to be closed, the recipient immune system can re-identify the cells with gene mutation presented by the HLA class I molecules in the transplanted cells on one hand, and eliminate diseased cells; on the other hand, the non-mutated part is not cleared by the recipient immune system due to the allogeneic HLA class i molecule tolerance produced by training with the above mentioned inducers. Thus, the recipient immune system can only eliminate the graft with harmful mutation, the graft with normal function is kept, and when the harmful graft is eliminated, the mode of HLA class I molecule silencing on the cell surface of the graft can be transferred. The graft tolerance program mediated by the exogenous inducer can also be used to implant a graft that does not induce or otherwise induce the turning on or off of the surface expression of HLA class i molecules after the recipient has become fully tolerant.
Drawings
FIG. 1 is a plasmid map of AAVS1 KI (Knock-in, the same below) Vector (shRNA, constitutive).
FIG. 2 is an AAVS1 KI Vector (shRNA, inducible) plasmid map.
FIG. 3 is an AAVS1 KI Vector (shRNA-miR, constitutive) plasmid map.
FIG. 4 is an AAVS1 KI Vector (shRNA-miR, inducible) plasmid map.
FIG. 5 is a sgRNA clone B2M-1 plasmid map.
FIG. 6 is a sgRNA clone B2M-2 plasmid map.
FIG. 7 is a sgRNA clone CIITA-1 plasmid map.
FIG. 8 is a sgRNA clone CIITA-2 plasmid map.
Fig. 9 is a Cas9(D10A) plasmid map.
FIG. 10 is a sgRNA Clone AAVS1-1 plasmid map.
FIG. 11 is a sgRNA Clone AAVS1-2 plasmid map.
Detailed Description
The present invention will be described in further detail with reference to the following specific embodiments and accompanying drawings.
It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.
1 Experimental materials and methods
1.1B 7-H5 blockers
The Heavy Chain (HC) sequence of the anti-B7-H5 antibody is shown as SEQ ID NO.1, and the Light Chain (LC) sequence is shown as SEQ ID NO. 2.
1.2 pluripotent Stem cells or derivatives thereof
The pluripotent stem cells can be selected from Embryonic Stem Cells (ESCs), Induced Pluripotent Stem Cells (iPSCs) and other forms of pluripotent stem cells, such as hPSCs-MSCs, NSCs, EBs cells. Wherein:
ESCs: HN4 cells were selected and purchased from Shanghai department of sciences.
And (3) iPSCs: using a third generation highly efficient and safe episomal-iPSCs induction system (6F/BM1-4C) established by us, pE3.1-OG-KS and pE3.1-L-Myc-hmiR 302 cluster are transferred into somatic cells through electricity, RM1 is cultured for 2 days, BioCISO-BM1 containing 2uM Parnate is cultured for 2 days, BioCISO-BM1 containing 2uM Parnate, 0.25mM sodium butyrate, 3uM CHIR99021 and 0.5uM PD03254901 is cultured for 2 days, iPSCs clones can be picked up after being cultured to about 17 days by using a dry cell culture medium BioCISO, and the picked iPSCs clones are purified, digested and passaged to obtain stable iPSCs. The specific construction method is as follows: stem Cell Res ther.2017nov 2; 8(1):245.
hPSCs-MSCs: iPSCs are cultured for 25 days by using a stem cell culture medium (BioCISO containing 10uM TGF beta inhibitor SB431542), during which digestion passage (2mg/mL Dispase digestion) is carried out at 80-90 confluence, passage is carried out at 1:3 into a Matrigel coated culture plate, then ESC-MSC culture medium (knockkockout DMEM culture medium containing 10% KSR, NEAA, diabody, glutamine, beta-mercaptoethanol, 10ng/mL bFGF and SB-431542) is cultured, fluid is changed every day, passage is carried out at 80-90 confluence (passage is carried out at 1: 3), and continuous culture is carried out for 20 days. The specific construction method is as follows: proc Natl Acad Sci U S A.2015; 112(2):530-535.
NSCs: iPSCs are cultured for 14 days by using an induction medium (a knockout DMEM medium containing 10% KSR, a TGF-beta inhibitor and a BMP4 inhibitor), rose annular nerve cells are picked to a low-adhesion culture plate for culture, the culture medium is cultured by using DMEM/F12 (containing 1% N2 and Invitrogen) and Neurobasal medium (containing 2% B27 and Invitrogen) in a ratio of 1:1 and also contains 20ng/ml bFGF and 20ng/ml EGF, and digestion is carried out by using Accutase for digestion and passage. The specific construction method is as follows: FASEB J.2014; 28(11):4642-4656.
EBs cells: and digesting iPSCs with the confluence of 95% for 6min by using a BioC-PDE1, scraping the cells into blocks by using a mechanical scraping method, settling and reducing cell masses, transferring the settled cell masses into a low-adhesion culture plate, culturing for 7 days by using a BioCISO-EB1, and changing the liquid every other day. After 7 days, the cells were transferred to a Matrigel-coated plate and adherent culture was continued using BioCISO, and Embryoid Bodies (EBs) having an inner, middle and outer mesoderm structure were obtained after 7 days. The specific construction method is as follows: stem Cell Res ther.2017Nov 2; 8(1):245.
The pluripotent stem cell derivative also includes adult stem cells, each germ layer cell or tissue, organ into which the pluripotent stem cells are differentiated; the adult stem cells include mesenchymal stem cells or neural stem cells.
1.3 genomic safety sites
In the technical scheme of the invention, the genome safety locus for knocking-in the gene can be selected from an AAVS1 safety locus, an eGSH safety locus or other safety loci:
(1) AAVS1 safety site
The AAVS1 site (the alias "PPP 1R2C site") is located on chromosome 19 of the human genome and is a verified "safe harbor" site that ensures the desired function of the transferred DNA fragment. The site is an open chromosome structure, can ensure that the transgene can be normally transcribed, and has no known side effect on cells when the exogenous target segment is inserted into the site.
(2) eGSH safe site
The eGSH safe site is located on chromosome 1 of the human genome, and is another 'safe harbor' site which can ensure the expected function of the transferred DNA fragment after the paper verifies.
(3) Other safety sites
The H11 safe site (also called Hipp11) is located on the number 22 chromosome of a human, is a site between two genes Eif4enif1 and Drg1, is discovered and named in 2010 by Simon Hippenmeyer, and has little risk of influencing endogenous gene expression after the insertion of a foreign gene because the H11 site is located between the two genes. The H11 site was verified to be a safe transcription activation region between genes, a new "safe harbor" site outside the AAVS1, eGSH sites.
1.4 inducible Gene expression System
The inducible gene expression system is selected from: tet-Off system or dimer-Off expression system:
(1) tet-Off system
In the absence of tetracycline, the tTA protein continues to act on the tet promoter, resulting in sustained gene expression. This system is very useful in situations where it is desirable to maintain the transgene in a sustained expression state. When tetracycline is added, the tetracycline can change the structure of the tTA protein, so that the tTA protein cannot be combined with a promoter, and the expression level of a gene driven by the tTA protein is reduced. To keep the system in an "off" state, the tetracycline must be added continuously.
The invention knocks the sequence of the tet-Off system and one or more immune compatible molecules into the genome safety site of the pluripotent stem cell, and accurately turns on or Off the expression of the immune compatible molecules through the addition of tetracycline, thereby reversibly regulating the expression of major histocompatibility complex related genes in the pluripotent stem cell or the derivative thereof.
(2) Dimer-switched off expression system
Dimer-mediated gene expression regulation system: there are many ways of chemically regulating transcription of target genes, most commonly regulated using allosteric modulators that influence the activity of transcription factors. One such method is the use of dimerizing inducers or dimers to recombine active transcription factors on inactive fusion proteins. The most commonly used system is rapamycin (rapamydn), a natural product, or an analog that is biologically inactive, as the drug for dimerization. The rapamycin (or analog) sibling protein FKBP12 (the protein to which FKBP binds to FK 506) and a large serine-threonine protein kinase, known as FRAP [ FRBP-rapamycin associated protein, mTOR (mammalian target of rapamycin), have high affinity and function to bind to both proteins, thus bringing them together as a heterologous dimer. To regulate transcription of a target gene, a DNA binding domain is fused to one or more FKBP domains and a transcription repressing domain is fused to amino acid position 93 of FRAP, designated FRB, which is sufficient to bind the FKBP-rapamycin complex. Dimerization of these two fusion proteins can only occur in the presence of rapamycin. Thus inhibiting transcription of genes having sites that bind to the DNA binding region.
1.5 immune compatible molecules
The immune compatible molecule can regulate the expression of allogeneic immune rejection related genes in the pluripotent stem cells or derivatives thereof.
The types and sequences of specific immune-compatible molecules are shown in table 1.
TABLE 1 immune compatible molecules
The target sequences of the shRNA or shRNA-miR immune compatible molecules are shown in Table 2.
TABLE 2 target sequences for shRNA or shRNA-miR
In the immune compatible molecule knock-in schemes of tables 5-6 below, the shRNA or shRNA-miR sequences of each experimental group are shRNA or shRNA-miR immune compatible molecules constructed by using the target sequence 1 in table 2. Those skilled in the art will understand that: the shRNA or shRNA-miR immune compatible molecule constructed by other target sequences can also realize the technical effect of the invention and all fall into the protection scope of the claims of the invention.
1.6shRNA/miRNA processing Complex genes and anti-interferon effector molecules
The primary miRNA (pri-miRNA) in the nucleus is microprocessed through the complex Drosha-DGCR8, which cleaves the pri-miRNA into a precursor miRNA (pre-miRNA), which then forms a hairpin. Then, the pre-miRNA is transported out of the nucleus via the Exportin-5-Ran-GTP complex. The RNase Dicer enzyme, which binds to the double-stranded RNA-binding protein TRBP (TARBP2) in the cytoplasm, breaks down the pre-miRNA into mature lengths, at which point the miRNA is still in a double-stranded state. Finally, it is transported into AGO2 to form RISC (RNA-induced silencing complex). Finally, one strand of the miRNA double strand is retained in the RISC complex, and the other strand is eliminated and rapidly degraded. While DGCR8, the main binding protein of Drosha, can bind to pri-miRNA through two double-stranded RNA binding regions at its C-terminal end, recruit and guide Drosha to cut at the right position of pri-miRNA to produce pre-miRNA, which is further cut by Dicer and TRBP/PACT processing to form mature miRNA. Deletion or abnormal expression of DGCR8 affects the cleavage activity of Drosha, which in turn affects the activity of miRNA, leading to disease. TRBP is able to recruit Dicer complex mirnas to form RISC Ago 2.
According to the invention, by using a gene knock-in technology, when the shRNA-miR expression sequences aiming at HLA class I molecules, HLA class II molecules and the like which can be induced to close expression are knocked in at a genome safety site, preferably, shRNA and/or miRNA processing machines which can be induced to close expression are knocked in at the same time, wherein the shRNA and/or miRNA processing machines comprise Drosha (access number: NM-001100412), Ago1(access number: NM-012199), Ago2(access number: NM-001164623), Dicer1(access number: NM-001195573), export-5 (access number: NM-020750), TRBP (access number: NM-134323), PACT (access number: NM-003690) and DGCR8(access number: NM-022720), so that cells do not occupy the processing of other miRNAs and influence the cell functions.
In addition, during IFN induction, double-stranded RNA-dependent Protein Kinase (PKR), which is a key factor of the whole cell signal transduction pathway, and 2 ', 5' Oligoadenylate Synthetase (2,5-Oligoadenylate Synthetase,2-5As), which are closely related to dsRNA-induced IFN, are involved. PKR can inhibit protein synthesis by phosphorylating eukaryotic cell transcription factors, arrest cells in G0/G1 and G2/M phases and induce apoptosis, while dsRNA can promote synthesis of 2-5As, which results in nonspecific activation of RNase, RNaseL, degradation of all mRNA in cells and cell death. The specificity of induction of type I interferons is achieved by members of the IRF transcription factor family, which are not inducible to be secreted in many viral infections in the absence of IRF-3 and IRF-7 expression in cells. Lack of IFN response, in order to recover, requires the two proteins were expressed together.
According to the invention, by utilizing a gene knock-in technology, when an immune compatible molecule shRNA-miR expression sequence is knocked in at a genome safety site, shRNA and/or shRNA-miR expression sequences which can induce closed expression and aim at suppressing PKR, 2-5As, IRF-3 and IRF-7 genes are preferably knocked in at the same time, so that interferon reaction induced by dsRNA is reduced, and cytotoxicity is avoided.
The sequence of the insertion positions of the shRNA/miRNA processing complex related gene, the anti-interferon effector molecule and the immune compatible molecule at the genome safety site is not limited, and the shRNA/miRNA processing complex related gene, the anti-interferon effector molecule and the immune compatible molecule can be arranged in any sequence without mutual interference or influence on the structure and the function of other genes of the genome.
Specific target sequences for anti-interferon effector molecules are shown in table 3.
TABLE 3 target sequences for anti-interferon effector molecules
In the anti-interferon effector molecule knock-in schemes of tables 5 to 6 below, the anti-interferon effector molecules of each experimental group were all anti-interferon effector molecules constructed using target sequence 1 in table 3. Those skilled in the art will understand that: the anti-interferon effector molecules constructed by other target sequences can also achieve the technical effects of the invention and fall into the protection scope of the claims of the invention.
1.7 Universal framework for Immunocompatible molecules, shRNA or shRNA-miR of anti-Interferon Effector molecules
The general framework sequences of the shRNA or shRNA-miR of the immune compatible molecules and the anti-interferon effector molecules are as follows:
(1) the constitutive expression framework of shRNA is:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGCTAGCGCCACC(SEQ ID NO.164)N1...N21TTCAAGAGA(SEQ ID NO.165)N22...N42TTTTTT；
wherein:
a、N1...N21shRNA target sequence for the corresponding Gene, N22...N42Is a reverse complementary sequence of the shRNA target sequence of the corresponding gene;
b. if the plasmid needs to express shRNAs of a plurality of genes, each gene corresponds to a shRNA expression frame and then is connected seamlessly;
c. constitutive shRNA plasmids with different resistance genes only have different resistance genes and have the same other sequences;
d. n represents A, T, G, C bp;
e. SEQ ID No.164 is the U6 promoter sequence;
f. SEQ ID NO.165 is a stem-loop sequence.
(2) The shRNA inducible expression framework is as follows:
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTGCTAGCGCCACC(SEQ ID NO.166)N1...N21TTCAAGAGA(SEQ ID NO.165)N22...N42TTTTTT；
wherein:
a、N1...N21shRNA target sequence of the corresponding Gene, N22...N42Is a reverse complementary sequence of the shRNA target sequence of the corresponding gene;
b. if the plasmid needs to express shRNAs of a plurality of genes, each gene corresponds to a shRNA expression frame and then is connected seamlessly;
c. constitutive shRNA plasmids with different resistance genes only have different resistance genes and have the same other sequences;
d. n represents A, T, G, C bp;
e. SEQ ID No.166 is the H1 TO promoter sequence;
f. SEQ ID NO.165 is a stem-loop sequence.
(3) The shRNA-miR constitutive or inducible expression framework is as follows:
the shRNA-miR target sequence is used for replacing a target sequence in microRNA-30 to obtain the shRNA-miR target sequence, and the specific sequence is as follows:
GAGGCTTCAGTACTTTACAGAATCGTTGCCTGCACATCTTGGAAACACTTGCTGGGATTACTTCTTCAGGTTAACCCAACAGAAGGCTAAAGAAGGTATATTGCTGTTGACAGTGAGCG(SEQ ID NO.167)M1N1...N21TAGTGAAGCCACAGATGTA(SEQ ID NO.168)N22...N42M2TGCCTACTGCCTCGGACTTCAAGGGGCTACTTTAGGAGCAATTATCTTGTTTACTAAAACTGAATACCTTGCTATCTCTTTGATACATTTTTACAAAGCTGAATTAAAATGGTATAAAT(SEQ ID NO.169)；
wherein:
a、N1...N21shRNA-miR target sequence, N, of the corresponding Gene22...N42Is a reverse complementary sequence of shRNA-miR target sequence of a corresponding gene;
b. if the plasmid needs to express shRNA-miR of a plurality of genes, each gene corresponds to a shRNA-miR expression frame and is then connected seamlessly;
c. constitutive shRNA-miR plasmids with different resistance genes only have different resistance genes and have the same other sequences;
d. m is A or C, N is A, T, G, C;
e. if N is present1Is a G base, then M1Is A base; otherwise M1Is a C base;
f、M1base and M2And (3) base complementation.
1.8 Gene editing System, Gene editing method and inspection method
1.8.1 Gene editing System
The gene editing technology of the patent adopts a CRISPR-Cas9 gene editing system. The Cas9 protein used was Cas9(D10A), Cas9(D10A) bound to sgrnas which were responsible for specific recognition of the target sequence (genomic DNA) which was then single-stranded cleaved by Cas9 (D10A). Double Strand breaks in genomic DNA (DSB) must occur, and two Cas 9(D10A)/sgRNA must cleave the two strands of genomic DNA separately, and not too far apart. The Cas 9(D10A)/sgRNA scheme has the advantage of higher specificity and lower probability of off-target compared to the Cas 9/sgRNA scheme. The plasmids or Donor fragments used in the gene editing system were: cas9(D10A) plasmid, sgRNA clone plasmid, Donor fragment.
(1) Cas9(D10A) plasmid: a plasmid expressing the Cas9(D10A) protein, specifically single-stranded cleaving genomic DNA under the direction of sgrnas.
(2) sgRNA plasmid: a plasmid for expressing sgRNA, sgRNA (small guide RNA), is a guide RNA (guide RNA, gRNA) responsible for directing targeted cleavage of the expressed Cas9(D10A) protein at gene editing.
(3) Donor fragment: the two ends contain recombination arms which are respectively positioned at the left side and the right side of the breaking position of the genome DNA, and the middle part contains genes, fragments or expression elements needing to be inserted. In the presence of the Donor fragment, the cells undergo a Homologous Recombination (HR) reaction at the site of the genomic break. If the Donor fragment is not added, Non-homologous End Joining-NHEJ reaction occurs at the site of the genomic break in the cell. This fragment was obtained by digesting KI (Knock-in, the same applies hereinafter) Vector plasmid and recovering it.
1.8.2 constitutive plasmids and inducible plasmids
Constitutive plasmid: the expression function of the Donor fragment obtained from the constitutive plasmid cannot be regulated after knocking in the genomic DNA.
Inducible plasmids: after knocking in the genomic DNA, the expression function of the Donor fragment obtained from the inducible plasmid can be controlled by adding an inducer, which is equivalent to adding a switch for turning on or off the expression function.
1.8.3 plasmid construction method
(1) Cas9(D10A) plasmid: this Plasmid no longer needs to be constructed and is ordered directly from Addgene (Plasmid 41816, Addge ne).
(2) sgRNA plasmid: the original blank Plasmid is ordered from Addge (Plasmid 41824, Addge), then the DNA sequence is input in the website (URL: https:// ccttop. cos. uni-heidelberg. de) to design the target sequence, and finally different target sequences are respectively put into the blank sgRNA Plasmid to complete the construction.
(3) KI Vector plasmid:
acquisition of Amp (R) -pUC origin fragment: designing PCR primers, and amplifying and recovering the fragment by using a high fidelity enzyme (Nanjing Nozaki organism, P505-d1) through a PCR method by using a pUC18 plasmid as a template;
acquisition of aavs1 or eGSH recombination arms: extracting genome DNA of human cells and designing corresponding primers, and then amplifying and recovering the fragments by using the human genome DNA as a template and using a high fidelity enzyme (Nanjing Novozam organism, P505-d1) through a PCR method;
c. acquisition of the individual plasmid elements: designing PCR amplification primers of each element, and then respectively amplifying and recovering each plasmid element by using a plasmid containing the element as a template and using a high fidelity enzyme (Nanjing NuoZanza, P505-d1) through a PCR method;
d. assembly into a complete plasmid: the fragments obtained in the previous step were ligated together using a multi-fragment recombinase (Nanjing Nozam, C113-02) to form a complete plasmid.
1.8.4 Gene editing Process
Firstly, the single cell cloning operation procedure of AAVS1 gene knock-in (1) electrotransfer program:
donor cell preparation: human pluripotent stem cells.
The kit comprises: human Stem Cell
Kit 1。
The instrument comprises the following steps: an electrotransformation instrument.
Culture medium: BioCISO.
Induction of plasmid: cas9D10A, sgRNA clone AAVS1-1, sgRNA clone AAVS1-2, AAVS1 neo Vector I and AAVS1 neo Vector II.
Note: induction plasmid used for the knock-in of the eGSH gene: cas9D10A, sgRNA clone eGSH-1, sgRNA clone eGSH-2, eGSH-neo/eGSH-puro (donor) comparison of the donor plasmid with AAVS1 shows that only the right and left recombination arms are different, and the other elements are the same. Since the gene editing process of eGSH is the same as that of AAVS1, the following description will not be repeated.
(2) The transformed human pluripotent stem cells are screened in a double antibiotic medium containing G418 and puro.
(3) And (4) carrying out single cell clone screening and culture to obtain a single cell clone strain.
Second, AAVS1 gene knock-in single cell clone strain culture reagent
(1) Culture medium: BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro (should be placed at room temperature in advance, protected from light for 30-60 minutes until room temperature is restored. Note that BioCISO should not be placed at 37 ℃ for preheating to avoid reduction of the activity of the biomolecule.).
(2) Matrix glue: hESC grade Matrigel (before passage or cell recovery, the Matrigel working solution is added into a cell culture bottle dish and is shaken up to ensure that the Matrigel completely sinks to the bottom of the culture bottle dish and any Matrigel cannot be dried before use. to ensure that the cells can be attached to the wall and survive better, the Matrigel is put into a 37 ℃ culture box for 1:100X Matrigel cannot be less than 0.5 hour and 1:200X Matrigel cannot be less than 2 hours.).
(3) Digestion solution: EDTA was dissolved using DPBS to a final concentration of 0.5mM, pH7.4 (note: EDTA cannot be diluted with water, otherwise the cells die due to reduced osmotic pressure).
(4) Freezing and storing liquid: 60% BioCISO + 30% ESCs grade FBS + 10% DMSO (frozen stock is preferably ready for use).
Thirdly, the conventional maintenance subculture process
(1) Optimal time of passage and passage ratio
a. The best passage time: the overall confluency of the cells reaches 80 to 90 percent;
b. the optimal ratio of passage: the optimal confluence degree of the passage is maintained at 20-30% in the next day after passage of 1: 4-1: 7.
(2) Passage process
a. The Matrigel in the coated cell culture flask dishes was previously aspirated away, and an appropriate amount of medium (BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro) was added to the flask and placed at 37 ℃ in 5% CO2Incubation in an incubator;
b. when the cells meet the requirement of passage, sucking the supernatant of the culture medium, and adding a proper amount of 0.5mM EDTA digestive solution into a cell bottle dish;
c. the cells were incubated at 37 ℃ with 5% CO2Incubating in an incubator for 5-10 minutes (digesting until most cells are observed to shrink and become round under a microscope but not float, gently blowing the cells to separate the cells from the wall, sucking the cell suspension into a centrifugal tube, and centrifuging for 5 minutes at 200 g;
d. centrifuging, discarding the supernatant, suspending the cells by using a culture medium, gently and repeatedly blowing the cells for several times until the cells are uniformly mixed, and then transferring the cells to a bottle dish prepared for coating Matrigel in advance;
e. after the cells were transferred to the cell flask, the cells were horizontally shaken up all around, observed under a mirror to be free from abnormality, and then shaken up and placed at 37 ℃ with 5% CO2Culturing in an incubator;
f. observing the adherent survival state of the cells the next day, and normally and regularly changing the culture medium every day by sucking off the culture medium.
Fourthly, freezing and storing cells
(1) According to the conventional passage operation steps, digesting the cells by using 0.5mM EDTA until most cells shrink and become round but do not float, gently blowing and beating the cells, collecting cell suspension, centrifuging for 5 minutes at 200g, removing supernatant, adding a proper amount of freezing medium to resuspend the cells, and transferring the cells to a freezing tube (suggesting that one frozen cell with 80% confluence degree of a six-well plate and 0.5 mL/cell of freezing medium is frozen);
(2) placing the freezing tube in a programmed cooling box, and immediately placing the freezing tube at-80 ℃ overnight (ensuring that the temperature of the freezing tube is reduced by 1 ℃ per minute);
(3) the next day the cells were immediately transferred into liquid nitrogen.
Fifth, cell recovery
(1) Preparing a Matrigel-coated cell bottle dish in advance, sucking out the Matrigel before recovering the cells, adding a proper amount of BioCISO into the cell bottle dish, placing at 37 ℃ and 5% CO2Incubation in an incubator;
(2) taking out the cryopreservation tube from liquid nitrogen quickly, immediately putting the tube into a 37 ℃ water bath kettle for quick shaking to quickly melt the cells, carefully observing, stopping shaking after the ice crystals completely disappear, and transferring the cells to a biological safety cabinet;
(3) adding 10mL of DMEM/F12(1:1) basal medium into a 15mL centrifuge tube in advance, balancing to room temperature, sucking 1mL of DMEM/F12(1:1) by using a Pasteur pipette, slowly adding the DMEM/F12(1:1) into a freezing tube, gently mixing, transferring the cell suspension into a prepared 15mL centrifuge tube containing DMEM/F12(1:1), and centrifuging for 5 minutes at 200 g;
(4) carefully removing supernatant, adding appropriate amount of BioCISO, gently mixing cells, seeding into a cell bottle dish prepared in advance, shaking up horizontally, and observing under the mirror, shaking up, and standing at 37 deg.C and 5% CO2Culturing in an incubator;
(5) the adherent survival state of the cells is observed the next day, and the liquid is normally changed on time every day. If the adherence is good, the BioCISO is changed to BioCISO + 300. mu.g/mL G418+ 0.5. mu.g/mL puro.
1.8.5AAVS1 gene knock-in detection method
First, single cell clone AAVS1 gene knock-in detection
(1) AAVS1 Gene knock-in assay
a. The purpose of the test is as follows: detecting the cells subjected to the gene knock-in treatment by PCR, and testing whether the cells are homozygotes; since the two Donor segments only have the difference in the sequences of the resistance genes, it is necessary to determine whether the cell is homozygous (the two chromosomes knock in the Donor segments of different resistance genes respectively), and it is only possible that the double-knocked-in cell is the correct homozygous by detecting whether the genome of the cell contains the Donor segments of the two resistance genes;
b. firstly, designing a primer in the Donor plasmid (non-recombinant arm part), and then designing another primer in the genome PPP1R12C (non-recombinant arm part); if the Donor fragment can be correctly inserted into the genome, a target band appears, otherwise no target band appears);
c. test protocol primer sequences and PCR protocols are shown in Table 4.
TABLE 4 test protocol primer sequences and PCR protocol
Second, the detection method of eGSH gene knock-in is the same as the detection principle and method of AAVS1 gene knock-in, and will not be described here.
1.8.6 inspection method of knock-in Gene method at genomic safety site
(1) The purpose of the test is as follows: the cells treated by knock-in were tested for homozygote by PCR. Since the two Donor fragments have only difference in the sequences of the resistance genes, it is necessary to determine whether the cell is homozygous (the two chromosomes knock in the Donor fragments of different resistance genes), and it is only possible that the double-knocked-in cell is the correct homozygous by determining whether the genome of the cell contains the Donor fragments of the two resistance genes.
(2) The test method comprises the following steps: first, one primer was designed inside the Donor plasmid (non-recombinant arm portion), and then the other primer was designed in the genome (non-recombinant arm portion). If the Donor fragment is inserted correctly in the genome, the target band will appear, otherwise no target band will appear.
1.9 assay method for expression of B7-H5 antibody by pluripotent Stem cells
B7-H5 antibody expressed by pluripotent stem cells was detected using ELISA (competitive assay). Collecting the culture supernatant of the pluripotent stem cells expressing the B7-H5 antibody, mixing the culture supernatant with the enzyme-labeled anti-B7-H5 antibody (1:1), loading the mixture on an enzyme-labeled plate coated with the B7-H5 antigen, adding the culture supernatant of the pluripotent stem cells not expressing the B7-H5 antibody to a control group, and gently mixing the mixture uniformly. Sealing the plate, incubating at 37 deg.C for 30min, washing for 5 times, adding color developing solution, developing for 15min, adding stop solution 50ul, and reading to measure absorbance value of 450 nm. (the expression level of B7-H5 antibody is inversely correlated with the color intensity).
1.10 51Cr release method for detecting influence of B7-H5 antibody on T cell killing tumor
(1) Preparation of effector cells:
t cell isolation: separating human Peripheral Blood Mononuclear Cells (PBMC) by Ficoll density gradient centrifugation (Ficoll-hypaque density gradient centrifugation), and subjecting to centrifugationUsing DynabeadsTM CD3(InvitrogenTMAnd the goods number: 11151D) T cells are isolated by the kit. The cells were resuspended in RPMI1640 medium containing 10% FBS, the cells were counted by trypan blue staining, and concentrated to 1X 107cells/mL.
(2) Preparation of target cells
Tumor (NIC lung cancer) cells were digested and resuspended, and cells were counted by trypan blue staining to 1X 107cells/mL of cell suspension.
(3)51Cr Release test
When tumor cells are first incubated with culture supernatants of pluripotent stem cells expressing anti-B7-H5 antibody for 30 minutes and then contacted with T cells, T attacks the tumor cells and causes cell lysis and death. While the tumor cells which are not incubated by the culture supernatant of the pluripotent stem cells expressing the anti-B7-H5 antibody are not easily recognized by T cells, and immune escape can occur. So by detecting in the medium51The amount of Cr reflects the ability of T cells to kill tumors.51The less the amount of Cr released into the culture medium, the more immune escape of the tumor cells will occur.
Quantitative determination of cell-mediated cytotoxicity with radioisotopes51Cr-labeled target cells, co-incubated with effector molecules or cells, released upon lysis of the target cells51Number of Cr radiation pulses (cpm) to determine cytotoxic activity:
a. target cells were treated with 100. mu. Ci (Ci, radioactivity units) of Na51CrO4Labeling at 37 deg.C for 120min, shaking every 15min, labeling, centrifuging with cleaning solution for 5 times, and resuspending in culture medium to obtain 1 × 106cells/mL for use;
b. target cells and T cells were added to 96-well plates at 100. mu.L per well (2.5X 10)3One) and 100 μ L of effector cells (E/T ═ 1:2, 1:5, 1:10, E/T is the ratio of target cells to effector cells T), while a natural release control well (100 μ L of target cells +100 μ L of medium) and a maximum release well (100 μ L of target cells +100 μ L of 2% SDS) were established; standing at 37 deg.C and 5% CO2The culture was incubated for 4 h. Taking out, sucking out supernatant from each hole with a pipettorThen, centrifuging to take 100 mu L of supernatant, and measuring the cpm value by using a gamma counter;
c. and (4) calculating a result: according to the formula51Natural Cr release rate and T cell activity:
note: general requirements51Natural release rate of Cr<10％。
1.11 mouse tumor treatment methods
In humanized NSG mice (The Jackson Laboratory (JAX)), The right axilla was injected subcutaneously with 5X 106Tumor cells (NIC lung cancer, HCC liver cancer, ACC colorectal cancer) cells, when the tumor grows to 60mm3In size, tail vein injection of 200uLPBS (containing human immune cells and 1X 10)6The pluripotent stem cell derivative expressing the B7-H5 blocker) for tumor treatment, wherein only the group containing human immune cells was injected as a control group. Mice were sacrificed after 20 days and tumor sizes were compared between groups and statistical analysis of differences was performed.
2. Experimental protocol
The experimental protocols for knocking in the B7-H5 blocker-expressing gene, one or more immune compatible molecules, shRNA and/or miRNA processing complex-associated genes, anti-interferon effector molecules into a safe site in the genome of pluripotent stem cells are shown in table 5-table 6, wherein the "+" symbol indicates the knock-in of a gene or nucleic acid sequence and the "-" symbol indicates the knock-out of a gene.
TABLE 5 constitutive expression protocol
The plasmids selected and the specific knock-in positions were as follows:
general principle:
the anti-B7-H5 antibody is a secretory antibody, and has the structure as follows: signal peptide 1(SEQ ID NO.183) + light chain (end of light chain sequence added with stop codon TGA) + EMCV IRESSwt (SEQ ID NO.178) + signal peptide 1+ heavy chain (end of heavy chain sequence added with stop codon TGA).
The anti-B7-H5 antibody sequence is placed at the position of MCS2 of the corresponding plasmid (signal peptide 1 is added in front of an LC light chain and an HC heavy chain of the antibody, the LC light chain and the HC heavy chain of the antibody are connected by using EMCV IRESKT in the sequence of LC light chain, EMCV IRESKT and HC heavy chain), the shRNA is placed in an shRNA expression frame of the corresponding plasmid, the shRNA-miR is placed in an shRNA-miR expression frame of the corresponding plasmid, and other genes are placed at the position of MCS1 of the corresponding plasmid. The maps of the plasmids are shown in FIGS. 1 to 11.
Note: the sgRNA clone B2M plasmid comprises the sgRNA clone B2M-1 and sgRNA clone B2M-2 plasmids. The sgRNA clone CIITA plasmid comprises sgRNA clone CIITA-1 and sgRNA clone CIITA-2 plasmids.
(1) A1 grouping
MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the antibody sequence.
(2) A2 grouping
MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid was placed into the antibody sequence. The shRNA expression frame is placed into the shRNA target sequence (seamlessly joined if multiple shrnas are present). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(3) A3 grouping
MCS2 of AAVS1 KI Vector (shRNA-miR, constitutive) plasmid was placed into the antibody sequence. The shRNA-miR expression framework is put into a shRNA-miR target sequence (if a plurality of shRNA-miR exist, the shRNA-miR target sequences are connected seamlessly). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(4) A4 grouping
The MCS2 of AAVS1 KI Vector (shRNA, constitutive) plasmid places the antibody sequence, the target sequence of sgRNA clone B2M plasmid places the sgRNA target sequence of B2M (SEQ ID No.179 and SEQ ID No.180), and the target sequence of sgRNA clone CIITA plasmid places the sgRNA target sequence of CIITA (SEQ ID No.181 and SEQ ID No. 182). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(5) A5 grouping
Methods grouped with a 2.
(6) A6 grouping
Methods grouped with a 3.
TABLE 6 Experimental protocol for inducible expression (immuno-compatible reversible)
(1) B1 grouping:
MCS2 of AAVS1 KI Vector (shRNA, inducible) plasmid was placed into the antibody sequence. The shRNA expression framework places the shRNA target sequence (if multiple shrnas are present, they are seamlessly joined). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(2) B2 grouping:
MCS2 of AAVS1 KI Vector (shRNA-miR, inducible) plasmid places antibody sequences. The shRNA-miR expression framework is put into a shRNA-miR target sequence (if a plurality of shRNA-miR exist, the shRNA-miR target sequences are connected seamlessly). MCS1 was placed into the gene sequence (if multiple genes were present, they were ligated using EMCV IRESWT).
(3) B3 grouping:
methods grouped with B1.
(4) B4 grouping:
methods grouped with B2.
3. Results of the experiment
3.1 detection of blocking Effect of B7-H5 antibody expressed by Stem cells or derivatives thereof
The experimental set protocols in tables 5 and 6 were knocked into the genomic safety sites of MSCs at 37 ℃ with 0.5% CO2Culturing in incubator, collecting culture supernatant, and mixing with enzyme-labeled anti-B7-H5 antibody (1:1) (control group added with antibody not expressing B7-H5)Culture supernatant of pluripotent stem cells, gently mixed), and then loaded on an ELISA plate coated with B7-H5 antigen. Sealing the plate, incubating at 37 deg.C for 30min, washing for 5 times, adding color developing solution, developing for 15min, adding stop solution 50uL, and reading to measure absorbance value of 450 nm. The results of the tests of the respective experimental groups are shown in Table 7.
TABLE 7 blocking Effect of B7-H5 antibodies expressed in each experimental group on B7-H5
| Grouping | OD450 | Deviation (+/-) | Independent sample T test (. p)<0.01) |
| N (control) | 1.439 | 0.083 | - |
| A1 | 0.622 | 0.026 | * |
| A2 | 0.650 | 0.030 | * |
| A3 | 0.664 | 0.031 | * |
| A4 | 0.644 | 0.030 | * |
| A5 | 0.644 | 0.040 | * |
| A6 | 0.649 | 0.021 | * |
| B1 | 0.622 | 0.014 | * |
| B2 | 0.653 | 0.031 | * |
| B3 | 0.633 | 0.021 | * |
| B4 | 0.636 | 0.022 | * |
As can be seen from the above table, the B7-H5 antibody expressed by the pluripotent stem cells or the derivatives thereof of the invention can effectively block the binding of the enzyme-labeled B7-H5 antibody and the cell surface B7-H5. And the expression level is relatively constant in each group, so that the B7-H5 antibody expressed by the pluripotent stem cells or the derivatives thereof is not influenced by cell differentiation morphology and other exogenous genes (immune compatibility modification).
3.2 antitumor Effect of pluripotent Stem cells expressing B7-H5 blocking substance or derivatives thereof
The experimental group protocols in tables 5 and 6 were knocked into the genomic safety sites of MSCs cells to obtain cells expressing B7-H5 blockers. Use of51The Cr release test examined its anti-tumor effect, and the results are shown in table 8.
TABLE 8 Effect of B7-H5 antibodies expressed in each experimental group on T cell killing of tumor cells
Note: the N (control) group refers to cells that were not treated with the culture supernatant of pluripotent stem cells expressing the B7-H5 antibody or derivatives thereof. Independent samples were tested for T (./p < 0.01).
Through the experiments, the stem cells expressing the B7-H5 blocker or the derivatives thereof prepared by the invention can be proved to be capable of effectively blocking and activating T cells to play an anti-tumor role.
3.3 antitumor therapeutic Effect of pluripotent Stem cells expressing B7-H5 Block or derivatives thereof
In a humanized NSG mouse tumor model, hPSCs (hPSCs-MSCs) capable of expressing B7-H5 antibody is injected, the tumor treatment effects of NIC lung cancer, HCC liver cancer and ACC colorectal cancer are observed, and only a group containing human immune cells is injected to serve as a control group. Note that in order to avoid the problem of immune compatibility, the immune cells and the derivative of hPSCs are all from the same person, and the immune compatibility scheme of B2M and CIITA gene knockout is adopted. The results of the experiment are shown in FIG. 9.
TABLE 9 tumor therapeutic Effect of pluripotent Stem cells expressing B7-H5 antibody or derivatives thereof
Note: the control group refers to the NSG mouse tumor model without injection of pluripotent stem cells expressing the B7-H5 antibody or derivatives thereof.
Through the experiments, the stem cells expressing the B7-H5 blocker or the derivatives thereof prepared by the invention can effectively block B7-H5 and play an anti-tumor role.
3.4 reversible expression assay for immune-compatible molecule-inducible expression sets
Through the above examples, hPSCs and hPSCs derived derivatives expressing B7-H5 blockers were able to effectively block B7-H5 and thus exert anti-tumor effects. We must also consider the issue of immune compatibility of hPSCs and derivatives of hPSCs origin. Therefore we chose a suitable combination to test for immune compatibility.
By utilizing the characteristic of low immunogenicity of the MSCs, hPSCs (human fibroblast growth factor) sources capable of expressing B7-H5 blockers (anti-B7-H5 antibodies) are injected into humanized NSG mouse tumor models to be immune-compatible with the MSCs, and the effect of treating tumors (NIC lung cancer) is observed. Note that the immunocytes used were derived from a non-identical human as the hPSCs-derived MSCs.
The control group refers to the NSG mouse tumor model without MSCs cell injection.
The process of adding the Dox group is: mice were fed with 0.5mg/mL Dox in the mouse diet, and the mice were used from the time of injection of the expression blocker cells until the end of the experiment. The results are shown in Table 10.
TABLE 10 reversible expression test results for immune-compatible molecule-inducible expression sets
The above experiments show that: MSCs expressing only blocking agents (group 2), which have low immunogenicity and can exist within a foreign body for a certain period of time, can exert a certain tumor treatment effect, while those that are immuno-compatibly engineered (groups 3-11, including constitutive and reversible inducible immuno-compatibility), which have better immuno-compatibility effects than MSCs that have not been immuno-compatibly engineered, exist in vivo for a longer period of time (or can achieve long-term co-existence), which exert a better tumor treatment effect, whereas group 5 is a B2M and CIITA gene knock-out group, which completely eliminates the effects of HLA-I and HLA-II molecules, and thus, has the best tumor treatment effect. However, there are group 8-15 protocols set up due to their constitutive immune compatible modifications (knock-in/knock-out) which cannot be cleared when the graft becomes mutated or otherwise unwanted. In groups 12-15, the mice injected with the expression blocker cells were shown to have abolished the immune compatibility effect by administering Dox inducer (always used) to the mice at the same time as the injection of the expression blocker cells into the mice, and the cells existed in vivo for a period of time comparable to that of the MSCs without immune compatibility modification, and the tumor treatment effect was comparable to that of the MSCs without immune compatibility modification.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> future Chile regenerative medicine research institute (Guangzhou) Inc.; king shower stand
<120> pluripotent stem cell expressing B7-H5 blocking substance or derivative thereof and application
<130>
<160> 183
<170> PatentIn version 3.5
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<210> 20
<211> 21
<212> DNA
<213> human
<400> 20
ggtatttcta cacctccgtg t 21
<210> 21
<211> 21
<212> DNA
<213> human
<400> 21
ggaccggaac acacagatct a 21
<210> 22
<211> 21
<212> DNA
<213> human
<400> 22
accggaacac acagatctac a 21
<210> 23
<211> 21
<212> DNA
<213> human
<400> 23
ggaacacaca gatctacaag g 21
<210> 24
<211> 21
<212> DNA
<213> human
<400> 24
gaacacacag atctacaagg c 21
<210> 25
<211> 21
<212> DNA
<213> human
<400> 25
ttcttacttc cctaatgaag t 21
<210> 26
<211> 21
<212> DNA
<213> human
<400> 26
aagttaagaa cctgaatata a 21
<210> 27
<211> 21
<212> DNA
<213> human
<400> 27
aacctgaata taaatttgtg t 21
<210> 28
<211> 21
<212> DNA
<213> human
<400> 28
acctgaatat aaatttgtgt t 21
<210> 29
<211> 21
<212> DNA
<213> human
<400> 29
aagcgttgat ggattaatta a 21
<210> 30
<211> 21
<212> DNA
<213> human
<400> 30
agcgttgatg gattaattaa a 21
<210> 31
<211> 21
<212> DNA
<213> human
<400> 31
gggtctggtg ggcatcatta t 21
<210> 32
<211> 21
<212> DNA
<213> human
<400> 32
ggtctggtgg gcatcattat t 21
<210> 33
<211> 21
<212> DNA
<213> human
<400> 33
gcatcattat tgggaccatc t 21
<210> 34
<211> 21
<212> DNA
<213> human
<400> 34
gcacatggag gtgatggtgt t 21
<210> 35
<211> 21
<212> DNA
<213> human
<400> 35
ggaggtgatg gtgtttctta g 21
<210> 36
<211> 21
<212> DNA
<213> human
<400> 36
gagaagatca ctgaagaaac t 21
<210> 37
<211> 21
<212> DNA
<213> human
<400> 37
gctttaatgg ctttacaaag c 21
<210> 38
<211> 21
<212> DNA
<213> human
<400> 38
ggctttacaa agctggcaat a 21
<210> 39
<211> 21
<212> DNA
<213> human
<400> 39
gctttacaaa gctggcaata t 21
<210> 40
<211> 21
<212> DNA
<213> human
<400> 40
gctccgtact ctaacatcta g 21
<210> 41
<211> 21
<212> DNA
<213> human
<400> 41
gatgaccaca ttcaaggaag a 21
<210> 42
<211> 21
<212> DNA
<213> human
<400> 42
gaccacattc aaggaagaac t 21
<210> 43
<211> 21
<212> DNA
<213> human
<400> 43
gctttcctgc ttggcagtta t 21
<210> 44
<211> 21
<212> DNA
<213> human
<400> 44
ggcagttatt cttccacaag a 21
<210> 45
<211> 21
<212> DNA
<213> human
<400> 45
gcagttattc ttccacaaga g 21
<210> 46
<211> 21
<212> DNA
<213> human
<400> 46
gcgtaagtct gagtgtcatt t 21
<210> 47
<211> 21
<212> DNA
<213> human
<400> 47
gacaatttaa ggaagaatct t 21
<210> 48
<211> 21
<212> DNA
<213> human
<400> 48
ggccatagtt ctccctgatt g 21
<210> 49
<211> 21
<212> DNA
<213> human
<400> 49
gccatagttc tccctgattg a 21
<210> 50
<211> 21
<212> DNA
<213> human
<400> 50
gcagatgacc acattcaagg a 21
<210> 51
<211> 21
<212> DNA
<213> human
<400> 51
gatgaccaca ttcaaggaag a 21
<210> 52
<211> 21
<212> DNA
<213> human
<400> 52
gaccacattc aaggaagaac c 21
<210> 53
<211> 21
<212> DNA
<213> human
<400> 53
gctttgtcag gaccaggttg t 21
<210> 54
<211> 21
<212> DNA
<213> human
<400> 54
gaccaggttg ttactggttc a 21
<210> 55
<211> 21
<212> DNA
<213> human
<400> 55
gaagcctcac agctttgatg g 21
<210> 56
<211> 21
<212> DNA
<213> human
<400> 56
gatggcagtg cctcatcttc a 21
<210> 57
<211> 21
<212> DNA
<213> human
<400> 57
ggcagtgcct catcttcaac t 21
<210> 58
<211> 21
<212> DNA
<213> human
<400> 58
gcagcaggat aagtatgagt g 21
<210> 59
<211> 21
<212> DNA
<213> human
<400> 59
gcaggataag tatgagtgtc a 21
<210> 60
<211> 21
<212> DNA
<213> human
<400> 60
ggttcctgca cagagacatc t 21
<210> 61
<211> 21
<212> DNA
<213> human
<400> 61
gcacagagac atctataacc a 21
<210> 62
<211> 21
<212> DNA
<213> human
<400> 62
gagacatcta taaccaagag g 21
<210> 63
<211> 21
<212> DNA
<213> human
<400> 63
gagtactgga acagccagaa g 21
<210> 64
<211> 21
<212> DNA
<213> human
<400> 64
gctttcctgc ttggctctta t 21
<210> 65
<211> 21
<212> DNA
<213> human
<400> 65
ggctcttatt cttccacaag a 21
<210> 66
<211> 21
<212> DNA
<213> human
<400> 66
gctcttattc ttccacaaga g 21
<210> 67
<211> 21
<212> DNA
<213> human
<400> 67
ggatgtggaa cccacagata c 21
<210> 68
<211> 21
<212> DNA
<213> human
<400> 68
gatgtggaac ccacagatac a 21
<210> 69
<211> 21
<212> DNA
<213> human
<400> 69
gtggaaccca cagatacaga g 21
<210> 70
<211> 21
<212> DNA
<213> human
<400> 70
ggaacccaca gatacagaga g 21
<210> 71
<211> 21
<212> DNA
<213> human
<400> 71
gagccaactg tattgcctat t 21
<210> 72
<211> 21
<212> DNA
<213> human
<400> 72
agccaactgt attgcctatt t 21
<210> 73
<211> 21
<212> DNA
<213> human
<400> 73
gccaactgta ttgcctattt g 21
<210> 74
<211> 21
<212> DNA
<213> human
<400> 74
gggtagcaac tgtcaccttg a 21
<210> 75
<211> 21
<212> DNA
<213> human
<400> 75
ggatttcgtg ttccagttta a 21
<210> 76
<211> 21
<212> DNA
<213> human
<400> 76
gcatgtgcta cttcaccaac g 21
<210> 77
<211> 21
<212> DNA
<213> human
<400> 77
gcgtcttgtg accagataca t 21
<210> 78
<211> 21
<212> DNA
<213> human
<400> 78
gcttatgcct gcccagaatt c 21
<210> 79
<211> 21
<212> DNA
<213> human
<400> 79
gcaggaaatc actgcagaat g 21
<210> 80
<211> 21
<212> DNA
<213> human
<400> 80
gctcagtgca ttggccttag a 21
<210> 81
<211> 21
<212> DNA
<213> human
<400> 81
ggtgagtgct gtgtaaataa g 21
<210> 82
<211> 21
<212> DNA
<213> human
<400> 82
gacatatata gtgatccttg g 21
<210> 83
<211> 21
<212> DNA
<213> human
<400> 83
ggaaagtcac atcgatcaag a 21
<210> 84
<211> 21
<212> DNA
<213> human
<400> 84
gctcacagtc atcaattata g 21
<210> 85
<211> 21
<212> DNA
<213> human
<400> 85
gccctgaaga cagaatgttc c 21
<210> 86
<211> 21
<212> DNA
<213> human
<400> 86
gcggaccatg tgtcaactta t 21
<210> 87
<211> 21
<212> DNA
<213> human
<400> 87
ggaccatgtg tcaacttatg c 21
<210> 88
<211> 21
<212> DNA
<213> human
<400> 88
gcgtttgtac agacgcatag a 21
<210> 89
<211> 21
<212> DNA
<213> human
<400> 89
ggctggctaa cattgctata t 21
<210> 90
<211> 21
<212> DNA
<213> human
<400> 90
gctggctaac attgctatat t 21
<210> 91
<211> 21
<212> DNA
<213> human
<400> 91
ggaccaggtc acatgtgaat a 21
<210> 92
<211> 21
<212> DNA
<213> human
<400> 92
ggaaaggtct gaggatattg a 21
<210> 93
<211> 21
<212> DNA
<213> human
<400> 93
ggcagattag gattccattc a 21
<210> 94
<211> 21
<212> DNA
<213> human
<400> 94
gcctgatagg acccatattc c 21
<210> 95
<211> 21
<212> DNA
<213> human
<400> 95
gcatccaata gacgtcattt g 21
<210> 96
<211> 21
<212> DNA
<213> human
<400> 96
gcgtcactgg cacagatata a 21
<210> 97
<211> 21
<212> DNA
<213> human
<400> 97
gctgtcacat aataagctaa g 21
<210> 98
<211> 21
<212> DNA
<213> human
<400> 98
gctaaggaag acagtatata g 21
<210> 99
<211> 21
<212> DNA
<213> human
<400> 99
gggatttcta aggaaggatg c 21
<210> 100
<211> 21
<212> DNA
<213> human
<400> 100
ggagttgaag agcagagatt c 21
<210> 101
<211> 21
<212> DNA
<213> human
<400> 101
gccagtgaac acttaccata g 21
<210> 102
<211> 21
<212> DNA
<213> human
<400> 102
gcttctctga agtctcattg a 21
<210> 103
<211> 21
<212> DNA
<213> human
<400> 103
ggctgcaact aacttcaaat a 21
<210> 104
<211> 21
<212> DNA
<213> human
<400> 104
ggatggattt gattatgatc c 21
<210> 105
<211> 21
<212> DNA
<213> human
<400> 105
ggaccttgga acaatggatt g 21
<210> 106
<211> 21
<212> DNA
<213> human
<400> 106
gctaattctt gctgaacttc t 21
<210> 107
<211> 21
<212> DNA
<213> human
<400> 107
gctgaacttc ttcatgtatg t 21
<210> 108
<211> 21
<212> DNA
<213> human
<400> 108
gcctcatctc tttgttctaa a 21
<210> 109
<211> 21
<212> DNA
<213> human
<400> 109
gctctggaga agatatattt g 21
<210> 110
<211> 21
<212> DNA
<213> human
<400> 110
gctcttgagg gaactaatag a 21
<210> 111
<211> 21
<212> DNA
<213> human
<400> 111
gggacggcat taatgtattc a 21
<210> 112
<211> 21
<212> DNA
<213> human
<400> 112
ggacaaacat gcaaactata g 21
<210> 113
<211> 21
<212> DNA
<213> human
<400> 113
gcagcaacca gctaccattc t 21
<210> 114
<211> 21
<212> DNA
<213> human
<400> 114
gcagttctgt tgccactctc t 21
<210> 115
<211> 21
<212> DNA
<213> human
<400> 115
gggagagttc atccaggaaa t 21
<210> 116
<211> 21
<212> DNA
<213> human
<400> 116
ggagagttca tccaggaaat t 21
<210> 117
<211> 21
<212> DNA
<213> human
<400> 117
gagagttcat ccaggaaatt a 21
<210> 118
<211> 21
<212> DNA
<213> human
<400> 118
gcctgtcaaa gagagagagc a 21
<210> 119
<211> 21
<212> DNA
<213> human
<400> 119
gctcagcttc gtactgagtt c 21
<210> 120
<211> 21
<212> DNA
<213> human
<400> 120
gcttcacaga actacagaga g 21
<210> 121
<211> 21
<212> DNA
<213> human
<400> 121
gcatctactg gacaaagtat t 21
<210> 122
<211> 21
<212> DNA
<213> human
<400> 122
ggctgaatta cccatgcttt a 21
<210> 123
<211> 21
<212> DNA
<213> human
<400> 123
gctgaattac ccatgcttta a 21
<210> 124
<211> 21
<212> DNA
<213> human
<400> 124
gggttggttt atccaggaat a 21
<210> 125
<211> 21
<212> DNA
<213> human
<400> 125
ggatcagaag agaagccaac g 21
<210> 126
<211> 21
<212> DNA
<213> human
<400> 126
ggttcaccat ccaggtgttc a 21
<210> 127
<211> 21
<212> DNA
<213> human
<400> 127
gctctcttct ctggaactaa c 21
<210> 128
<211> 21
<212> DNA
<213> human
<400> 128
gctagagtga ctccatctta a 21
<210> 129
<211> 21
<212> DNA
<213> human
<400> 129
gctgaccacc aattataatt g 21
<210> 130
<211> 21
<212> DNA
<213> human
<400> 130
gcagaatatt taaggccata c 21
<210> 131
<211> 21
<212> DNA
<213> human
<400> 131
gcccacttaa aggcagcatt a 21
<210> 132
<211> 21
<212> DNA
<213> human
<400> 132
ggtcatcaat accactgtta a 21
<210> 133
<211> 21
<212> DNA
<213> human
<400> 133
gcattcctcc ttctcctttc t 21
<210> 134
<211> 21
<212> DNA
<213> human
<400> 134
ggaggaactt tgtgaacatt c 21
<210> 135
<211> 21
<212> DNA
<213> human
<400> 135
gctgtaagaa ggatgctttc a 21
<210> 136
<211> 21
<212> DNA
<213> human
<400> 136
gctgcaggca ggattgtttc a 21
<210> 137
<211> 21
<212> DNA
<213> human
<400> 137
gcagttcgag gtcaagtttg a 21
<210> 138
<211> 21
<212> DNA
<213> human
<400> 138
gccaattagc tgagaagaat t 21
<210> 139
<211> 21
<212> DNA
<213> human
<400> 139
gcaggtttac agtgtatatg t 21
<210> 140
<211> 21
<212> DNA
<213> human
<400> 140
gcctacagag actagagtag g 21
<210> 141
<211> 21
<212> DNA
<213> human
<400> 141
gcagttgggt accttccatt c 21
<210> 142
<211> 21
<212> DNA
<213> human
<400> 142
gcaactcagg tgcatgatac a 21
<210> 143
<211> 21
<212> DNA
<213> human
<400> 143
gcatggcgct ggtacgtaaa t 21
<210> 144
<211> 19
<212> DNA
<213> human
<400> 144
gcctcgagtt tgagagcta 19
<210> 145
<211> 19
<212> DNA
<213> human
<400> 145
agacattctg gatgagtta 19
<210> 146
<211> 19
<212> DNA
<213> human
<400> 146
gggtctgtta cccaaagaa 19
<210> 147
<211> 19
<212> DNA
<213> human
<400> 147
ggtctgttac ccaaagaat 19
<210> 148
<211> 19
<212> DNA
<213> human
<400> 148
ggaaggaagc ggacgctca 19
<210> 149
<211> 19
<212> DNA
<213> human
<400> 149
ggaggcagta cttctgata 19
<210> 150
<211> 19
<212> DNA
<213> human
<400> 150
cgctctagag ctcagctga 19
<210> 151
<211> 19
<212> DNA
<213> human
<400> 151
ccaccacctc aaccaataa 19
<210> 152
<211> 19
<212> DNA
<213> human
<400> 152
atttcaagaa gtcgatcaa 19
<210> 153
<211> 19
<212> DNA
<213> human
<400> 153
gaagatctga ttaccttca 19
<210> 154
<211> 21
<212> DNA
<213> human
<400> 154
ggacactggt tcaacacctg t 21
<210> 155
<211> 21
<212> DNA
<213> human
<400> 155
ggttcaacac ctgtgacttc a 21
<210> 156
<211> 21
<212> DNA
<213> human
<400> 156
acctgtgact tcatgtgtgc g 21
<210> 157
<211> 21
<212> DNA
<213> human
<400> 157
gctggacgtg accatcatgt a 21
<210> 158
<211> 21
<212> DNA
<213> human
<400> 158
ggacgtgacc atcatgtaca a 21
<210> 159
<211> 21
<212> DNA
<213> human
<400> 159
gacgtgacca tcatgtacaa g 21
<210> 160
<211> 21
<212> DNA
<213> human
<400> 160
acgtgaccat catgtacaag g 21
<210> 161
<211> 21
<212> DNA
<213> human
<400> 161
acgctatacc atctacctgg g 21
<210> 162
<211> 21
<212> DNA
<213> human
<400> 162
gcctctatga cgacatcgag t 21
<210> 163
<211> 21
<212> DNA
<213> human
<400> 163
gacatcgagt gcttccttat g 21
<210> 164
<211> 253
<212> DNA
<213> Artificial sequence
<400> 164
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgctagcgcc acc 253
<210> 165
<211> 9
<212> DNA
<213> Artificial sequence
<400> 165
ttcaagaga 9
<210> 166
<211> 686
<212> DNA
<213> Artificial sequence
<400> 166
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
ctttaccact ccctatcagt gatagagaaa agtgaaagtc gagtttacca ctccctatca 300
gtgatagaga aaagtgaaag tcgagtttac cactccctat cagtgataga gaaaagtgaa 360
agtcgagttt accactccct atcagtgata gagaaaagtg aaagtcgagt ttaccactcc 420
ctatcagtga tagagaaaag tgaaagtcga gtttaccact ccctatcagt gatagagaaa 480
agtgaaagtc gagtttacca ctccctatca gtgatagaga aaagtgaaag tcgagctcgg 540
tacccgggtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 600
aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg 660
gaccgatcca gcctgctagc gccacc 686
<210> 167
<211> 119
<212> DNA
<213> Artificial sequence
<400> 167
gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt gctgggatta 60
cttcttcagg ttaacccaac agaaggctaa agaaggtata ttgctgttga cagtgagcg 119
<210> 168
<211> 19
<212> DNA
<213> Artificial sequence
<400> 168
tagtgaagcc acagatgta 19
<210> 169
<211> 119
<212> DNA
<213> Artificial sequence
<400> 169
tgcctactgc ctcggacttc aaggggctac tttaggagca attatcttgt ttactaaaac 60
tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat ggtataaat 119
<210> 170
<211> 22
<212> DNA
<213> Artificial sequence
<400> 170
ccatagctca gtctggtcta tc 22
<210> 171
<211> 22
<212> DNA
<213> Artificial sequence
<400> 171
tcaggatgat ctggacgaag ag 22
<210> 172
<211> 20
<212> DNA
<213> Artificial sequence
<400> 172
ccggtcctgg actttgtctc 20
<210> 173
<211> 20
<212> DNA
<213> Artificial sequence
<400> 173
ctcgacatcg gcaaggtgtg 20
<210> 174
<211> 20
<212> DNA
<213> Artificial sequence
<400> 174
cgcattggag tcgctttaac 20
<210> 175
<211> 24
<212> DNA
<213> Artificial sequence
<400> 175
cgagctgcaa gaactcttcc tcac 24
<210> 176
<211> 23
<212> DNA
<213> Artificial sequence
<400> 176
cacggcactt acctgtgttc tgg 23
<210> 177
<211> 23
<212> DNA
<213> Artificial sequence
<400> 177
cagtacaggc atccctgtga aag 23
<210> 178
<211> 590
<212> DNA
<213> Artificial sequence
<400> 178
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataatatggc cacaaccatg 590
<210> 179
<211> 23
<212> DNA
<213> Artificial sequence
<400> 179
cgcgagcaca gctaaggcca cgg 23
<210> 180
<211> 23
<212> DNA
<213> Artificial sequence
<400> 180
actctctctt tctggcctgg agg 23
<210> 181
<211> 23
<212> DNA
<213> Artificial sequence
<400> 181
acccagcagg gcgtggagcc agg 23
<210> 182
<211> 23
<212> DNA
<213> Artificial sequence
<400> 182
gtcagagccc caaggtaaaa agg 23
<210> 183
<211> 60
<212> DNA
<213> Artificial sequence
<400> 183
atgtccgtgc ccacccaggt gctgggattg ctgctgctgt ggctgaccga cgccagatgc 60
Claims (20)
1. A pluripotent stem cell expressing a B7-H5 blocker or a derivative thereof, wherein: the genome of the pluripotent stem cell or the derivative thereof is introduced with an expression sequence of a B7-H5 blocker, and the B7-H5 blocker is an anti-B7-H5 antibody.
2. The pluripotent stem cell or derivative thereof according to claim 1, wherein: the B2M and/or CIITA gene of the pluripotent stem cell or the derivative thereof is knocked out.
3. The pluripotent stem cell or derivative thereof according to claim 1, wherein: the genome of the pluripotent stem cell or the derivative thereof is further introduced with one or more immune compatible molecule expression sequences for regulating the expression of genes associated with an immune response in the pluripotent stem cell or the derivative thereof.
4. The pluripotent stem cell or derivative thereof according to claim 3, wherein: the genes associated with the immune response include:
(1) major histocompatibility complex genes including at least one of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB 1;
(2) major histocompatibility complex-associated genes including at least one of B2M and CIITA.
5. The pluripotent stem cell or derivative thereof according to claim 3, wherein: the immune-compatible molecule includes any one or more of:
(1) immune tolerance related genes including CD47 or HLA-G;
(2) HLA-C molecules, including HLA-C multiple alleles of which the proportion in the population is over 90 percent in total, or fusion protein genes consisting of the HLA-C multiple alleles of which the proportion is over 90 percent and B2M;
(3) shRNA and/or shRNA-miR of major histocompatibility complex genes including at least one of HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1 and HLA-DPB 1;
(4) shRNA and/or shRNA-miR of a major histocompatibility complex-associated gene that includes at least one of B2M and CIITA.
6. The pluripotent stem cell or derivative thereof according to claim 5, wherein:
the target sequence of the shRNA and/or shRNA-miR of B2M is at least one of SEQ ID NO. 3-SEQ ID NO. 5;
the target sequence of the shRNA and/or shRNA-miR of the CIITA is at least one of SEQ ID NO. 6-SEQ ID NO. 15;
the target sequence of the shRNA and/or shRNA-miR of the HLA-A is at least one of SEQ ID NO. 16-SEQ ID NO. 18;
the target sequence of the shRNA and/or shRNA-miR of the HLA-B is at least one of SEQ ID NO. 19-SEQ ID NO. 24;
the target sequence of the shRNA and/or shRNA-miR of the HLA-C is at least one of SEQ ID NO. 25-SEQ ID NO. 30;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRA is at least one of SEQ ID NO. 31-SEQ ID NO. 40;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB1 is at least one of SEQ ID NO. 41-SEQ ID NO. 45;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB3 is at least one of SEQ ID NO. 46-SEQ ID NO. 47;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB4 is at least one of SEQ ID NO. 48-SEQ ID NO. 57;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DRB5 is at least one of SEQ ID NO. 58-SEQ ID NO. 66;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DQA1 is at least one of SEQ ID NO. 67-SEQ ID NO. 73;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DQB1 is at least one of SEQ ID NO. 74-SEQ ID NO. 83;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DPA1 is at least one of SEQ ID NO. 84-SEQ ID NO. 93;
the target sequence of the shRNA and/or shRNA-miR of the HLA-DPB1 is at least one of SEQ ID NO. 94-SEQ ID NO. 103.
7. The pluripotent stem cell or derivative thereof according to claim 3, wherein: shRNA and/or miRNA processing complex related genes and/or anti-interferon effector molecules are also introduced into the genome of the pluripotent stem cell or the derivative thereof.
8. The pluripotent stem cell or derivative thereof according to claim 7, wherein: the shRNA and/or miRNA processing complex related gene comprises at least one of Drosha, Ago1, Ago2, Dicer1, Exportin-5, TRBP (TARBP2), PACT (PRKRA) and DGCR 8; the anti-interferon effector molecule is shRNA and/or shRNA-miR of at least one of PKR, 2-5As, IRF-3 and IRF-7.
9. The pluripotent stem cell or derivative thereof according to claim 8, wherein:
the target sequence of the shRNA and/or shRNA-miR of the PKR is at least one of SEQ ID NO. 104-SEQ ID NO. 113;
the target sequence of the shRNA and/or shRNA-miR of the 2-5As is at least one of SEQ ID NO. 114-SEQ ID NO. 143;
the target sequence of the shRNA and/or shRNA-miR of the IRF-3 is at least one of SEQ ID NO. 144-SEQ ID NO. 153;
the target sequence of the shRNA and/or shRNA-miR of the IRF-7 is at least one of SEQ ID NO. 154-SEQ ID NO. 163.
10. The pluripotent stem cell or the derivative thereof according to claim 6 or 9, wherein: the expression frameworks of the major histocompatibility complex gene, the major histocompatibility complex related gene, PKR, 2-5As, the shRNA and/or shRNA-miR of IRF-3 or IRF-7 are As follows:
(1) shRNA expression framework: 5 'to 3' sequentially comprises the shRNA target sequence of claim 6 or 9, a stem-loop sequence, a reverse complement of the shRNA target sequence of claim 6 or 9, and Poly T; the two reverse complementary target sequences are separated by a middle stem-loop sequence to form a hairpin structure, and finally Poly T is connected to be used as a transcription terminator of RNA polymerase III;
(2) shRNA-miR expression framework: the shRNA-miR target sequence of claim 6 or 9 is used for replacing a target sequence in microRNA-30 or microRNA-155.
11. The pluripotent stem cell or derivative thereof of claim 10, wherein: the length of a stem-loop sequence in the shRNA expression frame is 3-9 bases; the length of the Poly T is 5-6 bases.
12. The pluripotent stem cell or the derivative thereof according to claim 3 or 7, wherein: an inducible gene expression system is also introduced into the genome of the pluripotent stem cell or the derivative thereof.
13. The pluripotent stem cell or derivative thereof of claim 12, wherein: the inducible gene expression system is at least one of a Tet-Off system and a dimer inducible expression system.
14. The pluripotent stem cell or the derivative thereof according to claim 12, wherein:
the introduction of the B7-H5 blocker expression sequence, the expression sequence of immune compatible molecules, shRNA and/or miRNA processing complex related genes, anti-interferon effector molecules and inducible gene expression system adopts a method of viral vector interference, non-viral vector transfection or gene editing.
15. The pluripotent stem cell or the derivative thereof according to claim 14, wherein:
the introduction sites of the B7-H5 blocker expression sequence, the immune compatible molecule expression sequence, shRNA and/or miRNA processing complex related genes, the anti-interferon effector molecule and the inducible gene expression system are genome safety sites of the pluripotent stem cells or derivatives thereof.
16. The pluripotent stem cell or derivative thereof of claim 15, wherein: the genome safe site comprises one or more of an AAVS1 safe site, an eGSH safe site and an H11 safe site.
17. The pluripotent stem cell or the derivative thereof according to any one of claims 1 to 9, 11 or 13 to 16, wherein: the pluripotent stem cells comprise embryonic stem cells, embryonic germ cells, embryonic cancer cells, or induced pluripotent stem cells;
the pluripotent stem cell derivative includes an adult stem cell, each germ layer cell or tissue into which the pluripotent stem cell is differentiated;
the adult stem cells include mesenchymal stem cells or neural stem cells.
18. The pluripotent stem cell or derivative thereof of claim 17, wherein: the heavy chain sequence of the anti-B7-H5 antibody is shown as SEQ ID NO.1, and the light chain sequence is shown as SEQ ID NO. 2.
19. Use of the pluripotent stem cell or derivative thereof of any one of claims 1 to 18 for the preparation of a medicament for treating a B7-H5 high-expression tumor.
20. A formulation, characterized by: comprising the pluripotent stem cell according to any one of claims 1 to 18 or a derivative thereof.
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| US20170067021A1 (en) * | 2015-07-31 | 2017-03-09 | Regents Of The University Of Minnesota | Modified cells and methods of therapy |
| CN111556892A (en) * | 2017-12-08 | 2020-08-18 | 菲特治疗公司 | Immunotherapy using enhanced iPSC-derived effector cells |
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