Cells and methods of uses and making the same

Abstract

To provide genetically engineered stem cells, plasma cells, and B cells for avoiding immune rejection within a host, and methods of making the same and uses thereof.SOLUTION: Provided is a method of differentiating human embryonic stem cells (ES cells) into plasma cells, the method comprising producing precursor cells and differentiating the precursor cells into B cells.SELECTED DRAWING: Figure 10

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 473,564, filed March 20, 2017, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable

[0003] Materials Incorporated by Reference The Sequence Listing, which is part of this disclosure, contains computer readable forms containing 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.

[0004] FIELD OF THE INVENTION The present disclosure relates generally to cell therapy. [Background technology]

[0005] Background of the Invention Vaccines against devastating infectious diseases represent some of humanity's greatest medical breakthroughs. The fundamental principle of vaccines is the establishment of immunity using attenuated or inactivated vectors that mimic natural pathogens without the systemic damage that live infection can induce. Thus, a prerequisite for all successful vaccines to date is that the human immune system must be inherently capable of ultimately providing adaptive immunity when faced with a live, natural infection. In cases such as polio, damage inflicted before adaptive immunity can control the native virus is unacceptable. Nevertheless, durable immunity is ultimately achieved, preventing subsequent infections.

[0006] Currently, however, many of the world's most problematic infectious diseases cannot be effectively controlled by the human immune system and do not induce lasting immunity against reinfection. For example, due to its variability, HIV establishes a chronic infection that cannot be cleared without the aid of antiviral drugs and is inefficiently controlled. Dengue virus infection provides lasting immunity against reinfection with the same serotype, but in reality, reinfection with a different dengue serotype causes much more severe disease than if the individual were completely naive. Similarly, influenza strains continually genetically alter themselves, so immunity to a given strain rarely provides complete protection against all subsequent influenza viruses circulating in time and space. Natural infection with malaria does not necessarily lead to lasting immunity, as the same individual can be reinfected multiple times throughout their lifetime. Pathogens such as these pose a conundrum: how vaccines designed to mimic natural pathogens can induce immunity when the immune system is essentially unable to confer broad and effective immunity in the face of actual infection. Innovative alternative strategies such as construct-directed sequential immunization, gene therapy, and cell-based therapy have all been proposed, although the latter approach remains largely undeveloped.

[0007] The field of cell therapy also requires advances in immunosuppressive or cellular technologies to avoid immune rejection. Advances in understanding immune surveillance and genetic engineering have made it possible to engineer cellular modifications to tailor cells to avoid immune rejection. Summary of the Invention

[0008] Among various aspects of the present disclosure, there are provided the generation of plasma cells, genetically engineered stem cells, methods for making plasma cells and genetically modified stem cells, and uses thereof to avoid immune rejection in a host.

[0009] Aspects of the present disclosure include methods of differentiating human embryonic stem cells (ES cells) into transplantable plasma cells that produce a therapeutic agent (such as a broadly neutralizing antibody, protein, or enzyme). In some embodiments, the methods include generating progenitor cells (e.g., hematopoietic progenitor cells) and differentiating the progenitor cells into B cells.

[0010] In some embodiments, differentiating the progenitor cells into B cells includes expressing a B-lineage-promoting genetic factor driven by an inducible promoter (optionally PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8, or SPI1), co-culturing the progenitor cells with (i) a cytokine selected from one or more of the group consisting of Flt3L, SCF, and IL-7, and (ii) MS5 stromal cells for a period of time sufficient to promote B lymphopoiesis (optionally about 20 days), or activating the progenitor cells with a stimulus resulting in B cell differentiation.

[0011] In some embodiments, the genetic element is optionally selected from one or more of the group consisting of PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8, or SPI1.

[0012] In some embodiments, the stimulus is selected from doxycycline or tetracycline.

[0013] In some embodiments, the hematopoietic progenitor cells are hemogenic endothelial cells; the hematopoietic progenitor cells are transduced (optionally transduced with a lentiviral vector) with constitutively expressed rTTA-T2A-GFP; a stimulus-inducible transcriptional activator linked to a reporter by a ribosomal skipping 2A sequence; or the genetic element is selected from one or more of the group consisting of PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8, or SPI1 driven by an inducible promoter, resulting in transduced cells that constitutively express rTTA and GFP, but express the genetic element only upon treatment with a stimulus (e.g., doxycycline).

[0014] In some embodiments, modified RNAs are used to transiently introduce factors only when they are required for differentiation and lineage commitment.

[0015] In some embodiments, human ES cells are nucleofected with a pathogen-specific antibody gene-encoding cassette.

[0016] In some embodiments, the pathogen-specific antibody cassette comprises VDJ and VJ sequences selected from one or more of the group consisting of a flu antibody, an HIV antibody, or a flavivirus antibody.

[0017] In some embodiments, the specific antibody cassette comprising the VDJ and VJ sequences is selected from one or more of the group consisting of FI6, VRC07, 10E8, N6, 3BNC117, EDE1, and C10.

[0018] In some embodiments, Cas9, guide RNA (gRNA), ZFN, or TALEN are used to target endogenous immunoglobulin heavy chain and kappa light chain loci.

[0019] Another aspect of the present disclosure provides a method for generating long-lived plasma cells with increased glucose uptake, the method comprising administering IFNγ or IL-4 to B cells derived from human ES cells.

[0020] In some embodiments, long-lived plasma cells have increased antibody secretion and increased mitochondrial pyruvate for respiration.

[0021] Yet another aspect of the present disclosure provides a method for generating long-lived plasma cells, the method comprising providing primary tonsillar naive B cells or introducing IL-4 or IFNγ into primary tonsillar naive B cells.

[0022] Yet another aspect of the present disclosure provides a method of 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.

[0023] Yet another aspect of the present disclosure provides a method of treating a subject having a virus, the method comprising administering to the subject a therapeutically effective amount of plasma cells, wherein the plasma cells express sequences from antibodies that broadly neutralize the virus.

[0024] 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.

[0025] In some embodiments, plasma cells are engineered to secrete the enzyme via gene replacement or IRES knock-in downstream of the Aβ gene.

[0026] Yet another aspect of the present disclosure provides a B cell or plasma cell produced by the aforementioned method.

[0027] Yet another aspect of the present disclosure provides a method of treating an autoimmune disease or cancer, comprising plasma cells expressing an immunotherapeutic agent, such as Rituxan or eculizimab.

[0028] 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.

[0029] In some embodiments, the neurodegenerative disease is Alzheimer's disease or the immunotherapeutic agent is aducanumab.

[0030] Yet another aspect of the present disclosure provides genetically engineered stem cells comprising (i) one or more HLA-I and HLA-II genes whose expression is modulated relative to wild-type stem cells, or (ii) constructs encoding genes that result in evasion of complement fixation, evasion of NK cell recognition, or evasion of phagocytosis.

[0031] In some embodiments, the genetically engineered stem cells comprise one or more immune evasion factors that have modulated expression relative to wild-type stem cells, hi some embodiments, the modulated expression comprises an immune evasion factor that has increased expression.

[0032] 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 the AAVS locus.

[0033] In some embodiments, the immune evasion factor inhibits an immune rejection response or promotes immune evasion.

[0034] In some embodiments, the immune evasion factor is selected from the group consisting of HLA-E, HLA-G, CD46 / Crry, CD47, or CD55.

[0035] In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the stem cells are hypoimmunogenic. In some embodiments, the stem cells are human stem cells.

[0036] In some embodiments, the genetically engineered stem cells comprise a genetic modification in a gene encoding a gene selected from one or more of the group consisting of β2 microglobulin, TAP1, CD74, CIITA, and a ligand for NKG2D.

[0037] In some embodiments, the ligand for NKG2D is optionally selected from one or more of the group consisting of MICA, MICB, Raet1e, Raet1g, Raet1l, Ulbp1, Ulbp2, and Ulbp3.

[0038] In some embodiments, the genetic modification comprises an inactivating mutation.

[0039] In some embodiments, the genetically engineered stem cells comprise enhanced expression of one or more of HLA-E and HLA-G.

[0040] In some embodiments, the genetically engineered stem cells comprise one or more of HLA-E and HLA-G that have enhanced expression relative to wild-type stem cells.

[0041] In some embodiments, (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) the gene encoding CD74 is genetically modified to eliminate HLA-II expression, (iii) the gene encoding an NKG2D ligand is genetically modified to avoid natural killer cell recognition, or (iv) the stem cells exhibit reduced immunogenicity.

[0042] In some embodiments, (i) at least one immune evasion 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; (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; or (iii) optionally, at least one suicide gene.

[0043] In some embodiments, (i) the immune evasion gene or factor is delivered to a safe harbor locus that is protected from silencing, and the immune evasion factor is selected from one or more of the group consisting of 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 group consisting of iCasp9, HSV thymidine kinase, cytosine deaminase, and E. coli nitroreductase; or (iii) the suicide gene is selected from a suicide gene having an induction agent selected from the group consisting of AP1903, ganciclovir, 5-fluorocytosine, and CB1954.

[0044] Yet another aspect of the present disclosure is a CD8 + T cells, CD4 + Genetically engineered stem cells are provided that contain genetic modifications in coding genes that evade recognition by one or more immune cells selected from the group consisting of T cells and NK cells.

[0045] In some embodiments, the genetic modification is in a gene encoding a gene selected from one or more of the group consisting of beta-2 microglobulin, TAP1, CD74, CIITA, and a ligand of NKG2D, where 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.

[0046] In some embodiments, the genetic modification comprises an inactivating mutation.

[0047] Yet another aspect of the present disclosure provides genetically engineered stem cells that contain one or more immune evasion factors whose expression is modulated relative to wild-type human stem cells.

[0048] In some embodiments, the immune evasion factor having modulated expression comprises an immune evasion factor having increased expression.

[0049] In some embodiments, the immune evasion factor is inserted into a safe harbor locus of at least one allele of the cell.

[0050] In some embodiments, the safe harbor locus comprises the AAVS locus.

[0051] In some embodiments, the immune evasion factor inhibits an immune rejection response or promotes immune evasion.

[0052] In some embodiments, the stem cells are embryonic stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the stem cells are hypoimmunogenic. In some embodiments, the stem cells are human stem cells.

[0053] 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.

[0054] In some embodiments, the genetically engineered stem cells comprise one or more of HLA-I and HLA-II that have modulated expression relative to wild-type stem cells.

[0055] Yet another aspect of the present disclosure provides a method of generating genetically engineered stem cells, comprising delivering a construct to a safe harbor locus and encoding a gene that confers complement fixation, NK cell recognition, or evasion of phagocytosis. In some embodiments, the gene that confers complement fixation evasion is selected from one or more of the group consisting of CD46 / Crry, CD55, and CD59. In some embodiments, the gene that confers evasion of NK cell recognition is selected from one or more of the group consisting of HLA-E and HLA-G. In some embodiments, the gene that confers evasion of phagocytosis is CD47.

[0056] Yet another aspect of the present disclosure provides a method of treating a subject in need thereof with genetically engineered stem cells or genetically engineered stem cells.

[0057] In some embodiments, stem cells are transplanted into a subject; a subject's tissue is destroyed by an autoimmune disease; a subject's tissue destroyed by an autoimmune disease is regenerated; pancreatic cells or oligodendrocytes are regenerated; the subject has type 1 diabetes, multiple sclerosis, a pathogen or an infection; humoral immunity is generated; or antibody-mediated immunity is generated.

[0058] In some embodiments, gene editing was used to remove CD74 or CIITA, transcription factors required for HLA-II expression, where the engineered stem cells retained hematopoietic potential and the derived monocytes lacked detectable expression of HLA-II.

[0059] In some embodiments, there is no host immunosuppression.

[0060] Other objects and features will be in part apparent and in part pointed out hereinafter.

[0061] Those skilled in the art will appreciate that the drawings, described below, are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way. [Brief explanation of the drawings]

[0062] [Figure 1A-B] Generation of secondary hematopoietic precursors from hES cells. (A) Schematic of hES cell differentiation protocol. (B) Example of differentiation. H1 hES cells were differentiated into mesoderm followed by secondary hemogenic endothelium over 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. [Figure 2A-B] Figure 1 shows that doxycycline-induced Pax5 expression promotes B cell differentiation from hES-derived progenitor cells. (A) Schematic of the lentiviral vector used to transduce hemogenic endothelial cells. (B) Representative data showing B cell differentiation of doxycycline-treated lentiviral-transduced cells after 25 days of coculture with MS5 stromal cells. [Figure 3A-C]Figure 1 shows targeting of influenza-specific antibody genes to endogenous immunoglobulin loci. (A) Schematic of the IgH targeting strategy. (B) PCR screening of genomic DNA from Zeocin-resistant clones or K562 cell line positive control transfectants. A similar strategy was performed for Igκ targeting. (C) PCR screening of genomic DNA hES clones carrying influenza antibody genes for drug resistance cassettes after transfection with Cre recombinase. [Figure 4] We show that IFNγ promotes glucose uptake during in vitro plasma cell differentiation. Naive B cells were cultured for 3 days on NIH 3T3 cells expressing CD40L and BAFF in the presence or absence of the indicated cytokines. Cells were then incubated with 50 μM of the fluorescent glucose analog 2NBDG for 30 minutes and analyzed by flow cytometry. [Figure 5A-B] Engraftment and persistence of primary human bone marrow plasma cells in ossicles. 1-5 x 106 CD138-enriched human bone marrow plasma cells were injected into preformed human ossicles in NSG mice. Serum ELISA (A) was performed on days 7 and 28, and flow cytometry analysis (B) was performed on day 28. The lower limit of detection for (A) was 3 pg / ml. [Figures 6A-E]Generation of HLA-deficient hES cell lines. (A) Schematic diagram of the genome editing workflow. Cas9 and three gRNAs targeting genes essential for HLA expression were nucleofected into H1 hES cells. Two rounds of subcloning and MiSeq analysis resulted in clonal mutant cell lines. (B) Example of MiSeq analysis of targeted genes. Frameshift mutations were introduced into five of six alleles. (C) Wild-type or HLA-KO hES cells were stained for HLA-I expression with or without IFNγ treatment. HLA-I expression was absent in β2m-deficient and TAP1-deficient cells. (D) Monocytes and dendritic cells derived from HLA-KO hES cells or controls were mixed with CFSE-labeled allogeneic T cells. CFSE dilution was quantified after 4 days. (E) Monocytes and dendritic cells derived from HLA-KO or control hES cells were assayed for HLA-II expression by flow cytometry. [Figure 7A-B] Immune evasion genes delivered to the AAVS locus are shown. (A) Schematic of AAVS targeting constructs to evade human (top) or mouse (bottom) immune recognition. (B) AAVS constructs were transfected into HLA-KO hES cells and selected for puromycin or neomycin resistance. Expression of immune evasion genes was quantified by flow cytometry. [Figure 8A-C] These results demonstrate that AAVS constructs 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) CHO cells stably transfected with construct 1 (top row) or construct 2 (bottom row) from 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. [Figure 9A-B]Figure 1 shows the survival of HLA-KO grafts in humanized mice. (A) Robust engraftment in unconditioned NSG-W41 mice with cord blood CD34+ cells. Example of spleen chimerism 20 weeks after transplantation of 105 CD34+ cells from cord blood. (B) HLA-KO, but not wild-type hES cells, form teratomas in humanized NSG-W41 mice. [Figure 10] We demonstrate the development of cell-based therapies and strategies for engineering universally transplantable cells that provide durable antibody-mediated immunity, showing that stem cell-based therapies can generate immunity against variable viruses. [Figure 11A-B] Successful deletion of MICA and MICB in HLA-KO cells is demonstrated. (A) Two separate gRNAs targeting MICA and MICB were transfected, and clones were sequenced for frameshift mutations. Clones B05, H10, and F01 harbor frameshift mutations in MICA and MICB in one allele. (B) Targeting of the MICA-MICB fusion allele with gRNA. On the other chromosome, an in-frame fusion between MICA and MICB was observed in clones B05, H10, and F01, resulting from deletion of the intervening sequence. This fusion allele was retargeted with gRNA, and five clones harboring frameshift mutations were isolated. DETAILED DESCRIPTION OF THE INVENTION

[0063] Detailed Description of the Invention The present disclosure is based, at least in part, on the discoveries that (1) differentiation of human pluripotent stem cells into plasma cells that secrete transplantable antibodies or enzymes, and (2) mutations (e.g., using CRISPR / Cas9) in genes encoding ligands for NKG2D (which avoid natural killer cell recognition and result in substantially non-immunogenic or minimally immunogenic human pluripotent stem cells for transplantation) and expression of genes that prevent complement deposition can eliminate major determinants of immunogenicity from human pluripotent stem cells. Together, these enable scalable, off-the-shelf therapies for autoimmune diseases, neurodegenerative diseases, cancer, and infectious diseases, as well as the general application of ES cell-based therapies using cells modified to avoid immune rejection.

[0064] As described herein, methods and targets have been developed that can be used to modify human pluripotent stem cells to evade recognition by several therapeutic arms of the immune system. The present disclosure provides methods for generating minimally immunogenic donor pluripotent stem cell lines that can be used as off-the-shelf therapies for regenerative medicine without host immunosuppression, as well as sources of cells generated by such methods.

[0065] One aspect of the present disclosure provides for a scalable delivery of cells that have been modified to avoid immune rejection. Commercially viable cell-based therapies may require a scalable delivery of cells to reduce the cost of treatment and standardize manufacturing, as well as to serve as an allogeneic source of cells used to create the cell therapy.

[0066] Described herein is the development of individual steps for scalable cell therapy for infectious diseases that can be assembled into an integrated approach to provide protection against infectious pathogens and can be used in other cell therapy applications.

[0067] Minimally immunogenic or substantially non-immunogenic human embryonic stem cells The generation of minimally immunogenic, "universal" donor hES cell lines has been a goal in many areas of regenerative medicine. Decades of transplantation research have revealed substantial immunological barriers to engraftment, and the best method for achieving truly "universal" lines has not been fully elucidated.

[0068] Described herein are substantially or minimally immunogenic hES cells for transplantation, particularly stem cell-based immunotherapy for infectious diseases. The generation of such lines for transplantation would enable scalable, off-the-shelf cell therapies, which is desirable for most stem cell-based therapies being developed by commercial companies. Such lines could also facilitate regenerative medicine for tissues destroyed by autoimmunity, such as pancreatic beta cells in type 1 diabetes and oligodendrocytes in multiple sclerosis.

[0069] As described herein, this is the removal of several major determinants of immunogenicity.

[0070] Applications for disease treatment The present disclosure provides for the generation of a scalable source of off-the-shelf therapies, with applications ranging from immunotherapy, pancreatic beta cell replacement, or oligodendrocyte restoration for multiple sclerosis.

[0071] Different types of antibodies mediate long-term immunity against previously encountered viruses as well as against viruses that have mutated since the initial exposure. Described herein is the identification of the signals that give rise to these different antibodies and define how they protect against pathogens.

[0072] Described herein are compositions and methods for providing durable immunity against globally relevant pathogens. Infectious diseases are responsible for nearly one-third of all deaths and are therefore the leading cause of mortality worldwide. Many of the most persistent infectious diseases have proven resistant to vaccination, despite decades of research into the microorganisms that cause these diseases. Therefore, new approaches are required if successful vaccines are to be developed. Efforts to develop a vaccine against HIV serve as a useful example.

[0073] A small proportion of the HIV-seropositive population makes highly potent antibodies capable of neutralizing over 90% of clinical HIV isolates. Similar rare but broadly neutralizing antibodies have been found against influenza and dengue viruses. However, many of these antibodies have a large number of somatic mutations and are structurally unusual. Therefore, it is unclear how to generate these types of responses in the general population.

[0074] Several creative possibilities have been proposed and experimentally explored. First, the structure-based design and serial vaccination of HIV glycoprotein immunogens has been proposed, allowing antibody responses to be directed toward broadly neutralizing properties. Several studies have demonstrated the feasibility of this approach using either immunoglobulin knock-in mice or animals with more diverse repertoires. However, these studies are still in their early stages in mouse models, and it is unclear at this time whether this strategy will translate into human vaccines. Indeed, it is unclear whether similar resources devoted to the structural design of HIV epitopes will be available for other problematic pathogens. In a second approach, adeno-associated virus (AAV) vectors are used to intramuscularly deliver broadly neutralizing antibody genes for heterologous expression. Both mouse and primate models have shown promising results leading to clinical trials. While largely safe, a major problem highlighted by previous studies is that the duration of heterologous gene expression can be very transient unless the vector is delivered to the site that provides immunity. This is due to the widespread pre-existing immunity in humans and the CD8 receptors that respond to AAV. +This is likely due to a T cell response. In a third approach, lentiviral-based gene therapy is used to modify hematopoietic stem cells (HSCs) to express broadly neutralizing antibodies. Upon autologous transplantation, genetically modified B cells and plasma cells derived from HSCs can be generated to secrete these neutralizing antibodies. This last approach has received little attention due to concerns about its practical feasibility. Even in autologous transplants, hematopoietic stem cell transplantation requires cytoreductive conditioning to achieve efficient engraftment. Furthermore, random integration of lentiviral vectors into the host genome has been shown to induce leukemia. Finally, this approach is not scalable and is cost-prohibitive. As described herein, these issues are addressed in the latter approach by generating pathogen-specific B cells with minimal immunogenic input that is scalable.

[0075] tissue regeneration There is a need for inexpensive, easily manipulated stem cells that can be used to regenerate human tissues. Described herein are stem cell lines (e.g., genetically engineered) that are minimally immunogenic and considered "universal" donors. These cells can be used as a source of off-the-shelf therapy for regenerative medicine without host immunosuppression.

[0076] Plasma cells and serum antibodies The generation of antibody-mediated immunity using pluripotent stem cells is described herein, and it is believed that there are no prior papers on this topic, or other ongoing research projects elsewhere that pursue antibody-mediated immunity using pluripotent stem cells.

[0077] Described herein is a novel method for using human pluripotent stem cells to generate humoral immunity (see, eg, Example 1).

[0078] The component steps for achieving humoral immunity are widely accepted. The efficacy of serum antibodies in protecting against pathogens has been known for over a century (see, e.g., Behring, 1965). The existence of long-lived plasma cells and their role in maintaining serum antibodies is widely accepted in the field (see, e.g., Slifka et al., 1998).

[0079] The generation of secondary hematopoietic progenitors (DHPs) from hES cells is well known. See, for example, Kennedy et al., 2012. Therefore, unless otherwise specified herein, the processes of the present disclosure may be carried out according to such processes. As described herein, secondary hematopoietic progenitors (DHPs) are first differentiated from ES cells using methods such as those described by Kennedy et al., Cell Reports, 2012; and Sturgeon et al., Nat Biotech, 2014.

[0080] Differentiation of DHPs into B cells can be achieved by activating PAX5 by any method known in the art. For example, PAX5 can be activated by gene transduction or activation with a small molecule. As shown in Example 1, PAX5 was activated by transducing target cells with PAX5 using a lentiviral vector. Also, accessory stromal cells (e.g., 3T3 fibroblasts) can be engineered to express a subset of these factors by retroviral transduction (e.g., BAFF and CD40L). Human embryonic stem cells (ES cells) can be differentiated into plasma cells as described herein. By way of example, the ES cells can be H1 human embryonic stem cells.

[0081] A progenitor cell, like a stem cell, tends to differentiate into a specific type of cell, but is more specific than a stem cell and is a biological cell that can be driven to differentiate into its "target" cell. For example, a progenitor cell can be a hematopoietic progenitor cell (e.g., a hemogenic endothelial cell) or a hematopoietic progenitor cell.

[0082] As described herein, secondary hematopoietic progenitor (DHP) cells can be differentiated into B cells using ectopic expression of transcription factors or cytokines and B-lineage-promoting activating proteins or genetic factors. These cytokines include IL-7, Flt3L, and SCF in the range of 1-100 ng / μl. Other cytokines described herein are IFNγ or IL-4. B-lineage-promoting activating proteins (also called B-lineage-promoting transcription factors or genetic factors) include PAX5, EBF1, FOXO1A, BCL11A, TCF3, IKZF1, IRF4, IRF8, and SPI1. Genetic factors may be inducibly expressed by transfection with lentiviral vectors, modified RNA, or plasmids.

[0083] Plasma cell application As described herein, plasma cells can be used as prophylaxis for infectious diseases. For example, influenza antibody genes knocked into endogenous loci of hES cells have been described (see, e.g., Example 1, Figure 3). These antibodies bind to conserved portions of the influenza virus and neutralize nearly all flu strains (see, e.g., Example 1). As another example, plasma cells can be used to treat ongoing chronic infections, such as HIV (see, e.g., Example 1). As another example, plasma cells can be used for enzyme replacement therapy for enzyme deficiencies, such as Hurler syndrome (see, e.g., Example 1), or diseases associated with factor IX deficiency. As another example, plasma cells can be used to treat cancer (see, e.g., Example 1). As another example, plasma cells can be used to treat autoimmune diseases (see, e.g., Example 1). As another example, plasma cells can be used to treat Alzheimer's disease (see, e.g., Example 1). As another example, antibody-expressing plasma cells can be used to treat a subject in need of antibody therapy (e.g., with an immunotherapeutic agent). For example, the immunotherapeutic agent can be an antibody. By way of example, the immunotherapeutic agent can be Rituxan, eclizimab, or aducanumab.

[0084] As described herein, the compositions and methods can be used to treat neurodegenerative diseases or disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Alexander disease, Alpers disease, Alpers-Huttenlocher syndrome, and the like. syndrome), alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Aerts syndrome, ataxic neuropathy spectrum, ataxia (e.g., oculomotor apraxia, autosomal dominant cerebellar ataxia, hearing loss, and narcolepsy), autosomal recessive spastic ataxia of Charles-Saguenay, Batten disease, beta-propeller protein-associated neurodegeneration, cerebro-ocular-facial-skeletal 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 with anhidrosis, dementia, familial encephalopathy with neuroserpin inclusions, familial British dementia, familial Danish dementia, fatty acid hydroxylase-associated neurodegeneration, Gerstmann-Straussler-Scheinker disease Disease), GM2-gangliosidosis (e.g., AB variant), HMSN type 7 (e.g., with retinitis pigmentosa), Huntington's disease, infantile neuroaxonal dystrophy, infantile-onset hereditary spastic paraparesis, Huntington's disease (HD), infantile-onset spinocerebellar ataxia, juvenile primary lateral sclerosis, Kennedy's disease, kuru, Leigh's disease, Marinesco-Sjögren's syndrome, mild cognitive impairment (MCI), mitochondrial membrane protein-associated neurodegeneration, motor neuron disease, monomelic muscular atrophyThe neurodegeneration may be due to: motor neuron disease (MND), multiple system atrophy, multiple system atrophy with orthostatic hypotension (Shy-Drager syndrome), multiple sclerosis, multiple system atrophy, neurodegeneration in Down syndrome (NDS), neurodegeneration of aging, neurodegeneration with cerebral iron accumulation, neuromyelitis optica, pantothenate kinase-associated neurodegeneration, opsoclonus / myoclonus syndrome, prion disease, progressive multifocal leukoencephalopathy, Parkinson's disease (PD), PD-related disorders, multicystic lipomembranous dysplasia with sclerosing leukoencephalopathy, prion disease, progressive external ophthalmoplegia, neuronal disorder of riboflavin transporter deficiency, Sandhoff disease, spinal muscular atrophy (SMA), spinocerebellar ataxia (SCA), striatonigral degeneration, transmissible spongiform encephalopathy (prion disease), or Wallerian degeneration.

[0085] As described herein, the compositions and methods can be used to treat cancer, including, for example, acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancer, Kaposi's sarcoma (soft tissue sarcoma), AIDS-related lymphoma (lymphoma), primary CNS lymphoma (lymphoma), anal cancer, appendix cancer, gastrointestinal carcinoid tumor, astrocytoma, atypical teratoma / rhabdoid tumor, pediatric central nervous system (brain cancer), basal cell carcinoma of the skin, bile duct cancer, bladder cancer, osteosarcoma (including Ewing's sarcoma and osteosarcoma and malignant fibrous histiocytoma), brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor (gastrointestinal); Childhood Carcinoid Tumors; Cardiac (Heart) Tumors; Central Nervous System Cancers; Atypical Teratoma / Rhabdoid Tumors, Childhood (Brain Cancer); Embryonal Tumors, Childhood (Brain Cancer); Germ Cell Tumors, Childhood (Brain Cancer); Primary CNS Lymphoma; Cervical Cancer; Cholangiocarcinoma; Cholangiocarcinoma Chordoma; Chronic Lymphocytic Leukemia (CLL); Chronic Myeloid Leukemia (CML); Chronic Myeloproliferative Neoplasia; Colorectal Cancer; Craniopharyngioma (Brain Cancer); Cutaneous T-Cell; Ductal Carcinoma in Situ (DCIS); Embryonal Tumors, Central Nervous System, Childhood (Brain Cancer); Endometrial Cancer (Uterine Cancer); Ependymoma, Childhood (Brain Cancer); Esophageal Cancer ;Esthesioneuroblastoma;Ewing's sarcoma (bone cancer);Extracranial germ cell tumor;Extragonadal germ cell tumor;Eye cancer;Intraocular melanoma;Intraocular melanoma;Retinoblastoma;Fallen tube cancer;Fibrous histiocytoma, malignant, or osteosarcoma of bone;Gallbladder cancer;Gastric ((Stomach)) cancer;Gastrointestinal carcinoid tumor;Gastrointestinal stromal tumor (GIST) (soft tissue sarcoma);Germ cell tumor;Central nervous system germ cell tumor (brain cancer);Pediatric extracranial germ cell tumor;Extragonadal germ cell tumor;Ovarian germ cell tumor;Testicular cancer;Gestational trophoblastic disease;Hairy cell leukemia;Head and neck cancer;Cardiac tumor;Hepatocellular (liver) tumor pancreatic (hepatic) cancer; histiocytosis, Langerhans cell; Hodgkin's lymphoma; hypopharyngeal carcinoma (head and neck cancer); intraocular melanoma; pancreatic islet cell tumor; pancreatic neuroendocrine tumor; Kaposi's sarcoma (soft tissue sarcoma); kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer (head and neck cancer); leukemia; cancer of the lip and oral cavity (head and neck cancer); liver cancer; lung cancer (non-small cell and small cell); lymphoma; male breast cancer; malignant fibrous histiocytoma of bone or osteosarcoma; melanoma; intraocular (eye); Merkel cell carcinoma (skin cancer); mesothelioma, malignant; metastatic carcinoma; metastatic squamous cell carcinoma of unknown primary (head and neck cancer);NUT gene-related midline tract carcinoma; oral cancer (head and neck cancer); multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides (lymphoma); myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm; myeloid leukemia, chronic (CML); myeloid leukemia, acute (AML); myeloproliferative neoplasm; nasal cavity and paranasal sinus cancer (head and neck cancer); nasopharyngeal carcinoma (head and neck cancer); neuroblastoma; non-Hodgkin's lymphoma; non-small cell lung cancer; oral cavity cancer, lip or oral cavity cancer; Pharyngeal cancer (head and neck cancer); osteosarcoma 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 neoplasm / multiple myeloma; pleuropulmonary blastoma; breast cancer; primary central nervous system (CNS) lymphoma; primary peritoneal cancer; prostate cancer; rectal cancer; recurrent renal cell (kidney) cancer; retinoblastoma; Rhabdomyosarcoma, childhood (soft tissue sarcoma); salivary gland carcinoma (head and neck cancer); sarcoma; childhood rhabdomyosarcoma (soft tissue sarcoma); childhood vascular tumor (soft tissue sarcoma); Ewing's sarcoma (bone cancer); Kaposi's sarcoma (soft tissue sarcoma); osteosarcoma (bone cancer); uterine sarcoma; Sezary syndrome (lymphoma); skin cancer; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma of the skin; squamous neck carcinoma of unknown primary, metastatic (head and neck cancer); stomach (gastr ic)) cancer; T-cell lymphoma, skin; lymphoma; mycosis fungoides and Sézary syndrome; testicular cancer; throat cancer (head and neck cancer); nasopharyngeal carcinoma; oropharyngeal cancer; hypopharyngeal cancer; thymoma and thymic carcinoma; thyroid cancer; thyroid tumor; transitional cell carcinoma of the renal pelvis and ureter (renal (renal cell) carcinoma); ureter and renal pelvis; transitional cell carcinoma (renal (renal cell) carcinoma); urethral cancer; uterine cancer, endometrium; uterine sarcoma; vaginal cancer; vascular tumor (soft tissue sarcoma); vulvar cancer; or Wilms' tumor.

[0086] As described herein, the compositions and methods can be used to treat autoimmune diseases or disorders, such as achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathy (AMAN), Baro's disease, Behcet's disease, Benign mucous membrane pemphigoid; bullous pemphigoid; Castleman's disease (CD); celiac disease; Chagas' disease; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic relapsing multifocal osteomyelitis (CRMO); Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA); cicatricial pemphigoid; Cogan's syndrome; cold agglutinin disease; congenital heart block; Coxsackie myocarditis; CREST syndrome; Crohn's disease; dermatitis herpetiformis; dermatomyositis; Devic's disease (neuromyelitis optica); discoid lupus; Dressler's 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-Schönlein purpura (HSP); herpes gravidarum or pemphigoid of pregnancy (PG); hidradenitis suppurativa (HS) (acne contralateralis); hypogammaglobulinemia; IgA nephropathy; IgG4-related sclerosing disease; immune thrombocytopenic purpura 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; Lignin conjunctivitis; Linear IgA disease (LAD); Lupus; Chronic Lyme disease; Meniere's disease; Microscopic polyangiitis (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 rheumatoid arthritis (PR); PANDAS; paraneoplastic neurological syndromes (PCD); paroxysmal nocturnal hemoglobinuria (PNH); Parry-Romberg syndrome; pars planitis (peripheral uveitis); Parsonage-Turner syndrome; pemphigus; peripheral neuropathy; perivenous encephalomyelitis; pernicious anemia (PA); POEMS syndrome; polyarteritis nodosa; polyglandular syndrome types I, II, and III; polymyalgia rheumatica; polymyositis; post-myocardial infarction syndrome; post-pericardiotomy syndrome; primary biliary cirrhosis; primary sclerosing cholangitis; progestational dermatitis; psoriasis; psoriatic arthritis; pure red cell aplasia (PRCA); pyoderma gangrenosum; Raynaud's phenomenon; reactive arthritis; reflex sexual intercourse Neuropathic dystrophy; relapsing polychondritis; restless legs syndrome (RLS); retroperitoneal fibrosis; rheumatic fever; rheumatoid arthritis; sarcoidosis; Schmidt syndrome; scleritis; scleroderma; Sjögren's syndrome; sperm & testicular autoimmunity; stiff-person syndrome (SPS); subacute bacterial endocarditis (SBE); Susac syndrome; sympathetic ophthalmia (SO); Takayasu's arteritis; temporal arteritis / giant cell arteritis; thrombocytopenic purpura (TTP); Tolosa-Hunt syndrome (THS); transverse myelitis; type 1 diabetes; ulcerative colitis (UC); differentiated connective tissue disease (UCTD); uveitis; vasculitis; vitiligo; Vogt-Koyanagi-Harada disease; or Wegener's granulomatosis (or granulomatosis with polyangiitis (GPA)).

[0087] As described herein, antibody-expressing plasma cells can be generated, for example, from human ES cells nucleofected with a pathogen-specific cassette encoding an antibody gene.

[0088] As described herein, the plasma cells can express antibodies specific to a virus. For example, the virus can be influenza, HIV, malaria, or a flavivirus (e.g., dengue, Zika, West Nile, tick-borne encephalitis, yellow fever, cell-mediated encephalitis virus (CFAV), Palm Creek virus (PCV), or Parramatta River virus (PaRV)). For example, the plasma cells can express antibodies specific to FI6, VRC07, 10E8, N6, 3BNC117, EDE1, or C10.

[0089] Genetically modified stem cells using genome editing, a method for immune manipulation As described herein, genetically engineered pluripotent stem cell lines have been modified to allow the cells to avoid recognition by several arms of the immune system. Such genetic alterations are typically made in predefined groups of genes and targets, some of which have been described in the prior art. The significant novel features of the present invention, in addition to modifications of the prior art, will be readily apparent to those skilled in the art.

[0090] Cells containing the novel modifications of the present invention, alone or in combination with the foregoing, may be used to express CD8 + T cells, CD4 + The cells can avoid recognition by T cells, NK cells, complement, or phagocytes. Furthermore, the cells may contain an inducible suicide gene and a drug resistance cassette. This allows for selective removal of the graft in the event of adverse reactions and easy drug selection in culture to identify clonal cell lines. Taken together, the process allows for the generation of human pluripotent stem cells with significantly reduced immunogenicity for transplantation.

[0091] As described herein, it has been discovered that disrupting specific immune receptors and introducing specific transgenes into human stem cells (i.e., modified by gene deletion and / or transgene (cDNA) insertion) can result in universal donor pluripotent stem cells.

[0092] Provided herein are genetically engineered stem cells in which (i) the beta2 microglobulin and TAP1 encoding genes are genetically modified to eliminate HLA-I expression and prevent direct recognition by allogeneic CD8+ T cells, (ii) HLA-II expression is eliminated, thus avoiding direct recognition by CD4+ T cells, or (iii) the NKG2D ligand encoding gene is genetically modified to avoid natural killer (NK) cell recognition.

[0093] Also provided herein are stem cells, as well as the following immune cells: (i) CD8 + T cells (i.e., due to lack of MHC class I expression due to genetic alterations in the β2 microglobulin and TAP1-encoding genes), (ii) CD4 + A method for generating stem cell lines that avoid recognition by (iii) T cells (i.e., due to lack of MHC class II expression due to genetic modifications in the genes encoding CD74 and CIITA), and / or (iv) NK cells (i.e., due to genetic modifications in the genes encoding ligands for NKG2D (e.g., MICA, MICB, Raet1e, Raet1g, Raet1l, Ulbp1, Ulbp2, and Ulbp3)).

[0094] Also provided herein are methods for generating genetically engineered stem cells, comprising delivering constructs into the AAVS locus in ES cells to express, for the purposes indicated, the following genes (or immune evasion factors): (i) CD46 / Crry, CD55, and CD59, which confer evasion of complement fixation; (ii) HLA-E and HLA-G single-chain trimers; Kb single-chain trimers (as demonstrated in mice), which confer evasion of NK cell recognition; (iii) CD47, which confer evasion of phagocytic macrophages; (iv) icasp9 and HSV thymidine kinase, which confer inducible suicide genes (death by AP1903 and ganciclovir, respectively); and / or (v) puromycin and neomycin resistance cassettes, which confer drug resistance.

[0095] Also provided is a method of treating a subject with genetically engineered pluripotent stem cells, wherein the subject has an autoimmune disease such as type 1 diabetes or multiple sclerosis, or the patient has an infection (e.g., a tissue-destroying infection); the subject has damaged tissue, and the damaged tissue is regenerated by the genetically engineered stem cells (e.g., pancreatic cells, oligodendrocytes).

[0096] Genetic modifications to the β2-microglobulin and TAP1-encoding genes The present disclosure provides genetic modifications in the β2-microglobulin and TAP1-encoding genes that eliminate HLA-I expression and prevent direct recognition by allogeneic CD8+ T cells. As described herein, the genetic modifications can be inactivating mutations.

[0097] The present disclosure further provides mutations in the CD74 and CIITA-encoding genes, which eliminate HLA-II expression and prevent direct recognition by CD4+ T cells. Previous studies have described gene mutations to prevent HLA-I and HLA-II expression separately. However, as described herein (see, e.g., Example 2, Figure 6), two additional genes were identified for targeting. For example, the use of TAP1 mutations to prevent HLA-I expression is described. Because deletion of either gene by itself is not sufficient to completely eliminate HLA-I expression, deletion of TAP1 in addition to β2M was shown to be important. Previous studies have demonstrated residual CD8+ T cell reactivity to TAP1-deficient cells, and β2M-deficient grafts can re-express HLA-I expression in β2M-sufficient hosts through the acquisition of serum β2M. As another example, mutating CD74 to prevent HLA-II expression is described. Deletion of CD74 in addition to CIITA has been shown to be important because some cell types can express HLA-II independently of CIITA. Relying on key mutations to eliminate HLA-I and II, these additional genes can be removed to prevent leaky HLA expression.

[0098] As described herein, an inactivating mutation can be any mutation in a gene that results in reduced or eliminated expression of HLA-I or HLA-II (see, e.g., Figure 6). Inactivating mutations can include nucleotide insertions or deletions that alter the reading frame and prevent translation of a functional protein. For example, a two-base pair deletion in one allele of the TAP1 gene and a one-base pair insertion in the other allele of the TAP1 gene resulted in premature termination of translation and lack of HLA-I expression (see, e.g., Figure 6).

[0099] The present disclosure further demonstrates that these HLA-deficient cells generate teratomas in xenogeneic chimeric mice reconstituted with an allogeneic human immune system. As shown herein, the cells are demonstrated to lack expression of HLA-I and HLA-II. Furthermore, monocytes derived from these cells have been shown to be unable to stimulate the proliferation of allogeneic T cells. As further demonstrated herein, the modified cells have been shown to avoid rejection by mice reconstituted with a human immune system. It has also been demonstrated herein that the AAVS targeting construct correctly expresses all intended genes and confers resistance to natural killer cell recognition and complement deposition.

[0100] It is currently believed that none of the previous approaches combined disruption of HLA-I and HLA-II, and that the genes targeted in previous approaches were insufficient to mediate complete loss of HLA-I and HLA-II expression.

[0101] Prevention of phagocytosis and NK cell activation The present disclosure further provides mutations generated in genes encoding ligands for NKG2D to avoid natural killer (NK) cell recognition. NK cells have many different modes of recognition, and NKG2D is the only activating receptor known to be expressed on all NK cells. Elimination of the NKG2D ligand thus abolishes reactivity by all NK cells, in contrast to other alternative strategies described in the prior art.

[0102] This disclosure further provides the design and validation of constructs delivered to the AAVS locus in human ES cells. These constructs encode genes that confer evasion of NK cell recognition (HLA-E and HLA-G single-chain trimer) and phagocytosis (CD47 and HLA-G single-chain trimer). Expression of these genes substantially reduces NK cell activation (see, e.g., Example 2). These constructs also encode inducible suicide genes (e.g., icasp9 and HSV thymidine kinase) and drug resistance cassettes (e.g., puromycin and neomycin resistance). This allows for selective removal of the graft in the event of adverse reactions and easy drug selection during culture to identify clonal cell lines. Collectively, this process allows for the generation of human pluripotent stem cells with significantly reduced immunogenicity for transplantation.

[0103] Prevention of complement deposition The present disclosure further provides the design and validation of constructs delivered to the AAVS locus in human ES cells. These constructs encode genes that confer evasion of complement fixation (e.g., CD46 / Crry, CD55, and CD59). The use of CD46 / Crry, CD55, and CD59 expression to prevent classical and alternative complement deposition on cells is described herein (see, e.g., Example 2). Prevention of classical and alternative complement deposition is believed to be important for preventing antibody-dependent immune rejection.

[0104] molecular manipulation The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0105] As used herein, the terms "heterologous DNA sequence," "exogenous DNA segment," or "heterologous nucleic acid" each refer to a sequence derived from a source exogenous to a particular host cell, or, if derived from the same source, a sequence that is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell but that has been modified, for example, through the use of 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 exogenous or heterologous to the cell, or a DNA segment that is homologous to the cell but is in a location within the host cell nucleic acid where the element is not normally found. An exogenous DNA segment is expressed to produce an exogenous polypeptide. A "homologous" DNA sequence is a DNA sequence that is naturally associated with the host cell into which it is introduced.

[0106] An expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid created by human intervention, including by recombinant means or direct chemical synthesis, using, for example, a set of specific nucleic acid elements that allow for the transcription or translation of a particular nucleic acid in a host cell. An expression vector can be part of a plasmid, a virus, or a nucleic acid fragment. Typically, an expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0107] A "promoter" is generally understood to be a nucleic acid control sequence that directs transcription of a nucleic acid. An inducible promoter is generally understood to be a promoter that mediates transcription of an operably linked gene in response to a specific stimulus or activator (e.g., a doxycycline-inducible promoter or a tetracycline-inducible promoter). A promoter may include necessary nucleic acid sequences near the start site of transcription, such as a TATA element in the case of a polymerase II type promoter. A promoter may also include distal enhancer or repressor elements as needed, which may be located as much as several thousand base pairs from the start site of transcription.

[0108] As used herein, " transcribable nucleic acid molecule " refers to any nucleic acid molecule that can be transcribed into an RNA molecule.Methods are known for introducing constructs into cells so that transcribable nucleic acid molecules are transcribed into functional mRNA molecules, which are translated, and then expressed as protein products.Constructs can also be configured to express antisense RNA molecules to inhibit the translation of specific RNA molecules of interest. Conventional compositions and methods for preparing and using constructs and host cells for the practice of the present disclosure are well known to those of skill in the art (Sambrook and Russell (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, Latest Protocols, ISBN-10: 0471250929; Sambrook and Russell (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). Enzymology)167,747-754).

[0109] The "transcription start site" or "start site" is the position surrounding the first nucleotide that is part of the transcribed sequence; it is also defined as position +1. With respect to this site, all other sequences of the gene and its regulatory regions can be numbered. The downstream sequence (i.e., the additional protein-coding sequence in the 3' direction) can be designated positive, and the upstream sequence (most of the regulatory region in the 5' direction) is designated negative.

[0110] "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 is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked to" or "associated with" a DNA sequence encoding an RNA or polypeptide when the two sequences are positioned so 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). The coding sequence may be operably linked to the regulatory sequence in either sense or antisense orientation. The two nucleic acid molecules may be part of a single, contiguous nucleic acid molecule, or may be adjacent. For example, a promoter is operably linked to a gene of interest if it regulates or mediates the transcription of the gene of interest in a cell.

[0111] A "construct" is generally understood as any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, self-replicating nucleic acid molecule, phage, or linear or circular, single- or double-stranded DNA or single- or double-stranded RNA nucleic acid molecule, derived from any source capable of genomic integration or self-replication, comprising one or more operably linked nucleic acid molecules.

[0112] The constructs of the present disclosure may contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, the constructs may include additional regulatory nucleic acid molecules, for example, but not limited to, from the 3' untranslated region (3' UTR). The constructs may include the 5' untranslated region (5' UTR) of an mRNA nucleic acid molecule, which may play an important role in translation initiation and may also be a genetic component of the expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from sources that are native or heterologous to the other elements present on the promoter construct.

[0113] 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 fragments are referred to as "transgenic" cells, and organisms containing transgenic cells are referred to as "transgenic organisms."

[0114] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism, such as a bacterium, cyanobacterium, animal, or plant, 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 described 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, and partially mismatched primers. The term "untransformed" refers to a normal cell that has not undergone a transformation process.

[0115] "Wild-type" refers to a virus or organism as found in nature without any known mutations.

[0116] It is within the skill of the art to design, create, and test variant nucleotides and the polypeptides they encode that have the required percent identity and retain the desired activity of the expressed protein. For example, directed evolution and rapid isolation of variants may follow methods described in 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. Thus, one skilled in the art can create a large number of nucleotide and / or polypeptide variants that have, for example, at least 95-99% identity to the reference sequences described herein and screen them for the desired phenotype according to methods routine in the art.

[0117] The percent (%) of nucleotide and / or amino acid sequence identity is understood as the percentage of nucleotides or amino acid residues that are identical to the nucleotides or amino acid residues in a candidate sequence compared to a reference sequence when the two sequences are aligned. To determine percent identity, the sequences are aligned, and gaps are introduced, if necessary, to achieve the maximum percent sequence identity. Sequence alignment procedures for determining percent identity are well known to those skilled in the art. In many cases, publicly available computer software such as BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. Once the sequences are aligned, the percent sequence identity of a given sequence A with or relative to a given sequence B (which can alternatively be expressed as a given sequence A having or containing a particular percent sequence identity with or relative to a given sequence B) can be calculated as percent sequence identity = X / Y100, where X is the number of residues scored as identical matches by a sequence alignment program or algorithm aligning A and B, 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, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0118] Generally, conservative substitutions can be made at any position as long as the required activity is maintained. So-called conservative exchanges can be performed, in which the exchanged amino acid has similar properties to the original amino acid, such as Glu for Asp, Gln for Asn, Val for Ile, Leu for Ile, and Ser for Thr. 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). Deletions are substitutions of amino acids with direct bonds. Deletion positions include the termini of the polypeptide and the linkages between individual protein domains. An insertion is the introduction of an amino acid into a polypeptide chain, a direct bond that formally replaces one or more amino acids. The amino acid sequence can be adjusted, for example, with the aid of computer simulation programs known in the art, which can generate polypeptides with improved activity or altered regulation. Based on this artificially created polypeptide sequence, a corresponding nucleic acid molecule encoding such a regulatory polypeptide can be synthesized in vitro using the specific codon usage of the desired host cell.

[0119] Host cells can be transformed using a variety of standard techniques known in the art (e.g., Sambrook and Russell (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, Latest Protocols, ISBN-10: 0471250929; Sambrook and Russell (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). (See Enzymology, 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, and electroporation. Transfected cells can be selected and expanded to provide recombinant host cells containing the expression vector stably integrated into the host cell genome.

[0120] [Table 1] JPEG2025169335000003.jpg88148

[0121] Exemplary nucleic acids that can be introduced into a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences that are co-derived or present in the same species but are incorporated into the recipient cell by genetic engineering techniques. The term "exogenous" is also intended to refer to genes that are not normally present in the transformed cell, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes that are normally present and that are desired to be expressed in a manner different from their native expression pattern, e.g., to achieve overexpression. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in foreign DNA may include DNA already present in the cell, such as a DNA sequence containing an antisense message of the gene or a DNA sequence encoding a synthetic or modified version of the gene, DNA from another individual of the same species of organism, DNA from a different organism, or DNA produced exogenously.

[0122] Host strains developed according to the techniques described herein can be evaluated by several means known in the art (see, for example, 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).

[0123] Methods for downregulating or silencing genes are known in the art. For example, the activity of expressed proteins can be determined using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) (see, e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G describing hammerhead ribozymes and short hairpin RNAs; Helene, C, et al. (1992) Ann. NY Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12):807-15 describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8 describing aptamers; and Reynolds et al. (2004) Nature Biotechnology describing RNAi). 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 various sources (e.g., Ambion, TX; Sigma-Aldrich, MO; Invitrogen). Several siRNA molecule design programs using various algorithms are known in the art (e.g., Cenix algorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tool, Bioinformatics & Research Computing).Influential features in defining an optimal siRNA sequence include the G / C content of the siRNA termini, the Tm of specific internal domains of the siRNA, the length of the siRNA, the position of the target sequence within the CDS (coding region), and the nucleotide content of the 3' overhang.

[0124] formulation The agents and compositions described herein can be formulated by any conventional method using, for example, one or more pharmaceutically acceptable carriers or excipients described in Remington's Pharmaceutical Sciences (A.R. Gennaro, ed.), 21st Edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such formulations contain a therapeutically effective amount of a biologically active agent described herein, which may be in purified form, together with a suitable amount of carrier to provide the form for proper administration to a subject.

[0125] The term "formulation" refers to preparing a pharmaceutical agent in a form suitable for administration to a subject, such as a human. As such, a "formulation" may include pharmaceutically acceptable excipients, including diluents or carriers.

[0126] As used herein, the term "pharmaceutically acceptable" can describe a substance or ingredient that does not result in an unacceptable loss of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those listed in the United States Pharmacopeia (USP 29) and National Federation of Drugs (NF 24), United States Pharmacopeia Office, Rockville, MD, 2005 ("USP / NF"), or those with monographs in more recent editions, and in the FDA's continuously updated online database of inactive ingredients. Other useful ingredients not listed in the USP / NF can also be used.

[0127] As used herein, the term "pharmaceutically acceptable excipient" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, 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 (A.R. Gennaro, ed.), 21st ed., ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0128] A "stable" formulation or composition may refer to a composition that has sufficient stability to permit storage at a convenient temperature, such as from about 0°C to about 60°C, for a commercially reasonable period of time, such as at least about 1 day, at least about 1 week, at least about 1 month, at least 3 months, at least about 6 months, at least about 1 year, or at least about 2 years.

[0129] The formulation should be compatible with the mode of administration. The agents used in the present disclosure can be formulated by known methods for administration to a subject using several routes, including, but not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ocular, buccal, and rectal. Individual agents can also be combined with one or more additional agents or administered with other biologically active or biologically inactive agents. Such biologically active or inactive agents can be in fluid or mechanical communication with the agent(s) or can be bound to the agent(s) by ionic, covalent, van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0130] Controlled-release (or sustained-release) formulations can be formulated to extend the activity of a drug(s) and reduce dosing frequency. Controlled-release formulations are used to achieve other characteristics, such as the time of onset of action or blood levels of the drug, which can then affect the occurrence of side effects. Controlled-release formulations can be designed to initially release an amount of drug(s) that produces a desired therapeutic effect, and gradually and continuously release other amounts of drug to maintain that level of therapeutic effect over an extended period of time. To maintain a near-constant level of drug in the body, the drug can be released from the dosage form at a rate that replaces the amount of drug metabolized or excreted from the body. Controlled-release of a drug can be stimulated by various inducers, such as a change in pH, a change in temperature, enzymes, water, or other physiological conditions or molecules.

[0131] The agents or compositions described herein can also be used in combination with other treatment modalities, as further described below, so that, in addition to the treatments described herein, the subject can also be provided with other therapies known to be effective in treating the disease, disorder, or condition.

[0132] Treatment method Also provided is a process for treating a disease (e.g., an autoimmune disease, tissue destroyed by an autoimmune disease, a pathogen, cancer, an enzyme deficiency, or a neurodegenerative disease) in a subject in need thereof with a cell-based therapy (e.g., differentiated progeny of the genetically engineered stem cells) and administration of a therapeutically effective amount of a cell-based therapeutic agent to treat the disease (e.g., a pathogen, an infectious disease) with pluripotent stem cells while evading recognition by natural killer cells.

[0133] Furthermore, 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 use in treating patients, and such differentiated cells can be administered to patients in need of such cells with reduced or no immunosuppressive drugs.

[0134] The methods described herein are generally administered to a subject in need thereof. A subject in need of the treatment methods described herein may be at risk, diagnosed as at risk, suspected of being at risk, or at risk for developing tissue damage, a pathogen, or an infection. The need for treatment is typically assessed by medical history and a physical examination consistent with the disease or condition in question. Diagnosis of various conditions treatable by the methods described herein is within the skill of one of ordinary skill in the art. The subject may be an animal subject, including mammals such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, as well as humans. For example, the subject may be a human subject.

[0135] Generally, a safe and effective amount of genetically engineered stem cells (e.g., genetically engineered pluripotent stem cells) is an amount that produces a desired therapeutic effect in a subject, for example, while minimizing undesirable side effects. In various embodiments, an effective amount of plasma cells derived from genetically engineered stem cells described herein can substantially inhibit tissue damage, pathogens, or infections, slow the progression of tissue damage, pathogens, or infections, or limit the onset of tissue damage, pathogens, or infections.

[0136] 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 administration.

[0137] When used in the treatments described herein, therapeutically effective amounts of plasma cells derived from genetically engineered stem cells (e.g., genetically engineered pluripotent stem cells) can be used in pure form, or, if such a form exists, in a pharmaceutically acceptable salt form, with or without a pharmaceutically acceptable excipient. For example, compounds of the present disclosure can be administered in an amount sufficient to substantially inhibit, slow the progression of, or limit the onset of tissue damage, pathogens, or infections, at a reasonable benefit / risk ratio applicable to any medical treatment. Genetically engineered stem cells can be minimally immunogenic to avoid recognition by the immune system of some therapeutic groups.

[0138] 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. It will be understood by those skilled in the art that the unit content of drug contained in an individual dose of each dosage form need not itself constitute a therapeutically effective amount, since the required therapeutically effective amount may be achieved by administering several individual doses.

[0139] The toxicity and therapeutic efficacy of the compositions described herein have been measured in cell cultures or experimental animals using LD 50 (lethal dose for 50% of the population) and ED 50 The dose ratio between toxic and therapeutic effects can be determined by standard pharmaceutical procedures to determine the LD (therapeutically effective dose in 50% of the population). 50 / ED 50 where a larger therapeutic index is generally understood in the art to be optimal.

[0140] The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the particular composition used; 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 used; the duration of treatment; and drugs used in combination with or concomitantly with the specific compound used (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 Edition, Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / appleton & Lange, ISBN 0071375503). For example, it is well within the skill of those skilled in the art to start administering the composition at a lower dose than required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.If desired, the effective daily dose can be divided into multiple doses for administration purposes.As a result, a single dose composition can contain such an amount or a submultiple thereof to constitute a daily dose.However, it is understood that the total daily dose of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0141] Again, each of the conditions, diseases, disorders, and conditions described herein, as well as others, can benefit from the compositions and methods described herein. Generally, treating a condition, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or susceptible to the condition, disease, disorder, or condition, but that has not yet experienced or exhibited clinical or subclinical symptoms thereof. Treatment may also include inhibiting the condition, disease, disorder, or condition, e.g., preventing or reducing the onset of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treatment may include alleviating the disease, e.g., causing regression of the condition, disease, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to a treated subject may be either statistically significant or at least perceptible to the subject or the physician.

[0142] Administration of the progeny derived from the genetically engineered stem cells can occur as a single event or over a period of treatment. For example, the genetically engineered stem cell progeny can be administered daily, weekly, biweekly, or monthly. For treatment of acute conditions, the period of treatment is usually at least several days. For certain conditions, treatment may extend for several days to several weeks. For example, treatment may extend for one, two, or three weeks. For more chronic conditions, treatment may extend for several weeks to several months, or even for a year or more.

[0143] Treatment in accordance with the methods described herein can precede, coincide with, or follow conventional treatments for tissue damage, pathogens, or infections.

[0144] The progeny of the genetically engineered stem cells can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory agent, or another agent. For example, the progeny of the genetically engineered stem cells can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory agent. Simultaneous administration can occur through the administration of separate compositions, each containing one or more of the progeny of the genetically engineered stem cells, the antibiotic, the anti-inflammatory agent, or another agent. Simultaneous administration can occur through the administration of a single composition containing two or more of the progeny from the genetically engineered stem cells, the antibiotic, the anti-inflammatory agent, or another agent. The differentiated progeny of the genetically engineered stem cells can be administered sequentially with the antibiotic, the anti-inflammatory agent, or another agent. For example, cells derived from the genetically engineered stem cells can be administered before or after the administration of the antibiotic, the anti-inflammatory agent, or another agent.

[0145] Administration The agents and compositions described herein can be administered in accordance with the methods described herein by a variety of means known in the art. Agents and compositions can be used therapeutically as either exogenous or endogenous substances. Exogenous agents are those that are produced or manufactured outside the body and administered to the body. Endogenous agents are those that are produced or manufactured within the body by some type of device (biological or other) for delivery into or to other organs within the body.

[0146] As noted above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intraocular, ossicular, buccal, or rectal.

[0147] The agents and compositions described herein can be administered by a variety of methods known in the art. Administration may include, for example, oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete a factor of interest, drug-releasing biomaterials, polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, combinations of any of the above, or other suitable delivery vehicles to provide a desired release profile at various rates. Other methods of controlled-release delivery of agents or compositions are known to those skilled in the art and are within the scope of this disclosure.

[0148] Delivery systems may include, for example, infusion pumps, which can be used to administer drugs or compositions in a manner similar to those used to deliver insulin or chemotherapy agents to specific organs or tumors. Typically, using such systems, drugs or compositions are administered in combination with biodegradable, biocompatible polymeric implants that release the drug at a selected site over a controlled period of time. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. Additionally, controlled-release systems can be placed in the vicinity of the therapeutic target, thereby requiring only a fraction of the systemic dose.

[0149] Drugs can be encapsulated and administered using a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric 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 molecular or biomolecule drug delivery can provide intracellular delivery, modulate biomolecule / drug release rates, increase the proportion of biomolecules that reach their site of action, improve drug transport to their site of action, enable colocalized deposition with other drugs or excipients, improve drug stability in vivo, extend drug residence time at their site of action by reducing clearance, reduce nonspecific drug delivery to non-target tissues, reduce drug-induced inflammation, reduce toxicity due to high initial drug doses, modify drug immunogenicity, reduce dosing frequency, improve product taste, or increase product shelf life.

[0150] kit Also provided are kits. Such kits may include the agents or compositions described herein, and in certain embodiments, instructions for administration. Such kits can facilitate the practice of the methods described herein. When supplied as a kit, the composition or different components for making the composition are packaged in separate containers and can be mixed immediately before use. Components include, but are not limited to, progenitor cells, progeny of universal pluripotent stem cells, and reagents used to generate therapeutic cells in vitro, such as IL-4, IFNγ, BMP4, bFGF, VEGF, IL-6, IGF1, IL-11, SCF, or EPO. Such separate packaging of components can, if desired, be provided in a pack or dispenser device that can contain one or more unit dosage forms containing the composition. The pack can, for example, comprise metal or plastic foil, such as a blister pack. Such separate packaging of components can also, in certain cases, allow for long-term storage without loss of activity of the components.

[0151] The kit may also contain reagents in separate containers, such as sterile water or saline, to be added to the separately packaged lyophilized active ingredient. For example, a sealed glass ampoule may contain the lyophilized ingredients, each of which may be sterile water, sterile saline, or sterilely packaged under a neutral, non-reactive gas such as nitrogen, in a separate ampoule. The ampoules may be made of any suitable material, such as glass, organic polymers such as polycarbonate, polystyrene, ceramic, metal, or any other material typically used to hold reagents. Other examples of suitable containers include bottles made from materials similar to ampoules, and packaging materials that may be made from foil-lined interiors such as aluminum or alloys. Other containers include test tubes, vials, flasks, bottles, and syringes. The container may have a sterile access port, such as a bottle with a stopper that can be pierced by a hypodermic needle. Other containers may have two compartments separated by an easily removable membrane that allows the components to be mixed upon removal. The removable membrane may be glass, plastic, rubber, or the like.

[0152] In certain embodiments, the kit can be supplied with instructions. The instructions can be printed on paper or other substrate and / or can be supplied as an electronically readable medium, such as a floppy disk, mini CD-ROM, CD-ROM, DVD-ROM, zip disk, videotape, and audiotape. The detailed instructions need not physically accompany the kit; instead, the user can be directed to an internet website designated by the kit manufacturer or distributor.

[0153] The compositions and methods described herein that utilize molecular biology protocols may follow a variety of standard techniques known in the art (e.g., Sambrook and Russell (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 Russell (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) 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).

[0154] The definitions and methods set forth herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise specified, terms should be understood according to conventional usage by those of ordinary skill in the relevant art.

[0155] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, and reaction conditions, etc., used to describe and claim particular embodiments of the present disclosure should be understood as being modified in some cases by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the average standard deviation for the device or method being employed to determine that value. In some embodiments, the numerical parameters set forth in the written description and accompanying claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth some embodiments of the present disclosure in broad ranges are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practical. The numerical values ​​presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values ​​is intended herein merely to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein.

[0156] In some embodiments, the terms "a," "an," and "the," and similar references, as used in the context of describing particular embodiments (particularly in the particular context of the claims below), can be construed to encompass both the singular and the plural, unless specifically stated otherwise. In some embodiments, the term "or," as used herein, including the claims, is used to refer to "and / or," unless expressly indicated to refer to alternatives only, or the alternatives are not mutually exclusive.

[0157] The terms "comprise," "having," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to possessing only those one or more steps, but may also include other, unrecited steps. Similarly, any composition or device that "comprises," "has," or "includes" one or more features is not limited to possessing only those one or more features, but may also include other, unrecited features.

[0158] Unless otherwise indicated herein or otherwise clearly contradicted by context, all methods described herein can be performed in any suitable order. Any and all examples provided herein with respect to specific embodiments, or the use of exemplary language (e.g., "etc."), are intended merely to facilitate a better understanding of the disclosure and do not limit the scope of the disclosure as particularly claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0159] Groupings of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limitations. Each group member refers to other members of the group or other elements found herein, which may be claimed individually or in any combination thereof. One or more members of a group may be included in or deleted from a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification herein is considered modified to include the group thus fulfilling the description of all Markush groups used in the appended claims.

[0160] All publications, patents, patent applications, and other references cited in this application are herein incorporated 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 by reference in its entirety for all purposes. The citation of a reference herein should not be construed as an admission that such is prior art to the present disclosure.

[0161] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalents are possible without departing from the scope of the present disclosure as defined in the appended claims. Furthermore, it should be understood that all examples in the present disclosure are provided as non-limiting examples.

[0162] Example 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 approaches the inventors have found to work well in the practice of the present disclosure and can thus be considered to constitute examples of modes for carrying out the same. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0163] Example 1: Differentiation of human ES cells into transplantable plasma cells that produce broadly neutralizing antibodies The following example describes the differentiation of human ES cells into transplantable plasma cells that produce broadly neutralizing antibodies.

[0164] Bone marrow long-lived plasma cells sustain serum antibodies after infection or vaccination Most clinically used vaccines depend on the generation and maintenance of neutralizing antibodies. Consequently, key correlates of protection after vaccination are the quantity, affinity, epitope specificity, and duration of plasma cell-derived antibodies elicited by immunization. Plasma cells are terminally differentiated B lymphocytes that secrete thousands of antibody molecules per second and devote approximately 70% of their transcriptome to antibody synthesis. During the first few days of a T cell-dependent antibody response, plasma cells survive for only a few days and then undergo apoptosis. Other plasma cells expressing progressively higher affinity antibodies are subsequently generated from germinal center reactions. These affinity-matured plasma cells can persist for up to decades and reside primarily in the bone marrow. These long-lived plasma cells constitutively secrete antibodies regardless of the presence of antigen and without cell division, and are the primary determinant of humoral immunity. Due to their prolific antibody secretion rate, it has been shown that as little as 1 μl of immune serum, corresponding to antibodies derived from only approximately three antigen-specific long-lived plasma cells, is sufficient to protect naive mice from a particularly lethal West Nile virus infection. The data presented herein demonstrate the protective efficacy of these cells when they express neutralizing antibodies.

[0165] Methods for generating secondary hematopoietic precursors from hES cells To develop a scalable source of antigen-specific, transplantable, long-lived plasma cells, we utilized H1 human embryonic stem cells. These are the most well-characterized human ES cells, have minimal lineage bias, and readily generate hematopoietic cells. Hematopoiesis occurs in at least two distinct waves: primary and secondary. Primary hematopoiesis generates a restricted set of erythroid and myeloid lineages. In contrast, secondary hematopoiesis ultimately gives rise to adult blood cell types, including lymphocytes. Therefore, a primary therapeutic goal of hES cell function has been to generate secondary hematopoietic precursors. A method for reproducibly generating secondary hemogenic endothelium, a secondary hematopoietic cell precursor, using H1 hES cells was optimized as described in Sturgeon et al., Nat Biotech, 2014. The method employs sequential manipulation of signals during germ layer induction, including the critical step of canonical Wnt signaling activation on day 2 of the process (see, e.g., Figure 1A). This ultimately enables the generation of T cells, even from pluripotent lines that inherently lack this potential (see, e.g., Figure 1B). Importantly, these differentiations are performed under defined, serum-free conditions, thereby maximizing reproducibility and allowing for easy troubleshooting.

[0166] Development of a method for obtaining B cells from secondary hematopoietic progenitor cells We investigated methods to generate secondary hematopoietic precursors and differentiate these precursors into B cells. Few studies to date have reported robust B cell differentiation from human ES cells; these approaches either failed to generate mature B cells or were only feasible with one specific iPS cell line. None of these approaches used defined conditions, and we have not been successful in adapting them to any of the hES cell lines currently tested. Therefore, we generated a combination of hemogenic endothelial cells (see, e.g., Figure 1) followed by expression of the B-lineage-promoting transcription factor PAX5. Secondary hemogenic endothelial cells were transduced with a lentiviral vector encoding constitutively expressed rTTA-T2A-GFP, a doxycycline-inducible transcriptional activator linked to a reporter by a ribosomal skipping 2A sequence, and PAX5 driven by a doxycycline-inducible promoter (see, e.g., Figure 2A). Transduced cells thus constitutively express rTTA and GFP, but only express PAX5 upon doxycycline treatment. After 20 days of coculture with Flt3L, SCF, IL7, and MS5 stromal cells, B cells were observed only upon doxycycline treatment (see, e.g., Figure 2B ), thus demonstrating that defined transcription factor expression can promote B lymphopoiesis from otherwise recalcitrant progenitors.

[0167] CRISPR / Cas9-mediated transfer of influenza-specific antibody genes into endogenous loci in hES cells Previous attempts to generate B cells from hES cells resulted in immature lymphocytes lacking surface antigen receptor expression. To address this issue and provide pathogen specificity to developing B cells, we engineered targeting cassettes containing the VDJ and VJ sequences from FI6, a broadly neutralizing influenza-specific antibody, as well as zeocin and hygromycin resistance cassettes, respectively (see, e.g., Figure 3A). These targeting cassettes were nucleofected into H1 hES cells along with Cas9 and guide RNAs (gRNAs) for targeting the endogenous immunoglobulin heavy and kappa light chain loci (ZFNs or TALENs could also be used). Cells were selected for zeocin and blasticidin resistance, and PCR analysis was performed to confirm proper targeting (see, e.g., Figure 3B). These cells were then transfected with a construct encoding Cre recombinase, and subclones lacking the drug resistance cassette were isolated (see, e.g., Figure 3C, showing PCR confirmation of IgH and Igκ targeting at the FI6 gene and removal of the drug resistance cassette).

[0168] Method for generating long-lived plasma cells from B cells A key factor promoting the formation of long-lived, rather than short-lived, plasma cells from B cells in vivo has recently been identified. Long-lived plasma cells have been shown to import significantly more glucose than their short-lived counterparts. This enhanced glucose uptake promotes the elevation of mitochondrial pyruvate for antibody secretion and respiration, and is required for long-term plasma cell persistence. Enhanced glucose uptake is currently believed to be the only known characteristic that allows for the promising separation of long-lived and short-lived plasma cells. Through in vitro screening of exogenous factors, it was discovered that IFNγ and IL-4 promote glucose uptake (see, for example, Figure 4).

[0169] Xenograft model for assessing plasma cell longevity In addition to the studies mentioned above, several protocols have reported methods that allow for the long-term survival of human plasma cells in vitro. However, the lack of xenotransplantation models for human plasma cells has hindered the analysis of their survival in vivo. To address this issue, we transfected adherent fibroblasts / mesenchymal stem cells from human bone marrow into immunodeficient NOD-SCID IL2rγ mice. - / - A system for subcutaneous implantation into non-steroidal anti-inflammatory (NSG) mice was developed. Bone-like ossicles formed approximately 8 weeks after implantation, providing a fully human microenvironment. These ossicles robustly supported normal human hematopoiesis and primary myeloid malignancies, some of which had not previously grown in xenografts. To test whether these ossicles also supported the survival of normal plasma cells, 1–5 × 10 6 Primary human bone marrow CD138 + Plasma cells were injected directly into the ossicles. Serum human IgG was observed 2 and 4 weeks after transplantation, with no decline in antibody concentrations between these time points (see, e.g., Figure 5A). At 4 weeks, ossicles were harvested for flow cytometry analysis of plasma cells (see, e.g., Figure 5B) and ELISPOT analysis (data not shown). Antibody-secreting cells were observed in all injected ossicles. These data demonstrate that human ossicles support the survival and function of normal, long-lived plasma cells. This is believed to be the first such system of its kind.

[0170] Generation of pathogen-specific B cells from hES cells Defined transcription factor expression is used to promote B cell development from FI6 knock-in hES cells, as shown in Figures 2-3. The use of genome-integrating lentivirus may offer a challenging translational strategy. Non-integrating approaches, such as modified RNA, can be used to transiently introduce factors only when they are required for differentiation and lineage commitment. In support of this, PAX5 expression mediates a positive feedback loop with EBF1 to maintain B lineage commitment. Therefore, transient expression of exogenous PAX5 can induce stable maintenance of endogenous PAX5 expression. To define the window during which exogenous PAX5 expression promotes B cell commitment, doxycycline is administered only on days 1-7, 8-14, or 15-21 of coculture with MS5 cells. Similar experiments are performed using lentivirus encoding EBF1. CD19 + B cells are identified by flow cytometry and expression of influenza-specific IgM is confirmed using hemagglutinin tetramer reagents or using methods and reagents known in the art.

[0171] Pax5 expression promotes B lineage commitment in the current system, but preferential development of B-1a cells may occur. These CD5-expressing B-1a cells are derived from early fetal precursors, not adult stem cells. B-1a cells have not been shown to have the ability to generate long-lived plasma cells. If differentiation of primarily B-1a cells is observed in PAX5 cultures, Notch signaling can be used to further differentiate hemogenic endothelial cells toward definitive hematopoiesis. As described above, hemogenic endothelial cells give rise to T cells when cocultured with OP9 cells expressing the Notch ligand Delta-like 4 (DL4). Notch ligands promote T cell development in two steps. First, Notch signaling is essential for promoting definitive hematopoiesis. Second, Notch signaling promotes T cell commitment from hematopoietic progenitor cells. To define the window of Notch signaling required for definitive hematopoiesis, hemogenic endothelium was transduced with a doxycycline-inducible PAX5 lentiviral vector and then co-cultured on OP9-DL4 cells for 1-10 days. This transient period of Notch signaling has been shown to promote HSC-like cells, from which conventional B-2 and B-1b cells are derived. Transduced cells were purified by FACS and transferred to MS5 cells for B cell differentiation in the presence of doxycycline for 20 days. B cells were examined for CD5 expression to quantify B-1a frequency.

[0172] Generation of long-lived plasma cells from mature human B cells In addition to generating mature B cells as described above, primary tonsillar naive B cells are used to optimize differentiation into long-lived plasma cells using the methods of the present invention. + CD27 - B cells are selected on NIH 3T3 cells expressing human CD40L and BAFF. Either IL-4 or IFNγ is added to the culture at a concentration of 1-100 ng / ml. On day 6, CD38 高 CD27 高Plasmablasts / plasma cells are injected intravenously or directly into the ossicles in NSG mice as in Figure 5. Serum human IgG is sampled biweekly. When serum antibodies are maintained for 8-16 weeks, animals are sacrificed and ossicles are collected for flow cytometry and ELISPOT analysis as in Figure 5.

[0173] Despite enhanced glucose uptake, in vitro-derived plasma cells may not survive in vivo upon transplantation. In parallel with the in vitro culture approach outlined above, we use a culture method reported by others. A transwell culture method is available that can maintain plasma cells in vitro for several months. In this method, fibroblast L cells are engineered to express CD40L, and the cells are cultured in the presence of IL-21. Subsequently, plasma cells are separated from stromal cells via a transwell filter. With regular medium changes, these cells achieve quiescence and can be maintained for several months. Using this differentiation system, plasma cells are transplanted into ossicles for long-term in vivo maintenance.

[0174] Treating infectious diseases (e.g., influenza) To validate this method in an animal model, hES cells are differentiated into long-lived plasma cells and transplanted into NOD-SCID-IL2rg (NSG) mice, which contain human ossicles. These ossicles are formed after transplantation of mesenchymal cells from human bone marrow into the mice (as described above). After a week or more (sufficient time to allow flu-specific antibody concentrations to rise), the mice are infected with a dose of flu that is normally lethal to NSG mice. Mouse survival and influenza virus load are then measured.

[0175] For therapeutic use in humans, hES cells modified into long-lived plasma cells that can avoid immune rejection are administered prophylactically as a vaccine.

[0176] Treating chronic infection To form ossicles, young NSG mice are simultaneously reconstituted with umbilical cord blood CD34+ cells (intravenously) and mesenchymal cells (as described above). After T cell reconstitution, the mice are then infected with HIV to attack human CD4+ T cells, followed by transplantation of broadly neutralizing HIV-specific plasma cells. HIV titers and antibodies are measured to determine the extent to which ongoing infection is suppressed. Mouse models are available, as described in Halper-Stromberg et al. 2014 Cell 158(5)989-999. Once universal cells are developed, they will be tested in nonhuman primates and SIV.

[0177] For therapeutic use, the cells of the invention are administered to a patient with a chronic infection, whether the virus is active or dormant.

[0178] enzyme replacement therapy A mouse model of Hurler syndrome lacking α-L-iduronidase (Clark et al. 1997 Science 6(4)503-11) is used. Systemic enzyme replacement is the treatment for Hurler syndrome. The mouse model is crossed to NSG and transplanted with formed ossicles (as described above) and plasma cells engineered to secrete α-L-iduronidase instead of antibodies (through gene replacement or IRES knock-in downstream of the Aβ gene). Enzyme levels and brain neuropathology (e.g., lysosomal distension) are measured.

[0179] Cancer treatment NSG / ossicular mice (described above) are implanted with primary non-Hodgkin's lymphoma and then administered plasma cells expressing Rituxan (Chao et al. 2010 Cell 142(5)699-713). Tumor burden is quantified histologically and by flow cytometry. If a suicide gene is incorporated, the plasma cells can be removed whenever there is no residual disease.

[0180] Treatment of autoimmune diseases NSG / ossicular mice are transplanted with plasma cells expressing Soliris / Eclizimab. These mice are treated with anti-Gq1b antibodies and motor function and paralysis are measured. This mouse model of autoimmune disease is described in Halstead et al. 2008 131(Pt5)1197-208).

[0181] Treatment with therapeutic antibodies The cells and methods of the present disclosure can be used to treat subjects in need of a therapeutic antibody. For example, a therapeutic antibody can be overexpressed on modified ES cells as described herein. For example, treatment of Alzheimer's disease can be achieved by engineering the modified ES cells of the present invention to express a therapeutic antibody, such as aducanumab, to avoid the accumulation of Aβ plaques.

[0182] Materials and Methods The studies described herein use the male-derived line H1 (WA01, NIH accession number 0043) cells for modification and differentiation. However, all mouse experiments utilize both males and females, which are included as experimental variables. Additionally, donor xenografts are derived from both males and females. Y-chromosome PCR was performed to determine the gender of the anonymous donor, and again, these data are included as variables in the analysis.

[0183] Description of procedure: The majority of the work is performed in vitro for hES cell differentiation and genetic modification. Moderate numbers of immunodeficient NOD-SCID / IL2rγC-deficient (NSG) mice are used as recipients of ossicles and plasma cells, and teratomas. A smaller number of wild-type C57BI6 mice are used as recipients of teratomas. Recipients are 8 weeks old and of both sexes.

[0184] These studies in mice are a precursor to the clinical development of cell-based vaccines. Mouse studies have proven highly relevant to such applications in the past, and much is known about the cellular, molecular, and genetic aspects of mouse immunity. Given the potential advantages, mouse studies can be conducted efficiently and at relatively low cost.

[0185] Procedures for reducing pain or discomfort: Every effort will be made to ensure minimal pain and discomfort levels in the mice. Mice will be anesthetized with inhaled isofluorane prior to retro-orbital injection. Animals bearing teratomas 20 mm in diameter will be sacrificed. Three mice bearing teratomas will be sacrificed, and sentinel cages will be used routinely in the animal facility.

[0186] Animals are euthanized by CO inhalation in accordance with the recommendations of the American Veterinary Medical Association's Committee on Euthanasia.

[0187] Example 2: Genetic modification of human ES cells to reduce immunogenicity The following examples describe the genetic modification of human ES cells to reduce immunogenicity.

[0188] Universal donor hES cells are a goal of many fields but present significant immunological barriers. Human pluripotent stem cells can grow indefinitely, thus addressing one aspect of scalability for plasma cell therapy. However, unless the stem cell line is autologous, immunosuppression is necessarily required to prevent graft rejection. This almost certainly limits such therapy to immediately life-threatening complications. Autologous iPS cell therapy is potentially immune-free but cannot be scaled to the general population. To address these issues, we propose herein the creation of "universal" donor cell lines. These lines are genetically stripped of their immunogenicity, thereby allowing cells and tissues derived from a single line to be transplanted into any recipient. While removal of HLA expression has been shown to be a requirement for such universal donor lines, mouse allotransplantation studies have demonstrated that this alone is insufficient to prevent rejection. Subsequent studies have implicated antibodies, complement, NK cells, and phagocytes as other mediators of graft rejection. Starting with HLA-deficient lines, we confirmed the reactivity of hES cells to each of these other immune mediators of rejection.

[0189] Materials and methods were as described above unless otherwise noted.

[0190] Generation of HLA-deficient hESC lines We optimized a robust workflow to generate targeted mutations in human ES cells. A Cas9 expression construct and up to three different gRNA-encoding vectors were co-transfected into H1 human ES cells. Individual colonies were manually picked and subjected to Miseq analysis of the target gene to identify clones with frameshift mutations introduced by non-homologous end-joining errors. Candidate clones were then seeded at exactly 1 cell / well by fluorescence-activated cell sorting (FACS), and Miseq analysis was again performed to confirm the absence of mutations and mosaicism (see, for example, Figure 6A). Clones were then expanded and karyotyped to confirm their differentiation potential toward hematopoietic lineages. CRISPR / Cas9-based targeted mutagenesis generated karyotypically normal human hES cell lines lacking HLA expression. Miseq analysis of the target region identified one clone with inactivating mutations in both β2m and TAP1 alleles and one CD74 allele (see, for example, Figure 6B). This clone completely abolished interferon-γ (IFNγ)-induced HLA-I expression (see, e.g., Figure 6C), and a normal karyotype was confirmed. Monocytes from this HLA-I-deficient line were cloned into allogeneic primary CD8 + These clones failed to stimulate T cell proliferation (see, e.g., Figure 6D, showing that HLA-deficient hES cell progeny do not stimulate T cells). This HLA-I-deficient line was subsequently retargeted using CRISPR to remove the remaining allele of CD74 and both alleles of CIITA, a transcription factor required for HLA-II expression. This clone was also confirmed to possess a normal karyotype. Subsequent analysis confirmed that after each genetic modification, the cells retained hematopoietic potential and derived monocytes lacked detectable expression of HLA-II (see, e.g., Figure 6E, showing that HLA-II is not expressed by CIITA / CD74-deficient cells).

[0191] Generation of AAVS-targeted immune evasion cassette Loss of HLA-I expression is thought to render cells susceptible to NK cell lysis. This eliminated the ligand for the activating receptor NKG2D (see below). However, NKG2D blockade only eliminated approximately 50% of NK cell-mediated specific lysis. Therefore, to further eliminate NK cell reactivity, an additional step is taken, for example, through expression of HLA-E and HLA-G covalently linked to peptide and β2m to form single-chain trimers. These trimers do not exchange peptide or β2m with endogenous HLA and inhibit NKG2A- and ILT2-expressing NK cells. Other aspects of the immune response, including antibody binding, complement deposition, and macrophage phagocytosis, may also contribute to rejection. To address and circumvent each of these immune mechanisms, we constructed a set of multicistronic vectors targeting the human AAVS locus (see, e.g., Figure 7A and Table 1). This locus is considered a "safe harbor" protected from silencing. Flow cytometry analysis confirmed expression in HLA-deficient hES cells (see, e.g., Figure 7B). A summary of immune evasion and suicide genes and targets can be found in Table 1.

[0192] [Table 2]

[0193] The targeting construct was validated through transfection into CHO cells and evaluation of immune evasion gene expression by flow cytometry. Results showed that human CD55 and HLA-E single-chain trimers exhibited tight coexpression (see, e.g., Figure 8A), confirming the function of the 2A sequence in Figure 7 and construct 1 (SEQ ID NO: 1). To confirm the correct function of the inserted gene product, stable hCD55-expressing CHO lines were identified through 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 the remaining hCD55 cells in culture showed robust complement deposition (see, e.g., Figure 8B). These data confirm the functionality of the hCD55 transgene. To confirm the correct function of the HLA-E single-chain trimer, stable cell lines were generated in 721.221 cells, an NK cell-sensitive target cell line. Incubation of unmodified 721.221 cells with primary PBMCs resulted in robust NK cell degranulation, as measured by cell surface CD107a staining. However, co-incubation with HLA-E-expressing 721.221 cells specifically inhibited NKG2A+ NK cell degranulation (see, e.g., Figure 8C).

[0194] Generation of NKG2D ligand-deficient hES cells Because the absence of HLA-I can render target cells susceptible to NK cell-mediated cytolysis, NKG2D ligands are targeted via CRISPR / Cas9. Of these, RNA-seq analysis demonstrates that only MICA and MICB are expressed in long-lived plasma cells. Preliminary sequencing of approximately 400 nucleofected clones revealed one line harboring frameshift mutations in two alleles of MICA and MICB and a large deletion in another chromosome, creating an in-frame fusion between MICA and MICB (Figure 11A). This MICA / B fusion was retargeted with CRISPR / Cas9 to generate a clone with frameshift mutations in all four alleles of MICA and MICB (Figure 11B). This clone was verified for normal karyotype. HLA, MICA / B-deficient hES cells are hereafter referred to as HM-KO hES.

[0195] Generation of stable AAVS immune escape transformants in HLA / MICA+MICB KO (HM-KO) hES cells Starting with HM-KO hES cells, two sets of nucleofections were performed. One nucleofection was performed with the human immune evasion cassette shown in Figure 7A, and a separate nucleofection 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 were selected for neomycin and puromycin resistance, and gene expression was confirmed by flow cytometry (see, e.g., Figure 7B).

[0196] In vivo teratoma formation assay To test human immune evasion in vivo, humanized NSG W41 mice were generated. Approximately 2 × 10 5umbilical cord blood CD34+ cells (obtained from the St. Louis Cord Blood Bank) were transplanted into unconditioned NSG-W41 mice (see, e.g., Figure 9A). Blood was collected from the mice to confirm human reconstitution, and 1 million cells of each modified hESC version were embedded in Matrigel and subcutaneously implanted into humanized NSG-W41 mice. Teratoma growth was monitored for 12 weeks or until tumors reached a 20 mm mass, at which point the mice were euthanized. Humanized mice inoculated with HLA-deficient cells formed teratomas, whereas unmodified hES cells did not (see, e.g., Figure 9B, which demonstrates that HLA-KO hES cells circumvent rejection by humanized mice). In one experiment, mice were treated with AP1903 to activate iCasp9 and initiate teratoma regression. Some aspects of the immune response are not functional in humanized NSG-W41 mice. For example, NK cells are barely formed in this system, and antibody responses are minimal. Therefore, we will perform teratoma assays in C57BI6 mice using HM-KO cells stably expressing the mAAVS construct. Rejection is expected to be delayed in this xenogeneic setting.

[0197] The modified hES cells are expected to evade immune recognition and therefore form teratomas in humanized NSG-W41 mice.

Claims

[Claim 1] A method for differentiating human embryonic stem cells (ES cells) into plasma cells, comprising: generating progenitor cells; and differentiating said precursor cells into B cells. A method comprising:

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