WO2024251119A1 - Cellule nerveuse à immunité privilégiée différenciée à partir d'une cellule souche pluripotente induite et son utilisation - Google Patents

Cellule nerveuse à immunité privilégiée différenciée à partir d'une cellule souche pluripotente induite et son utilisation Download PDF

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WO2024251119A1
WO2024251119A1 PCT/CN2024/097338 CN2024097338W WO2024251119A1 WO 2024251119 A1 WO2024251119 A1 WO 2024251119A1 CN 2024097338 W CN2024097338 W CN 2024097338W WO 2024251119 A1 WO2024251119 A1 WO 2024251119A1
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cells
cell
culture medium
dopaminergic
fusion protein
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李翔
朱珉喆
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Xellsmart Biomedical Suzhou Co Ltd
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Xellsmart Biomedical Suzhou Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention belongs to the intersection field of genetic engineering and stem cell technology, and specifically relates to a neural cell differentiated from immune-immune induced pluripotent stem cells and an application thereof.
  • Stem cells are a type of "seed” cells that have the ability to self-renew and differentiate into specific functional somatic cells. Based on the degree of differences in stem cell characteristics, 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 good application prospects in the treatment of cancer, neurological, cardiovascular and other related diseases.
  • PSCs pluripotent stem cells
  • hESC Human embryonic stem cells
  • iPSC induced pluripotent stem cells
  • Parkinson's disease is a serious neurodegenerative disease, and its overall incidence is second only to Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • DAP transplanted dopaminergic neurons
  • the present invention successfully constructs B2M/CIITA biallelic knockout positive clones (DKO cells) by knocking out ⁇ -2-microglobulin (B2M) in the endoplasmic reticulum of induced pluripotent stem cells (iPSCs) and knocking out CIITA, a positive regulator of MHC-II gene transcription; then a lentiviral vector is used to overexpress a new gene based on the fusion of CD47 and CD24 domains in DKO cells, and the induced pluripotent stem cells thus obtained can further escape the killing of NK cells on the basis of escaping T cell attacks.
  • the cells are differentiated to obtain isolated immune-exempt dopaminergic nerve cells.
  • the dopaminergic nerve cells can significantly reduce or escape the recognition and attack of the immune system, especially the attack of natural killer cells, macrophages, etc., and effectively treat Parkinson's disease.
  • the present invention adopts the following technical solution:
  • the present invention provides an isolated low-immunogenic dopaminergic neural cell differentiated from an immunoprivileged induced pluripotent stem cell (i.e., a low-immunogenic iPS cell), comprising:
  • HLA-I and HLA-II human leukocyte antigens are reduced or absent;
  • the cells can escape T cell attack and NK cell killing.
  • gene editing tools are used in the immune privileged induced pluripotent stem cells to target one or more genes encoding one or more transcriptional regulators of HLA-I, and one or more genes encoding one or more transcriptional regulators of HLA-II, to achieve reduced or no expression of HLA-I and HLA-II genes.
  • the transcriptional regulatory factor of HLA-I may be preferably selected from: one or more of B2M, TAP1, TAP2, TAP-associated glycoprotein (Tapasin) or NLRC5; the transcriptional regulatory factor of HLA-II may be preferably selected from: one or more of CIITA, RFXANK, RFX5, RFXAP;
  • the transcriptional regulatory factors are preferably B2M and CIITA.
  • the immunoprivileged induced pluripotent stem cells further comprise a genetic modification targeting the CIITA gene by a rare-cutting endonuclease that selectively inactivates the CIITA gene.
  • the immunoprivileged induced pluripotent stem cells further include selectively inactivating B2M Genetic modification of the B2M gene by targeting a rare-cutting endonuclease.
  • the genetic modification for targeting the CIITA gene or the B2M gene by a 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 gene.
  • the CRISPR/CAS9 system is used to directly knock out the B2M and CIITA exon segments at both ends, respectively, wherein the target sequences of the gRNA for the B2M gene are SEQ ID NO: 2 and SEQ ID NO: 3, and the target sequences of the gRNA for the CIITA gene are SEQ ID NO: 4 and SEQ ID NO: 5.
  • the expression of HLA-I and/or HLA-II genes is reduced or eliminated in the immunoprivileged induced pluripotent stem cells by introducing gene expression modifying molecules targeting one or more genes encoding one or more transcriptional regulators of HLA-I, or one or more genes encoding one or more transcriptional regulators of HLA-II, wherein the gene expression modifying molecules include one selected from siRNA, shRNA, microRNA, antisense RNA and another RNA-mediated inhibitory molecule.
  • the functional domain of CD47 in the fusion protein comprising the functional domains of CD47 and CD24 is the CD47 transmembrane domain; preferably, the amino acid sequence of the CD47 transmembrane domain is as shown in any one of SEQ ID NO: 6-10.
  • the functional domain of CD24 in the fusion protein comprising the functional domains of CD47 and CD24 is the signal peptide sequence of CD24, the mature peptide of CD24, the extracellular peptide of CD24, the membrane anchoring sequence of CD24, and the extracellular mature peptide of CD24; preferably, the amino acid sequence of the functional domain of CD24 is as shown in any one of SEQ ID NO: 11-16.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is a protein that connects the CD47 SIRP ⁇ binding domain and the membrane anchoring sequence of CD24.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is a protein in which the SIRP ⁇ binding domain of CD47 is inserted into the linking site of the extracellular peptide and membrane anchor sequence of CD24.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is obtained by linking CD47 to the extracellular peptide of CD24.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is obtained by linking the CD24 mature peptide to the SIRP ⁇ binding domain of CD47.
  • 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 SIRP ⁇ binding domain and the transmembrane domain connection point of CD47.
  • the fusion protein constructed by the functional domains of CD47 and CD24 is a fusion protein of CD24.
  • the mature peptide sequence is connected to the CD47 transmembrane domain;
  • the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 has more than 70% homology with the sequence shown in SEQ ID NO: 1, such as more than 80% homology, and more than 90%, more than 95%, or more than 98% homology;
  • amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 is as shown in SEQ ID NO: 1.
  • the increased expression of the fusion protein constructed by the functional domains of CD47 and CD24 is produced by introducing at least one copy of the fusion protein gene into the iPSC under the control of a promoter in the immune privileged induced pluripotent stem cells.
  • 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.
  • the isolated low immunogenic dopaminergic neural cells are selected from dopaminergic neural precursor cells, dopaminergic neural progenitor cells, immature dopaminergic neural cells and mature dopaminergic neural cells; preferably dopaminergic neural precursor cells.
  • the present invention provides a method for isolating low-immunogenic dopaminergic neural cells described in the first aspect by in vitro differentiation from immunoprivileged induced pluripotent stem cells, wherein endogenous ⁇ -2 microglobulin (B2M) gene activity and endogenous class II transactivator (CIITA) gene activity have been eliminated in the immunoprivileged iPSC cells; and a fusion protein constructed by the functional domains of CD47 and CD24 is expressed; preferably, the amino acid sequence of the fusion protein constructed by the functional domains of CD47 and CD24 has more than 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;
  • the method comprises:
  • Immunoprivileged induced pluripotent stem cells were plated in the form of clumps on a matrix pre-coated with laminin at a number of about 100,000 per well, and the cells were cultured using E8 complete medium. The cells were digested and passaged every 4 to 5 days during the culture process.
  • DAP cells dopaminergic neural precursor cells
  • the first culture medium is a neural basal culture medium containing 1 ⁇ M-20 ⁇ M SB431542, 1 ⁇ M-3 ⁇ M DMH1, 0.2 ⁇ M-1 ⁇ M CHIR99021 and 1 ⁇ M-1.75 ⁇ M SAG;
  • the first culture medium is a neural basal medium containing 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021 and 1 ⁇ M SAG;
  • the second culture medium is a neural basal medium containing 5 ⁇ M-15 ⁇ M Y27632, 1 ⁇ M-20 ⁇ M SB431542, 1-3 ⁇ M DMH1, 0.2 ⁇ M-1 ⁇ M CHIR99021 and 1 ⁇ M-1.75 ⁇ M SAG;
  • the second culture medium is a neurobasal medium containing 10 ⁇ M Y27632, 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021 and 1 ⁇ M SAG;
  • the third culture medium is a neural basal medium containing 0.8 ⁇ M-1.2 ⁇ M DMH1, 0.1 ⁇ M-0.5 ⁇ M SAG, and 80ng/mL-120ng/mL fibroblast growth factor 8b (FGF8b);
  • the third culture medium is a neurobasal medium containing 1 ⁇ M DMH1, 0.2 ⁇ M SAG and 100 ng/mL fibroblast growth factor 8b (FGF8b);
  • the fourth culture medium is a neurobasal medium containing 0.1 ⁇ M-0.5 ⁇ M SAG and 80ng/mL-120ng/mL FGF8b;
  • the fourth culture medium is a neurobasal medium containing 0.2 ⁇ M SAG and 100 ng/mL FGF8b;
  • the fifth culture medium is a neurobasal medium containing 5 ⁇ M-15 ⁇ M Y27632, 0.1 ⁇ M-0.5 ⁇ M SAG, and 80ng/mL-120ng/mL FGF8b;
  • the fifth culture medium is a neurobasal medium containing 10 ⁇ M Y27632, 0.2 ⁇ M SAG and 100 ng/mL FGF8b;
  • the neural basal culture medium is selected from at least one of IMDM (Iscove's Modified Dulbecco's Medium) culture medium, Eagle's Basal Medium (BME) culture medium, GMEM culture medium, MEM culture medium, DMEM culture medium, Ham's F-12 culture medium, RPMI1640 culture medium and Neurobasal culture medium.
  • IMDM Iscove's Modified Dulbecco's Medium
  • BME Eagle's Basal Medium
  • GMEM culture medium GMEM culture medium
  • MEM culture medium MEM culture medium
  • DMEM culture medium Ham's F-12 culture medium
  • RPMI1640 culture medium RPMI1640 culture medium
  • Neurobasal culture medium Neurobasal culture medium.
  • the neural basal medium consists of the following components: 98% (v/v) DMEM/F12 (Gibco), 1% (v/v) non-essential amino acids (Non-essential amino acid, Gibco) and 1% (v/v) N2 supplement (N2 supplement, Gibco).
  • the present invention provides a composition comprising the isolated low immunogenic dopaminergic nerve cells according to the first aspect or the isolated low immunogenic dopaminergic nerve cells prepared according to the method of the second aspect.
  • the composition comprises the isolated low-immunogenic dopaminergic neural cells described in the first aspect and one or more therapeutic agents, wherein the therapeutic agents include peptides, cytokines, small molecule compounds, macromolecules, ADCs, antibodies, nanoparticles, biosimilars, mRNA, traditional Chinese medicine, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double-stranded RNA (dsRNA), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors comprising one or more polynucleic acids of interest, antibodies, etc.
  • the therapeutic agents include peptides, cytokines, small molecule compounds, macromolecules, ADCs, antibodies, nanoparticles, biosimilars, mRNA, traditional Chinese medicine, proteins, vaccines, checkpoint inhibitors, mitogens, growth factors, small RNAs, double-stranded RNA (dsRNA), mononuclear blood cells, feeder cells, feeder cell components or their replacement factors, vectors compris
  • the present invention provides a method for treating a patient suffering from a neurodegenerative disease or condition, the method comprising administering a therapeutically effective amount of the isolated low immunogenic dopaminergic neural cells described in the first aspect of the present invention or the isolated low immunogenic dopaminergic neural cells prepared according to the method of the second aspect or the composition described in the third aspect.
  • the method wherein the composition further comprises a therapeutically effective carrier.
  • the method wherein the isolated hypoimmune dopaminergic neural cells are on a biodegradable scaffold.
  • the method wherein the administering comprises implantation or injection.
  • the method wherein the neurodegenerative disease or condition is selected from Parkinson's disease, Huntington's disease disease and multiple sclerosis.
  • the present invention provides the use of the isolated low immunogenic dopaminergic nerve cells described in the first aspect, or the isolated low immunogenic dopaminergic nerve cells prepared according to the method of the second aspect, or the composition described in the third aspect in the preparation of a product for the treatment of neurodegenerative diseases or conditions.
  • the use, wherein the neurodegenerative disease or condition is selected from Parkinson's disease, Huntington's disease and multiple sclerosis; preferably Parkinson's disease.
  • the present invention inactivates the major histocompatibility complex MHC-I and class II genes in induced pluripotent stem cells, overexpresses the fusion protein XSG006 constructed by the functional domains of CD47 and CD24, and the obtained induced pluripotent stem cells are differentiated to obtain isolated low-immunogenic dopaminergic neural precursor cells, which can further escape the killing of NK cells and macrophages on the basis of escaping T cell attack.
  • the low-immunogenicity dopaminergic neural precursor cells separated by the present invention can effectively escape the attack of the immune system in vivo (humanized mice and non-human primates).
  • the low-immunogenicity dopaminergic neural precursor cells separated by the present invention were transplanted.
  • the test results after 4 months showed that the number of mouse rotations in the apomorphine (APO)-induced rotation experiment was significantly reduced, and it was statistically significant; in the cylinder experiment, the number of times the mouse's right forelimb hit the wall was significantly increased, basically restored to the level before modeling, and it was statistically significant; in the rotating rod experiment, the time the mouse stayed on the rotating rod was significantly increased, and it was statistically significant.
  • APO apomorphine
  • the monkey low-immunogenic dopaminergic neural precursor cells isolated by the present invention can survive and produce activity in the host body for a long time.
  • Allogeneic DAP cell transplantation was performed on non-human primates (NHP) with a complete immune system.
  • NEP non-human primates
  • monkey DKO+G6-DAP cells have low immunity and can escape the attack of the immune system, and survive for a long time in the brains of allogeneic monkeys with a complete immune system.
  • In vivo safety testing showed that low-immunogenic dopaminergic neural precursor cells (DKO+G6-DAP) have no difference in proliferation ability compared with unedited cells (WT-DAP), and both contain very small amounts of Ki67, indicating a low risk of tumorigenicity in vivo.
  • low-immunogenic DAP cells can differentiate into TH+, i.e., functional dopaminergic neurons in vivo.
  • FIG. 1 Schematic diagram of the CRISPR/CAS9 gene knockout strategy of B2M.
  • FIG. 1 Schematic diagram of CIITA’s CRISPR/CAS9 gene knockout strategy.
  • FIG. 1 Schematic diagram of the structure of pGC-EF1a plasmid.
  • FIG4 is a graph showing the expression of OCT-4 in various DAP cells detected by flow cytometry in Example 1;
  • FIG5 is a graph showing the expression of FOXA2, PAX6, OTX2, and LMX1A in each DAP cell detected by flow cytometry in Example 1;
  • FIG6 is a graph showing the expression of OTX2, EN1, LMX1A and FOXA2 in each DAP cell detected by immunofluorescence in Example 1;
  • FIG7 is a graph showing the expression of HLA-I/II in various cells detected by flow cytometry after stimulating each DAP cell with INF-gamma in Example 1;
  • FIG8 Sequential rt-PCR identification of the DKO+G6 cell line constructed in Example 1 and its derived DAP cells.
  • T cells are the positive control for detecting HLA class I/II molecules
  • FIG9 is a graph showing the results of using RTCA to detect the killing of WT, DKO and DKO+G6 differentiated DAP cells by PBNK cells in Example 2;
  • FIG10 is a photograph showing the killing of WT, DKO and DKO+G6 differentiated DAP cells by MAC in Example 2;
  • FIG11 is a graph showing the results of using RTCA to detect the killing of MAC cells against WT, DKO and DKO+G6 differentiated DAP cells in Example 2;
  • FIG. 12 is a diagram showing the results of validating the immune escape effect of DAP cells differentiated from DKO+G6 in mice with human immune system in Example 2;
  • FIG. 13 is the result of apomorphine (APO)-induced rotation experiment in the PD mouse model after transplantation of WT-DAP injection in Example 3;
  • Figure 16 shows the results of immunofluorescence staining of brain sections of PD mice transplanted with cells for 4 months in Example 3;
  • 16B is a 4x, 20x, and 60x magnification of the portion cut out in 16A.
  • FIG18 is a graph showing the expression of OTX2, EN1, LMX1A and FOXA2 in DAP cells derived from various monkey stem cells detected by immunofluorescence in Example 4;
  • FIG. 19 MRI results of monkey brain before and after WT-DAP (left brain) and DKO+G6-DAP (right brain) cell transplantation in Example 4;
  • Figure 20 Statistical graph of the left and right food intake rate of PD monkeys before and after WT-DAP and DKO+G6-DAP cell transplantation in Example 4;
  • Figure 21 Schematic diagram of WT-DAP and DKO+G6-DAP cell transplantation in Example 5 and MRI results of monkey brain before and after transplantation (A); MRI detection of cell size statistics (B).
  • FIG 22 Immunofluorescence staining of mouse brain in Example 5 (A), co-staining of hNA (red) & Ki67 (green) & DAPI (blue), where hNA stains transplanted cells and Ki67 marks proliferating cells; statistics of immunofluorescence staining (B).
  • FIG. 23 The results of brain immunofluorescence staining after PD-NOG transplantation into DKO+G6-DAP cells in Example 5.
  • FIG. 23 The results of brain immunofluorescence staining after PD-NOG transplantation into DKO+G6-DAP cells in Example 5.
  • 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 without specifying specific conditions are usually carried out 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.
  • immune-privileged pluripotent stem cells A detailed description of immune-privileged pluripotent stem cells, methods for producing them, and methods for using them are found in 202210806871.7 filed on July 8, 2022 and PCT/CN2023/083761 filed on March 24, 2023, and the disclosures including the sequence listing, drawings, and examples are incorporated herein by reference in their entirety.
  • pluripotent cell refers to a cell that can self-renew and proliferate while maintaining an undifferentiated state and can be induced to differentiate into a specialized cell type under appropriate conditions.
  • the term “pluripotent cell” encompasses embryonic stem cells and other types of stem cells, including fetal, amniotic or somatic stem cells.
  • Exemplary human stem cell lines include the H9 human embryonic stem cell line.
  • Other exemplary stem cell lines include those available through the National Institutes of Health Human Embryonic Stem Cell Registry and the Howard Hughes Medical Institute HUES collection (described in Cowan, CA et al., New England J. Med. 350: 13. (2004), which is incorporated by reference (incorporated herein in its entirety)
  • pluripotent stem cells have the potential to differentiate into any of the three germ layers: endoderm (e.g., gastric junction, gastrointestinal tract, lung, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epidermal tissue and nervous system tissue).
  • endoderm e.g., gastric junction, gastrointestinal tract, lung, etc.
  • mesoderm e.g., muscle, bone, blood, urogenital tissue, etc.
  • ectoderm e.g., epidermal tissue and nervous system tissue.
  • the term “pluripotent stem cell” also encompasses "induced pluripotent stem cells” or "iPSCs,” which are a type of pluripotent stem cell derived from non-pluripotent cells. Examples of parental cells include somatic cells that have been reprogrammed to induce a pluripotent undifferentiated phenotype in various ways.
  • iPS or iPSC cells can be produced by inducing the expression of certain regulatory genes or by applying certain proteins exogenously. Methods for inducing iPS cells are known in the art and are further described below. (See, for example, Zhou et al., Stem Cells 27(11):2667-74 (2009); Huangfu et al., Nature Biotechnol. 26(7):795 (2008); Woltjen et al., Nature 458(7239):766-770 (2009); and Zhou et al., Cell Stem Cell 8:381-384 (2009); each of which is incorporated herein by reference in its entirety).
  • the term "subject” or “patient” refers to any animal, such as a domestic animal, a zoo animal, or a human.
  • a “subject” or “patient” can be a mammal, such as a dog, a cat, a bird, a livestock, or a human.
  • Specific examples of “subjects” and “patients” include, but are not limited to, individuals (particularly humans) suffering from diseases or conditions related to the liver, heart, lungs, kidneys, pancreas, brain, nervous tissue, blood, bones, bone marrow, etc.
  • immunoprivileged induced pluripotent stem cells refer to pluripotent cells that retain their pluripotent characteristics and cause a reduced immune rejection reaction when transferred into an allogeneic host. In a preferred embodiment, the cells do not cause an immune response. Therefore, “immune privilege” refers to an immune response that is significantly reduced or eliminated compared to the immune response of the parental (i.e., "wild type” or "wt") cells before the immune engineering outlined herein. In many cases, the cells are immunologically low immunogenic, but still retain pluripotency and differentiation potential.
  • HLA human leukocyte antigen
  • MHC major histocompatibility complex
  • HLA-I major histocompatibility complex
  • HLA-I includes three proteins, HLA-A, HLA-B and HLAC, which present peptides from the inside of the cell, while the antigens presented by the HLA-I complex attract killer T cells (also known as CD8+T- cells or cytotoxic T cells).
  • B2M beta-2 microglobulin
  • HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ and HLA-DR, which present antigens to T lymphocytes from the outside of the cell. This stimulates CD4+ cells (also known as T helper cells).
  • MHC or “HLA” is not meant to be limiting, as it depends on whether the gene is from human (HLA) or mouse (MHC). Therefore, as it relates to mammalian cells, these terms can be used interchangeably herein.
  • CD47 is also called integrin-associated protein (IAP), which is widely expressed on the surface of various cells. It can interact with signal regulatory protein ⁇ (SIRP ⁇ ) and thrombospondin-1 (TSP-1), and plays an important role in mediating the oxidative stress response and immune repair process of certain cardiovascular diseases.
  • SIRP ⁇ signal regulatory protein ⁇
  • TSP-1 thrombospondin-1
  • CD47 contains three components: an amino-terminal extracellular immunoglobulin variable domain (IgV)-like domain, a 5-domain transmembrane region, and a carboxyl-terminal splice variant cytoplasmic tail.
  • IgV extracellular immunoglobulin variable domain
  • CD24 stands for cluster of differentiation 24, also known as heat-stable antigen. It is a highly glycosylated glycosylphosphatidylinositol-anchored surface protein, which is known to interact with Siglec-10 (sialic-acid-binding Ig-like lectin10) on innate immune cells to inhibit destructive inflammatory responses in response to infection, sepsis, liver injury and chronic graft-versus-host disease. CD24 is a small molecular weight sialic acid glycoprotein composed of 31 amino acids with a typical mucin structure and is located in lipid rafts through its glycosylated phosphatidylinositol (GPI) anchor.
  • GPI glycosylated phosphatidylinositol
  • Gene knockout herein refers to the process of inactivating a specific gene in the host cell in which it resides, resulting in the non-production of the protein of interest or the formation of an inactivated form. As will be appreciated by those skilled in the art and described further below, this can be accomplished in many different ways, including removing the nucleic acid sequence from the gene, or interrupting the sequence with other sequences, changing the reading frame, or changing the regulatory components of the nucleic acid. For example, all or part of the coding region of the gene of interest can be removed or replaced by a "nonsense" sequence, all or part of a regulatory sequence such as a promoter can be removed or replaced, a translation initiation sequence can be removed or replaced, etc.
  • Gene knock-in herein refers to the process of increasing genetic function to a host cell. This results in an increase in the level of the encoded protein. As will be appreciated by those skilled in the art, this can be achieved in several ways, including adding one or more other copies of the gene to the host cell or changing the regulatory components of the endogenous gene, thereby increasing the expression of the protein. This can be achieved by modifying the promoter, adding different promoters, adding enhancers or modifying other gene expression sequences.
  • Allogeneic in this context refers to the genetic dissimilarity between the host organism and the cell transplant, in which an immune response is generated.
  • identity percentage refers to two or more sequences or subsequences that have a specified percentage of identical nucleotides or amino acid residues when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection.
  • sequence comparison algorithms e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art
  • the "identity" percentage can exist over regions of the sequences being compared, such as over functional domains, or alternatively over the full length of the two sequences to be compared.
  • one sequence is typically used as a reference sequence to which a test sequence is compared.
  • test sequence and the reference sequence are entered into a computer, and subsequences are specified if necessary.
  • sequence coordinates are provided and sequence algorithm program parameters are specified.
  • sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.
  • Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., see below).
  • the BLAST algorithm is one example of an algorithm suitable for determining percent sequence identity and sequence similarity and is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
  • homologous or derived sequences are biologically active molecules that are similar to reference molecules at the nucleotide sequence, peptide sequence, function or structural level. Homologues can include sequence derivatives that have a certain percentage of identity with the reference sequence. Therefore, in one embodiment, homologous or derived sequences have at least 70% sequence identity. In a specific embodiment, homologous or derived sequences have at least 80% or 85% sequence identity. In a specific embodiment, homologous or derived sequences have at least 90% sequence identity. In a specific embodiment, homologous or derived sequences have at least 95% sequence identity.
  • homologous or derived sequences have at least 50, 55, 60, 65, 70, 75, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity.
  • Homologous or derived nucleic acid sequences can also be defined by their ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions.
  • a homologue having structural or functional similarity to a reference molecule may be a chemical derivative of the reference molecule. Methods for detecting, generating and screening structural and functional homologues and derivatives are known in the art.
  • Dopaminergic neural precursor cells herein generally refer to cells that can proliferate and/or differentiate into dopaminergic neurons in vitro or in vivo.
  • Dopaminergic neural precursor cells can be derived from ventral midbrain neurons and can be differentiated from pluripotent stem cells.
  • Dopaminergic neural precursor cells can also be differentiated or reprogrammed from other cell types.
  • the "dopaminergic nerve cell” herein is selected from dopaminergic nerve precursor cells, dopaminergic nerve progenitor cells, immature dopaminergic nerve cells and mature dopaminergic nerve cells; preferably, it is a dopaminergic nerve precursor cell.
  • Example 1 Differentiation of midbrain dopaminergic neural precursor (DAP) cells using low immunogenic stem cells (i.e., low immunogenic iPS cells)
  • WT, DKO and DKO+G6 cells were plated in culture flasks pre-coated with Matrigel. After 24 hours of culture, the cells were switched to pre-differentiation medium to induce differentiation into midbrain cells. After 9 days of differentiation, high-purity midbrain cells were obtained. Subsequently, the obtained midbrain cells were amplified by adding neural progenitor cell culture medium to obtain a large number of high-purity neural progenitor cells. Finally, neural progenitor cells were further differentiated into neural cells by adding neural precursor cell culture medium. The cells can differentiate into dopaminergic neural precursor (DAP) cells in vitro.
  • DAP dopaminergic neural precursor
  • the present invention uses human induced pluripotent stem cells (iPSC cells, human blood PBMCs are reprogrammed into iPSC cells, i.e., WT cells, through CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat.
  • iPSC cells human blood PBMCs are reprogrammed into iPSC cells, i.e., WT cells, through CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat.
  • B2M-gRNA1 and B2M-gRNA2 are used to directly knock out the B2M exon segment at both ends, and then two pairs of PCR primers B2M-F1/R1 and B2M-F2/R2 are used to verify the genome sequence knockout.
  • B2M-gRNA1 CGTGAGTAAACCTGAATCTT
  • B2M-gRNA2 AGTCACATGGTTCACACGGC
  • CRISPR/CAS9 gene knockout strategy of CIITA is shown in Figure 2 , where CIITA-gRNA1 and CIITA-gRNA2 were used to directly knock out both ends of the CIITA exon segment, and then two pairs of PCR primers, CIITA-F1/R1 and CIITA-F2/R2, were used to verify the genome sequence knockout.
  • CIITA-R1 CCTTCCATGTCACACAACAGCC
  • Neon transfection system 100 According to the Neon transfection system 100 ⁇ L electroporation system, add 15 ⁇ g TrueCut TM Cas9 Protein + 3 ⁇ g gRNA (B2MgRNA1 + B2MgRNA2 + CIITA gRNA1 + CIITA gRNA2) to form the RNP system, mix well and place at room temperature for 20 minutes.
  • qPCR was performed using the following B2M-F/R and CIITA-F/R primers to detect the expression of B2M and CIITA at the RNA level in the B2M/CIITA biallelic knockout clone DKO to confirm the knockout.
  • the fusion protein XSG006 was overexpressed in the DKO cells prepared in 1.2 by lentiviral infection to form the fusion protein stable cell line DKO+G6.
  • the fusion protein XSG006 is constructed by the functional domains of CD47 and CD24, which connects the mature peptide sequence of CD24 and the transmembrane domain of CD47, and its amino acid sequence is shown in SEQ ID NO.1.
  • nucleic acid sequence encoding XSG006 (the amino acid sequence of XSG006 is shown in SEQ ID NO: 1) was directly synthesized and constructed into a lentiviral plasmid (pGC-EF1a) driven by EF1a and carrying a puromycin selection marker, and the structure of the pGC-EF1a plasmid is shown in Figure 3.
  • the plasmid was digested with BamHI/NheI, and after successful ligation, Sanger sequencing was used to verify the correctness of the inserted sequence and perform virus packaging.
  • the DKO human induced pluripotent stem cells obtained in 1.2 were transfected and then screened with a medium containing puromycin.
  • XSG006 is a completely exogenous sequence.
  • the constructed DKO+G6 cells were tested for the insertion of the sequence in the genome using PCR. DKO cells were used as negative controls. After confirming that the expression was correct, cell amplification and subsequent Functional testing.
  • a kit for differentiating human induced pluripotent stem cells (WT cells), DKO cells and DKO+G6 cells into dopaminergic neural precursor cells comprising: a first culture medium, a second culture medium, a third culture medium, a fourth culture medium and a fifth culture medium;
  • the first culture medium is a neural basal medium containing 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021 and 1 ⁇ M SAG;
  • the second culture medium was a neurobasal medium containing 10 ⁇ M Y27632, 10 ⁇ M SB431542, 2 ⁇ M DMH1, 0.6 ⁇ M CHIR99021, and 1 ⁇ M SAG;
  • the third culture medium was a neurobasal medium containing 1 ⁇ M DMH1, 0.2 ⁇ M SAG, and 100 ng/mL fibroblast growth factor 8b (FGF8b);
  • the fourth culture medium is a neurobasal medium containing 0.2 ⁇ M SAG and 100 ng/mL FGF8b;
  • the fifth culture medium is a neurobasal medium containing 10 ⁇ M Y27632, 0.2 ⁇ M SAG, and 100 ng/mL FGF8b;
  • the neural basal medium is composed of the following components: 98% (v/v) DMEM/F12 (Gibco), 1% (v/v) non-essential amino acid (Non-essential amino acid, Gibco) and 1% (v/v) N2 supplement (N2 supplement, Gibco).
  • a method for differentiating human induced pluripotent stem cells (WT cells), DKO cells and DKO+G6 cells into dopaminergic neural precursor cells comprising the steps of using the kit of 2.1, specifically as follows:
  • Human induced pluripotent stem cells, DKO cells or DKO+G6 cells were plated in the form of clumps on a matrix pre-coated with Laminin at a number of about 100,000 per well, and the cells were cultured using E8 complete medium. The cells were digested and passaged every 4 to 5 days during the culture process;
  • the culture medium is replaced with the first culture medium in the kit of 2.1 (as the 0th day of differentiation, D0), and cultured in a carbon dioxide cell culture incubator at 37°C and 5% CO2 for 8 days (the medium is changed every two days);
  • DAP cells dopaminergic neural precursor cells
  • DAP dopaminergic neural precursor
  • the identity of the derived cells was confirmed by flow cytometry and immunofluorescence.
  • the expression of the stemness gene OCT4 in WT, DKO, and DKO+G6 differentiated DAP cells was verified to be ⁇ 1% ( Figure 4), confirming that they had exited pluripotency and were completely differentiated cells.
  • FACS was used to detect the expression of dopaminergic neural precursor cell marker genes FOXA2, PAX6, OTX2, and LMX1A in WT, DKO, and DKO+G6 differentiated DAP cells ( Figure 5).
  • Immunofluorescence was also used to detect the expression of dopaminergic neural precursor cell marker genes in WT, DKO and DKO+G6 derived cells.
  • the specific operation was to plate the cells in a 12-well plate, aspirate the culture medium after the cells grew to a density of 60-80%, and add 4% paraformaldehyde for fixation. After the cell membrane was broken, the primary antibodies of OTX2, EN1, LMX1A and FOXA2 were used for incubation at 4°C overnight. After washing away the primary antibodies, the secondary antibodies with fluorescent labels were incubated at room temperature, and then the fluorescence microscope was used for observation. The results are shown in Figure 6, and all cells expressed dopaminergic neural precursor cell marker genes.
  • INF-gamma was used to stimulate WT and DKO+G6 differentiated DAP cells: cells were plated in well plates, and the medium containing INF-gamma was added to the cells when the medium 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 Figure 7. After differentiation into DAP, the positive clones of DKO+G6 stem cells with B2M/CIITA biallelic knockout still did not express HLA-I/II, and could not upregulate HLA-I/II in response to INF-gamma stimulation.
  • the molecular sequence of XSG006 is a completely exogenous sequence.
  • RT-PCR was used to compare the stability of XSG006 expression between WT and dopamine neural progenitor cells differentiated from it, and between DKO+G6 and dopamine neural progenitor cells differentiated from it (Figure 8).
  • WT-DAP and DKO-DAP cells were used as negative controls. After confirming that the expression was correct, cell expansion and subsequent functional testing were performed.
  • Low immunogenic stem cells can be successfully differentiated into dopaminergic neural precursor (DAP) cells in vitro.
  • DAP dopaminergic neural precursor
  • the derived cells have the characteristics of DAP cells and retain the expression of low immunogenic molecules.
  • the human induced pluripotent stem cells selected by the present invention use CRISPR/CAS9 to knock out beta-2-microglobulin (B2M) in the endoplasmic reticulum, so that HLA-I on the cell surface cannot form functional molecules, thereby escaping the killing of allogeneic CD8+T cells; the killing of CD4+T cells is escaped by knocking out the positive regulator CIITA of HLA-II gene transcription, and reducing the expression of HLA-II class molecules.
  • XSG006 is expressed in cells (DKO) with B2M/CIITA biallelic knockout by lentivirus to escape the killing of innate immunity.
  • the cells successfully constructed are DKO+G6, which are low immunogenic stem cells.
  • the dopaminergic neural precursor cells (DKO+G6-DAP) derived from the low immunogenic stem cells of Example 1 should also have the ability to not activate the immune system and escape its killing.
  • NK activation factors were added to PBMC in advance to increase the NK ratio in PBMC and the killing performance of T cells.
  • Activated mixed lymphocytes (PBNK) were used as effector cells for RTCA experiments to comprehensively evaluate the immune escape ability of DKO+G6-DAP cells (PMID: 33309274). The specific operation was to perform PBNK cell killing assay on the XCelligence platform (ACEA BioSciences, San Diego, CA.).
  • WT-DAP, DKO-DAP and DKO+G6-DAP cells were resuspended in 100 ⁇ l cell-specific culture medium (Neurobasal TM culture medium, catalog number: 21103049, Thermofisher) and plated on a 96-well E-plate (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, T cells were added at an E:T ratio of 1:1. The data were standardized and analyzed using RTCA software (ACEA). The results are shown in Figure 9 , and only DKO+G6-DAP cells can escape the killing of PBNK.
  • WT, DKO and DKO+G6 differentiated DAP cells were plated in 12-well plates, and the culture medium was discarded after 24 hours, and MAC cells were added for co-culture. After 24 hours, the remaining WT, DKO and DKO+G6 differentiated DAP cells were observed after the removal of MAC cells, and the results are shown in FIG10 .
  • MAC cell killing assay was performed on the XCelligence platform (ACEA BioSciences, San Diego, CA.). WT-DAP, DKO-DAP, and DKO+G6-DAP cells were resuspended in 100 ⁇ l of cell-specific culture medium and plated on 96-well E-plates (ACEA BioSciences) coated with Matrigel (Sigma-Aldrich). After the cell index value reached 1, MAC cells were added at an E:T ratio of 1:1. The data were normalized and analyzed using RTCA software (ACEA). The results are shown in Figure 11.
  • the survival of DAP cells in vivo was observed by observing the bioluminescence detection (BLI) injected into the brain of humanized mice.
  • the specific operation is: the lentivirus carrying luciferase (luc) is used to infect WT and DKO+G6 differentiated DAP cells, and then the cells expressing luc are transplanted into the brain of CD34+HSC-reconstructed humanized immune system mice.
  • luc's luminescent substrate D-luciferin A025011, Shanghai Yishen Biotechnology Co., Ltd.
  • an in vivo imager iVIS spectrum, PerkinElmer
  • the fluorescence of the transplanted site was continuously detected for 51 days.
  • the fluorescence value of the WT differentiated DAP cells tended to the background value, while the DKO+G6 differentiated After DAP cell transplantation, continuously rising fluorescence values can be detected, indicating that DKO+G6 differentiated DAP cells can effectively escape the attack of the immune system in vivo compared with WT differentiated DAP cells ( FIG. 12 ).
  • This embodiment describes a study on the use of in vitro differentiated dopaminergic neural precursor cells (DAP) from stem cells for the treatment of Parkinson's disease in a mouse model.
  • DAP dopaminergic neural precursor cells
  • Scid beige mice were used for modeling, and three behavioral tests, namely apomorphine-induced rotation, cylinder test, and rotarod test, were performed to select modeling mice that met the standards for cell transplantation.
  • the experiment set up a solvent (cell preservation solution, artificial cerebrospinal fluid ACSF, Maokang Bio, MX0951) control group and a test group (WT-DAP injection and DKO+G6-DAP injection).
  • mice After administration, the behavior of the three groups of mice was tested by immunofluorescence staining of brain tissue pathological sections and three behavioral tests, and the behavioral differences of the three groups of mice, the behavioral differences of the mice themselves before and after administration, and the changes in cell retention and dopaminergic neurons at the administration site were compared.
  • Scid beige mice were anesthetized with isoflurane gas anesthesia machine, and the anesthetized mice were fixed on the mouse brain stereotaxic instrument, and 6-hydroxydopamine (6-OHDA) solution (H4381-100mg (sigma)) was slowly injected into the substantia nigra of the left brain of the mice in stereotaxic manner.
  • 6-hydroxydopamine (6-OHDA) solution H4381-100mg (sigma) was slowly injected into the substantia nigra of the left brain of the mice in stereotaxic manner.
  • the cylinder test, the rotarod test and the apomorphine-induced rotation behavioral test were performed to determine the model mice.
  • the model mice were divided into groups for injection, and 2 ⁇ L WT-DAP injection solution, 2 ⁇ L DKO+G6-DAP injection solution and 2 ⁇ L cell preservation solution were slowly injected into the left striatum of the mouse brain in a stereotaxic manner. Behavioral tests were performed once a month after surgery for 4 months. After the last test, the brain of the mouse was sampled.
  • mice in the test group showed significant behavioral improvement at 4 months (p ⁇ 0.05), and the proportion of the number of times the right forelimb hit the wall increased significantly, indicating that transplantation of WT-DAP injection solution is effective in improving the Parkinson's-related resting tremor behavior of model mice ( Figure 14).
  • mice in the test group had significant behavioral improvements at 4 months (p ⁇ 0.05), and the time they stayed on the rotarod was significantly increased; compared with the control group, the behavioral improvements were also significant (p ⁇ 0.05). This shows that transplantation of WT-DAP injection is effective in improving the posture and gait disorder behavior of model mice (Figure 15).
  • Immunofluorescence and immunohistochemical staining of frozen sections of PD mouse brain tissue tissue sections were rinsed with PBS for 5 minutes, 3 times; blocked for 1 hour at 37°C; the blocking solution was a PBS solution containing 5% BSA and 0.3% triton-100.
  • Add primary antibody react slowly in a humidified box at 4°C for 24 hours to 48 hours; the primary antibody diluent was a PBS solution containing 1% BSA and 0.3% triton-100.
  • the fluorescent secondary antibody was incubated at room temperature in the dark for 1 to 2 hours. Then rinsed with PBS for 5 minutes, 3 times, and sealed with DAPI, and photographed under a microscope.
  • a vehicle control group cell preservation solution
  • a test group WT-DAP injection and DKO+G6-DAP injection
  • This example describes an in vitro study of iPSC-derived primate (NHP) models.
  • DAP Differentiated dopaminergic neural precursor cells
  • NAP Non-human primates
  • the methods include MRI scanning and analysis, PET analysis, video analysis, and behavioral analysis (see, for example, Kikuchi et al., Nature, 2017, 548, 592-596).
  • NHP non-human primate
  • DAP cell transplantation in non-human primates (NHPs) with Parkinson’s disease (PD) DAP differentiated cells generated from stem cells were evaluated in a monkey model of Parkinson’s disease (PD).
  • monkey stem cells were differentiated according to the method described above to generate monkey WT (monkey iPSC cells, monkey blood PBMCs were prepared by CTS TM CytoTune TM -iPS2.1 Sendai virus reprogramming kit (Cat. No.: A34546)) and monkey DKO+G6 differentiated DAP cells (monkey DKO+G6-DAP cells).
  • the B2M exon segment was directly knocked out at both ends using NHP-B2M-gRNA1 and NHP-B2M-gRNA2, and then the genomic sequence knockout was verified using NHP-B2M-F/R pair PCR primers, respectively.
  • the CIITA exon segment was directly knocked out at both ends using NHP-CIITA-gRNA1 and NHP-CIITA-gRNA2, and then the genomic sequence knockout was verified using NHP-CIITA-F/R pair PCR primers, respectively, and the monkey DKO+G6 cells overexpressed NHP-XSG006 protein.
  • Monkey DAP cells were transplanted stereotaxically into the putamen of MPTP-treated monkeys on both sides, with monkey WT-DAP cells injected on the left side and monkey DKO+G6-DAP cells injected on the right side.
  • MRI was used to determine the survival of DAP cells in the left and right brains ( Figure 19). MRI results showed that monkey DKO+G6-DAP cells persisted in the brain during the 28-day observation period, while monkey WT-DAP cells were no longer observed on the fifth day.
  • the effects of the transplanted DA neurons were evaluated according to methods recognized by those skilled in the art.
  • the changes or differences in left and right absorptive behavior, movement, PD symptoms, dopaminergic function, etc. were evaluated in monkeys using DAP cells differentiated from monkey WT and monkey DKO+G6 ( Figure 20).
  • the results of feeding rate showed that after the successful modeling, the two forelimbs did not have any feeding behavior before the cell transplantation.
  • the left forelimb (the side affected by the injection of monkey DKO+G6-DAP) has shown signs of improvement; the right forelimb (the side affected by the injection of WT-DAP) has no obvious improvement.
  • Example 5 Low immunogenic dopaminergic neural precursor cells in a normal non-human primate (NHP) model
  • This embodiment describes a study on the immune immunity of dopaminergic neural precursor cells (DAP) differentiated in vitro from monkey stem cells (iPSC) in a non-human primate (NHP) model with a complete immune system.
  • DAP dopaminergic neural precursor cells
  • iPSC monkey stem cells
  • NHS non-human primate
  • Allogeneic transplantation of NHP-DAP cells was performed on normal non-human primates without modeling to confirm that DAP derived from low immunogenic iPS cells can resist immune system attacks, and MRI scanning and analysis were used to monitor the survival of cells in vivo (see, for example, Kikuchi et al., Nature, 2017, 548, 592-596).
  • the same batch of monkey WT-DAP cells and monkey DKO+G6-DAP cells were transplanted in immunodeficient NOG mice to confirm the in vivo safety of DAP cells.
  • monkey DKO+G6-DAP cells were injected into PD-NOG mice to confirm the differentiation characteristics of low immunogenic
  • Monkey DAP cells were transplanted into the putamen in a stereotaxic manner, with monkey WT-DAP cells injected on the left side and monkey DKO+G6-DAP cells injected on the right side.
  • MRI was used to determine the survival of DAP cells in the left and right brains ( Figure 21A&B). MRI scans were performed on the monkey brains before transplantation (pre-TX), on the day of transplantation (day0), and on days 7, 14, 21, 28, 42, 56, 90, 122, 152, 183, and 212 after transplantation. The results showed that during the 259-day observation period, monkey DKO+G6-DAP cells continued to exist in the brain, while monkey WT-DAP cells could not be observed on day 14-21.
  • monkey DKO+G6-DAP cells have low immunity and can escape immune system attacks, and survive for a long time in the brains of allogeneic monkeys with a complete immune system; at the same time, unmodified WT-DAP cells are rejected and cannot survive in the same environment.
  • the monkey WT-DAP cells and monkey DKO+G6-DAP cells were stereoscopically injected into the left striatum of different NOG mice using the method of Example 3B.
  • the mice were sampled for brain sampling 2 months after transplantation, and the frozen sections of NOG mouse brain tissue were immunofluorescently stained with reference to the method of Example 3D.
  • the coronal frozen sections of the brain tissue of mice 3 months after cell transplantation were co-stained with hNA (red) & Ki67 (green) & DAPI (blue) ( Figure 22A).
  • Example 3A The method of Example 3A was used to model PD in NOG mice, and the model mice were stereotactically slowly injected with monkey DKO+G6-DAP cells into the left striatum.
  • Brain samples were taken from mice 3 months after transplantation, and frozen sections of brain tissue of PD mice were immunofluorescently stained with reference to the method of Example 3D. Coronal frozen sections of brain tissue of mice 3 months after cell transplantation were co-stained with hNA (red) & TH (green) & DAPI (blue) ( Figure 23). The staining results showed that low immunogenic DAP cells can differentiate into TH+, i.e., functional dopaminergic neurons, in vivo.

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Abstract

La présente invention porte sur une cellule nerveuse à immunité privilégiée différenciée à partir d'une cellule souche pluripotente induite et sur son utilisation. Dans la présente invention, une cellule souche pluripotente induite obtenue par inactivation des gènes HLA-I et II du complexe majeur d'histocompatibilité, puis par surexpression de la protéine de fusion XSG006 construite à partir des domaines fonctionnels CD47 et CD24, est différenciée pour obtenir une cellule précurseur neuronale dopaminergique séparée à faible immunogénicité. La cellule précurseur de neurones peut échapper aux lymphocytes T, aux cellules NK et aux macrophages. La cellule précurseur de neurones échappe aussi efficacement à l'attaque du système immunitaire in vivo. Dans un modèle de souris et un modèle de primate non humain (PNH) atteints de la maladie de Parkinson, la cellule précurseur de neurones peut survivre et présenter une activité pendant une longue période dans un hôte, et inverser fondamentalement la maladie de Parkinson. La cellule précurseur neuronale constitue une base expérimentale et une base théorique pour l'établissement d'une stratégie de traitement de la maladie de Parkinson et la conception d'un nouveau médicament thérapeutique à base de cellules souches.
PCT/CN2024/097338 2023-06-05 2024-06-04 Cellule nerveuse à immunité privilégiée différenciée à partir d'une cellule souche pluripotente induite et son utilisation Ceased WO2024251119A1 (fr)

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WO2023019203A1 (fr) * 2021-08-11 2023-02-16 Sana Biotechnology, Inc. Systèmes inductibles pour modifier l'expression génique dans des cellules hypoimmunogènes

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