Patent Application
20250222039
The present invention claims the priority of the prior applications with a patent application number of 202210307870.8 submitted to China National Intellectual Property Administration on Mar. 25, 2022, entitled “Universal cell and method for preparation same”, and with a patent application number of 202210806871.7 submitted to China National Intellectual Property Administration on Jul. 8, 2022, entitled “Universal cell and method for preparation same”. The entire contents of these two prior applications are incorporated herein by reference.
The present invention belongs to the cross field of genetic engineering and stem cell technology, and specifically relates to a universal cell and a method for preparation the same.
Through in vitro cell culture or induced differentiation of stem cells, healthy functional cells can be regenerated in large quantities in vitro, and diseases can be treated through allogeneic functional cell transplantation. However, immune incompatibility and immune rejection of transplanted cells remain key obstacles to their clinical application. Stem cells are a kind of “seed” cells with the ability of self-renewal and differentiation into specific functional cells. According to the differences of the extent of stem cell properties, stem cells are mainly divided into Totipotent stem cells, Pluripotent stem cells (PSCs) and Adult stem cells. Human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC) have the potential to proliferate indefinitely, self-renew, and differentiate into various types of cells, and have important application prospects in the treatment of cancers, nerve-related diseases, cardiovascular diseases and the like.
Transplantation of autologous cells can avoid the problem of immune rejection, but the cost of manufacturing autologous cells from patients is high and the preparation process cycle is long (Khera et al., 2013), and the quality and effectiveness of cell products from individual sources are uncertain. Data show that the cells in patients' bodies are different from those in normal people, and the effectiveness of treatment may be affected.
Immunogenicity, or rejection reaction of the host immune system to allogeneic transplanted cells, can be reduced through immunosuppressive drugs, HLA matching, and gene editing. Immunosuppressive drugs have serious side effects and can cause bone marrow suppression, liver toxicity, hair loss and gastrointestinal adverse reactions. At present, in the United States, Japan and China, HLA-matched iPSC libraries are being established, but the construction and maintenance of the libraries are costly, because HLA antigen genes are the most polymorphic genes observed in the human genome. Currently, iPSC libraries in various regions cannot provide matches for most people in their respective countries and can only cover specific populations (Solomon et al., 2015; Turner et al., 2013). Allogeneic cell therapy for large patient groups may have very obvious advantages over matching libraries in terms of economy and construction and operating costs, but allogeneic cell therapy will be subject to strong immune rejection. Therefore, it is urgent to construct universal PSCs that are allogeneic immune-compatible.
The human major histocompatibility complex (MHC), i.e., the human leukocyte antigen (HLA), is the main cause of immune incompatibility. The HLA complex consists of a series of genes that can be divided into class I, class II and class III. The MHC-I gene is expressed in almost all tissue cell types, and transplanted cells expressing “non-self” MHC class I molecules will stimulate the activation of CD8+ T cells and be eliminated. CD4+ helper T cells recognize the MHC-II gene of “non-self” cells, and thus immune rejection happens, but the class III molecules do not participate in immune activities. The use of a gene editing method to modify immunogenic elements to produce cells with low immunogenicity makes it possible to manufacture “off-the-shelf” cell therapy products with immune privilege on a large scale.
In recent years, it has been previously reported that by knocking out genes such as B2M and CIITA, the expression of MHC-I and MHC-II on cell surfaces or genes themselves can be deleted, thereby making the cells have immune tolerance or escape T/B cell-specific immune responses, generating immune-compatible universal PSCs, which has laid an important foundation for the wider application of cells, tissues, and organs derived from the universal PSCs. However, HLA molecules are the major inhibitory ligands of natural killer cells (NK cells), and MHC class I-negative cells are susceptible to natural killer (NK) cell lysis. In vitro and in vivo data show that host NK cells can eliminate implanted B2M−/− donor cells (Flahou et al., 2021). Therefore, there is a need to improve previous methods to generate universal donor cells that can avoid immune responses. It has been reported that by disrupting the expression of MHC class I and MHC class II genes, cells can express non-classical HLA class I molecules such as HLA-E/G, or express immune inhibitory checkpoint proteins such as PD-L1, CTLA4-Ig, CD47, and CD24, and thus can effectively escape killing by NK cells (Zhao, W. et al., 2020; Ye, Q. et al., 2020; WO2021041316 A1).
These schemes have technical problems, such as incomplete, unclear or non-lasting immune compatibility, narrow effective dose window, etc., only based on direct application of common stellar molecules known in the art, thus indicating that more innovative and more optimized strategies are needed to achieve more optimized modification of stem cells to obtain better immune privilege schemes.
In response to the shortcomings of the existing technology, the present invention provides a new method for modifying cells to obtain low immunogenicity, which adopts a strategy of new genes with domain fusion for the first time, carries out a strategic practice of modifying human stem cells based on CD47 and CD24 to obtain low immunogenicity, and finally identifies a representative new fusion gene through multiple rounds of screening and sequence combination testing. The new fusion gene can significantly reduce or escape the recognition and attack of the immune system, especially the attack of natural killer cells, macrophages, etc. The present invention provides a feasible strategy to realize the immune privilege of cells by developing new genes with domain fusion.
In the present invention, by knocking out β-2-microglobulin (B2M) in the endoplasmic reticulum of human pluripotent stem cells and knocking out CIITA, a positive regulatory factor of the MHC-II gene transcription, a positive clone with B2M/CIITA double alleles knocked out (a DKO cell) is successfully constructed; then a lentivirus vector is used to overexpress a new fusion gene identified by the present invention in the DKO cell, such that the obtained human pluripotent stem cells can, on the basis of escaping T cell attack, further escape killing by NK cells, thus achieving an unexpectedly excellent immune escaping effect compared with DKO+CD47 (hereinafter referred to as “CD47 prior art”). Meanwhile, the pluripotent stem cells with low immunogenicity retain key biological functions of the pluripotent stem cells such as their stemness and differentiation capacity.
In order to achieve the above-mentioned object, the present invention adopts the following technical solutions:
In a first aspect, the present invention provides a universal cell, relative to a wild-type cell, comprising:
In certain aspects, the fusion protein comprising immune inhibitory checkpoints include two or more of PD-L1, CTLA4-Ig, CD47, and CD24. Preferably, a fusion protein comprising two of the immune inhibitory checkpoints is expressed.
According to the present invention, a fusion protein comprising functional domains of CD47 and/or CD24 is expressed.
In certain aspects, the cell includes reduced expression or no expression of MHC-I and MHC-II human leukocyte antigens.
In certain aspects, gene editing tools (such as the TALEN and/or CRISPR system) are used in the cell to target one or more genes encoding one or more transcriptional regulatory factors of MHC-I, and one or more genes encoding one or more transcriptional regulatory factors of MHC-II, so as to achieve reduced expression or no expression of MHC-I and MHC-II genes.
In certain embodiments, in order to achieve reduced expression or no expression of MHC-I and MHC-II genes, the transcriptional regulatory factor of MHC-I can be preferably selected from one or more of B2M, TAP1, TAP2, TAP-related glycoproteins (Tapasin) or NLRC5; the transcriptional regulatory factor of MHC-II can be preferably selected from one or more of CIITA, RFXANK, RFX5 and RFXAP;
In certain embodiments, the cell further includes a genetic modification targeting a CIITA gene by a rare cutting endonuclease that selectively inactivates the CIITA gene.
In certain embodiments, the cell further includes a genetic modification targeting a B2M gene by a rare cutting endonuclease that selectively inactivates the B2M gene.
In certain embodiments, the genetic modification targeting the CIITA gene or the B2M gene by the rare cutting endonuclease includes a CAS protein or a polynucleotide encoding a CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or the B2M.
In a specific embodiment, a CRISPR/CAS9 system is used to directly knock out the exon segments of B2M and CIITA at both ends, respectively, wherein the target sequences of gRNA for the B2M gene are SEQ ID NO: 2 and SEQ ID NO: 3, and the target sequences of gRNA for the CIITA gene are SEQ ID NO: 4 and SEQ ID NO: 5.
In certain aspects, gene expression modifying molecules for one or more genes encoding one or more transcriptional regulatory factors of MHC-I, or one or more genes encoding one or more transcriptional regulatory factors of MHC-II are introduced into the cells, so as to achieve reduced expression or no expression of MHC-I and/or MHC-II genes, wherein the gene expression modifying molecules comprise one selected from siRNA, shRNA, microRNA, antisense RNA and another RNA-mediated inhibitory molecule.
In certain aspects, the functional domain of CD47 in the fusion protein comprising the functional domains of CD47 and/or CD24 is a transmembrane domain of CD47; preferably, the amino acid sequences of the transmembrane domains of CD47 are as shown in any one of SEQ ID Nos: 6-10.
In certain aspects, the functional domain of CD24 in the fusion protein comprising the functional domains of CD47 and/or CD24 is a signal peptide sequence of CD24, a mature peptide of CD24, an extracellular peptide of CD24, a membrane anchoring sequence of CD24, and an extracellular mature peptide of CD24; preferably, the amino acid sequences of the functional domains of CD24 are as shown in any one of SEQ ID NOs:11-16.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which a SIRPα binding domain of CD47 is connected to the membrane anchoring sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is inserted into the linking site of the extracellular sequence and the membrane anchoring sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is linked after the extracellular sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide of CD24 is linked after the SIRPα binding domain of CD47.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the extracellular mature peptide of CD24 is inserted into the linking site of the SIRPα binding domain and the transmembrane domain of CD47.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide sequence of CD24 is connected to the transmembrane domain of CD47;
In certain aspects, the nucleic acid sequence encoding the fusion protein constructed by the functional domains of CD47 and CD24 can be obtained based on the amino acid sequence of the fusion protein.
In certain aspects, the cell further includes modifications to increase the expression of one or more of the following polypeptides: DUX4, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, the HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, the Cl-inhibitor, IL-10, IL-35, CCL21, Mfge8 and SerpinB9.
In certain aspects, the cell is an embryonic stem cell.
In certain aspects, the cell is a pluripotent stem cell.
In certain embodiments, the cell is a stem cell with low immunogenicity.
In certain embodiments, the cell is a human stem cell or a human somatic cell.
In a second aspect, the present invention provides a method for preparing the universal cell of the first aspect, comprising the following steps:
In certain aspects, the fusion protein comprising immune inhibitory checkpoints includes more of PD-L1, CTLA4-Ig, CD47, and CD24. Preferably, a nucleic acid sequence encoding a fusion protein comprising two of the immune inhibitory checkpoints is introduced.
According to the present invention, a nucleic acid sequence encoding a fusion protein constructed by comprising functional domains of CD47 and/or CD24 is introduced.
In certain aspects, the transcriptional regulatory factor of MHC-I can be preferably selected from one or more of B2M, TAP1, TAP2, TAP-related glycoproteins (Tapasin) or NLRC5; the transcriptional regulatory factor of MHC-II can be preferably selected from one or more of CIITA, RFXANK, RFX5 and RFXAP.
In certain embodiments, the transcriptional regulatory factors are selected from B2M and CIITA.
In certain aspects, the knocking out in steps 1) and 2) is a genetic modification targeting a CIITA gene or a B2M gene by a rare cutting endonuclease that selectively inactivates the CIITA gene or the B2M gene.
Preferably, the rare cutting endonuclease is selected from CAS proteins, the TALE-nucleases, zine finger nucleases, large nucleases and homing nucleases.
Further preferably, the genetic modification targeting the CIITA gene or the B2M gene by the rare cutting endonuclease comprises a CAS protein or a polynucleotide encoding a CAS protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene or the B2M.
In certain embodiments, a CRISPR system is used in steps 1) and 2) to directly knock out the exon segments of B2M and CIITA at both ends, respectively, wherein the target sequences of gRNA for the B2M gene are SEQ ID NO: 2 and 3, and the target sequences of gRNA for the CIITA gene are SEQ ID NO: 4 and 5.
In certain aspects, the knocking out in step 1) or 2) is introducing gene expression modifying molecules for one or more genes encoding one or more transcriptional regulatory factors of MHC-I, or for one or more genes encoding one or more transcriptional regulatory factors of MHC-II, so as to achieve reduced expression or no expression of MHC-I and/or MHC-II genes, wherein the gene expression modifying molecules comprise one selected from siRNA, shRNA, microRNA, antisense RNA and another RNA-mediated inhibitory molecule.
In certain aspects, in step 3), an expression vector is used to introduce a nucleic acid sequence encoding a fusion protein comprising functional domains of CD47 and/or CD24 into cells.
Preferably, the expression vector used in the step 3) is a viral vector.
In certain embodiments, the viral vector used in the step 3) is a lentivirus.
In certain aspects, step 3) introduces a nucleic acid sequence encoding a fusion protein comprising functional domains of CD47 and/or CD24 into a selected site of the cell; preferably, the selected site of the cell is a safe harbor gene site.
In certain aspects, the functional domain of CD47 in the fusion protein comprising the functional domains of CD47 and/or CD24 is a transmembrane domain of CD47; preferably, the amino acid sequences of the transmembrane domains of CD47 are as shown in any one of SEQ ID Nos: 6-10.
In certain aspects, the functional domain of CD24 in the fusion protein comprising the functional domains of CD47 and/or CD24 is a signal peptide sequence of CD24, a mature peptide of CD24, an extracellular peptide of CD24, a membrane anchoring sequence of CD24, and an extracellular mature peptide of CD24; preferably, the amino acid sequences of the functional domains of CD24 are as shown in any one of SEQ ID NOs:11-16.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which a SIRPα binding domain of CD47 is connected to the membrane anchoring sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is inserted into the linking site of the extracellular sequence and the membrane anchoring sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is linked after the extracellular sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide of CD24 is linked after the SIRPα binding domain of CD47.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the extracellular mature peptide of CD24 is inserted into the linking site of the SIRPα binding domain and the transmembrane domain of CD47.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide sequence of CD24 is connected to the transmembrane domain of CD47;
Preferably, the fusion protein constructed by the functional domains of CD47 and CD24 has more than at least 70% homology with the sequence shown in SEQ ID NO: 1, for example more than 80% homology, and for example more than 90%, more than 95%, or more than 98% homology; further preferably, the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 is as shown in SEQ ID NO:1.
In certain aspects, the universal cell further comprises a second expression vector, comprising a polynucleotide sequence encoding one selected from CD35, CD27, DUX4, CD26, CD200, HLA-C, HLA-E, the HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, the Cl-inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL-35.
In certain embodiments, the second expression vector is an inducible expression vector; preferably, the second expression vector is a viral vector.
In a third aspect, the present invention provides a method for preparing differentiated universal cells, including culturing the universal cells prepared according to the method according to the second aspect under differentiation conditions, thereby preparing differentiated cells with low immunogenicity.
In certain aspects, the differentiation conditions are suitable for differentiating cells into a cell type selected from cardiomyocytes, neurocytes, glial cells, endothelial cells, T cells, NK cells, NKT cells, macrophages, hematopoietic progenitor cells, mesenchymal cells, pancreatic islet cells, chondrocytes, retinal pigment epithelial cells, kidney cells, hepatocytes, thyroid cells, skin cells, blood cells and epithelial cells.
In a fourth aspect, the present invention provides a method for treating a patient in need of cell therapy, including administering the differentiated cell population with low immunogenicity prepared according to the method according to the third aspect.
In a fifth aspect, the present invention provides a composition, comprising the universal cell described in the first aspect.
In certain aspects, the composition comprises the universal cell described in the first aspect and one or more therapeutic agents, and the therapeutic agent comprises peptides, cytokines, small molecular compounds, macromolecules, ADC, antibodies, nanoparticles, biological analogues, mRNA, Chinese herbologies, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double stranded RNAs (dsRNAs), mononuclear blood cells, feeder cells, feeder cell components or replacement factors thereof, vectors comprising one or more polynucleic acids of interest, antibodies, etc.
In a sixth aspect, the present invention provides a fusion protein, comprising the functional domains of CD47 and/or CD24.
In certain aspects, the functional domain of CD47 in the fusion protein comprising the functional domains of CD47 and/or CD24 is a transmembrane domain of CD47; preferably, the amino acid sequences of the transmembrane domains of CD47 are as shown in any one of SEQ ID Nos: 6-10.
In certain aspects, the functional domain of CD24 in the fusion protein comprising the functional domains of CD47 and/or CD24 is a signal peptide sequence of CD24, a mature peptide of CD24, an extracellular peptide of CD24, a membrane anchoring sequence of CD24, and an extracellular mature peptide of CD24; preferably, the amino acid sequences of the functional domains of CD24 are as shown in any one of SEQ ID NOs:11-16.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which a SIRPα binding domain of CD47 is connected to the membrane anchoring sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the SIRPα binding domain of CD47 is inserted into the linking site of the extracellular sequence and the membrane anchoring sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which CD47 is linked after the extracellular sequence of CD24.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide of CD24 is linked after the SIRPα binding domain of CD47.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the extracellular mature peptide of CD24 is inserted into the linking site of the SIRPα binding domain and the transmembrane domain of CD47.
In certain embodiments, the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein in which the mature peptide sequence of CD24 is connected to the transmembrane domain of CD47; Preferably, the fusion protein constructed by the functional domains of CD47 and CD24 has more than at least 70% homology with the sequence shown in SEQ ID NO: 1, for example more than 80% homology, and for example more than 90%, more than 95%, or more than 98% homology; further preferably, the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 is as shown in SEQ ID NO:1.
In a seventh aspect, the present invention provides a nucleic acid sequence encoding the fusion protein of the sixth aspect, wherein the nucleic acid sequence can be obtained based on the amino acid sequence of the fusion protein.
In an eighth aspect, the present invention provides an expression vector or expression cassette, comprising the nucleic acid sequence of the seventh aspect.
In a ninth aspect, the present invention provides a cell, comprising the expression of the fusion protein of the sixth aspect, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In a tenth aspect, the present invention provides a cell, comprising no expression of CIITA, the expression of the fusion protein of the sixth aspect, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In an eleventh aspect, the present invention provides a cell, comprising no expression of B2M, the expression of the fusion protein of the sixth aspect, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In a twelfth aspect, the present invention provides a cell, comprising no expression of CIITA and B2M, the expression of the fusion protein of the sixth aspect, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In a thirteenth aspect, the present invention provides a cell, comprising the expression of the fusion protein of the sixth aspect and at least one polypeptide selected from DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, the Cl-inhibitor, CD46, CD55, CD59, and IL-35, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In a fourteenth aspect, the present invention provides a cell, comprising no expression of CIITA, the expression of the fusion protein of the sixth aspect and at least one polypeptide selected from CD35, CD27, DUX4, CD26, CD55, CD59, CD200, HLA-C, HLA-E, the HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, the Cl-inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL-35, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In a fifth aspect, the present invention provides a cell, comprising no expression of B2M, the expression of the fusion protein of the sixth aspect and at least one polypeptide selected from DUX4, HLA-C, HLA-E, HLA-G, PD-L1, CTLA4, the Cl-inhibitor, CD46, CD55, CD59, and IL-35, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In a sixteenth aspect, the present invention provides a cell, comprising no expression of CIITA and B2M, the expression of the fusion protein of the sixth aspect and at least one polypeptide selected from CD35, CD27, DUX4, CD26, CD55, CD59, CD200, HLA-C, HLA-E, the HLA-E heavy chain, HLA-G, PD-L1, IDO1, IDO2, TDO, CTLA4-IgG, the Cl-inhibitor, IL-10, CD46, CD55, CD59, CCL21, Mfge8, SerpinB9 and IL-35, and reduced expression or no expression of MHC class I and/or MHC class II human leukocyte antigens.
In a seventeenth aspect, the present invention provides a cell described in the ninth to sixteenth aspects above, wherein the cell is selected from stem cells, differentiated cells, pluripotent stem cells, induced pluripotent stem cells, adult stem cells, progenitor cells, somatic cells, primary T cells and chimeric antigen receptor T cells.
In an eighteenth aspect, the present invention provides use of the universal cell described in the first aspect, the composition described in the fifth aspect, or the expression vector or expression cassette described in the eighth aspect in the preparation of a product for cell therapy.
In a nineteenth aspect, the present invention provides use of the universal cell described in the first aspect, the composition described in the fifth aspect, or the expression vector or expression cassette described in the eighth aspect in the preparation of a product for organ transplantation.
In a twentieth aspect, the present invention provides use of the universal cell described in the first aspect, the composition described in the fifth aspect, or the expression vector or expression cassette described in the eighth aspect in the construction of cell libraries of universal PSCs.
In a twenty-first aspect, the present invention provides use of the universal cell described in the first aspect, the composition described in the fifth aspect, or the expression vector or expression cassette described in the eighth aspect as gene drug carriers.
After major histocompatibility complex class I (MHC-I) and class II genes are inactivated in stem cells in the present invention, the fusion protein XSG006 constructed by functional domains of CD47 and CD24 is overexpressed, such that the obtained human pluripotent stem cells can, on the basis of escaping T cell attack, further escape killing by NK cells, the effect of which is even better than that of the reported positive targets CD47 and CD24. Meanwhile, the pluripotent stem cells with low immunogenicity retain their stemness and differentiation capacity. Compared with neurocytes differentiated from WT cells, neurocytes differentiated from DKO+G6 cells overexpressing the fusion protein XSG006 can effectively escape the attack of the immune system in vivo.
The technical solution of the present invention will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only intended to illustrate and explain the present invention by way of example only, and should not be construed as limiting the scope of protection of the present invention. All technologies achieved based on the above contents of the present invention are covered in the scope that the present invention aims to protect.
Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.
Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in the present disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs.
In the present invention, the human pluripotent stem cell lines H1 (Wicell, WA01) or H9 (Wicell, WA09) were selected, and β-2-microglobulin (B2M) in the endoplasmic reticulum was knocked out by CRISPR/CAS9, so that MHC-I on the cell surface cannot form functional molecules, thereby escaping killing by allogeneic CD8+ T cells; the escape of killing by CD4+ T cell was achieved by knocking out the positive regulatory factor CIITA of MHC-II gene transcription, so as to reduce the expression of MHC class II molecules.
Wherein, the CRISPR/CAS9 knocking out strategy of B2M gene is shown in
gRNA Sequences:
B2M-F2+B2M-R2=608 bp
After knocking out: no band
After knocking out: 569 bp.
In addition, the CRISPR/CAS9 knocking out strategy of CIITA gene is shown in
gRNA Sequences:
After knocking out: no band
After knocking out: 459 bp
The specific operations were as follows:
The expression of B2M and CIITA at RNA level in the clone DKO with B2M/CIITA double alleles knocked out was detected by qPCR as shown in
The expression of B2M at protein level in the clone DKO with B2M/CIITA double alleles knocked out was detected by Western-Blot as shown in
Stimulation of WT and DKO with INF-gamma: The cells were plated, and the medium containing INF-gamma was added to the cells when the liquid was changed the next day. After 48 hours, the cells were digested and the expression of HLA-I/II was detected by flow cytometry. The Results are shown in
Karyotype detection of the obtained positive clones (DKO) with B2M/CIITA double alleles knocked out: the chromosome specimens fixed on the slide were treated with trypsin and then stained with Giemsa stain. The chromosome number and morphological structure of metaphase chromosomes were analyzed to determine whether their karyotypes were consistent with the normal karyotypes. The results are shown in
Immunofluorescence detection shows that WT and DKO cells express the stemness genes POU5F1 and NANOG at protein level: The cells were plated in a 12-well plate, the medium of which was pipetted off after the cells grew to 60-80% density, and into which was added 4% paraformaldehyde for fixation. After the cell membrane was broken, the primary antibody of POU5F1 and NANOG was used for overnight incubation at 4° C. After washing off the primary antibodies, the secondary antibody with a fluorescent label was incubated at room temperature, and then photos were taken with a fluorescent microscope. The results are shown in
Flow cytometry show that the sternness genes SSEA-4 and Tra1-81 are highly expressed on both of the surface of WT and DKO cells, accounting for 100%, 99.98%, 96.75%, and 99.13% respectively. The results are shown in
2. Differentiation Ability of Obtained Positive Clone with B2M/CIITA Double Alleles Knocked Out (DKO)
100 μL of a suspension containing 5E+5 DKO cells was injected subcutaneously into immunodeficient mice (SCID Beige). After the teratoma was larger than 1.5 cm3, it was taken out, sliced and stained.
The obtained positive clone with B2M/CIITA double alleles knocked out (DKO) can form a teratoma in vivo and differentiate into cells with three germ layers: an entoderm, a mesoderm, and an ectoderm. The results are shown in
The killing experiments of T cells and NK cells were carried out with the xCELLigence RTCA Instrument. The same number of WT and DKO cell lines were suspended in the Essential 8 medium containing IL-2, and inoculated into a 96-well E-plate coated with matrigel, into which were added activated T cells or NK cells for killing detection. The RTCA detection data were analyzed by the xCELLigence software to calculate the killing ratio and escape function.
As shown in the RTCA data of
CD47 (NM_198793) was overexpressed in the DKO cells obtained in Example 1 using a lentiviral vector. The amino acid sequence of CD47 was shown in SEQ ID NO.29. The cDNA of the overexpressed sequence (SEQ ID No. 30) was constructed in a lentiviral plasmid initiated by EF1a with a puromycin screening marker (pGC-EF1a), and the structure of the pGC-EF1a plasmid is shown in
Referring to Example 2, RTCA was used to detect whether the overexpressed DKO+CD47 cell line can escape the killing by NK cells successfully while escaping the killing by T cells. As shown in
CD24 is a precursor protein, which comprises a signal peptide region and a glycosyl phosphatidyl inositol (GPI) membrane anchoring sequence. After cleavage, it becomes a mature and functional CD24 protein (Chen et al., 2014; Pirruccello and LeBien, 1986). CD24 is a glycoprotein with multiple potential O- and N-glycosylation sites, which can be sialylated, thus binding to the sialic acid recognition receptor Siglec 10 (sialic acid binding Ig-like lectin 10) and interacting with macrophages to inhibit phagocytosis (Barkal et al., 2019).
CD47 is a transmembrane protein with an extracellular N-terminal IgV domain, five transmembrane domains and an intracellular C-terminal (Logtenberg et al., 2020). The N-terminal IgV domain can interact with the immunosuppressant receptor SIRPA (signal regulatory protein α), so as to inhibit immune responses (Jaiswal et al., 2009).
The strategy of the present invention is to combine known functional domains of CD24 and CD47 to form a variety of fusion proteins. Each fusion protein must comprise at least one membrane anchoring or transmembrane sequence selected from CD24 or CD47; meanwhile, each fusion protein must comprise at least one Siglec 10 or SIRPα receptor recognition sequence selected from CD24 or CD47. Each domain is linked by a flexible protein to finally form a fusion protein XSG01-XSG20. According to the method of Example 3, the fusion protein XSG001-XSG020 was overexpressed in the H1 DKO cells prepared in Example 2 by lentivirus infection, and a cell strain DKO+G1-G20 was formed, in which all fusion proteins were stably transfected.
The following are the construction strategies and specific sequences of 6 fusion proteins:
MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQST
MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQS
GGSTNATTKAAGGALQSTASLFVVSLSLLHLYS
MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQST
LLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVG
AILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIG
LTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLIS
GLSILALAQLLGLVYMKFV
VVSLSLLHLYS
The cell strains DKO+G1-G20 were subjected to the NK cell killing function detection as shown in Example 2, respectively, in order to screen out the fusion protein with the best degree of NK cell escape. The results are shown in
The nucleic acid sequence encoding XSG006 (the amino acid sequence of XSG006 is shown in SEQ ID NO: 1) was directly synthesized, and constructed in a lentiviral plasmid initiated by EF1a with a puromycin screening marker (pGC-EF1a), and the structure of the pGC-EF1a plasmid is shown in
Referring to Example 2, in the NK cell killing experiment detected by RTCA, the DKO cells constructed in Example 2 are completely killed by NK cells, and H1WT and DKO+G6 escape successfully. DKO+G6 has a degree of escape which is slightly better than that of H1 WT. The results are shown in
Cell lines involved in this example: DKO+CD47 and DKO+CD24 in Example 3 and DKO+CD47 in the prior art (WO2020018615A2) (hereinafter referred to as “CD47 prior art”), wherein CD47 of the DKO+CD47 cell line in the prior art has an amino acid sequence shown in SEQ ID NO.40, and promotes immune escaping by interacting with a signal regulatory protein α (SIRPα) on the surface of immune cells. The construction of the “CD47 prior art” cell line is completed according to the records in WO2020018615A2. Overexpression of CD47 in cells with B2M and CIITA double knocked out (DKO) can successfully escape killing by T and NK cells. (PMID: 32433947/PMID: 30778232).
CD24 promotes immune escaping by interacting with sialic acid binding Ig-like lectin 10 (Siglec-10), an inhibitory receptor on the surface of immune cells (PMID: 31367043). CD24 of the cell line DKO+CD24 in this example has an amino acid sequence shown in SEQ ID NO.41. The construction method of the cell line refers to Example 3.
Meanwhile, the repeated experiments were normalized into each DKO killing, and the summary of the killing results shows that the killing ratio of DKO+G6 is the smallest, which is significantly different from that of WT, DKO+CD24 and “CD47 prior art”. The results are shown in
The DKO+CD47+CD24 cell line was obtained by further overexpressing CD24 by lentivirus transfection on the basis of Example 3. For lentivirus construction and overexpression operation, refer to Example 3. The amino acid sequence of CD24 is MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAAGG ALQSTASLFVVSLSLLHLYS. Comparing DKO+G6 with DKO+CD47+CD24 through the NK cell killing experiment, it is found that DKO+G6 cells have a smaller killing ratio and a better escaping effect. The results are shown in
After passage, the WT and DKO+G6 cells were plated in a culture bottle that had been pre-coated with Matrigel, and after 24 h of culture, they were switched to a pre-differentiation medium to induce the cells to differentiate into midbrain cells. (Nolbrant S, Heuer A, Parmar M, Kirkeby A. Generation of high-purity human ventral midbrain dopaminergic progenitors for in vitro maturation and intracerebral transplantation. Nat Protoc. 2017 September; 12(9):1962-1979. doi: 10.1038/nprot.2017.078. Epub 2017 Aug. 31. PMID: 28858290); after differentiation for 9 d, high-purity midbrain cells can be obtained. Then, a large number of neural progenitor cells with high purity could be obtained by adding a neural progenitor cell medium to amplify the obtained midbrain cells; finally, the neural progenitor cells were further differentiated into neurocytes by adding a neural precursor cell medium. The differentiated WT and DKO+G6 cells were infected by lentivirus carrying luciferase (luc), and then the cells expressing luc were transplanted into mice with humanized immune system with CD34+HSC reconstructed. The survival of the cells in mice can be indicated by intraperitoneal injection of D-fluorescein (A025011, Shanghai Yisheng Biotechnology Co., Ltd.), a luminescent substrate of luc, and detection of the fluorescence intensity of the cells by iVIS spectrum (PerkinElmer). After continuous detection of the fluorescence at the transplantation site for 51 days, the fluorescence value of the neurocytes differentiated from WT tends to the background value, while the continuously rising fluorescence value of the neurocytes differentiated from DKO+G6 can be detected after transplantation, indicating that neurocytes differentiated from DKO+G6 can effectively escape the attack of immune system in vivo compared with the neurocytes differentiated from WT. The results are shown in
The embodiments of the present invention have been described above. However, the present invention is not limited to the above-mentioned embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be comprised in the protection scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210307870.8 | Mar 2022 | CN | national |
| 202210806871.7 | Jul 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/083761 | 3/24/2023 | WO |
