Cells and methods of use and preparation thereof

Abstract

In various aspects of the present disclosure, genetically engineered stem cells, plasma cells, B cells, methods of making them, and uses thereof are provided for avoiding immunological rejection in a host.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 201880032723.6, application date March 20, 2018, and invention name “Cells and their use and preparation methods”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 62 / 473,564, filed on March 20, 2017, which is incorporated herein by reference in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] not applicable.

[0006] Materials Incorporated by Reference

[0007] The sequence listing is part of this disclosure and includes a computer readable form of the nucleotide and / or amino acid sequences of the present invention. The subject matter of the sequence listing is incorporated herein by reference in its entirety. Technical Field

[0008] The present disclosure relates generally to cell therapy. Background of the Invention

[0010] Vaccines against devastating infectious diseases represent some of humanity’s greatest medical breakthroughs. The basic principle of vaccines is to establish immunity using attenuated or inactivated vectors that mimic the natural pathogen but do not cause the systemic damage that infection with live vectors can cause. Therefore, a prerequisite for all successful vaccines to date is that the human immune system must be inherently capable of eventually generating adaptive immunity in the face of infection with a natural live vector. In cases like polio, the damage inflicted before adaptive immunity can control the natural virus is unacceptable; nevertheless, long-lasting immunity is ultimately achieved and prevents subsequent infection.

[0011] However, many of the world's most difficult infectious diseases cannot be effectively controlled by the human immune system and cannot induce long-lasting immunity against reinfection. For example, HIV uses its mutability to establish chronic infections that cannot be cleared and cannot be effectively controlled without the help of antiviral drugs. Dengue virus infection leads to long-lasting immunity to reinfection caused by the same serotype, but in fact, if the individual is reinfected with a different dengue serotype, it will cause a disease that is much more severe than the disease caused by his / her complete initial infection. Similarly, influenza strains constantly change their genes; therefore, immunity against a specific strain rarely provides complete protection against all influenza viruses that subsequently spread in time and space. Natural infection with malaria does not necessarily produce long-lasting immunity because the same individual can be repeatedly infected many times in a lifetime. Pathogens such as these pose a difficult problem: how can vaccines designed to mimic natural pathogens induce immunity when the immune system is essentially unable to generate broadly effective immunity when faced with actual infection? Innovative alternative strategies such as structure-guided sequential immunization, gene therapy, and cell-based therapy have all been proposed, but the latter approach has been the least developed.

[0012] In addition, the field of cell therapy requires advances in immunosuppressive techniques or cell technologies to avoid immune rejection. Advances in understanding immune surveillance and genetic manipulation have made it possible to engineer cells for immune rejection. Summary of the invention

[0013] In various aspects of the present disclosure, methods of generating plasma cells, genetically engineered stem cells, preparing plasma cells and genetically engineered stem cells for avoiding immune rejection in a host, and methods of using the same are provided.

[0014] One aspect of the present disclosure includes a method for differentiating human embryonic stem cells (ES cells) into transplantable plasma cells that produce therapeutic agents (e.g., broadly neutralizing antibodies, proteins, or enzymes). In some embodiments, the method includes generating progenitor cells (e.g., hematopoietic progenitor cells) and differentiating the progenitor cells into B cells.

[0015] In some embodiments, differentiating the progenitor cells into B cells comprises: expressing a B lineage promoting genetic factor driven by an inducible promoter (optionally, PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8 or SPI1); culturing the progenitor cells with (i) one or more cytokines selected from the group consisting of Flt3L, SCF and IL-7 and (ii) MS5 stromal cells for a period of time sufficient to promote B lymphocyte production (optionally, up to about 20 days); or activating the progenitor cells with a stimulator, thereby differentiating the B cells.

[0016] In some embodiments, the genetic factor is optionally selected from one or more of the group consisting of: PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8, or SPI1.

[0017] In some embodiments, the irritant is selected from doxycycline or tetracycline.

[0018] In some embodiments, the hematopoietic progenitor cells are hematopoietic endothelial cells; the hematopoietic progenitor cells are transduced (optionally with a lentivirus) to encode rTTA-T2A-GFP, which is constitutively expressed; a stimulus-inducible transcriptional activator linked to a reporter gene via a ribosomal skipping 2A sequence; or a genetic factor driven by an inducible promoter and selected from one or more of the group consisting of PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8 or SPI1, to generate transduced cells that constitutively express rTTA and GFP, but only express the genetic factor after treatment with a stimulus (e.g., doxycycline).

[0019] In some embodiments, modified RNA is used to transiently introduce factors only when they are required for differentiation and lineage commitment.

[0020] In some embodiments, human ES cells are nucleofected with a pathogen-specific antibody gene encoding cassette.

[0021] In some embodiments, the pathogen-specific antibody cassette comprises VDJ and VJ sequences selected from one or more of the group consisting of influenza antibodies, HIV antibodies, or flavivirus antibodies.

[0022] In some embodiments, the specific antibody cassette comprising VDJ and VJ sequences is selected from one or more of the group consisting of FI6, VRC07, 10E8, N6, 3BNC117, EDE1, and C10.

[0023] In some embodiments, Cas9, guide RNA (gRNA), ZFNs, or TALENs are used to target endogenous immunoglobulin heavy chain and kappa light chain loci.

[0024] Another aspect of the present disclosure provides a method of generating long-lived plasma cells with enhanced glucose uptake, the method comprising administering IFNγ or IL-4 to B cells derived from human ES cells.

[0025] In some embodiments, the long-lived plasma cells have increased antibody secretion and increased mitochondrial pyruvate for respiration.

[0026] Yet another aspect of the present disclosure provides a method for generating long-lived plasma cells, the method comprising: providing primary tonsil naive B cells; or introducing IL-4 or IFNγ into primary tonsil naive B cells.

[0027] Yet another aspect of the present disclosure provides a method for generating long-lived plasma cells, the method comprising: providing 3T3 fibroblasts engineered to express CD40L and BAFF; or culturing the cells in the presence of IL-21.

[0028] Yet another aspect of the present disclosure provides a method of treating a subject carrying a virus, the method comprising administering to the subject a therapeutically effective amount of plasma cells, wherein the plasma cells express sequences of antibodies that broadly neutralize the virus.

[0029] Yet another aspect of the present disclosure provides a method of treating a subject in need of enzyme replacement therapy, the method comprising administering plasma cells engineered to secrete an enzyme.

[0030] In some embodiments, the plasma cells are engineered to secrete the enzyme by gene replacement or IRES knock-in downstream of the A[beta] gene.

[0031] Yet another aspect of the present disclosure provides a B cell or plasma cell generated by the aforementioned method.

[0032] Yet another aspect of the present disclosure provides a method of treating an autoimmune disease or cancer, the method comprising plasma cells expressing an immunotherapeutic agent such as rituxan or eculizimab.

[0033] Yet another aspect of the present disclosure provides a method of treating a neurodegenerative disease, the method comprising administering to a subject a therapeutically effective amount of plasma cells expressing an immunotherapeutic agent.

[0034] In some embodiments, the neurodegenerative disease is Alzheimer's disease, or the immunotherapeutic agent is aducanumab.

[0035] Yet another aspect of the present disclosure provides a genetically engineered stem cell comprising: (i) regulated expression of one or more HLA-I and HLA-II relative to wild-type stem cells; or (ii) a construct encoding a gene that results in escape from complement fixation, escape from NK cell recognition, or escape from phagocytosis.

[0036] In some embodiments, the genetically engineered stem cells include regulated expression of one or more immune evasion factors relative to wild-type stem cells. In some embodiments, the regulated expression of immune evasion factors includes increased expression of immune evasion factors.

[0037] In some embodiments, the immune evasion factor is inserted into a safe harbor locus of at least one allele of the cell. In some embodiments, the safe harbor locus comprises an AAVS locus.

[0038] In some embodiments, the immune evasion factor suppresses immune rejection or promotes immune evasion.

[0039] In some embodiments, the immune evasion factor is selected from the group consisting of HLA-E, HLA-G, CD46 / Crry, CD47 or CD55.

[0040] In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the stem cell is low immunogenic. In some embodiments, the stem cell is a human stem cell.

[0041] In some embodiments, the genetically engineered stem cells comprise a genetic modification in one or more genes encoding a ligand selected from the group consisting of β2 microglobulin, TAP1, CD74, CIITA, and NKG2D.

[0042] In some embodiments, the ligand of NKG2D is optionally selected from one or more of the group consisting of MICA, MICB, Raet1e, Raet1g, Raet11, Ulbp1, Ulbp2, and Ulbp3.

[0043] In some embodiments, the genetic modification comprises an inactivating mutation.

[0044] In some embodiments, the genetically engineered stem cells comprise enhanced expression of one or more of HLA-E and HLA-G.

[0045] In some embodiments, the genetically engineered stem cells comprise enhanced expression of one or more of HLA-E and HLA-G relative to wild-type stem cells.

[0046] In some embodiments, (i) the β2 microglobulin and TAP1 encoding genes are genetically modified to eliminate HLA-I expression and prevent direct recognition by allogeneic CD8+ T cells; (ii) the CD74 encoding gene is genetically modified to eliminate HLA-II expression; (iii) the NKG2D ligand encoding gene is genetically modified to escape recognition by natural killer cells; or (iv) the stem cells exhibit reduced immunogenicity.

[0047] In some embodiments, (i) at least one immune escape gene is selected from the group consisting of: HLA-E single chain trimer, HLA-G single chain trimer, Kb single chain trimer, CD46 / Crry, CD55 and CD47; (ii) at least one immune escape gene inhibits a pathway or cell type selected from the group consisting of: NKG2A+NK cells, ILT2 / KIR2DL4+NK cells, Ly49C+NK cells, complement / C3b and C4b, complement / C3 convertase, complement / C9 and phagocytosis; (iii) optionally, at least one suicide gene.

[0048] In some embodiments, (i) an immune escape gene or immune escape factor is delivered to a safe harbor locus that prevents silencing, and the immune escape factor is selected from one or more of the following groups: HLA-E single chain trimer, HLA-G single chain trimer, Kb single chain trimer, CD46 / Crry, CD55 and CD47; (ii) the suicide gene is selected from one or more of the following groups: iCasp9, HSV thymidine kinase, cytosine deaminase and Escherichia coli nitroreductase; or (iii) the suicide gene is selected from a suicide gene with an induction drug, and the induction drug is selected from the group consisting of AP1903, ganciclovir, 5-fluorocytosine and CB1954.

[0049] Yet another aspect of the present disclosure provides a genetically engineered stem cell comprising a genetic modification in a gene encoding an ability to escape recognition by one or more immune cells selected from the group consisting of: CD8 + T cells, CD4 + T cells and NK cells.

[0050] In some embodiments, the genetic modification is in a gene encoding one or more of the groups consisting of: β2 microglobulin, TAP1, CD74, CIITA, and a ligand of NKG2D, wherein the ligand of NKG2D is optionally selected from one or more of the groups consisting of: MICA, MICB, Raet1e, Raet1g, Raet1l, Ulbp1, Ulbp2, and Ulbp3.

[0051] In some embodiments, the genetic modification comprises an inactivating mutation.

[0052] Yet another aspect of the present disclosure provides a genetically engineered stem cell comprising regulated expression of one or more immune evasion factors relative to wild-type human stem cells.

[0053] In some embodiments, the modulated expression of the immune evasion factor comprises increased expression of the immune evasion factor.

[0054] In some embodiments, the immune evasion factor is inserted into a safe harbor locus of at least one allele of the cell.

[0055] In some embodiments, the safe harbor locus comprises an AAVS locus.

[0056] In some embodiments, the immune evasion factor suppresses immune rejection or promotes immune evasion.

[0057] In some embodiments, the stem cell is an embryonic stem cell. In some embodiments, the stem cell is a pluripotent stem cell. In some embodiments, the stem cell is low immunogenic. In some embodiments, the stem cell is a human stem cell.

[0058] In some embodiments, the immune evasion factor is selected from one or more of the group consisting of HLA-E, HLA-G, CD46 / Crry, CD47, and CD55.

[0059] In some embodiments, the genetically engineered stem cells comprise modulated expression of one or more of HLA-I and HLA-II relative to wild-type stem cells.

[0060] Another aspect of the present disclosure provides a method for preparing a genetically engineered stem cell, the method comprising delivering a construct to a safe harbor locus and encoding a gene that causes escape from complement fixation, NK cell recognition or engulfment. In some embodiments, the gene that causes escape from complement fixation is selected from one or more groups consisting of CD46 / Crry, CD55 and CD59. In some embodiments, the gene that causes escape from NK cell recognition is selected from one or more groups consisting of HLA-E and HLA-G. In some embodiments, the gene that causes escape from engulfment is CD47.

[0061] Yet another aspect of the present disclosure provides a method of treating a subject in need thereof with a genetically engineered stem cell, or a genetically engineered stem cell.

[0062] In some embodiments, the stem cells are transplanted into a subject; the subject's tissue is destroyed by an autoimmune disease; the subject's tissue destroyed by an autoimmune disease is regenerated; pancreatic cells or oligodendrocytes are regenerated; the subject suffers from type 1 diabetes, multiple sclerosis, is a carrier of a pathogen, or has an infectious disease; humoral immunity is generated; or antibody-mediated immunity is generated.

[0063] In some embodiments, gene editing is used to remove the transcription factors CD74 or CIITA required for HLA-II expression, wherein the genetically engineered stem cells retain hematopoietic potential and the derived monocytes lack detectable HLA-II expression.

[0064] In some embodiments, host immunosuppression is absent.

[0065] Other objects and features will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Those skilled in the art will appreciate that the following drawings are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.

[0067] Figure 1A to Figure 1B The generation of definitive hematopoietic precursors from hES cells is shown. (A) Schematic diagram of the hES cell differentiation protocol. (B) Example of differentiation. H1 hES cells differentiated into mesoderm and then into definitive hemogenic endothelium for 8 days. These cells were co-cultured with OP9-DLL4 cells for an additional 3 weeks to generate committed CD7+CD5+CD4-CD8-'DN' T cells.

[0068] FIG. 2A to FIG. 2B Doxycycline-induced Pax5 expression promotes B cell differentiation of hES-derived progenitor cells. (A) Schematic representation of lentiviral vectors used to transduce hemogenic endothelium. (B) Representative data showing B cell differentiation of doxycycline-treated lentiviral-transduced cells after 25 days of co-culture with MS5 stromal cells.

[0069] FIG. 3A to FIG. 3C Targeting of influenza-specific antibody genes to endogenous immunoglobulin loci is shown. (A) Schematic representation of the IgH targeting strategy. (B) PCR screening of genomic DNA from zeocin-resistant clones or positive control transfectants of the K562 cell line. A similar strategy was adopted for Igκ targeting. (C) PCR screening of genomic DNA hES clones carrying influenza antibody genes for resistance cassettes after Cre recombinase transfection.

[0070] Figure 4 IFNγ was shown to promote glucose uptake during in vitro plasma cell differentiation. Naive B cells were cultured on NIH 3T3 cells expressing CD40L and BAFF for 3 days in the presence or absence of the indicated cytokines. Cells were then incubated with 50 μM 2NBDG (a fluorescent glucose analog) for 30 minutes and analyzed by flow cytometry.

[0071] FIG. 5A to FIG. 5BThe engraftment and persistence of primary human bone marrow plasma cells in the auditory ossicles is shown. 1-5 x 10 6 Human bone marrow plasma cells enriched for CD138 were injected into preformed human auditory ossicles of NSG mice. Serum ELISA analysis was performed on days 7 and 28 (A), and flow cytometry analysis was performed on day 28 (B). The detection limit of (A) was 3 pg / ml.

[0072] FIG. 6A to FIG. 6E The generation of HLA-deficient hES cell lines is shown. (A) Schematic diagram of the genome editing workflow. Cas9 and three gRNAs targeting genes essential for HLA expression were nuclear transfected into H1 hES cells. Two rounds of subcloning and MiSeq analysis resulted in clonal mutant cell lines. (B) Example of Miseq analysis of target genes. Frameshift mutations were introduced in 5 of 6 alleles. (C) Wild-type or HLA-KO hES cells were stained for HLA-I expression in the presence or absence of IFNγ treatment. HLA-I expression was absent in β2m and TAP1-deficient cells. (D) Monocytes and dendritic cells from HLA-KO hES cells or control sources were mixed with allogeneic T cells labeled with CFSE. CFSE dilutions were quantified after 4 days. (E) Monocytes and dendritic cells were derived from HLA-KO hES cells or control hES cells, and HLA-II expression was measured by flow cytometry.

[0073] FIG. 7A to FIG. 7B Immune escape genes to be delivered to the AAVS locus are shown. (A) Schematic diagram of AAVS targeting constructs that evade human (upper panel) or mouse (lower panel) immune recognition. (B) AAVS constructs were transfected into HLA-KO hES cells and selected for puromycin or neomycin resistance. Expression of immune escape genes was quantified by flow cytometry.

[0074] FIG. 8A to FIG. 8C AAVS constructs are shown to mediate immune evasion. (A) CHO cells were transfected with construct 1 (SEQ ID NO: 1) or 2 (SEQ ID NO: 2) in Figure 7 and analyzed by flow cytometry. (B) Stably transfected CHO cells containing construct 1 (upper row) or construct 2 (lower row) in Figure 7 were tested for complement deposition. (C) 721.221 cells transfected with construct 1 in Figure 7 were cultured with primary human NK cells. NK cell degranulation was measured as a function of CD107a expression.

[0075] FIG. 9A to FIG. 9B The survival of HLA-KO transplants in humanized mice is shown. (A) Robust engraftment of untreated NSG-W41 mice containing cord blood CD34+ cells.5 (B) HLA-KO, but not wild-type hES cells, formed teratomas in humanized NSG-W41 mice.

[0076] Fig.10 The development of cell-based therapies and strategies for engineering universal transplantable cells that can provide durable antibody-mediated immunity are shown, indicating that stem cell-based therapies can generate immunity against mutable viruses.

[0077] FIG. 11A to FIG. 11B Successful deletion of MICA and MICB in HLA-KO cells is shown. (A) Two separate gRNAs targeting MICA and MICB were transfected and clones were sequenced for frameshift mutations. Clones B05, H10, and F01 carried frameshift mutations in MICA and MICB of one allele. (B) MICA-MICB fusion alleles were targeted with gRNA. On another chromosome, in-frame fusions caused by deletion of intervening sequences were observed between MICA and MICB in clones B05, H10, and F01. This fusion allele was re-targeted with gRNA, and 5 clones carrying frameshift mutations were isolated. DETAILED DESCRIPTION

[0078] The present disclosure is based at least in part on the discovery that (1) differentiation of human pluripotent stem cells into transplantable antibody-secreting or enzyme-secreting plasma cells, and (2) mutations (e.g., using CRISPR / Cas9) in genes encoding ligands for NKG2D that evade natural killer cell recognition and render human pluripotent stem cells for transplantation essentially non-immunogenic or minimally immunogenic, as well as expression of genes that prevent complement deposition, can eliminate major determinants of human pluripotent stem cell immunogenicity. In summary, this allows for the scalability of existing therapies for autoimmune diseases, neurodegenerative diseases, cancer, and infectious diseases, as well as the general application of ES cell-based therapies that use altered cells to avoid immune rejection.

[0079] As described herein, methods and targets for modifying human pluripotent stem cells to allow them to evade recognition by multiple arms of the immune system have been developed. The present disclosure provides methods for generating minimally immunogenic donor pluripotent stem cell lines that can be used as a source of "off-the-shelf" therapies for regenerative medicine without host immunosuppression, and cells generated by such methods.

[0080] One aspect of the present disclosure provides for scalable input of cells altered to avoid immune rejection. Commercially viable cell-based therapies may require scalable cell input to reduce treatment costs and enable standardized production, and can serve as an allogeneic source of cells for establishing cell therapies.

[0081] This article describes the steps for the development of a scalable cell therapy for infectious diseases that can be assembled into an integrated pathway to provide protection against infectious pathogens and can also be used in other cell therapy applications.

[0082] Minimally immunogenic or essentially non-immunogenic human embryonic stem cells

[0083] The generation of minimally immunogenic "universal" donor hES cell lines has become a goal in many areas of regenerative medicine. Decades of transplantation research have shown that the immunological barriers to transplantation are formidable, and how best to achieve a truly "universal" cell line is not fully understood.

[0084] This article describes hES cells that are essentially non-immunogenic or minimally immunogenic for transplantation, in particular, for hepatocyte-based immunotherapy for infectious diseases. The creation of such cell lines for transplantation can enable scalable, off-the-shelf cell therapies. This is required for most stem cell-based therapies being developed by private industry. Such cell lines can also promote regenerative medicine for tissues destroyed by autoimmunity, such as pancreatic beta cells in type I diabetes and oligodendrocytes in multiple sclerosis.

[0085] This article describes the elimination of several major determinants of immunogenicity.

[0086] Application in disease treatment

[0087] The present disclosure provides for the production of a scalable source of off-the-shelf therapeutics with applications ranging from immunotherapy, pancreatic beta cell replacement, or restoration of oligodendrocytes for multiple sclerosis.

[0088] Different types of antibodies mediate long-term immunity against previously encountered viruses, compared to viruses that have mutated after initial exposure. This paper describes the identification of the signals that lead to these different antibodies and determines how they protect against pathogens.

[0089] Compositions and methods for providing lasting immunity against globally relevant pathogens are described herein. Infectious diseases cause nearly 1 / 3 of all deaths and are therefore the leading cause of death worldwide. Despite decades of research into the microorganisms that cause these diseases, many of the most difficult infectious diseases have proven difficult to combat through vaccination. Therefore, new approaches are needed if successful vaccines are to be developed. Efforts to develop an anti-HIV vaccine are a useful example. A small portion of the HIV seropositive population produces very potent antibodies that are able to neutralize greater than 90% of clinical HIV isolates. Similar rare but broadly neutralizing antibodies have been found for both influenza and dengue viruses. However, many of these antibodies have abnormal structures and very high numbers of somatic mutations. Therefore, it is unclear how these types of responses are generated in the general population.

[0090] Several creative possibilities have been proposed and explored experimentally. First, it has been proposed that antibody responses could be directed toward broadly neutralizing properties through structure-based design of HIV glycoprotein immunogens and sequential vaccination. Several studies have demonstrated the feasibility of this approach using immunoglobulin knock-in mice or animals carrying more diverse lineages. However, these studies are still in the early stages of mouse models, and it remains to be seen whether this strategy can be translated into human vaccines. Indeed, for other intractable pathogens, it is unclear whether similar resources are available specifically for HIV epitope structural design. In the second approach, adeno-associated virus (AAV) vectors are used to deliver broadly neutralizing antibody genes intramuscularly for heterologous expression. Both mouse and primate models have shown encouraging results, so clinical trials are being conducted. Although generally safe, a major problem revealed by previous studies is that the duration of heterologous gene expression can be quite short-lived unless the vector is delivered to immune-privileged areas. This may be due to the extensive pre-existing immunity in humans and the CD8 + In the third approach, lentiviral-based gene therapy is used to modify hematopoietic stem cells (HSCs) so that they express broadly neutralizing antibodies. Upon autologous transplantation, HSC-derived genetically modified B cells and plasma cells can be formed to secrete these neutralizing antibodies. This last approach has received much less attention due to issues of practical feasibility. Hematopoietic stem cell transplantation, even autologous transplantation, requires cytoreductive conditioning to achieve effective graft engraftment. In addition, random integration of lentiviral vectors in the host genome has been shown to induce leukemia. Finally, this approach is not scalable, resulting in high costs. As described here, these issues are addressed in the latter approach by generating pathogen-specific B cells using scalable minimally immunogenic inputs.

[0091] Tissue regeneration.

[0092] There is a need for low-cost and easily manipulated stem cells that can be used to regenerate human tissues. Stem cell lines (e.g., genetically engineered) that have minimal immunogenicity and are considered "universal" donors are described herein. These cells can be used as a source of "off-the-shelf" therapies for regenerative medicine without host immunosuppression.

[0093] Plasma cells and serum antibodies

[0094] This paper describes the use of pluripotent stem cells to generate antibody-mediated immunity. It is believed that there are no previous papers on this topic, nor are there other ongoing research projects using pluripotent stem cells to generate antibody-mediated immunity.

[0095] Described herein are novel methods for generating humoral immunity using human pluripotent stem cells (see, eg, Example 1).

[0096] The component steps to achieve humoral immunity are well-recognized. The effectiveness of serum antibodies in protecting against pathogens has been known for more than a hundred years (see, e.g., Behring, 1965). The existence of long-lived plasma cells and their role in maintaining serum antibodies are widely accepted in the art (see, e.g., Slifka et al., 1998).

[0097] The generation of definitive hematopoietic precursors (DHP) from hES cells is well known; see, e.g., Kennedy et al., 2012. Therefore, unless otherwise specified herein, the processes disclosed herein can be performed in accordance with such processes. As described herein, the definitive hematopoietic precursors (DHP) are first differentiated from ES using the methods described by Kennedy et al. (Cell Reports, 2012) and Sturgeon et al. (Nat Biotech, 2014).

[0098] By activating PAX5 by any method known in the art, differentiation from DHP to B cell can be achieved. For example, PAX5 can be activated by gene transduction or with small molecule activation. As shown in Example 1, PAX5 is activated by transduction to target cells using a lentiviral vector. In addition, auxiliary matrix cells (such as 3T3 fibroblasts) can be engineered to express a subgroup of these factors (such as BAFF and CD40L) by retroviral transduction. As described herein, human embryonic stem cells (ES cells) can be differentiated into plasma cells. For example, ES cells can be H1 human embryonic stem cells.

[0099] A progenitor cell is a biological cell that, like a stem cell, has a tendency to differentiate into a specific type of cell, but has become more specific than a stem cell and is forced to differentiate into its "target" cell. For example, a progenitor cell can be a hemogenic progenitor cell (e.g., a hemogenic endothelial cell) or a hematopoietic progenitor cell.

[0100] As described herein, transcription factors or cytokines and B lineages can be used to promote ectopic expression of activating proteins or genetic factors to differentiate final hematopoietic progenitor cells (DHP) into B cells. These cytokines include IL-7, Flt3L and SCF in a concentration range of 1-100ng / μl. Other cytokines described herein are IFNγ or IL-4. B lineages promote activating proteins (also called B lineages promoting transcription factors or genetic factors) including PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8 and SPI1. Genetic factors can be induced to express using lentiviral vectors, modified RNA or plasmid transfection.

[0101] Application of plasma cells.

[0102] As described herein, plasma cells can be used as preventive treatments for infectious diseases. For example, it is described that influenza antibody genes are knocked into the endogenous locus of hES cells (see, for example, Example 1, Fig. 3). These antibodies are combined with the conservative part of influenza virus and neutralize almost all influenza virus strains (see, for example, Example 1). As another example, plasma cells can be used for treating progressive chronic infections such as HIV (see, for example, Example 1). As another example, plasma cells can be used for enzyme replacement therapy for diseases such as Hurler syndrome (see, for example, Example 1) or factor IX deficiency related to enzyme deficiency. As another example, plasma cells can be used for treating cancer (see, for example, Example 1). As another example, plasma cells can be used for treating autoimmune diseases (see, for example, Example 1). As another example, plasma cells can be used for treating Alzheimer's disease (see, for example, Example 1). As another example, plasma cells expressing antibodies can be used for treating subjects (for example, using immunotherapeutic agents) who need antibody therapy. For example, immunotherapeutic agents can be antibodies. As an example, the immunotherapeutic agent can be rituxan, eculizimab, or aducanumab.

[0103] As described herein, the compositions and methods can be used to treat a neurodegenerative disease or condition. For example, the neurodegenerative disease or condition can be Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Alexander disease, Alpers disease, Alpers-Huttenlocher syndrome, alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Arts syndrome, ataxia neuropathy spectrum, ataxia (e.g., with oculomotor ataxia, autosomal dominant cerebellar ataxia, deafness, and narcolepsy), autosomal recessive spastic ataxia of Charlevoix-Saguenay, Batten disease, beta-propeller protein-associated neurodegenerative disease, brain-eye-brain ... Facial-musculoskeletal syndrome (COFS), Corticobasal degeneration, CLN1 disease, CLN10 disease, CLN2 disease, CLN3 disease, CLN4 disease, CLN6 disease, CLN7 disease, CLN8 disease, Cognitive impairment, Congenital insensitivity to pain and anhidrosis, Dementia, Familial encephalopathy with neuroserpin inclusions, Familial English dementia, Familial Danish dementia, Fatty acid hydroxylase-related neurodegenerative diseases, Gerstmann-Straussler-Scheinker disease, GM2-gangliosidosis (e.g., AB variant), HMSN type 7 (e.g., with retinitis pigmentosa), Huntington's disease, Neuroaxonal dystrophy, Infantile, Infantile-onset ascending hereditary spastic palsy, Huntington's disease (HD), Infantile-onset spinocerebellar ataxia, Juvenile primary lateral sclerosis, Kennedy disease, Kuru, Leigh's disease, Marinesco- syndrome, mild cognitive impairment (MCI), mitochondrial membrane protein-associated neurodegenerative disease, motor neuron disease, monomelic muscular atrophy, motor neuron disease (MND), multiple system atrophy, multiple system atrophy with orthostatic hypotension (Shy-Drager syndrome), multiple sclerosis, multiple system atrophy, neurodegenerative disease in Down syndrome (NDS), senile neurodegeneration, neurodegeneration with brain iron accumulation, neuromyelitis optica, pantothenate kinase-associated neurodegenerative disease, oculomotor myoclonus, prion disease, progressive multifocal leukoencephalopathy, Parkinson's disease (PD), PD-related diseases, multicystic lipomembranous dysplasia with sclerosing leukoencephalopathy, prion disease, progressive external ophthalmoplegia, riboflavin transporter-deficient neuropathy, Sandhoff disease, spinal muscular atrophy (SMA), spinocerebellar ataxia (SCA), striatonigral degeneration, transmissible spongiform encephalopathy (or prion disease), or Wallerian degeneration.

[0104] As described herein, the compositions and methods can be used to treat cancer. For example, the cancer can be acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); adrenocortical carcinoma; AIDS-related cancer; Kaposi's sarcoma (soft tissue sarcoma); AIDS-related lymphoma (lymphoma); primary central nervous system (CNS) lymphoma (lymphoma); anal cancer; appendix cancer; gastrointestinal carcinoid; astrocytoma; childhood atypical teratoid / rhabdoid tumor of the central nervous system (brain cancer); basal cell carcinoma of the skin; bile duct cancer; bladder cancer; bone cancer (including Ewing's sarcoma and osteogenic sarcoma and malignant fibrous histiocytoma); brain tumors; breast cancer; bronchial tumors; Burkitt's lymphoma; carcinoid (gastrointestinal); childhood carcinoid; cardiac (heart) tumors; central nervous system cancer; Childhood atypical teratoid / rhabdoid tumor (brain cancer); Childhood embryonal tumor (brain cancer); Childhood germ cell tumor (brain cancer); Primary central nervous system (CNS) lymphoma; Cervical cancer; Biliary epithelial cell carcinoma; Cholangiocarcinoma chordoma; Chronic lymphocytic leukemia (CLL); Chronic myeloid leukemia (CML); Chronic myeloproliferative neoplasms; Colorectal cancer; Craniopharyngioma (brain cancer); Cutaneous T-cell tumor; Ductal carcinoma in situ (DCIS); Childhood CNS embryonal tumor (brain cancer); Endometrial cancer (uterine cancer); Childhood ependymoma (brain cancer); Esophageal cancer; Esthenoblastoma; Ewing sarcoma (bone cancer); Extracranial germ cell tumor; Extragonadal germ cell tumor; Eye cancer; Intraocular melanoma; intraocular melanoma; retinoblastoma; fallopian tube cancer; malignant fibrous histiocytoma or osteosarcoma of bone; gallbladder cancer; stomach (gastric) cancer; gastrointestinal carcinoid; gastrointestinal stromal tumor (GIST) (soft tissue sarcoma); germ cell tumor; central nervous system germ cell tumor (brain cancer); extracranial germ cell tumor of childhood; extragonadal germ cell tumor; ovarian germ cell tumor; testicular cancer; gestational trophoblastic disease; hairy cell leukemia; head and neck cancer; heart tumor; hepatocellular (liver) carcinoma; Langerhans cell histiocytosis; Hodgkin lymphoma; hypopharyngeal cancer (head and neck cancer); intraocular melanoma; islet cell tumor; pancreatic neuroendocrine tumor; Kaposi sarcoma (soft tissue sarcoma); kidney (renal cell ) cancer; Langerhans cell histiocytosis; Laryngeal cancer (head and neck cancer); Leukemia; Lip and oral cancer (head and neck cancer); Liver cancer; Lung cancer (non-small cell and small cell); Lymphoma; Male breast cancer; Malignant bone fibrous histiocytoma or osteosarcoma; Melanoma; Intraocular (eye); Merkel cell carcinoma (skin cancer); Malignant mesothelioma; Metastatic cancer; Occult primary metastatic squamous neck cancer (head and neck cancer); NUT gene-associated midline cancer; Oral cancer (head and neck cancer); Multiple endocrine neoplasm syndrome; Multiple myeloma / plasma cell neoplasms; Mycosis fungoides (lymphoma); Myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms; Chronic myeloid leukemia (CML); Acute myeloid leukemia (AML);Myeloproliferative neoplasms; Nasal and paranasal sinus cancer (head and neck cancer); Nasopharyngeal cancer (head and neck cancer); Neuroblastoma; Non-Hodgkin lymphoma; Non-small cell lung cancer; Cancer of the oral cavity, lip, or mouth; Oropharyngeal cancer (head and neck cancer); Osteogenic sarcoma and malignant fibrous histiocytoma of bone; Ovarian cancer; Pancreatic cancer; Pancreatic neuroendocrine tumors (islet cell tumors); Papillomatosis; Paraganglioma; Paranasal sinus and nasal cancer (head and neck cancer); Parathyroid cancer; Penile cancer; Pharyngeal cancer (head and neck cancer); Pheochromocytoma; Pituitary tumor; Plasma cell neoplasms / multiple myeloma; Pleuropulmonary blastoma; Breast cancer; Primary central nervous system (CNS) lymphoma; Primary peritoneal cancer; Prostate cancer; Rectal cancer; Recurrent cancer; Renal cell (kidney) cancer; Retinoblastoma; Childhood rhabdomyosarcoma (soft tissue sarcoma); Salivary gland cancer (head and neck cancer); Sarcoma; Childhood rhabdomyosarcoma (soft tissue sarcoma) sarcoma); childhood vascular tumors (soft tissue sarcoma); Ewing sarcoma (bone cancer); Kaposi sarcoma (soft tissue sarcoma); osteogenic sarcoma (bone cancer); uterine sarcoma; Sézary syndrome (lymphoma); skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; occult primary metastatic squamous neck cancer (head and neck cancer); gastric (stomach) cancer; cutaneous T-cell lymphoma; lymphoma; mycosis fungoides and Sèzary syndrome; testicular cancer; pharyngeal cancer (head and neck cancer); nasopharyngeal cancer; oropharyngeal cancer; hypopharyngeal cancer; thymoma and thymic cancer; thyroid cancer; thyroid tumors; transitional cell carcinoma of the renal pelvis and ureter (kidney (renal cell) cancer); transitional cell carcinoma of the ureter and renal pelvis (kidney (renal cell) cancer); urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vascular tumors (soft tissue sarcomas); vulvar cancer; or renal bud cell tumor. ;

[0105] As described herein, the compositions and methods can be used to treat autoimmune diseases or conditions. For example, the autoimmune disease or condition can be apathy; Addison's disease; Adult Still's disease; Agammaglobulinemia; Alopecia areata; Amyloidosis; Ankylosing spondylitis; Anti-GBM / Anti-TBM nephritis; Antiphospholipid syndrome; Autoimmune angioedema; Autoimmune dysautonomia; Autoimmune encephalomyelitis; Autoimmune hepatitis; Autoimmune inner ear disease (AIED); Autoimmune myocarditis; Autoimmune oophoritis; Autoimmune orchitis; Autoimmune pancreatitis; Autoimmune retinopathy; Autoimmune urticaria; Axonal and neuronal neuropathy (AMAN); Baló disease; Behcet's disease; disease); benign mucous membrane pemphigoid; bullous pemphigoid; Castorman syndrome (CD); celiac disease; Chagas disease; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing multifocal osteomyelitis (CRMO); Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis with pancreatic eosinophilia (EGPA); scarring pemphigoid; Cogan syndrome; cold agglutinin disease; congenital heart block; coxsackievirus myocarditis; CREST syndrome; Crohn's disease; dermatitis herpetiformis; dermatomyositis; Devic's disease (optical Neuromyelitis; discoid lupus; Dressler syndrome; endometriosis; eosinophilic esophagitis (EoE); eosinophilic fasciitis; erythema nodosum; essential mixed cryoglobulinemia; Evans syndrome; fibromyalgia; fibrosing alveolitis; giant cell arteritis (temporal arteritis); giant cell myocarditis; glomerulonephritis; Goodpasture syndrome; granulomatosis with polyangiitis; Graves disease; Guillain-Barré syndrome; Hashimoto's thyroiditis; hemolytic anemia; Henoch-Schonlein purpura purpura)(HSP); herpes gestationis or pemphigoid gestationis (PG); hidradenitis suppurativa (HS) (acne inversa); hypogammaglobulinemia; IgA nephropathy; IgG4-related sclerosing disease; immune thrombocytopenic purpura (ITP); inclusion body myositis (IBM); interstitial cystitis (IC); juvenile arthritis; juvenile diabetes mellitus (type 1 diabetes); juvenile myositis (JM); Kawasaki disease; Lambert-Eaton syndrome; Leukocytoclastic vasculitis; lichen planus; lichen sclerosus; woody conjunctivitis; linear IgA disease (LAD); lupus; chronic Lyme disease; Meniere's disease; microscopic polyarteritis (MPA); mixed connective tissue disease (MCTD); Mooren's ulcer; Mucha-Habermann disease; multifocal motor neuropathy (MMN) or MMNCB; multiple sclerosis; myasthenia gravis; myositis; narcolepsy; neonatal lupus; neuromyelitis optica; neutropenia;Ocular cicatricial pemphigoid; optic neuritis; relapsing rheumatic disease (PR); PANDAS; paraneoplastic cerebellar degeneration (PCD); paroxysmal nocturnal hemoglobinuria (PNH); Parry Romberg syndrome; pars plana ciliitis (peripheral uveitis); Parsonnage-Turner syndrome; pemphigus; peripheral neuropathy; peripheral encephalomyelitis; pernicious anemia (PA); POEMS syndrome; polyarteritis nodosa; polyglandular syndromes I, II, and III; polymyalgia rheumatica; polymyositis; postmyocardial infarction syndrome; postpericardiotomy syndrome; primary biliary cirrhosis; primary sclerosing cholangitis; progestogen dermatitis; psoriasis; psoriatic arthritis; pure red cell aplasia (PRCA); pyoderma gangrenosum; Raynaud's phenomenon; reactive arthritis; reflex sympathetic dystrophy; relapsing polychondritis; restless legs syndrome (RLS); retroperitoneal fibrosis; rheumatic fever; rheumatoid arthritis; sarcoidosis; Schmidt syndrome; scleritis; scleroderma; syndrome; sperm and testicular autoimmunity; stiff-man syndrome (SPS); subacute bacterial endocarditis (SBE); Susac syndrome; sympathetic ophthalmia (SO); Takayasu arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS); transverse myelitis; type 1 diabetes mellitus; ulcerative colitis (UC); undifferentiated connective tissue disease (UCTD); uveitis; vasculitis; vitiligo; Vogt-Koyanagi-Harada disease; or Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

[0106] As described herein, plasma cells expressing antibodies can be generated. For example, plasma cells can be generated from human ES cells nucleofected with a pathogen-specific antibody gene encoding cassette.

[0107] As described herein, plasma cells can express antibodies specific to viruses. For example, the virus can be influenza virus, HIV, malaria or flavivirus (e.g., dengue, Zika, West Nile, tick-borne encephalitis, yellow fever, cell fusion factor virus (CFAV), Palm Creek virus (PCV), Parramatta River virus (PaRV)). For example, plasma cells can express antibodies specific to FI6, VRC07, 10E8, N6, 3BNC117, EDE1 or C10.

[0108] Stem cells genetically engineered through genome editing, an immunoengineering approach

[0109] As described herein, genetically engineered pluripotent stem cell lines are modified to allow the cells to escape recognition by multiple branches of the immune system. Such gene replacements are typically performed on a set of genes and targets, some of which have been described in the prior art. The key novel features of the present invention that are added to the prior art modifications will be apparent to those skilled in the art.

[0110] Cells containing the novel modifications of the present invention may escape CD8 + T cells, CD4 + The recognition of T cells, NK cells, complement or phagocytes. In addition, the cells can contain inducing suicide genes and drug resistance boxes. This allows selective elimination of transplants when adverse reactions occur, and easily carries out drug selection in culture to identify clonal cell lines. In short, the method allows the generation of human pluripotent stem cells with greatly reduced immunogenicity for transplantation.

[0111] As described herein, it was discovered that disruption of specific immune receptors and introduction of specific transgenes into human stem cells (ie, modification by gene knockout and / or transgene (cDNA) insertion) can generate universal donor pluripotent stem cells.

[0112] Provided herein is a genetically engineered stem cell, wherein (i) the genes encoding β2 microglobulin and TAP1 are genetically modified to eliminate HLA-I expression and prevent direct recognition by allogeneic CD8+ T cells; (ii) HLA-II expression is eliminated to evade direct recognition by CD4+ T cells; or (iii) the gene encoding NKG2D ligand is genetically modified to evade recognition by natural killer (NK) cells.

[0113] Also provided herein is a stem cell line and a method for generating a stem cell line that evades recognition by the following immune cells: (i) CD8 + T cells (i.e., with genetically modified MHC class I expression due to lack of genes encoding β2-microglobulin and TAP1); (ii) CD4 + T cells (i.e., due to lack of MHC class II expression with genetic modifications in the genes encoding CD74 and CIITA); and / or (iii) NK cells (i.e., due to genetic modifications in the genes encoding NKG2D ligands (e.g., MICA, MICB, Raet1e, Raet1g, Raet1l, Ulbp1, Ulbp2 and Ulbp3)).

[0114] Also provided herein is a method for preparing genetically engineered stem cells, the method comprising delivering a construct to the AAVS locus in ES cells to express the following genes (or immune escape factors) for a specified purpose: (i) CD46 / Crry, CD55 and CD59 that lead to escape from complement fixation; (ii) HLA-E and HLA-G single chain trimers, and Kb single chain trimer (shown in mice) that lead to escape from NK cell recognition; (iii) CD47 that leads to escape from phagocytic macrophages; (iv) icasp9 and HSV thymidine kinase that lead to induction of suicide genes (death by AP1903 and ganciclovir, respectively); and / or (v) puromycin and neomycin resistance cassettes that lead to drug resistance.

[0115] Also provided is a method for treating a subject with genetically engineered pluripotent stem cells, wherein: the subject suffers from an autoimmune disease, such as type 1 diabetes or multiple sclerosis, or the patient suffers from an infectious disease (such as an infectious disease that destroys tissue); the subject has damaged tissue, and the damaged tissue is regenerated by genetically engineered stem cells (e.g., pancreatic cells, oligodendrocytes).

[0116] Genetic modification of genes encoding β2-microglobulin and TAP1.

[0117] The present disclosure provides genetic modifications in genes encoding β2 microglobulin and TAP1. This eliminates HLA-I expression and prevents direct recognition of allogeneic CD8+ T cells. As described herein, the genetic modification can be an inactivating mutation.

[0118] The disclosure further provides mutations in genes encoding CD74 and CIITA. This eliminates HLA-II expression and escapes direct recognition of CD4+T cells. Previous studies have described gene mutations that prevent HLA-I and HLA-II expression, respectively. However, as described herein (see, e.g., Example 2 FIG6), two additional genes as targets have been identified. For example, in order to prevent HLA-I expression, the use of TAP1 mutations is described. In addition to β2M, the deletion of TAP1 has also been shown to be important, because the deletion of either gene in these two genes is not sufficient to completely remove HLA-I expression alone. Previous studies have confirmed that residual CD8+T cells are reactive to TAP1-deficient cells, and β2M-deficient grafts can re-express HLA-I expression in β2M-sufficient hosts by obtaining serum β2M. As another example, in order to prevent HLA-II expression, mutated CD74 is described. In addition to CIITA, the deletion of CD74 has also been shown to be important, because some cell types can express HLA-II independently of CIITA. Depending on the major mutations that remove HLA-I and II, these additional genes can be removed to prevent omission of HLA expression.

[0119] As described herein, an inactivating mutation can be any mutation in a gene that causes a reduction or elimination of HLA-I or HLA-II expression (see, e.g., FIG. 6 ). An inactivating mutation can include nucleotide insertions or deletions that change the reading frame and prevent functional protein translation. For example, 2 base pairs are deleted in one allele of the TAP1 gene and 1 base pair is inserted in another allele of the TAP1 gene, resulting in premature termination of translation and lack of HLA-I expression (see, e.g., FIG. 6 ).

[0120] The disclosure further shows that these HLA-deficient cells produce teratomas in xenogeneic chimeric mice reconstituted with an allogeneic human immune system. As shown herein, it has been demonstrated that the cells lack expression of HLA-I and HLA-II. It has been further demonstrated that monocytes derived from these cells are unable to stimulate allogeneic T cell proliferation. As further demonstrated herein, the modified cells have been demonstrated to escape rejection by mice reconstituted with a human immune system. It is also demonstrated herein that the AAVS targeting construct correctly expresses all the expected genes and confers resistance to natural killer cell recognition and complement deposition.

[0121] It is currently believed that none of the previous approaches combine disruption of both HLA-I and HLA-II. Furthermore, it is believed that the genes targeted in previous approaches are insufficient to mediate complete loss of HLA-I and HLA-II expression.

[0122] Prevents phagocytosis and NK cell activation.

[0123] The disclosure further provides mutations produced in genes encoding NKG2D ligands to escape natural killer (NK) cell recognition. NK cells have many different recognition modes, and NKG2D is the only activating receptor known to be expressed on all NK cells. Therefore, in contrast to other alternative strategies described in the prior art, the removal of NKG2D ligands eliminates the reactivity of all NK cells.

[0124] The disclosure further provides the design and verification of the construct of the AAVS locus to be delivered to human ES cells. These constructs encode genes (HLA-E and HLA-G single-chain trimers) that cause evasion of NK cell recognition and genes (CD47 and HLA-G single-chain trimers) that cause evasion of engulfment. The expression of these genes significantly reduces the activation of NK cells (see, for example, Example 2). These constructs encode suicide-inducing genes (such as icasp9 and HSV thymidine kinase) and drug resistance boxes (such as puromycin and neomycin resistance) simultaneously. This allows selective elimination of transplants when adverse reactions occur, and is easy to perform drug selection in culture to identify clonal cell lines. In short, this process allows the generation of human pluripotent stem cells with significantly reduced immunogenicity for transplantation.

[0125] Prevent complement deposition.

[0126] The disclosure further provides the design and verification of constructs to be delivered to the AAVS locus in human ES cells. These constructs encode genes (e.g., CD46 / Crry, CD55, and CD59) that lead to escape complement fixation. The expression of CD46 / Crry, CD55, and CD59 is described herein to prevent classical and alternative complement deposition on cells (see, e.g., Example 2). It is believed that preventing classical and alternative complement deposition is essential for preventing antibody-dependent immune rejection.

[0127] Molecular Engineering

[0128] The following definitions and methods are provided to better define the present invention and guide those skilled in the art to implement the present invention. Unless otherwise indicated, terms should be understood by those of ordinary skill in the relevant art according to conventional usage.

[0129] As used herein, the terms "heterologous DNA sequence," "exogenous DNA fragment," or "heterologous nucleic acid" all refer to sequences that are derived from a source foreign to a particular host cell, or, if derived from the same source, are modified from their original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified, for example, using DNA shuffling. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA segment that is foreign or heterologous to a cell, or a DNA segment that is homologous to a cell but occurs at a position within a host cell nucleic acid where the element is not normally found. The exogenous DNA segment is expressed to produce an exogenous polypeptide. A "homologous" DNA sequence is a DNA sequence naturally associated with the host cell into which it is introduced.

[0130] Expression vectors, expression constructs, plasmids or recombinant DNA constructs are generally understood to refer to nucleic acids with a series of specific nucleic acid elements produced by human intervention (including by recombinant means or direct chemical synthesis), which allow, for example, transcription or translation of a specific nucleic acid in a host cell. The expression vector can be a part of a plasmid, a virus or a nucleic acid fragment. Typically, the expression vector can include a nucleic acid to be transcribed that is operably linked to a promoter.

[0131] "Promoter" is generally understood to be a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood to be a promoter that mediates the transcription of an operably linked gene in response to a specific stimulus or activator (e.g., a doxycycline or tetracycline inducible promoter). A promoter may include an essential nucleic acid sequence close to the transcription start site, such as a TATA element for a polymerase II type promoter. A promoter may optionally include a distal enhancer or repressor element, which may be located up to several thousand base pairs from the transcription start site.

[0132] As used herein, "transcribable nucleic acid molecule" refers to any nucleic acid molecule that can be transcribed into an RNA molecule. A known method is to introduce a construct into a cell so that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule, which is translated and thus expressed as a protein product. The construct can also be constructed to express an antisense RNA molecule to inhibit the translation of a specific target RNA molecule. For the practice of the present disclosure, conventional compositions and methods for making and using constructs and host cells are well known to those of skill in the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th edition, Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754).

[0133] The "transcription start site" or "promoter site" is the position around the first nucleotide that is part of the transcribed sequence, also defined as position +1. All other sequences of the gene and its control regions can be numbered relative to this site. Downstream sequences (i.e., other protein coding sequences in the 3' direction) can be designated as positive, while upstream sequences (mostly control regions in the 5' direction) are designated as negative.

[0134] "Operably linked" or "functionally linked" preferably refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one nucleic acid sequence is affected by the other nucleic acid sequence. For example, for a regulatory DNA sequence and a DNA sequence encoding an RNA or polypeptide, if the position of the two sequences is such that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter), then the two sequences are said to be "operably linked" or "associated" with each other. The coding sequence can be operably linked to the regulatory sequence in sense or antisense orientation. The two nucleic acid molecules can be part of a single continuous nucleic acid molecule and can be adjacent. For example, if the promoter regulates or mediates the transcription of the target gene in a cell, then the promoter is operably linked to the target gene.

[0135] "Construct" is generally understood to be any recombinant nucleic acid molecule derived from any source, capable of genomic integration or autonomous replication, such as a plasmid, a cosmid, a virus, an autonomously replicating nucleic acid molecule, a bacteriophage, or a linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, including nucleic acid molecules in which one or more nucleic acid molecules have been operably linked.

[0136] Constructs disclosed herein may contain a promoter operably connected to a transcribable nucleic acid molecule, which is operably connected to a 3' transcription termination nucleic acid molecule. In addition, constructs may include, but are not limited to, additional regulatory nucleic acid molecules derived from, for example, 3' untranslated regions (3'UTRs). Constructs may include, but are not limited to, 5' untranslated regions (5'UTRs) of mRNA nucleic acid molecules, which may play an important role in translation initiation and may also be genetic components in expression constructs. These additional upstream and downstream regulatory nucleic acid molecules may be derived from natural or heterologous sources relative to other units present on the promoter construct.

[0137] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragment are called "transgenic" cells, and organisms containing transgenic cells are called "transgenic organisms."

[0138] "Transformed", "transgenic" and "recombinant" refer to a host cell or organism, such as bacteria, cyanobacteria, animals or plants, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome as generally known and disclosed in the art (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, etc. The term "untransformed" refers to a normal cell that has not undergone the transformation process.

[0139] “Wild type” refers to a virus or organism found in nature without any known mutations.

[0140] The design, generation and testing of variant nucleotides and their encoded polypeptides having the above-mentioned desired identity percentages and retaining the desired activity of the expressed protein are within the technical scope of the art. For example, directed evolution and rapid isolation of mutants can be performed according to the methods described in the references, including but not limited to Link et al. (2007) Nature Reviews 5 (9), 680-688; Sanger et al. (1991) Gene 97 (1), 119-123; Ghadessy et al. (2001) Proc Natl Acad Sci USA 98 (8) 4552-4557. Therefore, a person skilled in the art can generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity with the reference sequences described herein, and screen them for the desired phenotype according to conventional methods in the art.

[0141] Nucleotide and / or amino acid sequence identity percentage (%) is understood as the percentage of nucleotides or amino acid residues in the candidate sequence that are identical with the nucleotides or amino acid residues in the reference sequence when comparing two sequences. In order to determine the identity percentage, the sequences are compared, and if necessary, spaces are introduced to obtain maximum sequence identity percentage. Sequence alignment programs that determine the identity percentage are well known to those skilled in the art. Compare sequences using computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) that are usually publicly available. Those skilled in the art can determine the appropriate parameters for measuring the comparison, including any algorithm required for maximum comparison over the full length of the compared sequence. When aligning sequences, the percentage of sequence identity between a given sequence A and a given sequence B, and a given sequence B, or relative to a given sequence B (or can be described as the percentage of sequence identity between a given sequence A and a given sequence B, and a given sequence B, or relative to a given sequence B having or comprising a certain percentage of sequence identity) can be calculated as follows: percentage of sequence identity = X / Y×100, where X is the number of residues that are assessed as identical matches based on the alignment of A and B by a sequence alignment program or sequence alignment algorithm, and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percentage of sequence identity between A and B will not be equal to the percentage of sequence identity between B and A.

[0142] In general, conservative substitutions can be made at any position as long as the desired activity is retained. So-called conservative exchanges can be made, in which the replaced amino acid has similar properties to the original amino acid, for example, Asp for Glu, Asn for Gln, Ile for Val, Ile for Leu and Thr for Ser. For example, amino acids with similar properties can be aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine); hydroxyl or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine); cyclic amino acids (e.g., proline); aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan); basic amino acids (e.g., histidine, lysine, arginine); or acidic amino acids and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion is the replacement of an amino acid with a direct bond. The deleted positions include the polypeptide end groups and the bonds between the various protein domains. Insertion is the introduction of an amino acid into a polypeptide chain, with the direct bond formally replaced by one or more amino acids. The amino acid sequence can be adjusted with the aid of computer simulation programs known in the art, which can generate polypeptides with, for example, improved activity or altered regulatory properties. Based on such artificially generated polypeptide sequences, the corresponding nucleic acid molecules encoding such regulatory polypeptides can be synthesized in vitro using the specific codons of the desired host cell.

[0143] Host cells can be transformed using a variety of standard techniques known in the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th edition, Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microparticle-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, etc. Transfected cells can be selected and propagated to provide recombinant host cells comprising the expression vector stably integrated into the host cell genome.

[0144]

[0145]

[0146]

[0147]

[0148] Exemplary nucleic acids that can be introduced into host cells include, for example, DNA sequences or genes from another species, or even genes or sequences derived from or present in the same species but incorporated into recipient cells by genetic engineering methods. The term "exogenous" is also intended to refer to genes that are not normally present in transformed cells, or may not simply exist in the form, structure, etc. found in the transformed DNA segments or genes, or genes that normally exist and need to be expressed in a manner different from the natural expression pattern, such as overexpressed genes. Therefore, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment introduced into the recipient cell, regardless of whether similar genes already exist in such cells. The DNA type included in the exogenous DNA can include DNA already present in the cell, DNA of another individual of the same type of organism, DNA of different organisms, or externally produced DNA (e.g., a DNA sequence containing gene antisense information or a DNA sequence encoding a synthetic or modified DNA pattern).

[0149] Host strains developed according to the approaches described herein can be evaluated by various methods known in the art (see, e.g., Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

[0150] Methods for downregulating or silencing genes are known in the art. For example, antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) can be used to downregulate or eliminate the activity of expressed proteins (see, e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G, which describes hammerhead ribozymes and small hairpin RNAs; Helene, C. et al. (1992) Ann. NY Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14 (12): 807-15, which describes targeted deoxynucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, which describes aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326–330, which describes RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504–510, which describes RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147–173, which describes RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401–423, which describes RNAi). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programs using a variety of algorithms are known in the art (see, e.g., Cenix algorithm, Ambion; BLOCK-iT TM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing). Features that have an impact on defining the optimal siRNA sequence include the G / C content of the siRNA ends, the Tm of a specific internal domain of the siRNA, the siRNA length, the position of the target sequence in the CDS (coding region), and the nucleotide content of the 3' overhang.

[0151] preparation

[0152] The agents and compositions described herein can be formulated in any conventional manner using, for example, one or more pharmaceutically acceptable carriers or excipients described in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such preparations will contain a therapeutically effective amount of a bioactive agent described herein, which may be in pure form, or may be in combination with an appropriate amount of a carrier to provide a form suitable for administration to a subject.

[0153] The term "formulation" refers to a drug prepared in a form suitable for administration to a subject (eg, a human). Therefore, a "formulation" may include a pharmaceutically acceptable excipient, including a diluent or carrier.

[0154] As used herein, the term "pharmaceutically acceptable" can describe a substance or component that does not cause unacceptable loss of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those with monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 (" USP / NF ") or updated versions, and components listed in the inactive ingredient retrieval online database in the constantly updated FDA. Other available components not described in USP / NF etc. can also be used.

[0155] As used herein, the term "pharmaceutically acceptable excipient" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005). Unless any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic composition is contemplated. Supplementary active compounds may also be incorporated into the composition.

[0156] A "stable" formulation or composition can refer to a composition that has sufficient stability to be stored at a convenient temperature, such as between about 0°C and about 60°C, for a commercially reasonable period of time, e.g., at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0157] Preparation should be suitable for mode of administration. According to the agent used in the present disclosure, it can be prepared by known methods so that it is applied to the subject by several approaches, including but not limited to parenteral, pulmonary, oral, external, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, eye, cheek and rectum. Various agents can also be used in combination with one or more additional agents or used together with other biologically active or biologically inert agents. Such biologically active or inert agents can be connected to one or more agent fluids or mechanically connected or connected to one or more agents by ions, covalent, van der Waals forces, hydrophobic, hydrophilic or other physical forces.

[0158] Controlled release (or sustained release) products can be formulated to extend the activity of one or more agents and reduce the frequency of administration. Controlled release products can also be used to affect the onset of action or other characteristics, such as the blood level of the agent, thereby affecting the appearance of side effects. Controlled release products can be designed to initially release a certain amount of one or more agents that produce the desired therapeutic effect, and gradually and continuously release other amounts of the agent, thereby maintaining the therapeutic effect level over a long period of time. In order to maintain a near constant agent level in the body, the agent can be released from the dosage form at a certain rate, and the released agent will replace the amount of the agent metabolized by the body or excreted from the body. The controlled release of the agent can be stimulated by various inducing factors such as pH changes and changes in temperature, enzymes, water or other physiological conditions or molecules.

[0159] The agents or compositions described herein may also be used in combination with other treatment modalities as further described below. Thus, in addition to the therapies described herein, other therapies known to be effective in treating the disease, disorder or condition may also be provided to the subject.

[0160] Treatment

[0161] Also provided is a method of treating a disease (e.g., an autoimmune disease, tissue destroyed by an autoimmune disease, a pathogen, cancer, an enzyme deficiency, or a neurodegenerative disease) with a cell-based therapy (e.g., the differentiated progeny of genetically engineered stem cells) in a subject in need thereof, and administering a therapeutically effective amount of a cell-based therapy so as to treat a disease (e.g., a pathogen, an infectious disease) with pluripotent stem cells while evading recognition by natural killer cells.

[0162] Further, ES cells modified using the methods of the present invention to avoid immune rejection can be used to generate any other cell type currently being developed for patient treatment.Such differentiated cells are administered to patients in need of such cells with reduced or no need for immunosuppressants.

[0163] The methods described herein are generally performed on subjects in need. Subjects in need of the methods described herein may be subjects suffering from, diagnosed with, suspected of having, or at risk of tissue damage, pathogens, or infectious diseases. The determination of treatment needs is usually assessed based on a medical history and physical examination consistent with the disease or condition in question. The diagnosis of various conditions that can be treated by the methods described herein belongs to the technical scope of the art. Subjects may be animal subjects (including mammals, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens) and humans. For example, the subject may be a human subject.

[0164] In general, a safe and effective amount of a genetically engineered stem cell (e.g., a genetically engineered pluripotent stem cell) is an amount that, for example, produces a desired therapeutic effect in a subject while minimizing adverse side effects. In various embodiments, an effective amount of plasma cells derived from genetically engineered stem cells as described herein can substantially inhibit tissue damage, pathogens, or infectious diseases, slow the progression of tissue damage, pathogens, or infectious diseases, or limit the development of tissue damage, pathogens, or infectious diseases.

[0165] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, ossicular, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ocular, buccal, or rectal.

[0166] When used in the treatments described herein, an effective amount of plasma cells derived from genetically engineered stem cells (e.g., genetically engineered pluripotent stem cells) can be used in pure form, or in the form of a pharmaceutically acceptable salt (if such a form exists) and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered in sufficient amounts to substantially inhibit tissue damage, pathogens, or infectious diseases, slow the progression of tissue damage, pathogens, or infectious diseases, or limit the development of tissue damage, pathogens, or infectious diseases at a reasonable benefit / risk ratio suitable for any medical treatment. Genetically engineered stem cells can have minimal immunogenicity, thereby escaping recognition by multiple branches of the immune system.

[0167] The amount of the compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. Those skilled in the art will appreciate that the unit content of the agent contained in a single dose of each dosage form need not constitute a therapeutically effective amount in itself, since the necessary therapeutically effective amount can be achieved by administering multiple single doses.

[0168] The toxicity and therapeutic efficacy of the compositions described herein can be determined by measuring the LD in cell culture or experimental animals. 50 (the dose that causes 50% of the population to die) and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD 50 / ED 50 , where larger therapeutic indices are generally understood in the art to be optimal.

[0169] The specific therapeutically effective dosage level for any particular subject will depend on a variety of factors, including the condition being treated and the severity of that condition; the activity of the specific compound employed; the specific composition employed; the age, weight, general health, sex, and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; the drugs used in combination or concomitantly with the specific compound employed; and other factors well known in the medical arts (see, e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is entirely within the capabilities of those skilled in the art to control the initial dose of the composition to a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. If necessary, the effective daily dose can be divided into multiple doses for ease of administration. Therefore, a single dose composition may contain such an amount or a submultiple thereof constituting the daily dose. However, it should be understood that the total daily dosage of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of reasonable medical judgment.

[0170] Likewise, each morbid state, disease, disorder and condition described herein and other situations can benefit from the compositions and methods described herein. In general, treating a morbid state, disease, disorder or condition includes preventing or delaying the appearance of clinical symptoms of a mammal that may suffer from the morbid state, disease, disorder or condition or be susceptible to the morbid state, disease, disorder or condition but has not yet appeared or shows its clinical or subclinical symptoms. Treatment can also include suppressing the morbid state, disease, disorder or condition, such as preventing or reducing the development of the disease or at least one clinical or subclinical symptom thereof. In addition, treatment can include alleviating the disease, such as causing a morbid state, disease, disorder or condition, or at least one of its clinical symptoms or subclinical symptoms to disappear. The benefit of the subject to be treated can be statistically significant, or at least can be perceived by the subject or doctor.

[0171] The administration of the offspring derived from genetically engineered stem cells can occur as a single event, or over a period of time. For example, the offspring of genetically engineered stem cells can be administered daily, weekly, biweekly, or monthly. For the treatment of acute conditions, the time course of treatment is generally at least a few days. Certain conditions may extend the treatment from a few days to a few weeks. For example, the treatment may last for a week, two weeks, or three weeks. For more chronic conditions, the treatment time may extend from a few weeks to a few months, or even a year or more.

[0172] Treatment according to the methods described herein can be performed prior to, concurrently with, or after conventional treatment modalities for tissue damage, pathogens, or infectious diseases.

[0173] The offspring of genetically engineered stem cells can be administered simultaneously or sequentially with another agent such as an antibiotic, an anti-inflammatory drug, or another agent. For example, the offspring of genetically engineered stem cells can be administered simultaneously with another agent such as an antibiotic or an anti-inflammatory drug. Simultaneous administration can be performed by administering a separate composition, each composition containing one or more of the offspring of genetically engineered stem cells, an antibiotic, an anti-inflammatory drug, or another agent. Simultaneous administration can be performed by administering a composition containing two or more of the offspring of genetically engineered stem cells, an antibiotic, an anti-inflammatory drug, or another agent. The differentiated offspring of genetically engineered stem cells can be administered sequentially with an antibiotic, an anti-inflammatory drug, or another agent. For example, cells derived from genetically engineered stem cells can be administered before or after administering an antibiotic, an anti-inflammatory drug, or another agent.

[0174] Application

[0175] The agents and compositions described herein can be administered in various ways known in the art according to the methods described herein. The agents and compositions can be used therapeutically as exogenous materials or endogenous materials. Exogenous agents are agents produced or manufactured outside the body and applied to the body. Endogenous agents are agents produced or manufactured inside the body by some type of device (biological or other device) for delivery to the body or to other organs in the body.

[0176] As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, intraossicular, buccal, or rectal.

[0177] Agents and compositions as described herein can be used by various methods well known in the art.Using can include, for example, being related to oral intake, direct injection (for example, whole body or stereotactic), being engineered into the cell implantation of secretion target factor, releasing the biomaterial of medicine, polymer matrix, gel, permeable membrane, osmotic system, multilayer coating, microparticle, implantable matrix device, micro-osmotic pump, implantable pump, injection gel and hydrogel, liposome, micelle (for example, at most 30 μ m), nanosphere (for example, less than 1 μ m), microsphere (for example, 1-100 μ m), reservoir device, any combination of the above, or other suitable delivery vehicles, provide required release curve with different ratios.Other methods of controlled release delivery of agent or composition will be known to technicians, and within the scope of the present disclosure.

[0178] Delivery system can comprise for example infusion pump, and described infusion pump can be used agent or composition in the mode similar to the mode used by insulin or chemotherapeutic drug delivery to specific organ or tumor.Usually, use such system, agent or composition can be used in combination with biodegradable biocompatible polymer implant, and described implant releases described agent in selected position in controlled time period.The example of polymeric material comprises polyanhydride, polyorthoester, polyglycolic acid, polylactic acid, polyethylene vinyl acetate and their copolymer and combination.In addition, controlled release system can be placed near treatment target, therefore only need a part of whole body dose.

[0179] The agent can be encapsulated in a variety of carrier delivery systems and administered. Examples of carrier delivery systems include microspheres, hydrogels, polymer implants, smart polymer carriers, and liposomes (see generally Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-based systems for the delivery of molecular or biomolecular agents can: provide intracellular delivery; adjust the release rate of biomolecules / agents; increase the proportion of biomolecules reaching their site of action; improve the transport of drugs to their site of action; allow for co-local deposition with other agents or excipients; improve the in vivo stability of agents; extend the residence time of agents at their site of action by reducing clearance; reduce nonspecific delivery of agents to non-target tissues; reduce irritation caused by agents; reduce toxicity caused by high initial doses of agents; change the immunogenicity of agents; reduce the frequency of administration, improve the taste of products; or increase the shelf life of products.

[0180] Pill Box

[0181] Also provided herein is a medicine box. Such medicine boxes may include agents or compositions as described herein, and in certain embodiments, may include instructions for use. Such medicine boxes may make the methods described herein easy to perform. When provided in the form of a medicine box, the different components of the composition or the different components for preparing the composition may be packaged in separate containers and mixed before use. Components include, but are not limited to, progeny of progenitor cells, universal pluripotent stem cells, and reagents for in vitro generation of therapeutic cells, such as IL-4, IFNγ, BMP4, bFGF, VEGF, IL-6, IGF1, IL-11, SCF or EPO. Such individual packaging of components, if necessary, may be presented in the form of a pouch or a dispensing device, which may include one or more unit dosage forms containing the composition. The pouch may, for example, include metal or plastic foil, such as a blister bag. Such individual packaging of components may also allow long-term storage without loss of component activity in some cases.

[0182] Medicine box can also include reagent in separate container, such as sterile water or saline to be added to the freeze-dried active component of separate packaging.For example, sealed glass ampoule can contain freeze-dried component, and in separate ampoule, contain sterile water, sterile saline or aseptic, they are all packaged under neutral non-reactive gas such as nitrogen.Ampoule can be made of any suitable material, such as glass, organic polymer (such as polycarbonate, polystyrene), ceramic, metal or any other material that is generally used to hold reagent.Other examples of suitable container include bottles that can be made with materials similar to ampoule, and can be made of the shell of aluminum or alloy that is lined with foil.Other containers include test tube, vial, flask, bottle, syringe etc.Container can have aseptic access port, such as bottle with stopper that can be pierced with hypodermic needle.Other containers can have two compartments separated by easily removable film, and this easily removable film allows component mixing after removal.Removable film can be glass, plastic, rubber etc.

[0183] In certain embodiments, the kit may be supplied with instructional materials. The instructions may be printed on paper or other substrates, and / or may be provided as an electronically readable medium, such as a floppy disk, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disk, videotape, audiotape, etc. The detailed instructions may not be physically associated with the kit; instead, the user may be directed to an Internet website designated by the manufacturer or distributor of the kit.

[0184] The compositions and methods described herein that utilize molecular biology protocols can be prepared according to various standard techniques known in the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th edition, Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif.41(1),207–234; Gellissen,ed.(2005)ProductionofRecombinant Proteins:Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH,ISBN-10:3527310363;Baneyx(2004)Protein Expression Technologies, Taylor & Francis, ISBN-10:0954523253).

[0185] The definitions and methods described herein are provided to better define the present disclosure and guide those of ordinary skill in the art to implement the present disclosure. Unless otherwise noted, terms should be understood according to conventional usage by those of ordinary skill in the relevant technical field.

[0186] In some embodiments, the numbers representing the number of components, properties (such as molecular weight), reaction conditions, etc., used to describe certain embodiments of the present disclosure and to claim them should be understood to be modified by the term "about" in some cases. In some embodiments, the term "about" is used to indicate that the numerical value includes the mean value standard deviation of the device or method used to determine the value. In some embodiments, the numerical parameters shown in the written specification and the attached claims are approximate values, which can vary according to the desired properties sought to be obtained in a specific embodiment. In some embodiments, the numerical parameters should be interpreted according to the number of reported significant digits and by applying the usual rounding method. Although the numerical ranges and parameters shown in some embodiments of the present disclosure are approximate values ​​with a wide range, the numerical values ​​shown in the specific examples are reported as accurately as possible. The numerical values ​​shown in some embodiments of the present disclosure may include certain errors that are inevitably caused by the standard deviation present in its corresponding test measurement. The enumeration of the range of values ​​herein is intended only to be used as a shorthand method for individually referring to each independent value falling into the range. Unless otherwise specified herein, each individual value is incorporated in this specification as if it were individually enumerated herein.

[0187] In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing specific embodiments (especially in the context of certain claims below) may be understood to cover both the singular and the plural, unless otherwise specifically stated. In some embodiments, the term "or" as used herein (including the claims) is used to mean "and / or" unless it is explicitly stated to refer only to alternatives or that the alternatives are mutually exclusive.

[0188] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprise," "comprising," "has," "having," "includes," and "including," are also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps, and may also encompass other unlisted steps. Similarly, any composition or device that "comprises," "has," and "includes" one or more features is not limited to having only those one or more features, and may encompass other unlisted features.

[0189] All methods described herein can be performed in any suitable order, except where otherwise specified herein or clearly contradictory in context. The use of any and all examples or exemplary language (e.g., "such as" or "such as") provided in connection with certain embodiments herein is intended only to better illustrate the present disclosure and is not intended to limit the scope of the present disclosure otherwise claimed. The language in the specification should not be understood to indicate that any unclaimed element is necessary to implement the present disclosure.

[0190] The grouping of alternative elements or embodiments of the present disclosure should not be understood as limiting. Each group member may be mentioned separately and claimed separately or claimed in any combination with other members of the group or other elements found herein. For convenience or patentability reasons, one or more members of the group may be included in a group or deleted from the group. When any such inclusion or deletion occurs, this specification sheet shall be considered to contain the changed group in this article, thereby satisfying the written description of all Markush groups used in the appended claims.

[0191] All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated herein by reference in its entirety for all purposes. Citation of references herein should not be construed as an admission that such references are prior art to the present disclosure.

[0192] After describing the present disclosure in detail, it will be apparent that modifications, variations and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. In addition, it should be recognized that all examples in the present disclosure are provided as non-limiting examples.

[0193] Example

[0194] The following non-limiting examples are provided to further illustrate the present disclosure. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent methods that the inventors have found to work well in the practice of the present disclosure and therefore can be considered to constitute examples of ways to practice the present disclosure. However, it should be understood by those skilled in the art in light of the present disclosure that many changes may be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtaining similar or similar results.

[0195] Example 1: Differentiation of human ES cells into transplantable plasma cells producing broadly neutralizing antibodies

[0196] The following examples describe the differentiation of human ES cells into transplantable plasma cells that produce broadly neutralizing antibodies.

[0197] Long-lived plasma cells of the bone marrow maintain serum antibodies after infection or vaccination.

[0198] Most clinically used vaccines depend on the production and maintenance of neutralizing antibodies. Therefore, the main relevant factors for the protective effect after vaccination are the number, affinity, epitope specificity and duration of antibodies derived from plasma cells triggered by immunity. Plasma cells are terminally differentiated B lymphocytes that secrete thousands of antibody molecules per second and use nearly 70% of their transcriptome for antibody synthesis. In the first few days of T cell-dependent antibody response, plasma cells can only survive for a few days before apoptosis. Then, other plasma cells expressing antibodies with gradually increasing affinity are produced from germinal center reactions. These affinity-mature plasma cells can last for up to decades and are mostly present in the bone marrow. These long-lived plasma cells can constitutively secrete antibodies, regardless of the presence of antigens and without cell division, and are the main determinants of humoral immunity. Due to their high production rate of antibody secretion, it has been confirmed that only 1 μl of immune serum (equivalent to antibodies derived from only about 3 antigen-specific long-lived plasma cells) is sufficient to protect naive mice from lethal West Nile virus infection. The data described herein demonstrate their protective efficacy when these cells express neutralizing antibodies.

[0199] Methods for generating definitive hematopoietic precursors from hES cells.

[0200] To develop an expandable source of antigen-specific transplantable long-lived plasma cells, H1 human embryonic stem cells were used. These are the best characterized human ES cells, have minimal lineage bias, and readily generate hematopoietic cells. Hematopoiesis occurs in at least two distinct waves: primitive and final. Primitive hematopoiesis generates a restricted set of erythroid and myeloid lineages. In contrast, final hematopoiesis ultimately produces adult-type blood cells, including lymphocytes. Therefore, the primary therapeutic goal of hES cell work is to generate final hematopoietic precursors. As described in Sturgeon et al., Nat Biotech, 2014, a method for reproducibly generating final hematopoietic endothelium (precursors of final hematopoietic cells) using H1 hES cells has been optimized. The method employs sequential manipulation of signals during mesoderm induction, including the critical step of canonical WNT signaling activation on day 2 of the process (see, e.g. Figure 1A This ultimately allows the generation of T cells even from pluripotent cell lines lacking this potential (see e.g. Figure 1B ). Importantly, these differentiations were performed under defined serum-free conditions to maximize reproducibility and allow for easy troubleshooting.

[0201] Developing a pathway for generating B cells from definitive hematopoietic precursors.

[0202] After generating the final hematopoietic progenitor cells, the method for differentiating these precursors into B cells was explored. So far, few studies have reported differentiation into robust B cells from human ES cells, and these approaches either cannot generate mature B cells or can only be performed on a specific iPS system. None of these approaches use limited conditions, and these approaches are adjusted to any hES cell line that is currently tested without success. Therefore, a combination of hemogenic endothelial cells (see, e.g., Fig. 1) was generated, and PAX5 (B lineage promoting transcription factor) was then expressed. The final hemogenic endothelial cells (see, e.g., PAX5) were transduced with a lentiviral vector encoding constitutively expressed rTTA-T2A-GFP (doxycycline-inducible transcription activator connected to a reporter gene via a ribosome skipping 2A sequence) and PAX5 driven by a doxycycline-inducible promoter. Figure 2A ). Thus, transduced cells constitutively expressed rTTA and GFP, but only expressed PAX5 after doxycycline treatment. After 20 days of co-culture with Flt3L, SCF, IL7, and MS5 stromal cells, B cells were observed only after doxycycline treatment (see, e.g. Figure 2B ). Thus, restricted transcription factor expression can promote B lymphopoiesis by otherwise tenacious progenitor cells.

[0203] CRISPR / Cas9-mediated introduction of influenza-specific antibody genes into endogenous loci in hES cells.

[0204] Previous attempts to generate B cells using hES cells have resulted in immature lymphocytes without surface antigen receptor expression. To address this issue and provide pathogen specificity to developing B cells, a targeting cassette containing VDJ and VJ sequences from FI6 (a broadly neutralizing influenza-specific antibody) and a zeocin and hygromycin resistance cassette were generated, respectively (see e.g. Figure 3A ). These targeting cassettes were nucleofected into H1 hES cells along with Cas9 and guide RNA (gRNA) to target the endogenous immunoglobulin heavy chain and kappa light chain loci (ZFNs or TALENs could also be used). Cells that were resistant to zeocin and blasticidin were selected and PCR analysis was performed to confirm correct targeting (see, e.g. Figure 3B These cells are then transfected with a construct encoding the Cre recombinase, and subclones lacking the resistance cassette are isolated (see, e.g. Figure 3C , which shows PCR confirmation of IgH and Igk targeted by FI6 gene and removal of the resistance cassette).

[0205] Methods for generating long-lived plasma cells from B cells.

[0206] Signals that promote the formation of long-lived rather than short-lived plasma cells from B cell bodies have recently been identified. The results show that long-lived plasma cells import much higher amounts of glucose than their short-lived counterparts. This elevated glucose uptake promotes increased antibody secretion and the use of mitochondrial pyruvate for respiration, which is required for long-term persistence of plasma cells. It is currently believed that elevated glucose uptake is the only known property that allows prospective separation of short-lived from long-lived plasma cells. Through in vitro screening of exogenous factors, IFNγ and IL-4 were found to promote glucose uptake (see Figure 4 ).

[0207] Xenograft Model for Assessing Plasma Cell Lifespan.

[0208] In addition to the above work, several protocols have reported methods that extend the survival of human plasma cells in vitro. However, the lack of xenograft models for human plasma cells has hampered the analysis of in vivo survival. To address this issue, a system was developed in which adherent fibroblasts / mesenchymal stem cells from human bone marrow were transplanted subcutaneously into immunodeficient NOD-SCID IL2rγ - / - (NSG) mice. Bone-like ossicles formed approximately 8 weeks after transplantation, providing a complete human microenvironment. These ossicles robustly supported normal human hematopoiesis as well as primary myeloid malignancies, some of which had never been grown in xenografts before. To test whether these ossicles also supported normal plasma cell survival, 1-5 x 10 6 Primary human bone marrow CD138 + Plasma cells. Serum human IgG was observed at 2 and 4 weeks post-transplantation, and antibody concentrations did not decrease between these time points (see, e.g. Figure 5A At week 4, the ossicles were harvested for flow cytometry of plasma cells (see e.g. Figure 5B ) and ELISPOT (data not shown) analysis. Antibody secreting cells were observed in all injected ossicles. These data demonstrate that human ossicles support normal long-lived plasma cell survival and function. It is believed that this is the first such system of its kind.

[0209] Generation of pathogen-specific B cells from hES cells.

[0210] B cell development from FI6 knock-in hES cells was driven using defined transcription factor expression, as shown in Figures 2 to 3. The use of genomic integration of lentiviruses can provide a tricky translation strategy. Such non-integrated approaches (e.g., modified RNA) can be used to temporarily introduce factors only when differentiation and lineage commitment are required. To this end, PAX5 expression mediates a positive feedback loop with EBF1 to maintain B lineage commitment. Therefore, transient expression of exogenous PAX5 can trigger stable maintenance of endogenous PAX5 expression. In order to determine the window in which exogenous PAX5 expression promotes B cell commitment, doxycycline is only applied at D1-7, D8-14, or D15-21 co-cultured with MS5 cells. Similar experiments will be performed with lentivirus encoding EBF1. CD19 will be identified by flow cytometry + B cells and confirm the expression of influenza-specific IgM using a hemagglutinin tetramer reagent or using methods and reagents known in the art.

[0211] If preferential development of B-1a cells can occur, then Pax5 expression can promote B lineage commitment in this system. These CD5-expressing B-1a cells are derived from early fetal precursors rather than from adult stem cells. B-1a cells have not yet been shown to have the ability to generate long-lived plasma cells. If dominant B-1a cell differentiation is observed in PAX5 cultures, Notch signaling can be used to further differentiate hemogenic endothelial cells toward definitive hematopoiesis. As shown above, hemogenic endothelial cells generate T cells when co-cultured with OP9 cells expressing the Notch ligand delta-like 4 (DL4). Notch ligands promote T cell development in two stages. First, Notch signaling is essential for promoting definitive hematopoiesis. Second, Notch signaling can promote T cell commitment from hemogenic progenitors. To determine the Notch signaling window required for definitive hematopoiesis, hemogenic endothelial cells will be transduced with a doxycycline-inducible PAX5 lentiviral vector and then co-cultured on OP9-DL4 cells for 1-10 days. This short cycle of Notch signaling has been demonstrated to promote HSC-like cells from which conventional B-2 and B-1b cells are derived. Transduced cells will be purified by FACS and transferred into MS5 cells for B cell differentiation in the presence of doxycycline for 20 days. B cells will be examined for CD5 expression to quantify B-1a frequency.

[0212] Generation of long-lived plasma cells from mature human B cells.

[0213] In addition to generating mature B cells as described above, primary tonsil naive B cells were also used to optimize differentiation into long-lived plasma cells using the methods of the present invention. + CD27 -B cells were sorted onto NIH3T3 cells expressing human CD40L and BAFF. IL-4 or IFNγ was added to the culture at a concentration of 1-100 ng / ml. On day 6, CD38 高 CD27 高 Plasmablasts / plasma cells were injected intravenously or directly into the ossicles of NSG mice as shown in Figure 5. Serum human IgG was sampled every two weeks. If serum antibodies were maintained over 8-16 weeks, the animals were sacrificed and the ossicles were harvested for flow cytometry and ELISPOT analysis as shown in Figure 5.

[0214] Although glucose uptake increases, there is the possibility that plasma cells derived in vitro cannot last after in vivo transplantation. The culture method reported by others will be adopted in parallel with the in vitro culture approach summarized above. The Transwell culture method can be used, in which plasma cells can be maintained for several months in vitro. In this approach, fibroblast L cells are engineered to express CD40L, and cells are cultured in the presence of IL-21. At the later time point, plasma cells are separated from stromal cells by a Transwell filter. By regularly changing the culture medium, these cells can reach a quiescent state and can be maintained for several months. Using this differentiation system, plasma cells will be transplanted into the ossicles for long-term in vivo maintenance.

[0215] Treatment of infections (such as influenza).

[0216] hES cells were differentiated into long-lived plasma cells and then transplanted into NOD-SCID-IL2rg (NSG) mice carrying human auditory ossicles to validate the method in an animal model. These ossicles were formed after transplanting mesenchymal cells from human bone marrow into mice (as described above). After one or more weeks (enough time for influenza-specific antibody concentrations to rise), these mice were infected with a dose of influenza that would normally be lethal to NSG mice. The survival rate and influenza virus load of the mice were then determined.

[0217] For therapeutic use in humans, hES cells modified to become long-lived plasma cells (capable of avoiding immune rejection) are administered prophylactically as a vaccine.

[0218] Treatment of chronic infections.

[0219] NSG pups will be reconstituted simultaneously with cord blood CD34+ cells (intravenously) and mesenchymal cells (as described above) to form the ossicles. After T cell reconstitution, the mice will then be infected with HIV to hit human CD4+ T cells, and then transplanted with broadly neutralizing HIV-specific plasma cells. HIV titers and antibodies will be measured to understand the extent to which ongoing infection is suppressed. The mouse model described by Halper-Stromberg et al. in 2014 Cell 158(5)989-999 can be used. Once universal cells are developed, they can be tested in non-human primates and SIV.

[0220] For therapeutic use, the cells of the invention are administered to chronically infected patients, regardless of whether the virus is active or dormant.

[0221] Enzyme replacement therapy.

[0222] A mouse model of Hurler syndrome (Clark et al., 1997 Science 6 (4) 503-11) was used, in which there is a deficiency of α-L-iduronidase. Systemic enzyme replacement has a therapeutic effect on Hurler syndrome. The mouse model was crossed with NSG, the auditory ossicles were formed (as described above), and plasma cells engineered to secrete α-L-iduronidase instead of antibodies were transplanted (by gene replacement or IRES knock-in downstream of the Aβ gene). Enzyme concentrations and brain neuropathology (e.g., lysosomal dilation) were measured.

[0223] Cancer treatment.

[0224] NSG / ossicular mice (described above) were transplanted with primary non-Hodgkin lymphoma and then given plasma cells expressing rituximab (Chao et al. 2010 Cell 142(5)699-713). Tumor burden was quantified by histology and flow cytometry. If a suicide gene is built in, plasma cells can be removed when residual disease disappears.

[0225] Treatment of autoimmune diseases.

[0226] NSG / ossicular mice were transplanted with plasma cells expressing Serulerin / eculizumab. These mice were treated with anti-Gq1b antibodies and motor function and paralysis were measured. Halstead et al. (2008 131(Pt5)1197-208)) described this mouse model of autoimmune disease.

[0227] Treatment with therapeutic antibodies.

[0228] The cells and methods disclosed herein can be used to treat subjects in need of therapeutic antibodies. For example, therapeutic antibodies can be overexpressed on modified ES cells, as described herein. For example, by engineering the modified ES cells of the present invention to express therapeutic antibodies, such as aducanumab, to avoid the accumulation of Aβ plaques, treatment of Alzheimer's disease can be achieved.

[0229] Materials and methods.

[0230] These studies described herein used H1 (WA01, NIH Accession #0043) cells, a male-derived cell line, for modification and differentiation. However, all mouse experiments used both males and females and were included as variables in the experiments. In addition, donor xenografts were from both males and females. Y chromosome PCR was performed to determine the sex of the anonymous donor and these data were again included as variables in the analysis.

[0231] Description of procedures: Most of the hES cell differentiation and genetic modification work is performed in vitro. An appropriate number of immunocompromised NOD-SCID / IL2rγc null (NSG) mice will be used as recipients for ossicles and plasma cells and teratomas. A smaller number of wild-type C57Bl6 mice will be used as recipients for teratomas. Recipients will be 8 weeks old and of both sexes.

[0232] These studies in mice are a precursor to clinical development of cell-based vaccines. Previous studies in mice have proven highly relevant for such applications, and much is known about the cellular, molecular, and genetic aspects of mouse immunity. Given the potential benefits, studies in mice can be performed efficiently at relatively low cost.

[0233] Procedures to relieve pain or discomfort: Every effort was made to ensure that the level of pain and discomfort in mice was minimal. Mice were anesthetized with inhaled isoflurane followed by retro-orbital injection. Mice with teratomas 20 mm in diameter were sacrificed. Mice that developed disease were sacrificed and sentinel cages were used as routine in the animal facility.

[0234] Mice were euthanized by carbon monoxide inhalation according to the recommendations of the American Veterinary Medical Association Euthanasia Panel.

[0235] Example 2: Genetic modification of human ES cells to reduce immunogenicity

[0236] The following examples describe genetic modifications of human ES cells to reduce immunogenicity.

[0237] Universal donor hES cells are a goal in many fields, but the immunological obstacles are formidable. Human pluripotent stem cells can grow indefinitely, thus addressing one aspect of the scalability of plasma cell therapy. However, unless the stem cell line is autologous, immunosuppression must be performed to prevent graft rejection. This will almost certainly limit such therapies to immediately life-threatening diseases. Autologous iPS therapies, while likely to be immune-free, cannot be expanded to the general population. To address these issues, this paper proposes the creation of "universal" donor cell lines. These cell lines have been genetically removed of their immunogenicity, allowing cells and tissues derived from a single cell line to be transplanted into any recipient. It has been shown that the removal of HLA expression is a requirement for such universal donor cell lines, but mouse allograft studies have shown that this alone is not sufficient to prevent rejection. Subsequent studies have shown that antibodies, complement, NK cells, and phagocytes are other mediators of graft rejection. Starting with HLA-deficient cell lines, hES cell responses to each of these other immune rejection mediators have been identified.

[0238] Materials and methods were as described above unless otherwise noted.

[0239] Generation of HLA-deficient hESC cell lines.

[0240] A robust workflow was optimized for generating targeted mutations in human ES cells. Cas9 expression constructs and up to 3 different gRNA encoding vectors were co-transfected into H1 human ES cells. Single colonies were manually picked and subjected to Miseq analysis of the target gene to identify clones carrying frameshift mutations mis-introduced by non-homologous end joining. Candidate clones were then precisely plated 1 cell / well by fluorescence activated cell sorting (FACS) and again subjected to Miseq analysis to confirm mutations and lack of mosaicism (see e.g. Fig. 6A ). Next, the clones were expanded, karyotyped, and their potential to differentiate into the hematopoietic lineage was confirmed. Karyotypically normal hES cell lines lacking HLA expression were generated by CRISPR / Cas9-based targeted mutagenesis. One clone was identified by MiSeq analysis of the targeted regions, which carried inactivating mutations in both alleles of β2m and TAP1 and one allele of CD74 (see, e.g. Figure 6B In this clone, interferon-γ (IFNγ)-induced HLA-I expression was completely abolished (see, e.g. Figure 6C ) and confirmed normal karyotype. The monocytes derived from this HLA-I-deficient cell line failed to stimulate allogeneic primary CD8 + T cell proliferation (see e.g. Fig.6D, which shows that HLA-deficient hES cell offspring cannot stimulate T cells). Subsequently, this HLA-I-deficient cell line was retargeted using CRISPR to eliminate the remaining allele of CD74 and both alleles of CIITA, a transcription factor required for HLA-II expression. This clone was also confirmed to have a normal karyotype. Subsequent analysis confirmed that the cells retained hematopoietic potential after each genetic modification and that the derived monocytes lacked detectable HLA-II expression (see, e.g. Fig. 6E , which shows that CIITA / CD74-deficient cells cannot express HLA-II).

[0241] Generation of immune evasion cassettes targeting AAVS.

[0242] It is thought that elimination of HLA-I expression renders cells susceptible to NK cell lysis. The ligand for the activating receptor NKG2D is eliminated (see below). However, NKG2D blockade only removes approximately 50% of NK cell-mediated specific lysis. Therefore, additional steps are taken to further eliminate NK cell reactivity, such as by expressing HLA-E and HLA-G that covalently link to peptide and β2m to form single-chain trimers. These trimers do not exchange peptide or β2m with endogenous HLA and inhibit NK cells expressing NKG2A and ILT2. Other aspects of the immune response, including antibody binding, complement deposition, and macrophage phagocytosis, may also contribute to rejection. To address and evade each of these immune mechanisms, a set of polycistronic vectors were constructed to target the human AAVS locus (see, e.g., Table 1 of FIG7A ). This locus is considered a "safe harbor" from silencing. Flow cytometric analysis confirmed expression in HLA-deficient hES cells, see, e.g. Figure 7B ). A summary of immune escape and suicide genes and their targets can be found in Table 1.

[0243] Table 1. Human and mouse genes that can be expressed in the AAVS locus.

[0244]

[0245] The targeting construct was validated by transfection into CHO cells and evaluation of immune evasion gene expression by flow cytometry. The results showed that human CD55 and HLA-E single chain trimers showed tight co-expression (see e.g. Fig. 8A), confirming the functionality of the 2A sequence in Figure 7 and construct 1 (SEQ ID NO: 1). To confirm the correct functionality of the inserted gene product, a stable CHO cell line expressing hCD55 was identified by neomycin selection. These cells were then stained with anti-CHO antibodies, incubated with human C7-deficient serum, and finally stained for C3c, C3d, and C4c deposition. Expression of hCD55 and hCD46 abolished complement deposition, while residual hCD55- cells in culture showed robust complement deposition (see, e.g. Figure 8B ). These data confirm the functionality of the hCD55 transgene. To confirm that the HLA-E single-chain trimer is functioning correctly, a stable cell line was generated in 721.221 cells (a target cell line susceptible to NK cells). As measured by cell surface CD107a staining, incubation of unmodified 721.221 cells with primary PBMCs results in robust NK cell degranulation. However, co-incubation with 721.221 cells expressing HLA-E can specifically inhibit NKG2A+ NK cell degranulation (see, e.g. Figure 8C ).

[0246] Generation of NKG2D ligand-deficient hES cells.

[0247] Since the lack of HLA-I may make target cells susceptible to NK cell-mediated cell lysis, NKG2D ligands were targeted by CRISPR / Cas9. Among them, RNA-seq analysis showed that only MICA and MICB were expressed in long-lived plasma cells. Preliminary sequencing of about 400 nuclear transfection clones showed that one cell line carried frameshift mutations in 2 alleles of MICA and MICB and a large deletion in another chromosome, resulting in an in-frame fusion between MICA and MICB ( Fig.11A ). This MICA / B fusion was retargeted with CRISPR / Cas9 to generate a clone with a frameshift mutation in all four alleles of MICA and MICB ( Fig. 11B ). The normal karyotype of this clone was verified. From then on, HLA and MICA / B-deficient hES cells were called HM-KO hES.

[0248] Generation of stable AAVS immune evasion transfectants in HLA / MICA+MICBKO (HM-KO) hES cells.

[0249] Starting with HM-KO hES cells, two sets of nucleofections were performed. One set of nucleofections was performed with Fig. 7AIn one set of experiments, the human immune evasion cassette shown in was used, while another set of nuclear transfections was performed with the mouse immune evasion cassette (mAAVS). These constructs were co-transfected with a Cas9 expression construct and a gRNA targeting the AAVS locus. Cells with neomycin and puromycin resistance were selected, and expression of the genes was confirmed by flow cytometry (see, e.g., Figure 7B ).

[0250] Testing of teratoma formation in vivo.

[0251] To test human immune evasion in vivo, humanized NSG W41 mice were generated. Approximately 2 x 10 5 Cord blood CD34+ cells (obtained from Saint Louis Cord Blood Bank) were transplanted into untreated NSG W41 mice (see e.g. Fig.9A ). Mice were bled to confirm human recombination, and 1 million cells of each form of modified hESC were embedded in Matrigel and transplanted subcutaneously into humanized NSG-W41 mice. Teratoma growth was monitored over the course of 12 weeks, or until the tumor reached a mass of 20 mm, and then the mice were euthanized. Humanized mice inoculated with HLA-deficient cells developed teratomas, but humanized mice inoculated with unmodified hES cells did not (see, e.g., Fig. 9B , which shows that HLA-KO hES cells escape rejection generated by humanized mice). In some experiments, mice will be treated with AP1903 to activate iCasp9 and trigger teratoma regression. Certain aspects of the immune response do not work in humanized NSG W41 mice. For example, NK cells are poorly formed in this system and antibody responses are minimal. Therefore, teratoma assays in C57Bl6 mice will be performed using HM-KO cells stably expressing the mAAVS construct. In this xenogeneic environment, rejection reactions are expected to be delayed.

[0252] The modified hES cells are expected to escape immune recognition and thus form teratomas in humanized NSG-W41 mice.

Claims

1. A method for differentiating human embryonic stem cells (ES cells) into plasma cells, the method comprising: Generate progenitor cells; and Differentiate progenitor cells into B cells.

2. The method of claim 1, wherein The progenitor cell is a hematopoietic progenitor cell or a hematopoietic endothelial cell; or The plasma cells produce a therapeutic agent selected from a protein, a broadly neutralizing antibody, or an enzyme.

3. The method of claim 1, wherein differentiating the progenitor cells into B cells comprises: Expression of B-lineage-promoting genetic factors driven by an inducible promoter; and Stimulants are used to activate progenitor cells, thereby causing B cell differentiation.

4. The method of claim 3, wherein the genetic factor is selected from one or more of the group consisting of PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8 and SPI1; or the stimulant is selected from doxycycline or tetracycline.

5. The method of claim 3, comprising co-culturing the progenitor cells with (i) one or more cytokines selected from the group consisting of Flt3L, SCF, and IL-7, and (ii) MS5 stromal cells for a period of time sufficient to promote B lymphopoiesis.

6. The method of claim 1, wherein the human ES cells are nucleofected with a pathogen-specific antibody gene encoding cassette.

7. The method of claim 6, wherein the pathogen-specific antibody cassette comprises VDJ and VJ sequences selected from one or more of the group consisting of influenza antibodies, HIV antibodies, or flavivirus antibodies.

8. The method of claim 7, wherein the pathogen-specific antibody cassette comprising VDJ and VJ sequences is selected from one or more of the group consisting of FI6, VRC07, 10E8, N6, 3BNC117, EDE1 and C10.

9. A method for generating long-lived plasma cells, the method comprising: (i) administering IFNγ or IL-4 to B cells derived from human ES cells; or (ii) IL-21 was administered to 3T3 fibroblasts engineered to express CD40L and BAFF.

10. The method of claim 9, comprising: Provide primary tonsil naive B cells; and introducing IL-4 or IFNγ into the primary tonsil naive B cells.

11. The method of claim 9, wherein the long-lived plasma cells have increased antibody, protein, or enzyme secretion; increased mitochondrial pyruvate for respiration; or elevated glucose uptake relative to wild-type plasma cells.

12. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of plasma cells, in, The subject has a virus, and the plasma cells express sequences from an antibody that broadly neutralizes the virus; The subject suffers from an enzyme deficiency and the plasma cells secrete the enzyme; or The subject has an autoimmune disease, a neurodegenerative disease, or cancer, and the plasma cells express an immunotherapeutic agent, optionally selected from aducanumab, rituximab, or eculizumab.

13. A B cell or plasma cell generated according to the method of any one of claims 1 to 12.

14. A genetically engineered stem cell, comprising: (i) reduced expression of one or more of HLA-I and HLA-II relative to wild-type stem cells; or (ii) Constructs encoding genes that result in escape from complement fixation, escape from NK cell recognition, or escape from phagocytosis.

15. The genetically engineered stem cell of claim 14, comprising increased expression of one or more immune evasion factors relative to wild-type stem cells.

16. The genetically engineered stem cell of claim 15, wherein the immune escape factor is selected from one or more of the group consisting of HLA-E, HLA-G, CD46 / Crry, CD47 and CD55.

17. The genetically engineered stem cell of claim 16, wherein the immune escape factor is selected from one or more of the group consisting of CD46 / Crry and CD55.

18. The genetically engineered stem cell of claim 15, wherein the immune escape factor suppresses immune rejection or promotes immune escape.

19. The genetically engineered stem cell according to any one of claims 14 to 18, wherein the stem cell is a human stem cell, an embryonic stem cell, a pluripotent stem cell, or has low immunogenicity.

20. The genetically engineered stem cell of claim 14, comprising an inactivating mutation in a gene encoding a ligand selected from one or more of the group consisting of β2 microglobulin, TAP1, CD74, CIITA, and NKG2D.

21. The genetically engineered stem cell of claim 20, wherein (i) the inactivating mutation is in the genes encoding TAP1 or CD74; or (ii) The ligand of NKG2D is optionally selected from one or more of the group consisting of MICA, MICB, Raet1e, Raet1g, Raet1l, Ulbp1, Ulbp2 and Ulbp3.

22. The genetically engineered stem cell of claim 15, comprising enhanced expression of one or more of HLA-E and HLA-G relative to wild-type stem cells.

23. The genetically engineered stem cell according to any one of claims 20 to 21, wherein: (i) Genetic modification of the genes encoding β2-microglobulin and TAP1 to eliminate HLA-I expression and prevent direct recognition by allogeneic CD8+ T cells; (ii) genetically modifying the gene encoding CD74 to eliminate HLA-II expression; or (iii) genetic modification of genes encoding NKG2D ligands to evade natural killer cell recognition; wherein the genetically engineered stem cells produce progeny and the progeny exhibit reduced immunogenicity.

24. The genetically engineered stem cell of claim 14, wherein the genetically engineered stem cell comprises (i) at least one immune escape gene selected from the group consisting of: HLA-E single chain trimer, HLA-G single chain trimer, Kb single chain trimer, CD46 / Crry, CD55 and CD47; or (ii) at least one immune evasion gene that inhibits a pathway or cell type selected from the group consisting of NKG2A+ NK cells, ILT2 / KIR2DL4+ NK cells, Ly49C+ NK cells, complement / C3b and C4b, complement / C3 convertase, complement / C9, and phagocytosis; and (iii) optionally, at least one suicide gene.

25. The genetically engineered stem cell of claim 14, wherein the genetically engineered stem cell comprises a drug-inducible suicide gene selected from one or more of the group consisting of: iCasp9, HSV thymidine kinase, cytosine deaminase, and Escherichia coli nitroreductase, and the drug is selected from the group consisting of AP1903, ganciclovir, 5-fluorocytosine, and CB1954.

26. A method for preparing genetically engineered stem cells, the method comprising: (i) introducing genetic modifications or inactivating mutations to reduce immunogenicity; or (ii) Constructs are delivered to the safe harbor locus and encode genes that lead to escape from complement fixation, NK cell recognition or phagocytosis.

27. The method of claim 26, wherein (i) reducing the immunogenicity of the cells by eliminating HLA-I expression, eliminating HLA-II expression, and eliminating natural killer cell recognition; (ii) the inactivating mutation is in a coding gene selected from one or more of the group consisting of β2 microglobulin, TAP1, CD74, CIITA and a ligand of NKG2D, and the ligand of NKG2D is optionally selected from one or more of the group consisting of MICA, MICB, Raet1e, Raet1g, Raet1l, Ulbp1, Ulbp2 and Ulbp3; (iii) the gene that causes escape from complement fixation is selected from one or more of the group consisting of CD46 / Crry, CD55 and CD59; (iv) the gene that causes escape from NK cell recognition is selected from one or more of the group consisting of HLA-E and HLA-G; or (v) The gene that causes escape from phagocytosis is CD47.

28. A method of treating a subject in need thereof with the genetically engineered stem cell or progeny of a genetically engineered stem cell according to any one of claims 14 to 25 or the method according to any one of claims 26 to 27.

29. The method of claim 28, wherein: (i) the stem cells are transplanted into a subject; (ii) the subject's tissue is destroyed by an autoimmune disease; (iii) regeneration of tissues in a subject destroyed by an autoimmune disease; (iv) pancreatic cells or oligodendrocytes are regenerated; (v) the subject suffers from an autoimmune disease, cancer, or a neurodegenerative disease; (vi) the subject suffers from type I diabetes, multiple sclerosis, pathogens or infectious diseases; (vii) produce humoral immunity; or (viii) Producing antibody-mediated immunity.

30. The method of any one of claims 12-13 and 28-29, wherein host immunosuppression is absent.

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