Compositions and Methods for Enhancing Immunological Tolerance

Abstract

This specification discloses methods for generating low immunogenic cells and methods for mediating localized immune tolerance. In some embodiments, the invention relates to cells engineered (i.e., modified) to induce a tolerogenic local microenvironment. In some embodiments, the cells are induced pluripotent cells, and in some embodiments, the cells are stem cells. In some embodiments, the cells are progenitor or precursor cells, e.g., β-cell progenitor or precursor cells.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 340,453, filed May 10, 2022, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] Type 1 diabetes (T1D) is an autoimmune disease that results in the destruction of insulin-producing β cells in the pancreas. Transplantation of whole pancreases or pancreatic islets from cadavers has been successful in treating T1D. However, limited donor availability and the need for lifelong immunosuppression hinder these treatments (Shapiro et al., 2006). To address the shortage of islet material, several protocols have been developed to induce in vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional SC islets, including glucose-responsive β cells (D'Amour et al., 2006; Millman et al., 2016; Nair et al., 2019; Pagliuca et al., 2014; Rezania et al., 2014; Russ et al., 2015; Veres et al., 2019).

[0003] Current strategies to protect allografted islet cells include encapsulation ( Alagpulinsa et al., 2019 , Bochenek et al., 2018 ), modification of the patient's immune system by coadministration of low-dose IL-2 and biologics such as anti-CD3 (tepulzimab) ( Hartemann et al., 2013 , Herold et al., 2019 ), and / or genetic modification of SC islets. Because human leukocyte antigens (HLA) are major contributors to immune recognition and rejection, HLA targeting has been performed in iPSCs to reduce or eliminate immune responses to foreign cells (Castro-Gutierrez et al., 2021; Deuse et al., 2019; Gornalusse et al., 2017; Han et al., 2019; Harding et al., 2019; Riolobos et al., 2013; Xu et al., 2019; Yoshihara et al., 2020). To date, engineering strategies that have shown some degree of immune protection for SC islet cells involve lentiviral overexpression of PD-L1 and selective retention of a single HLA-A2 allele in HLA-B / C-deficient cells (Parent et al., 2021; Yoshihara et al., 2020). However, lentiviral transgene overexpression is limited by transgene silencing (Herbst et al., 2012;Wen et al., 2021), and retention of a single HLA-A2 allele on SC islet cells can result in recurrent autoimmune rejection mediated by antigen-specific and tissue-resident memory T cells (Abou-Daya et al., 2021;Monti et al., 2008;Stegall et al., 1996). Summary of the Invention [Means for solving the problem]

[0004] In some embodiments, the invention relates to cells that have been engineered (i.e., modified) to induce a tolerogenic local microenvironment. In some embodiments, the cells are induced pluripotent cells, and in some embodiments, the cells are stem cells. In some embodiments, the cells are progenitor or precursor cells, e.g., beta cell progenitor or beta cell precursor cells. The cells are engineered to express one or more tolerogenic genes, e.g., one or more transgenes, and / or one or more immunomodulatory agents, from at least one locus within the cell that is not substantially silenced upon differentiation of the cell into a desired cell type. As used herein, a locus that is not substantially silenced is a locus that is measurably expressed after differentiation of the cell into a desired cell type. In some embodiments, the locus is measurably expressed both during and after differentiation of the cell into the desired cell type. The invention relates to both progenitor or precursor cells engineered as described, and differentiated cells derived from the engineered progenitor or precursor cells and comprising the engineered locus. The invention further relates to methods of making the cells and to methods of using the cells in cell therapy methods, for example, to treat a subject in need of cell therapy.

[0005] In some embodiments, the present invention relates to a method for generating hypoimmunogenic cells, the method comprising: engineering stem or progenitor cells to express one or more tolerogenic transgenes from a locus within the stem or progenitor cell that is not silenced during differentiation of the cell into a desired cell type; and exposing the stem or progenitor cells to conditions suitable for the differentiation of the stem or progenitor cells into the desired cell type. The present invention also relates to cells generated by this method, including engineered stem cells, engineered progenitor cells, and engineered differentiated cells. In some embodiments, the stem cells are induced pluripotent cells. In some embodiments, the progenitor cells are beta cell precursors. In some embodiments, the desired cell type is a beta cell.

[0006] In some aspects, the locus is a housekeeping gene locus. In some embodiments, the locus is constitutively expressed in all cells of a pancreatic islet, e.g., a human pancreatic islet. In some embodiments, the locus is constitutively expressed in beta cells, e.g., human beta cells. In some embodiments, the housekeeping gene is selected from the group consisting of actin, ubiquitin, and GAPDH.

[0007] In some aspects, the one or more tolerogenic agents / transgenes are selected from the group consisting of PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single chain fusion, soluble PDL1-Ig, and CTLA4-Ig. In some aspects, the tolerogenic agent is an HLA-E single chain fusion. In some embodiments, the single chain fusion is loaded with a peptide that can activate NK cells, for example, via interaction with the inhibitory receptors NKG2A and / or NKG2C. In some embodiments, the peptide is selected from the group consisting of VMAPRTLLL (SEQ ID NO: 2), VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12) and VMAPRALLL (SEQ ID NO: 13).

[0008] In another aspect, the present invention relates to a method of mediating localized immune tolerance, comprising: engineering stem or progenitor cells to express one or more immunomodulatory agents from a locus within the stem or progenitor cell that is not silenced during differentiation of the cell into a desired cell type; and exposing the stem or progenitor cells to conditions suitable for the stem or progenitor cells to differentiate into the desired cell type, thereby producing the desired cell type that secretes the one or more immunomodulatory agents. The present invention also relates to cells produced by this method, including engineered stem cells, engineered progenitor cells, and engineered differentiated cells. In some embodiments, the stem cells are induced pluripotent cells. In some embodiments, the progenitor cells are beta cell precursors. In some embodiments, the desired cell type is a beta cell. The present invention also relates to cells produced by this method, including engineered stem cells, engineered progenitor cells, and engineered differentiated cells.

[0009] In some aspects, the locus is a housekeeping gene locus. In some embodiments, the locus is constitutively expressed in all cells of a pancreatic islet, e.g., a human pancreatic islet. In some embodiments, the locus is constitutively expressed in a beta cell, e.g., a human beta cell. In some embodiments, the housekeeping gene is selected from the group consisting of actin, ubiquitin, and GAPDH. In some aspects, the one or more immunomodulatory agents are cytokines. In some aspects, the one or more immunomodulatory agents are selected from the group consisting of IL-10, TGF-β, and IL-2, and modified IL-2.

[0010] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0011] [Figure 1A]1 shows the generation of immune-evasive SC islet cells. Schematic diagram of the genetic engineering strategy to generate hypoimmunogenic hESCs. [Figure 1B] Figure 1 shows the generation of immune-evasive SC islet cells. Figure 2 shows FACS analysis of Nkx6.1 / C-peptide SC-β cells derived from hypoimmunogenic hESCs (S6d14). [Figure 1C] Figure 1 shows the generation of immune-evasive SC islet cells. Figure 2 shows the expression of PDL1 and HLA-ABC on SC-β cells. Data are shown as MFI (gated on C-pep+ / Nkx6.1+ SC-β cells) normalized to mode. [Figure 1D] Figure 1 shows the generation of immune-evasive SC islet cells. Figure 2 shows the expression of HLA-ABC and HLA-E on SC-β cells. Data are shown as MFI (gated on C-pep+ / Nkx6.1+ SC-β cells) normalized to mode. [Figure 1E] Figure 1 shows the generation of immune-evasive SC islet cells. Immunofluorescence staining of HLA-E, Nkx6.1, and C-peptide in CD49a+ enriched SC-β cells (S6d14). Size bar = 100 μm. [Figure 1F] 1 shows the generation of immune-evasive SC islet cells. Schematic of an in vivo SC islet cell xeno-rejection assay. [Figure 1G] Figure 1 shows the generation of immune-evasive SC islet cells. Quantitative analysis of xenograft rejection is shown. Data are shown as mean ± SEM (n=5 / group). The dashed line represents background luminescence. [Figure 2A] Figure 1 shows the in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. A schematic diagram of the in vitro immune SC islet cell co-culture assay is shown. [Figure 2B] Figure 1 shows in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Quantification of SC islet cell viability when co-cultured with primary human PBMCs at a 1:1 ratio is shown. Cell viability is expressed as mean ± SD (n=5). [Figure 2C]Figure 1 shows in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Quantification of SC islet cell viability when co-cultured with primary human PBMCs at a 3:1 ratio is shown. Cell viability is expressed as mean ± SD (n=5). [Figure 2D] Figure 1 shows in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Figure 2 shows heat maps of T cell ligand gene expression in IFN-γ-treated CD49a+ SC-β cells. Ligand expression levels are expressed as fold change (log2). [Figure 2E] Figure 1 shows in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Figure 2 shows differential expression of T cell co-inhibitory ligands in IFN-γ-treated CD49a+ SC-β cells. Ligand expression is expressed as fold change (log2). [Figure 2F] Figure 1 shows in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Figure 2 shows differential expression of T cell co-activation ligands in IFN-γ-treated CD49a+ SC-β cells. Ligand expression is expressed as fold change (log2). [Figure 2G] Figure 1 shows in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Quantification of SC-β cell viability when co-cultured with primary human CD56+ NK cells is shown. Cell viability is expressed as mean ± SD (n=5). [Figure 2H] Figure 1 shows the in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Figure 2 shows a schematic diagram of an in vivo NK cell cytotoxicity assay. [Figure 2I] Figure 1 shows in vitro co-culture of hypoimmunogenic SC islet cells with allogeneic human immune cells. Quantitative analysis of in vivo NK cell assays is shown. Data are presented as mean ± SEM. The dashed line represents background luminescence. [Figure 3A] Figure 1 shows the characterization of SC islet cell-specific NK cell ligand profiles. Figure 2 shows a schematic diagram of the bulk RNA sequencing workflow for SC-β and SC-endothelial cells. [Figure 3B]Characterization of SC islet cell-specific NK cell ligand profiles. Heatmap of NK cell ligand expression (-IFN-γ vs +IFN-γ) in SC-β and SC-endothelial cells. Ligand expression is expressed as fold change (log2). [Figure 3C] Figure 1 shows characterization of SC islet cell-specific NK cell ligand profiles. Expression of NK cell activating ligands on SC-β and SC-endothelial cells is shown. Data are shown as MFI normalized to mode and are representative of three independent experiments. [Figure 3D] Figure 1 shows the characterization of SC islet cell-specific NK cell ligand profiles. Expression of NK cell inhibitory ligands on SC-β and SC-endothelial cells is shown. Data are shown as MFI normalized to mode and are representative of three independent experiments. [Figure 3E] Figure 1 shows the characterization of SC islet cell-specific NK cell ligand profiles.Figure 2 shows a schematic diagram of SC islet cell transplantation in diabetic humanized NSG-DKO mice. [Figure 3F] Figure 1 shows the characterization of SC islet cell-specific NK cell ligand profiles. Non-fasting blood glucose concentrations after SC islet transplantation and PBMC injection are shown. Data are presented as mean ± SD (n=12 / group). [Figure 3G] Figure 1 shows the characterization of SC islet cell-specific NK cell ligand profiles. Figure 2 shows in vivo GSIS before and after PBMC injection. Plasma insulin concentrations were measured at t = 0 and 30 min after glucose injection. Data are presented as mean ± SD (n = 5 / group). [Figure 4A] Immune-tolerized SC islet cells survive xenorejection. Schematic of the genetic engineering strategy to generate immunomodulatory hESCs and a schematic of the GAP-2B10 HDR plasmid. [Figure 4B] Tolerized SC islet cells survive xenorejection. FACS analysis of Nkx6.1 / C-peptide SC-β cells derived from immunomodulatory hESCs (S6d8). [Figure 4C]Tolerized SC islet cells survive xenorejection. In vitro GSIS of 2B10 SC islet cells is shown. Data are presented as mean ± SD (n=4). [Figure 4D] Tolerized SC islet cells survive xenorejection. Quantification of cytokine secretion from 2B10 SC islet cells is shown. Data are presented as mean ± SD (n=2). [Figure 4E] Tolerized SC islet cells survive xenorejection. Quantification of SC islet cell viability when co-cultured with primary human PBMCs at a 1:1 ratio is shown. Cell viability is expressed as the mean ± SD (n=5). [Figure 4F] Tolerized SC islet cells survive xenorejection. In vivo bioluminescence imaging of B6 / albino mice transplanted with 2B10 SC islet cells (n=3 / group). [Figure 4G] Tolerized SC islet cells survive xenorejection. Quantitative analysis of in vivo graft survival in B6 / albino mice. Data are presented as mean ± SD (n=3 / group). [Figure 4H] Tolerized SC islet cells survive xenorejection. Immunostaining of 2B10 and WT SC islet grafts showing the presence of INS+ cells and recruitment of Tregs 5 weeks after transplantation. Size bar = 100 μm. [Figure 5A] t-SNE projections of primary human islet cells are shown. [Figure 5B] t-SNE projections of GAPDH expression across each assigned population are shown. Cells are color-coded according to their assigned cluster (adapted from Segerstolpe et al., 2016). [Figure 5C] Schematic diagram of the homology-directed repair plasmid for integration of Luc2 and PD-L1 into the GAPDH locus. [Figure 5D] FIG. 1 is a schematic diagram of the GAPDH-targeted Luc2 and peptide::B2M::HLA-E HDR plasmids. [Figure 5E] FIG. 1 is a schematic diagram of a peptide::B2M::HLA-E long fusion. [Figure 5F] Figure 1 shows a PD1-Fc binding assay. PD-L1 and WT SC islet cells were dissociated and stained with a two-fold dilution series of PE-conjugated human and mouse PD1-Fc. Data are shown as MFI normalized to the mode. [Figure 5G] 1 shows the FACS sorting strategy for HLA-ABC− / − hESCs. [Figure 5H] Figure 1 shows the FACS gating strategy for quantification of Nkx6.1+ / C-pep+ SC-β cells after in vitro differentiation. [Figure 6A] FACS profiling of HLA-A2 status of five PBMC donors is shown. [Figure 6B] Figure 1 shows the FACS gating strategy for CD4+ and CD8+ T cells and CD56+ NK cells in PBMCs enriched by human apheresis leukocyte reduction. Plots represent five donors. [Figure 6C] Quantification of SC islet cell viability when co-cultured with purified human CD8+ T cells at a 1:1 ratio. Cell viability is expressed as the mean ± SD (n=5). [Figure 6D] Quantification of SC islet cell viability when co-cultured with purified human CD8+ T cells at a 3:1 ratio. Cell viability is expressed as the mean ± SD (n=5). [Figure 6E] Quantification of SC islet cell viability when co-cultured with purified human CD4+ T cells at a 1:1 ratio. Cell viability is expressed as the mean ± SD (n=5). [Figure 6F] Quantification of SC islet cell viability when co-cultured with purified human CD4+ T cells at a 3:1 ratio. Cell viability is expressed as the mean ± SD (n=5). [Figure 6G]

[0023] Figure 1 shows the FACS gating strategy for CD4+ T cells enriched by human apheresis leukocyte reduction. Plots represent five donors. [Figure 6H]

[0023] Figure 1 shows the FACS gating strategy for CD8+ T cells enriched by human apheresis leukocyte reduction. Plots represent five donors. [Figure 7A]Quantification of SC-β cell viability when co-cultured with NK92mi cells. K562 and Raji cells were used as positive and negative controls, respectively. Cell viability is expressed as mean ± SD (n = 5). [Figure 7B] 1 shows the expression of NKG2A / NKG2C in NK92mi cells. [Figure 7C] Figure 1 shows the FACS gating strategy for CD56+ NK cells enriched by human apheresis leukopenia. Specific population gates include all CD56+ NK cells (red), CD56 high (green), and CD56 low (pink). Plots represent five donors. [Figure 7D] Quantitative analysis of CD56 expression on enriched primary human NK cells. Data are presented as % CD56 expression (n=5). [Figure 7E] In vivo NK cell assay. Bioluminescence imaging was performed on days 1 and 5 after transplantation. [Figure 7F] FIG. 1 is a schematic diagram of the SC-endothelial cell differentiation protocol. [Figure 7G] FACS analysis of the endothelial cell marker CD31 in SC endothelial cells derived from WT and B2M- / - hESCs is shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] Immunoprotection of transplanted stem cell-derived islet (SC islet) cells without long-term immunosuppression or encapsulation has yet to be achieved. Conventional genetic engineering approaches to generate hypoimmunogenic SC islet cells have shown mixed results. Herein, we show that targeting human leukocyte antigen (HLA) and PD-L1 alone does not sufficiently protect SC islet cells from xeno- or allo-rejection. Herein, we describe genetically engineered SC islet cells that secrete the cytokines IL-10, TGF-β, and modified IL-2, thereby promoting a tolerogenic local microenvironment by activating and expanding regulatory T cells (Tregs). These cytokine-secreting human SC islet cells prevented xenorejection in B6 / albino mice for up to 9 weeks after transplantation. Therefore, hESCs engineered to induce a tolerogenic local microenvironment may be an alternative source of SC islet cells that does not require encapsulation or immunosuppression for diabetic cell replacement therapy. The described cell modifications can be used alone or in addition to other approaches to generate hypoimmunogenic SC islet cells.

[0013] To address the issue of transgene silencing during differentiation into SC islet cells, we used a transgene targeting strategy utilizing the GAPDH gene, which is constitutively expressed in primary human islets and SC islet cells (Gerace et al., 2021; Sintov et al., 2021). We generated hypoimmunogenic SC islet cells by overexpressing PD-L1 and HLA-E long chain fusions in both HLA-competent and HLA-deficient backgrounds. In these studies, PD-L1 overexpression did not protect SC islet cells from xenorejection, and overexpression of HLA-E short chain fusions was not required to inhibit primary human NK cells. In addition to immune evasion, we also addressed immune modulation and showed that constitutive secretion of IL-10, TGF-β, and IL-2 mutant proteins protected SC islet cells from xenorejection for up to 9 weeks after transplantation without impairing SC islet cell function in vitro. These immunomodulatory SC islet cells therefore represent a further advance in providing a source of islets for the treatment of T1D with the long-term goal of obviating the need for encapsulation or systemic immunosuppression.

[0014] The utility of immune evasion / tolerogenic islet cell replacement therapy relies heavily on maintaining transgene expression throughout cell differentiation and after transplantation. Therefore, to analyze the effects of various gene editing strategies for immune protection of SC islet cells, SC islet cells were engineered to constitutively express tolerogenic molecules, including PD-L1 (Castro-Gutierrez et al., 2021; Yoshihara et al., 2020) and HLA-E long chain fusions (Gornalusse et al., 2017), at the GAPDH locus in both HLA-competent and HLA-deficient backgrounds. We have reported that PD-L1 overexpression does not confer xenoprotective effects in SC islet cells. This may be explained by species-specific differences in PD-L1 / PD-1 binding (Viricel et al., 2015) and the fact that human and mouse PD-1 share only 60% homology at the amino acid level (Finger et al., 1997). Indeed, reduced binding of soluble mouse PD-1 to human PD-L1 expressed on SC islet cells has been observed. Although these results suggest that overexpression of human PD-L1 is not sufficient to overcome xenorejection, PD-L1 overexpression may still be beneficial in allogeneic conditions.

[0015] Consistent with previous studies, HLA-deficient SC islet cells were resistant to PBMC cytotoxicity in vitro (Han et al., 2019; Leite et al., 2022). We also found that SC-β cells modulate T cell ligand profiles in response to partial inflammatory stimuli. While we and others have attempted to engineer immune-evasive SC islet cells using the PD-L1 / PD-1 T cell signaling axis (Yoshihara et al., 2020), our transcript analysis of T cell ligands in SC-β cells suggests that the LGALS9 / TIM-3 signaling axis may also be an interesting target. Indeed, similar to PD-L1, LGALS9 is frequently upregulated in cancer cells and contributes to tumor progression by inhibiting T cell function (Heusschen et al., 2013; Yang et al., 2021). Furthermore, we identified the T cell activation ligands HVEM, CD40, and BTN3A1 as potential targets to knock out in SC islet cells to further affect T cell function.

[0016] The studies described herein further demonstrate that HLA-deficient SC islet cells are resistant to preactivated NK cell cytotoxicity both in vitro and in vivo, and that overexpression of HLA-E long fusions does not confer any additional protective benefit. This may be due to the specific lack of NK cell-activating ligands, such as MIC and ULBP proteins, expressed on SC endothelial cells, in SC islet cells, which may explain why SC islet cells are resistant to preactivated NK cell cytotoxicity while SC endothelial cells are susceptible to NK cell cytotoxicity (Deuse et al., 2021). However, because many immunodeficient mouse models lack factors critical for NK cell survival and function, such as SIRPα and IL-15 (Herndler-Brandstetter et al., 2017), these models do not support long-term engraftment of human NK cells. We could not rule out the possibility of disrupting HLA-deficient SC islet cells in mouse models that better support NK cell engraftment. Furthermore, transplantation of HLA-deficient SC islet cells was able to normalize blood glucose levels in diabetic mice, but after PBMC injection, the cells were ultimately rejected, albeit at a slower rate. This may be explained by the presence of other immune cell subsets, such as macrophages, monocytes, and dendritic cells, which may play a role in indirect allograft rejection (Oberbarnscheidt et al., 2014; Wyburn et al., 2005; Zhuang et al., 2016). Finally, this highlights the limitations of solely using in vitro immune cell coculture assays to assess the effects of genetic modifications on protecting SC islet cells from immune destruction (Castro-Gutierrez et al., 2021; Leite et al., 2022).

[0017] Finally, the studies described herein are the first to demonstrate that SC islet cells engineered to secrete modified IL-2, TGF-β, and IL-10 are protected from xenorejection. This finding has several important implications. First, because β cells are specialized secretory cells that require extensive translation, this demonstrates that SC islet cells can be adapted to secrete other proteins while maintaining their intended function (Lim et al., 2020). Second, because some cell types may not require extensive genetic engineering, the unique ligand profile of the desired cell type must be considered when determining the set of genetic modifications required to generate immune-evading cells. Because the safety of immune-evasion / immune-tolerizing cell therapies is crucial for their clinical application, ideally, engineered products should be generated with minimal genetic perturbations to limit off-target events and chromosomal instability. Third, these results provide validation for the use of immune-tolerizing approaches (alone or in combination with immune evasion) as a method to protect SC islet cells from the immune system. In one variation of this approach, some, but not all, of the cells secreting these tolerizing molecules could be included in the graft. Overall, this approach could obviate the need for encapsulation or immunosuppression, a long-standing challenge in the field of islet transplantation.

[0018] Although HLA-deficient SC islet cells showed improved survival in in vitro PBMC coculture assays (Figure 2B and 2C), these results did not translate to in vivo studies. Selecting an appropriate humanized mouse model is essential for evaluating immune evasion / tolerization genetic engineering strategies. Many PBMC-humanized mouse models, such as the one used in this study, do not fully recapitulate human allograft rejection due to biased CD3+ cell engraftment and lack of other immune cell subsets. Therefore, while HLA-deficient SC islets exhibited delayed rejection in the PBMC-humanized mouse model (Figure 3F), we cannot exclude the possibility that these cells are rejected earlier in CD34+ or BLT mice (humanized mouse models that better recapitulate components of human immunity). For these reasons, validation of the protective effects of immune evasion / tolerization genetic engineering strategies should ideally be performed in humanized mouse models that more accurately reflect allograft rejection. Although xenorejection does not mimic allorejection, the xenorejection model is a more robust model of immune rejection and eliminates the immune cell bias of humanized mouse models, so we used a xenorejection model to evaluate this immune tolerization genetic manipulation strategy.

[0019] The use of such a strong graft rejection model introduces an additional immune barrier, requiring complex genetic engineering approaches for solutions. This means combining gene knockout and knockin to generate the desired immune-evading / tolerizing cell product. Knocking in multiple genes into specific loci within the genome requires large homology-directed repair templates, which are associated with poor integration efficiency and the recovery of genetically unstable clones. Exploring the possibility of introducing tolerogenic molecules into multiple constitutively expressed loci may reduce the size of the HDR template and allow the recovery of genetically stable homozygous knockin clones. Achieving a balance between the length of HDR and the number of editing events may allow for the introduction of more foreign genetic material into the genome without destabilizing effects.

[0020] Furthermore, because tolerized SC islets secrete cytokines from all cells within the islet, there is a risk that the constitutively secreted cytokine levels may lead to chronic immunosuppression. Therefore, chronic immunosuppression must be avoided, and it is therefore important to evaluate the immune status of mice receiving tolerized SC islet cells. However, if transplantation of tolerized SC islets leads to chronic immunosuppression, the ability to enrich for specific endocrine cell populations may allow for the adjustment of cytokine secretion levels by creating designer islets composed of cytokine-secreting and non-cytokine-secreting endocrine cells so that local graft tolerance is achieved.

[0021] definition

[0022] For convenience, certain terms used in the specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0023] The term "differentiated cell," as defined herein, refers to any primary cell that is not pluripotent in its native form. Alternatively stated, the term "differentiated cell" refers to a cell of a more specialized cell type that is derived from a cell of a less specialized cell type (e.g., a stem cell, such as an induced pluripotent stem cell) during the cell differentiation process. Without wishing to be limited by theory, pluripotent stem cells can differentiate during normal ontogeny first into endodermal cells that can form pancreatic cells and other endodermal cell types. Further differentiation of endodermal cells leads to the pancreatic pathway, where approximately 98% of cells become exocrine, ductal, or matrix cells, and approximately 2% become endocrine cells.

[0024] As used herein, the term "somatic cell" refers to any cell that forms the body of an organism, as opposed to a germ cell. In mammals, germ cells (also called "gametes") are sperm and eggs, which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Apart from sperm and eggs, the cells from which they arise (gametocytes), and undifferentiated stem cells, all other cell types in a mammal's body are somatic cells: internal organs, skin, bone, blood, and connective tissue are all made up of somatic cells. In some embodiments, the somatic cell is a "non-embryonic somatic cell," which refers to a somatic cell that is not present in or obtained from an embryo and that does not result from the propagation of such a cell in vitro. In some embodiments, the somatic cell is an "adult somatic cell," which refers to a cell that is present in or obtained from an organism other than an embryo or fetus, or a cell that results from the propagation of such a cell in vitro. Unless otherwise specified, the methods described herein can be performed in vivo or in vitro.

[0025] As used herein, the term "adult cell" refers to a cell found throughout the body after embryonic development.

[0026] As used herein, the term "endodermal cell" refers to a cell derived from one of the three major germ cell layers in the very early embryo (the other two being mesoderm and ectoderm). The endoderm is the innermost of the three layers. Endodermal cells differentiate to give rise first to the embryonic gut, then to the lining of the respiratory and digestive tracts (such as the intestine), the liver, and the pancreas.

[0027] As used herein, the term "cell of endodermal origin" refers to any cell that develops or differentiates from an endodermal cell. For example, cells of endodermal origin include cells of the liver, lung, pancreas, thymus, intestine, stomach, and thyroid. Without wishing to be limited by theory, liver and pancreatic progenitor cells (also called pancreatic progenitor cells) develop from endodermal cells in the embryonic foregut. Shortly after their specification, liver progenitor cells and pancreatic progenitor cells rapidly acquire significantly different cellular functions and regenerative capabilities. These changes are driven by inductive signals and genetic regulatory factors that are highly conserved among vertebrates.

[0028] The terms "stem cell-derived β cells," "SC-β cells," and "mature SC-β cells" refer to cells (e.g., pancreatic β cells) that display at least one marker indicative of pancreatic β cells, express insulin, and exhibit a GSIS response characteristic of endogenous mature β cells. In some embodiments, "SC-β cells" include mature pancreatic β cells. It is understood that SC-β cells need not be derived (e.g., directly) from stem cells. It should further be understood that the SC-β cells of the present invention are non-native, i.e., non-naturally occurring, non-endogenous cells, and have at least one characteristic that differs from native / naturally occurring / endogenous cells. Examples of SC-β cells and methods for obtaining such SC-β cells are described in WO2015 / 002724 and WO2014 / 201167 (Millman et al., 2016 and Pagliuca et al., 2014), both of which are incorporated herein by reference in their entireties. In some embodiments, "SC-beta cells" are stem cell-derived beta cells that are hypoimmunogenic, eg, beta cells that elicit only a limited or no immune response.

[0029] As used herein, the term "exocrine cells" refers to cells of exocrine glands, i.e., glands that excrete secretions through ducts. In certain embodiments, exocrine cells refer to exocrine pancreatic cells, which are pancreatic cells that produce enzymes that are secreted into the small intestine. These enzymes aid in the digestion of food as it passes through the digestive tract. Exocrine pancreatic cells, also known as the islets of Langerhans, secrete two hormones: insulin and glucagon.

[0030] As used herein, the term "pluripotent" refers to cells that have the ability to differentiate under different conditions into more than one differentiated cell type, preferably into cell types characteristic of all three germ layers. Pluripotent cells are primarily characterized by their ability to differentiate into more than one cell type, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, a preferred test of pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. Note that simply culturing such cells does not render them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as the term is defined herein) are also characterized by the ability to be passaged for extended periods without loss of growth potential, compared to their parent primary cells, which generally undergo only a limited number of divisions in culture.

[0031] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells that have been artificially derived (e.g., by induction or complete reversal) from non-pluripotent cells, typically adult somatic cells, for example, by inducing forced expression of one or more genes.

[0032] The terms "progenitor cell" or "precursor" cell are used interchangeably herein and refer to a cell that has a more primitive cellular phenotype (i.e., earlier in a developmental pathway or progression than a fully differentiated cell) compared to the cell it can give rise to by differentiation. In many cases, progenitor cells also have significant, or even very high, proliferative potential. Progenitor cells can give rise to multiple different differentiated cell types, or a single differentiated cell type, depending on the developmental pathway and environment in which the cell develops and differentiates.

[0033] As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of proliferation and can give rise to more progenitor cells, which in turn have the capacity to give rise to numerous mother cells, which in turn can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and subsequently generate progeny that differentiate into one or more mature cell types, while simultaneously retaining one or more cells of the parent's developmental potential. The term "stem cell" refers to a subset of progenitor cells that, under certain circumstances, have the capacity or potential to differentiate into a more specialized or differentiated phenotype and, under certain circumstances, retain the ability to proliferate without substantial differentiation. In one embodiment, the term stem cell refers to a naturally occurring mother cell whose progeny (descendant cells) often specialize in different directions through differentiation, e.g., by acquiring entirely distinct characteristics, as they occur in the progressive diversification of embryonic cells and tissues. Cell differentiation is a complex process, typically occurring through many cell divisions. Differentiated cells may be derived from multipotent cells that are themselves derived from multipotent cells, etc. Although each of these multipotent cells may be considered a stem cell, the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells of greater developmental potential. This ability can be natural or induced artificially by treatment with various factors. In many biological examples, stem cells can generate progeny of more than one different cell type and are therefore also "multipotent," but this does not require "stemness." Self-renewal is another classic part of the definition of a stem cell and, as used herein, is essential. Theoretically, self-renewal can occur primarily through one of two main mechanisms: stem cells can divide asymmetrically, with one daughter maintaining the stem cell state and the other expressing some other specific function and phenotype. Alternatively, some stem cells in a population can divide symmetrically into two stem cells, thus maintaining some stem cells in the population as a whole, while other cells in the population give rise only to differentiated progeny.Formally, cells that begin as stem cells progress toward a differentiated phenotype but may subsequently "reverse" and re-express the stem cell phenotype, a term often referred to by those skilled in the art as "dedifferentiation" or "reprogramming," or "reverse differentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0034] The term "embryonic stem cells" is used to refer to pluripotent stem cells of the inner cell mass of blastocysts (see U.S. Patent Nos. 5,843,780 and 6,200,806). Such cells can also be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, e.g., U.S. Patent Nos. 5,945,577, 5,994,619, and 6,235,970). The distinguishing characteristics of embryonic stem cells define their phenotype. Thus, a cell has an embryonic stem cell phenotype if it possesses one or more of the distinctive characteristics of embryonic stem cells that distinguish it from other cells. Exemplary distinguishing characteristics of embryonic stem cells include, but are not limited to, gene expression profile, proliferation potential, differentiation potential, karyotype, and responsiveness to specific culture conditions.

[0035] The term "adult stem cell" or "ASC" refers to any multipotent stem cell derived from non-embryonic tissue, including fetal, juvenile, and adult tissue. Stem cells have been isolated from a variety of adult tissues, including blood, bone marrow, brain, olfactory epithelium, skin, pancreas, skeletal muscle, and cardiac muscle. Each of these stem cells has been characterized based on gene expression, factor responsiveness, and morphology in culture. Exemplary adult stem cells include neural stem cells, neural crest stem cells, mesenchymal stem cells, hematopoietic stem cells, and pancreatic stem cells. As noted above, stem cells have been found to be present in virtually every tissue. Accordingly, the present invention recognizes that stem cell populations can be isolated from virtually any animal tissue.

[0036] As used herein, the term "reprogramming" refers to the process of changing or reversing the differentiation state of a somatic cell. Prior to reprogramming, a cell may be partially or terminally differentiated. Reprogramming includes the complete reversal of the differentiation state of a somatic cell to a pluripotent cell. Such a complete reversal of differentiation generates induced pluripotent (iPS) cells. As used herein, reprogramming also includes the partial reversal of the differentiation state of a cell, e.g., to a multipotent state, or to a somatic cell that is neither pluripotent nor multipotent but has lost one or more specific characteristics of the differentiated cell from which it was derived, e.g., direct reprogramming of a differentiated cell into a different somatic cell type. Reprogramming generally involves changing, e.g., reversing, at least a portion of the genetic patterns, such as nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic changes, and genomic imprinting, that occur during cell differentiation as a zygote develops into an adult.

[0037] As used herein, the term "agent" refers to any compound or substance, such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An "agent" can be any chemical entity or moiety, including, but not limited to, synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, an agent is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, oligomer of nucleic acid, amino acid, or carbohydrate, including, but not limited to, proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNA, lipoproteins, aptamers, and modifications and combinations thereof. In certain embodiments, an agent is a small molecule bearing a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclic moieties, including macrolides, leptomycin, and related natural products or their analogs. The compound may be known to have a desired activity and / or characteristics, or may be selected from a diverse library of compounds.

[0038] As used herein, a "cell culture medium" (also referred to herein as a "culture medium" or "culture medium") is a medium for culturing cells, containing nutrients that maintain cell viability and support growth. Cell culture media may contain any of the following in appropriate combinations: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum substitutes, and other components such as peptide growth factors. Cell culture media commonly used for particular cell types are known to those of skill in the art.

[0039] The term "cell line" refers to a population of largely or substantially identical cells, typically derived from a single ancestral cell or from a population of distinct and / or substantially identical ancestral cells. A cell line may have been or may be maintainable in culture for extended periods of time (e.g., months, years, or an indefinite period). A cell line may have undergone a spontaneous or induced process of transformation that confers indefinite culture lifespan to the cells. A cell line includes all cell lines recognized in the art as such. It is recognized that at least some characteristics of individual cells of a cell line may differ from one another because cells acquire mutations over time and, in some cases, undergo epigenetic changes. In some embodiments, a cell line comprises cells derived from stem cells as described herein.

[0040] The term "exogenous" refers to a substance present in a cell or organism other than its natural source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein that has been introduced by human-mediated process into a biological system, such as a cell or organism, in which it is not normally found, or is found in reduced amounts. A substance is considered exogenous if it was introduced into a cell or an ancestor of a cell that inherits the substance. In contrast, the term "endogenous" refers to a substance that is natural to a biological system.

[0041] The term "expression" refers to the cellular processes involved in producing RNA and proteins and, if appropriate, secreting the proteins, including, but not limited to, transcription, translation, folding, modification, and processing, as applicable. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.

[0042] As used herein, the term "genetically modified" or "engineered" refers to a cell into which an exogenous nucleic acid has been introduced by a human process (or the progeny of such a cell that inherits at least a portion of the nucleic acid). The nucleic acid may, for example, comprise a sequence exogenous to the cell, a native sequence (i.e., a sequence naturally found in the cell), or a non-naturally occurring arrangement (e.g., a coding region linked to a promoter from a different gene), or a modified version of a native sequence. Introducing a nucleic acid into a cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate- or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, a polynucleotide, or a portion thereof, is integrated into the genome of the cell. The nucleic acid may then be removed or excised from the genome, provided that such removal or excision results in a detectable change in the cell compared to an unmodified but otherwise equivalent cell. It should be recognized that the term genetically modified is intended to include the direct introduction of modified RNA into a cell (e.g., synthetically modified RNA). Such synthetic modified RNAs include modifications that prevent rapid degradation by endonucleases and exonucleases and avoid or reduce cellular innate immune or interferon responses to RNA. Modifications include, for example, (a) terminal modifications, such as 5'-end modifications (phosphorylation, dephosphorylation, conjugation, inverted ligation, etc.), 3'-end modifications (conjugation, DNA nucleotides, inverted ligation, etc.), (b) base modifications, such as substitution with modified bases, stabilizing bases, destabilizing bases, or bases that base-pair with an expanded repertoire of partners, or conjugate bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions, and (d) internucleoside linkage modifications, including phosphodiester linkage modifications or substitutions. If such modifications prevent translation (i.e., translation is reduced by 50% or more relative to the absence of the modification), the modifications are not suitable for the methods and compositions described herein.

[0043] As used herein, the terms "isolated" or "partially purified," in the case of a nucleic acid or polypeptide, refer to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and / or that is present with the nucleic acid or polypeptide when expressed by a cell or secreted, in the case of a secreted polypeptide. Chemically synthesized nucleic acids or polypeptides or those synthesized using in vitro transcription / translation are considered "isolated."

[0044] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally, the cell is later introduced into a second organism, or the cell (or its progeny) is reintroduced into the organism from which it was isolated.

[0045] As used herein, the term "isolated population," with respect to an isolated population of cells, refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells compared to the heterogeneous population from which the cells are isolated or enriched.

[0046] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the total cell population.

[0047] The terms "enrich" or "enriched" are used interchangeably herein and mean that the yield (proportion) of a type of cell is increased by at least 10% over the proportion of that type of cell in the starting culture or preparation.

[0048] As used herein, the terms "renewal" or "self-renewal" or "proliferation" are used interchangeably to refer to the ability of stem cells to renew themselves over extended periods of time and / or over periods of months to years by dividing into the same unspecialized cell type. In some instances, proliferation refers to the expansion of a cell by the repeated division of a single cell into two identical daughter cells.

[0049] As used herein, the term "lineage" refers to cells that share a common ancestor or a common developmental fate. For example, in the context of a cell of endodermal origin or a cell that is of "endodermal lineage," it means that the cell is derived from an endodermal cell and can differentiate along one or more developmental lineage pathways that are restricted to the endodermal lineage, e.g., give rise to definitive endoderm cells, which can then differentiate into liver cells, thymus, pancreas, lung, and intestine.

[0050] As used herein, the term "marker" is used to describe the characteristics and / or phenotype of a cell. Markers can be used to select cells containing a desired characteristic. Markers vary depending on the specific cell. A marker is either a morphological, functional, or biochemical (enzymatic) characteristic of a cell of a particular cell type or a molecule expressed by a cell type. Preferably, such a marker is a protein, more preferably having an epitope for an antibody or other binding molecule available in the art. However, a marker may be composed of any molecule found within a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nucleus-to-cytoplasm ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to take up or exclude a particular dye, the ability to migrate under certain conditions, and the ability to differentiate along a particular lineage. Markers can be detected by any method available to those skilled in the art. A marker can also be the absence of a morphological characteristic or the absence of a protein, lipid, etc. Markers can be a combination of a panel of unique features relating to the presence and absence of polypeptides, as well as other morphological features.

[0051] The term "modulate" is used consistent with its use in the art, i.e., to cause or promote a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such a change can be an increase, decrease, or change in the relative strength or activity of different components or branches of the process, pathway, or phenomenon. A "modulator" is an agent that causes or promotes a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.

[0052] The term "polynucleotide" is used interchangeably with "nucleic acid" herein to refer to a polymer of nucleosides. Typically, polynucleotides of the invention are composed of nucleosides naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) linked by phosphodiester bonds. However, the term also encompasses molecules containing nucleosides, whether found in naturally occurring nucleic acids, or nucleoside analogs, including chemically or biologically modified bases, modified backbones, and the like, which may be preferred for particular applications. When the present application refers to polynucleotides, it is understood that DNA, RNA, and in each case both single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information (i.e., the sequence of letters used as abbreviations for bases) that biochemically characterizes a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' direction unless otherwise specified.

[0053] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2 to 60 amino acids in length. As used herein, a polypeptide typically contains amino acids, such as the 20 L-amino acids most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a conjugation linker, or functionalization. Polypeptides to which non-polypeptide moieties are covalently or non-covalently attached are also considered "polypeptides." Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized by chemical means such as conventional solid-phase peptide synthesis. As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide material itself and / or to the sequence information that biochemically characterizes the polypeptide (i.e., the sequence of letters or three-letter codes used as abbreviations for amino acid names). The sequences of polypeptides presented herein are presented in the N-terminal to C-terminal direction unless otherwise specified.

[0054] The term "variant" when referring to a polypeptide can be, for example, a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to the full-length polypeptide. A variant can be a fragment of a full-length polypeptide. A variant can be a naturally occurring splice variant. A variant can be a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, or 99% identical to a fragment of a polypeptide, where the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of the length of the full-length wild-type polypeptide or a domain thereof that has the desired activity. In some embodiments, the domain is at least 100, 200, 300, or 400 amino acids in length, starting at any amino acid position in the sequence and extending toward the C-terminus. Mutations known in the art that eliminate or substantially reduce the activity of a protein are preferably avoided. In some embodiments, a variant lacks the N-terminal and / or C-terminal portions of the full-length polypeptide, e.g., up to 10, 20, or 50 amino acids from either end. In some embodiments, the polypeptide has the sequence of a mature (full-length) polypeptide, meaning a polypeptide from which one or more portions, such as a signal peptide, have been removed during normal intracellular proteolytic processing (e.g., during co-translational or post-translational processing). In some embodiments, where the protein is produced by means other than purification from cells that naturally express it, the protein is a chimeric polypeptide, meaning that it contains portions from two or more different species. In some embodiments, where the protein is produced by means other than purification from cells that naturally express it, the protein is a derivative, meaning that it contains additional sequences not associated with the protein, so long as those sequences do not substantially reduce the biological activity of the protein.

[0055] As used herein, the term "functional fragment" refers to a polypeptide that is smaller in size than but has a substantially homologous amino acid sequence than the polypeptide from which it is derived, wherein the functional fragment polypeptide sequence has at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold the effective biological activity of the polypeptide from which it is derived. Functional fragment polypeptides can have additional functions that may include reduced antigenicity, increased DNA binding (such as transcription factors), or altered RNA binding (such as regulating RNA stability or degradation).

[0056] The term "vector" refers to a carrier DNA molecule into which a DNA sequence can be inserted for introduction into a host cell. Preferred vectors are capable of autonomous replication and / or expression of a nucleic acid to which they are linked. A vector capable of directing the expression of an operably linked gene is referred to herein as an "expression vector." Thus, an "expression vector" is a specialized vector containing the regulatory regions necessary for the expression of a gene of interest in a host cell. In some embodiments, the gene of interest is operably linked to another sequence in the vector. Vectors can be viral or non-viral. When a viral vector is used, it is preferable that the viral vector is replication-deficient, which can be achieved, for example, by removing all viral nucleic acid encoding replication. Replication-deficient viral vectors still retain infectious properties and enter cells in a manner similar to replicating adenoviral vectors, but once inside the cell, replication-deficient viral vectors do not replicate or propagate. Vectors also include liposomes and nanoparticles, as well as other means for delivering DNA molecules to cells.

[0057] The term "operably linked" means that control sequences required for expression of a coding sequence are positioned within a DNA molecule in the appropriate position relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g., promoters, enhancers, and termination elements) in an expression vector. The term "operably linked" includes having an appropriate initiation signal (e.g., ATG) preceding the polynucleotide sequence to be expressed, and maintaining the correct reading frame to allow expression of the polynucleotide sequence under the control of the expression control sequences and production of the desired polypeptide encoded by the polynucleotide sequence.

[0058] The term "viral vector" refers to the use of a virus or virus-related vector as a carrier of a nucleic acid construct into a cell. The construct can be incorporated into and packaged in a non-replicating defective viral genome, such as adenovirus, adeno-associated virus (AAV), or herpes simplex virus (HSV), or others, including retroviral and lentiviral vectors, for infection or transduction of the cell. The vector may or may not integrate into the genome of the cell. The construct may optionally include viral sequences for transfection. Alternatively, the construct can be incorporated into a vector capable of episomal replication, such as EPV and EBV vectors.

[0059] The terms "control sequence" and "promoter" are used interchangeably herein and refer to nucleic acid sequences, such as initiation signals, enhancers, and promoters, that induce or control the transcription of protein-coding sequences to which they are operably linked. In some examples, transcription of a recombinant gene is under the control of a promoter sequence (or other transcription control sequence) that controls expression of the recombinant gene in a cell-type in which expression is intended. It is also understood that a recombinant gene can be under the control of transcription control sequences that are the same or different from those that control transcription of the naturally occurring form of the protein. In some cases, a promoter sequence is recognized by the synthetic machinery of the cell, or introduced synthetic machinery, necessary to initiate transcription of a particular gene.

[0060] As used herein, the term "transcription factor" refers to a protein that binds to a specific portion of DNA using a DNA-binding domain and is part of a system that controls the transfer (or transcription) of genetic information from DNA to RNA. As used herein, "proliferate" and "proliferation" refer to an increase in the number of cells in a population (growth) through cell division. Cell proliferation is generally understood to result from the coordinated activation of multiple signaling pathways in response to an environment containing growth factors and other mitogens. Cell proliferation can also be promoted by intracellular or extracellular signals and by relief from the action of mechanisms that block or negatively affect cell proliferation.

[0061] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers on a cell a selectable phenotype, e.g., resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), prototrophy, or expression of a specific protein that can be used as the basis for distinguishing between cells that express the protein and cells that do not. Proteins whose expression can be easily detected ("detectable markers"), such as fluorescent or luminescent proteins or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance, constitute a subset of selectable markers. The presence of a selectable marker linked to the native expression control elements for a gene that is normally selectively or exclusively expressed in pluripotent cells allows for the identification and selection of somatic cells that have been reprogrammed to the pluripotent state. Various selectable marker genes can be used, such as the neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, and mutants of any of these. Luminescent proteins, such as luciferase (e.g., firefly or Renilla luciferase), are also useful. As will be apparent to those skilled in the art, the term "selectable marker," as used herein, can refer to a gene or the expression product of a gene, e.g., an encoded protein.

[0062] In some embodiments, a selectable marker confers a growth and / or survival advantage to cells expressing it compared to cells that do not express it or express it at significantly lower levels. Such a growth and / or survival advantage typically occurs when cells are maintained under specific conditions, i.e., "selective conditions." To ensure effective selection, a population of cells can be maintained under conditions for a sufficient period of time such that cells that do not express the marker do not grow and / or survive and are eliminated from the population or their numbers are reduced to a small fraction of the population. The process of selecting cells that express a marker that confers a growth and / or survival advantage by maintaining a cell population under selective conditions that largely or completely eliminate cells that do not express the marker is referred to herein as "positive selection," and a marker is said to be "useful for positive selection." Negative selection and markers useful for negative selection are also of interest in certain methods described herein. Expression of such a marker confers a growth and / or survival disadvantage on cells that express the marker relative to cells that do not express the marker or express it at significantly lower levels (or, viewed another way, cells that do not express the marker have a growth and / or survival advantage relative to cells that express the marker). Thus, cells that express the marker can be largely or completely eliminated from a cell population if maintained under selective conditions for a sufficient period of time.

[0063] As used herein, "reporter gene" encompasses any gene genetically introduced into a cell that adds to the stem cell phenotype. The reporter genes disclosed herein are intended to encompass fluorescent genes, luminescent genes, enzyme genes, and resistance genes, but also encompass other genes that can be easily detected by those skilled in the art. In some embodiments of the present invention, reporter genes are used as markers to identify specific stem cells, cardiovascular stem cells, and their differentiated progeny. Reporter genes are generally operably linked to sequences that control their expression in a manner that depends on one or more conditions monitored by measuring reporter gene expression. In some cases, reporter gene expression is determined in living cells. When live cell reporter gene assays are used, reporter gene expression can be monitored at multiple time points, for example, 2, 3, 4, 5, 6, 8, or 10 or more time points. In some cases, when a live cell reporter assay is used, reporter gene expression is monitored at a frequency of at least about 10 minutes to about 24 hours, e.g., 20 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, or any other frequency from about 10 minutes to about 24 hours.

[0064] The terms "subject" and "individual" are used interchangeably herein and refer to an animal, e.g., a human, from which cells can be obtained and / or to which cell therapy (including prophylactic therapy) as described herein is provided. For the treatment of an infection, condition, or disease state specific to a particular animal, such as a human subject, the term subject refers to that particular animal. As used interchangeably herein, "non-human animal" and "non-human mammal" include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Preferably, however, the subject is a mammal, such as a human, or a domesticated mammal, e.g., a dog, cat, horse, or other mammal, such as a production mammal, e.g., a cow, sheep, or pig.

[0065] When applied to isolated cells, the terms "treat," "treating," "treatment," and the like include subjecting the cells to any type of process or condition, or performing any type of manipulation or treatment on the cells. When applied to a subject, these terms refer to the medical or surgical attention, care, or management of an individual who is typically ill or injured, or at increased risk of developing a disease compared to the average member of the population, and who is in need of such attention, care, or management.

[0066] As used herein, the terms "treating" and "treatment" refer to administering an effective amount of a composition to a subject so that the subject experiences at least one symptom of a disease or an improvement in the disease, e.g., a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, a decrease in the extent of the disease, a stabilized (i.e., not worsening) state of the disease, a delay or slowing of the progression of the disease, an improvement or palliation of the disease state, and a detectable or undetectable remission (whether partial or complete). Treating can also refer to prolonging survival compared to expected survival in the absence of treatment. Thus, those skilled in the art will recognize that treatment may improve the disease state but may not result in a complete cure of the disease. As used herein, the term "treatment" includes prophylaxis. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment.

[0067] As used herein, the terms "administering," "introducing," and "implanting" are used interchangeably in the context of placing the cells of the present invention into a subject by a method or route that results in at least partial localization of the introduced cells to a desired site. Cells can be directly implanted into the pancreas or digestive tract, or can be administered by a suitable route that results in delivery to a desired location in a subject while at least a portion of the implanted cells or cell components remain viable. The viability of cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to as long as several days or years. In some cases, cells can be administered subcutaneously, e.g., in a capsule (e.g., microcapsule) to maintain the implanted cells at the implantation site and prevent migration of the implanted cells.

[0068] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to administration of stem cell-derived cells and / or their progeny and / or compounds and / or other substances by a method other than direct administration to the central nervous system, such that it enters the animal's system and therefore will undergo metabolic and other similar processes, for example, subcutaneous administration.

[0069] The term "tissue" refers to a group or layer of specialized cells that together perform a particular specialized function. The term "tissue-specific" refers to the source of cells that are derived from a specific tissue.

[0070] As used herein, the terms "reduce," "reduced," "reduction," "reduction," or "inhibit" are all generally used to mean a statistically significant reduction. However, for the avoidance of doubt, "reduced," "reduction," "reduction," or "reduce," or "inhibit" refers to a reduction of at least 10% compared to the reference level, for example, a reduction of at least about 20%, or a reduction of at least about 30%, or a reduction of at least about 40%, or a reduction of at least about 50%, or a reduction of at least about 60%, or a reduction of at least about 70%, or a reduction of at least about 80%, or a reduction of at least about 90%, or a reduction of up to 100% (i.e., absent levels compared to the reference sample), or any reduction between 10% and 100% compared to the reference level.

[0071] The terms "increased," "increase," "enhance," or "activate" are all used herein to generally mean an increase by a statistically significant amount. For the avoidance of doubt, the terms "increase," "increase," "enhance," or "activate" mean an increase of at least 10% compared to a base level, e.g., at least about a 20% increase, or at least about a 30% increase, or at least about a 40% increase, or at least about a 50% increase, or at least about a 60% increase, or at least about a 70% increase, or at least about an 80% increase, or at least about a 90% increase, or up to a 100% increase, or any increase between 10-100% compared to a base level, or at least about a 2-fold increase compared to a base level, or at least about a 3-fold increase, or at least about a 4-fold increase, or at least about a 5-fold increase, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or more compared to a base level.

[0072] The term "statistically significant" or "significantly" refers to statistical significance, generally meaning two standard deviations (2SD) or lower than the normal concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is in fact true. This decision is often made using a p-value.

[0073] stem cells

[0074] Stem cells are cells that retain the ability to renew themselves through mitotic cell division and can differentiate into a variety of specialized cell types. There are two broad types of mammalian stem cells: embryonic stem (ES) cells, found in blastocysts, and adult stem cells, found in adult tissues. In the developing embryo, stem cells can differentiate into all specialized embryonic tissues. In adult organisms, stem and progenitor cells serve as the body's repair system, not only replenishing specialized cells but also maintaining the normal turnover of regenerative organs such as blood, skin, and intestinal tissue. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0075] While specific embodiments are described below with reference to the use of stem cells, germ cells can be used in place of or in conjunction with stem cells to provide at least one differentiated cell using protocols similar to the exemplary protocols described herein. Suitable germ cells can be prepared, for example, from primordial germ cells present in human fetal material collected approximately 8-11 weeks after the last menstrual period. Exemplary methods for preparing germ cells are described, for example, in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S. Patent No. 6,090,622.

[0076] ES cells, such as human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs), have a virtually limitless replicative capacity and the potential to differentiate into most cell types, providing in principle an unlimited starting material for generating differentiated cells for clinical therapy (stemcells.nih.gov / info / scireport / 2006report.htm, 2006).

[0077] hESC cells are described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998), and embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol. Reprod. 55:254, 1996), and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998), can also be used in the methods disclosed herein. mESCs are described, for example, in Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1:Unit 1C.4, 2008). Stem cells may be, for example, unipotent, totipotent, multipotent, or pluripotent. In some examples, any cell of primate origin that can generate progeny that are derivatives of at least one germ cell layer, or all three germ cell layers, can be used in the methods disclosed herein.

[0078] In certain instances, ES cells can be isolated as described, for example, by Cowan et al. (N Engl. J. Med. 350:1353, 2004), and U.S. Patent No. 5,843,780, and by Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995. For example, hESC cells can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Patent No. 6,200,806, Science 282:1145, 1998, Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Equivalent cell types to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as reviewed in, for example, WO 01 / 51610 (Bresagen). hESCs can also be derived from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or human one-cell embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). The inner cell mass can be isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes, washed three times with DMEM for 5 minutes, and then exposed to a 1:5 dilution of guinea pig complement (Gibco) for 3 minutes (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes with DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM is plated onto mEF feeder layers.After 9–15 days, inner cell mass-derived outgrowths can be dissociated into clumps by exposure to phosphate-buffered saline (PBS) containing 1 mM EDTA without calcium or magnesium, by exposure to dispase or trypsin, or by mechanical dissociation using a micropipette, and then replated onto mEFs in fresh medium. Growing colonies with undifferentiated morphology can be individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized by small colonies with a clearly high nucleus-to-cytoplasm ratio and prominent nucleoli. The resulting hESCs can be split periodically every 1–2 weeks, for example, by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (approximately 200 U / mL; Gibco), or by selecting individual colonies with a micropipette. In some examples, clump sizes of approximately 50–100 cells are optimal. mESC cells can be prepared, for example, using the technique described by Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[0079] Embryonic stem cells can be isolated from blastocysts of members of primate species (U.S. Patent No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastocyst cells using the techniques described by Thomson et al. (U.S. Patent No. 6,200,806, Science 282:1145, 1998, Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Cell types equivalent to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as outlined in WO01 / 51610 (Bresagen).

[0080] Alternatively, in some embodiments, hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or human one-cell embryos can be expanded to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Embryos are cultured to the blastocyst stage in G1.2 and G2.2 media (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developed blastocysts by brief exposure to pronase (Sigma). The inner cell mass is isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes, followed by three 5-minute washes with DMEM, and a 3-minute exposure to a 1:5 dilution of guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes with DMEM, lysed trophectoderm cells are removed from the intact inner cell mass (ICM) by gentle pipetting, and the ICM is plated onto mEF feeder layers.

[0081] After 9–15 days, the inner cell mass-derived outgrowths are dissociated into clumps by exposure to phosphate-buffered saline (PBS) containing 1 mM EDTA without calcium or magnesium, by exposure to dispase or trypsin, or by mechanical dissociation using a micropipette, and then replated onto mEFs in fresh medium. Growing colonies with undifferentiated morphology are individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized by small colonies with a clearly high nucleus-to-cytoplasm ratio and prominent nucleoli. The resulting ES cells are split periodically every 1–2 weeks by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (approximately 200 U / mL; Gibco), or by selecting individual colonies with a micropipette. Clump sizes of approximately 50–100 cells are optimal.

[0082] In some embodiments, human embryonic germ (hEG) cells are pluripotent stem cells that can be used to differentiate into primitive endoderm cells using methods as disclosed herein. hEG cells can be prepared from primordial germ cells present in human fetal material collected approximately 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998, and U.S. Patent No. 6,090,622, which are incorporated herein by reference in their entireties.

[0083] Briefly, genital ridges are treated to form disaggregated cells. EG growth medium is DMEM, 4500 mg / L D-glucose, 2200 mg / L mM NaHCO3, 15% ES-qualified fetal bovine serum (BRL), 2 mM glutamine (BRL), 1 mM sodium pyruvate (BRL), 1000-2000 U / mL human recombinant leukemia inhibitory factor (LIF, Genzyme), 1-2 ng / mL human recombinant bFGF (Genzyme), and 10 μM forskolin (10% in DMSO). A 96-well tissue culture plate is prepared with a subconfluent layer of feeder cells (e.g., STO cells, ATCC No. CRL1503) cultured for 3 days in modified EG growth medium without LIF, bFGF, or forskolin inactivated by 5000 rad γ-irradiation. Approximately 0.2 mL of primary germ cell (PGC) suspension is added to each well. The first passage is performed after 7-10 days in EG growth medium, transferring each well into one well of a 24-well culture dish previously prepared with irradiated STO mouse fibroblasts. Culture the cells with daily changes of medium until a cell morphology consistent with EG cells is observed, typically after 7-30 days or 1-4 passages.

[0084] In some examples, stem cells may be undifferentiated (e.g., cells not committed to a particular lineage) before being exposed to at least one maturation factor according to the methods disclosed herein, while in other examples, it may be desirable to differentiate stem cells into one or more intermediate cell types before being exposed to at least one maturation factor(s) described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics of undifferentiated cells that can be used to distinguish them from differentiated cells of embryonic or adult origin. In some examples, undifferentiated cells may be visible in colonies of cells viewed two-dimensionally under a microscope, with a high nuclear-to-cytoplasmic ratio and prominent nucleoli. Stem cells may be used by themselves (e.g., in the substantial absence of undifferentiated cells) or in the presence of differentiated cells. In some examples, stem cells may be cultured in the presence of appropriate nutrients and, optionally, other cells, to allow the stem cells to proliferate and, optionally, differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to support stem cell growth. Fibroblasts may be present during some stages of stem cell proliferation, but not necessarily during all stages. For example, fibroblasts may be added to the stem cell culture during an initial culture step, but not during one or more subsequent culture steps.

[0085] Stem cells used in all aspects of the present invention can be any cell derived from any type of tissue (e.g., embryonic tissue, such as fetal or prefetal tissue, or adult tissue), characterized in that, under appropriate conditions, they can generate progeny of different cell types, e.g., derivatives of all three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines, or can be obtained directly from primary embryonic tissue and immediately used for differentiation. Cells listed in the NIH Human Embryonic Stem Cell Registry include hESBGN-01, hESBGN-02, hESBGN-03, and hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, and HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1 and HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, and H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into stem cell-derived cells did not involve the destruction of human embryos.

[0086] In another embodiment, stem cells can be isolated from tissues, including solid tissues. In some embodiments, the tissue is skin, fat tissue (e.g., adipose tissue), muscle tissue, heart, or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0087] Stem cells of interest also include various types of embryonic cells, exemplified by human embryonic stem (hES) cells, as described by Thomson et al. (1998) Science 282:1145; embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineage-committed stem cells, such as mesodermal stem cells and other early cardiac progenitor cells (see, e.g., Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) Circ Res. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, horse, cow, pig, dog, cat, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos were not destroyed for the source of pluripotent cells used in the methods and compositions as disclosed herein.

[0088] ES cells are considered undifferentiated when they are not committed to a specific lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated ES cells are easily recognized by those skilled in the art and typically appear in colonies of cells with a high nuclear-to-cytoplasmic ratio and prominent nucleoli when viewed two-dimensionally under a microscope. Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells, and their polypeptide products can be used as markers for negative selection. See, e.g., U.S. Patent Application No. 2003 / 0224411 A1; Bhattacharya (2004) Blood 103(8):2956-64; and Thomson (1998), supra, each of which is incorporated herein by reference. Human ES cell lines express cell surface markers characteristic of undifferentiated non-human primate ES cells and human EC cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. GL7, a globo-series glycolipid carrying the SSEA-4 epitope, is formed by the addition of sialic acid to GbS, a globo-series glycolipid carrying the SSEA-3 epitope. GL7 therefore reacts with antibodies against both SSEA-3 and SSEA-4. Undifferentiated human ES cell lines did not stain with SSEA-1, whereas differentiated cells stained strongly with SSEA-1. Methods for expanding undifferentiated hES cells are described in WO99 / 20741, WO01 / 51616, and WO03 / 020920.

[0089] A mixture of cells from a suitable source of endothelial, muscle, and / or neural stem cells can be harvested from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (i.e., recruited) circulating peripheral blood, may be removed from a subject. Alternatively, bone marrow may be harvested from a mammal, such as a human patient undergoing an autologous transplant. In some embodiments, stem cells can be obtained from a subject's adipose tissue using, for example, Cytori's CELUTION™ SYSTEM, as disclosed in U.S. Pat. Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entireties.

[0090] In some embodiments, human umbilical cord blood cells (HUCBCs) are useful in the methods disclosed herein. Human UBC cells are recognized as a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Until now, umbilical cord and placental blood have been considered waste products that are typically discarded at the birth of an infant. Umbilical cord blood cells have been used as a source of transplantable stem and progenitor cells and as a source of bone marrow-repopulating cells for the treatment of malignant diseases (i.e., acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, and neuroblastoma) and non-malignant diseases such as Fanconi anemia and aplastic anemia (Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79;1874-1881; Lu et al., 1996 Crit.Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503). A distinct advantage of HUCBCs is that these cells have an immature immune response very similar to fetal cells, which significantly reduces the risk of rejection by the host (Taylor & Bryson, 1985 J. Immunol. 134:1493-1497).Human umbilical cord blood contains mesenchymal and hematopoietic progenitor cells, as well as endothelial cell precursors that can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113; Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422; Wagner et al., 1992 Blood 79;1874-1881; Lu et al., 1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503; Taylor & Bryson, 1985 J. Immunol. 134:1493-1497; Broxmeyer, 1995 Transfusion 35:694-702;Chen et al., 2001 Stroke 32:2682-2688;Nieda et al., 1997 Br. J. Haematology 98:775-777, Erices et al., 2000 Br. J. Haematology 109:235-242). The total hematopoietic progenitor cell content in umbilical cord blood is comparable to or exceeds that of bone marrow, and highly proliferative hematopoietic cells express hematopoietic markers such as CD14, CD34, and CD45 at an eight-fold higher concentration in HUCBC than in bone marrow (Sanchez-Ramos et al., 2001 Exp. Neur. 171:109-115; Bicknese et al., 2002 Cell Transplantation 11:261-264; Lu et al., 1993 J. Exp Med. 178:2089-2096).

[0091] In another embodiment, the pluripotent cells are cells in a hematopoietic microenvironment such as circulating peripheral blood, preferably the mononuclear fraction of mammalian peripheral blood, umbilical cord blood, bone marrow, fetal liver, or yolk sac. Stem cells, particularly neural stem cells, may be derived from the central nervous system, including the meninges.

[0092] In another embodiment, pluripotent cells present in embryoid bodies are formed by harvesting ES cells through brief protease digestion and growing small aggregates of undifferentiated human ESCs in suspension culture. Differentiation is induced by removing the conditioned medium. The resulting embryoid bodies are plated on a semi-solid substrate. The formation of differentiated cells is observed after about 7 days to about 4 weeks. To select viable differentiated cells from in vitro cultures of stem cells, embryoid bodies or similar structures are partially dissociated to obtain cell aggregates. Aggregates containing cells of interest are then selected for phenotypic characteristics using methods that substantially maintain contact between cells within the aggregates.

[0093] In another embodiment, the stem cells can be reprogrammed stem cells, such as stem cells derived from somatic cells or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic, tumor, and cancer cells, or alternatively derived reprogrammed cells, such as induced pluripotent stem cells, or iPS cells.

[0094] Cloning and cell culture

[0095] Exemplary methods of molecular genetics and genetic engineering that can be used in the techniques described herein can be found, for example, in the current editions of Molecular Cloning: A Laboratory Manual (Sambrook et al., Cold Spring Harbor), Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.), and Current Protocols in Molecular Biology (F.M. Ausubel et al. eds., Wiley & Sons). Methods for cell biology, protein chemistry, and antibody techniques can be found, for example, in Current Protocols in Protein Science (J.E. Colligan et al. eds., Wiley & Sons), Current Protocols in Cell Biology (J.S. Bonifacino et al., Wiley & Sons), and Current Protocols in Immunology (J.E. Colligan et al. eds., Wiley & Sons). Exemplary reagents, cloning vectors, and kits for genetic manipulation can be obtained commercially from, for example, BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0096] Suitable cell culture methods can be found, for example, in the latest editions of "Culture of Animal Cells: A Manual of Basic Technique" (RI Freshney ed., Wiley & Sons), "General Techniques of Cell Culture" (MA Harrison & IF Rae, Cambridge University Press), and "Embryonic Stem Cells: Methods and Protocols" (K. Turksen ed., Humana Press). Suitable tissue culture supplies and reagents are commercially available from, for example, Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

[0097] Pluripotent stem cells can be continuously propagated in culture by those skilled in the art using culture conditions that promote proliferation without promoting differentiation. An exemplary serum-containing ES medium is made with 80% DMEM (e.g., Knock-Out DMEM, Gibco), 20% defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO98 / 30679), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Human bFGF is added to a concentration of 4 ng / mL immediately before use (WO99 / 20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.

[0098] Multipotent SCs can be maintained in an undifferentiated state even without feeder cells. The environment for feeder-free culture includes an appropriate culture substrate, particularly an extracellular matrix such as Matrigel® or laminin. Enzymatic digestion is typically stopped before the cells are completely dispersed (e.g., about 5 minutes with collagenase IV). Subsequently, clumps of approximately 10–2,000 cells are plated directly onto the substrate without further dispersion.

[0099] Feeder-free culture is supported by a nutrient medium containing factors that support cell growth without differentiation. Such factors can be introduced into the medium by culturing the medium with cells that secrete such factors, such as irradiated (approximately 4,000 rad) primary mouse embryonic fibroblasts, telomerized mouse fibroblasts, or fibroblast-like cells derived from pPS cells. The medium is cultured at approximately 5-6 x 10 cells / well in a serum-free medium such as KO DMEM supplemented with 20% serum replacement and 4 ng / mL bFGF. 4 cm -2 The medium can be conditioned by plating feeders at a density of 1000-150 ...

[0100] Under a microscope, ES cells appear with a high nuclear-to-cytoplasmic ratio, prominent nucleoli, and small colonies with poorly discernible cell junctions. Primate ES cells express markers detectable using antibodies designated stage-specific embryonic antigens (SSEA) 3 and 4, as well as Tra-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998). Mouse ES cells can be used as a positive control for SSEA-1 and a negative control for SSEA-4, Tra-1-60, and Tra-1-81. SSEA-4 is consistently present in human embryonic carcinoma (hEC) cells. In vitro differentiation of pluripotent stem cells results in the loss of expression of SSEA-4, Tra-1-60, and Tra-1-81, while increased expression of SSEA-1, which is also seen in undifferentiated hEC cells, is observed.

[0101] Method for producing hypoimmunogenic cells

[0102] Aspects of the present disclosure relate to producing hypoimmunogenic cells. Hypoimmunogenic cells can include pancreatic islet cells or β cells, for example, stem cell-derived β cells. In some embodiments, stem cells or progenitor cells are engineered to express one or more tolerogenic transgenes from intracellular loci, and the tolerogenic transgenes are not silenced when the cells differentiate into the desired cell type. The engineered stem cells or progenitor cells can be exposed to appropriate conditions to differentiate the cells into the desired cell type, for example, SC islet cells or SC-β cells.

[0103] In some embodiments, the stem or progenitor cells are pluripotent stem cells, e.g., iPSCs, pancreatic progenitor cells, non-native endocrine cells, or beta cell progenitors. In some embodiments, the stem or progenitor cells can be engineered to express one or more tolerogenic transgenes from a locus within the stem or progenitor cell, where the tolerogenic transgenes are not silenced during differentiation. Non-limiting examples of tolerogenic transgenes include PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single-chain fusion, soluble PDL1-Ig, and CTLA4-Ig. In one embodiment, the tolerogenic agent is an HLA-E single-chain fusion. The single-chain fusion can be loaded with a peptide capable of activating NK cells, for example, through interaction with the inhibitory receptors NKG2A and / or NKG2C. In some embodiments, the peptide can be VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12) and / or VMAPRALLL (SEQ ID NO: 13).

[0104] In some embodiments, stem or progenitor cells can be engineered to express one or more immunomodulatory agents from a locus within the stem or progenitor cell, wherein the one or more immunomodulatory agents are not silenced during differentiation of the cells. In some embodiments, the immunomodulatory agent comprises a cytokine. In particular embodiments, the immunomodulatory agent comprises IL-10, TGF-β, and / or IL-2. In one embodiment, the immunomodulatory agent is a cytokine. In one embodiment, the immunomodulatory agent is IL-10. In one embodiment, the immunomodulatory agent is TGF-β. In one embodiment, the immunomodulatory agent is IL-2.

[0105] In some embodiments, the expression of one or more genes in stem cells or progenitor cells is modified by using any gene editing tool known to those skilled in the art (for example, TALENS, CRISPR, etc.).In some embodiments, the gene editing tool is delivered to stem cells using retrovirus (for example, lentivirus).In some embodiments, gene editing (for example, CRISPR) can be used to target one or more genes to regulate the expression of one or more genes.

[0106] In some embodiments, the engineered stem or progenitor cells, e.g., iPSC cells or beta cell precursor cells, are differentiated into beta cells, e.g., SC-beta cells. In some embodiments, the engineered stem or progenitor cells, e.g., iPSC cells or beta cell precursor cells, are differentiated into pancreatic islets, e.g., SC islets. In some embodiments, the engineered stem or progenitor cells are differentiated into a desired cell type, e.g., beta cells, that secrete one or more immunomodulatory agents from an intracellular locus.

[0107] In some embodiments, the stem or progenitor cells are engineered to express one or more tolerogenic transgenes from a locus within the stem or progenitor cells. In some embodiments, the stem or progenitor cells are engineered to express one or more immunomodulatory agents from a locus within the stem or progenitor cells. In some embodiments, the locus is constitutively expressed in all cells of a pancreatic islet, e.g., a human pancreatic islet. In some embodiments, the locus is constitutively expressed in beta cells, e.g., human beta cells. The locus can be a locus for a housekeeping gene. In some embodiments, the housekeeping gene comprises actin, ubiquitin, and / or GAPDH. In one embodiment, the housekeeping gene is GAPDH.

[0108] The at least one hypoimmunogenic cell or its precursor, e.g., stem or progenitor cell, can be derived from any mammalian species, including, but not limited to, murine, bovine, simian, porcine, equine, ovine, or human cells. While the method descriptions herein refer to mammalian hypoimmunogenic cells or their precursors for clarity and simplicity, it should be understood that all methods described herein can be readily applied to hypoimmunogenic cells or their precursors of other cell types. In some embodiments, the hypoimmunogenic cells or their precursors are derived from a human individual.

[0109] In some embodiments, the hypoimmunogenic cells or their precursors are a substantially pure population of hypoimmunogenic cells or their precursors. In one embodiment, the hypoimmunogenic cells or their precursors are a substantially pure population of hypoimmunogenic beta cells or their precursors. In some embodiments, the population of hypoimmunogenic cells or their precursors comprises a mixture of pluripotent or differentiated cells (e.g., a mixture of SC-beta cells and / or other differentiated cell types). In some embodiments, the SC islet (e.g., hypoimmunogenic SC islet) comprises a mixture of pluripotent or differentiated cells (e.g., a mixture of SC-beta cells and / or other differentiated cell types). In some embodiments, the population of hypoimmunogenic SC islet cells or their precursors is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0110] Low immunogenic cells

[0111] In some aspects of the present disclosure, hypoimmunogenic cells (e.g., hypoimmunogenic beta cells) are provided. The hypoimmunogenic cells disclosed herein share many distinguishing characteristics of native pancreatic cells, but differ in certain aspects. In some embodiments, the hypoimmunogenic cells are non-native, i.e., non-endogenous cells that do not occur in nature. As used herein, "non-native" means that the modified hypoimmunogenic cells differ significantly in some aspects from cells found in nature, i.e., native cells. However, it should be recognized that although hypoimmunogenic cells may exhibit certain differences due to such significant differences, they may behave similarly to native cells (e.g., with respect to insulin secretion in the case of beta cells), with certain functions altered (e.g., improved) compared to native cells.

[0112] The present invention is not intended to be limited to the starting cells from which the hypoimmunogenic cells are derived; hypoimmunogenic cells can be differentiated in vitro from any starting cell. Exemplary starting cells include, but are not limited to, endocrine cells or their precursors, such as NKX6-1+ pancreatic progenitor cells and Pdx1+ pancreatic progenitor cells, as well as pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. In some embodiments, hypoimmunogenic cells are differentiated in vitro from reprogrammed cells, partially reprogrammed cells (i.e., somatic cells, e.g., fibroblasts partially reprogrammed to exist in an intermediate state between induced pluripotent cells and the somatic cells from which they were derived), or transdifferentiated cells. In some embodiments, the hypoimmunogenic cells disclosed herein can be differentiated in vitro from endocrine cells or their precursors. In some embodiments, hypoimmunogenic cells are differentiated in vitro from stem or progenitor cells, which may include beta cell precursors or pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. In some embodiments, the hypoimmunogenic cells or the pluripotent stem cells from which the hypoimmunogenic cells are derived are human. In some embodiments, the hypoimmunogenic cells are human.

[0113] In some embodiments, hypoimmunogenic cells (e.g., SC-β cells) are engineered to express or overexpress one or more tolerogenic transgenes from a locus within the stem or progenitor cells. Non-limiting examples of tolerogenic transgenes include PDL1, HLA-E / G, CD47, SERPINB9, CCL21, FASL, CD200, MFGE8, CD55, CD46, HLS-G single-chain fusion, soluble PDL1-Ig, and CTLA4-Ig. In one embodiment, the tolerogenic agent is an HLA-E single-chain fusion. The single-chain fusion can be loaded with a peptide capable of activating NK cells, for example, through interaction with the inhibitory receptors NKG2A and / or NKG2C. In some embodiments, the peptide can be VMAPRTLL (SEQ ID NO: 3), VMAPRTLFL (SEQ ID NO: 4), VMAPRTLVL (SEQ ID NO: 5), VMAPRTLIL (SEQ ID NO: 6), IMAPRTLVL (SEQ ID NO: 7), VMPPRTLLL (SEQ ID NO: 8), VMAPRTVLL (SEQ ID NO: 9), VTAPRTLLL (SEQ ID NO: 10), VTAPRTVLL (SEQ ID NO: 11), VMAPRTLTL (SEQ ID NO: 12) and / or VMAPRALLL (SEQ ID NO: 13).

[0114] In some embodiments, hypoimmunogenic cells (e.g., SC-β cells) are engineered to express or overexpress one or more immunomodulatory agents from a locus within the stem or progenitor cell. In some embodiments, the immunomodulatory agent comprises a cytokine. In particular embodiments, the immunomodulatory agent comprises IL-10, TGF-β, and / or IL-2. In one embodiment, the immunomodulatory agent is a cytokine. In one embodiment, the immunomodulatory agent is IL-10. In one embodiment, the immunomodulatory agent is TGF-β. In one embodiment, the immunomodulatory agent is IL-2.

[0115] In some embodiments, the locus is constitutively expressed in all cells of a pancreatic islet, e.g., a human pancreatic islet. In some embodiments, the locus is constitutively expressed in beta cells, e.g., human beta cells. The locus can be a housekeeping gene locus. In some embodiments, the housekeeping gene comprises actin, ubiquitin, and / or GAPDH. In one embodiment, the housekeeping gene is GAPDH.

[0116] Hypoimmunogenic cells may exhibit a reduced risk of autologous and / or allogeneic rejection upon transplantation. In some embodiments, hypoimmunogenic cells secrete one or more immunomodulatory agents. For example, hypoimmunogenic cells (e.g., hypoimmunogenic beta cells) secrete cytokines such as IL-10, TGF-β, and IL-2, or modified IL-2.

[0117] In some aspects, the present disclosure provides cell lines comprising the hypoimmunogenic cells (e.g., hypoimmunogenic beta cells) described herein. In some aspects, the present disclosure provides SC islets comprising hypoimmunogenic stem cell-derived beta cells.

[0118] In some embodiments, the cells described herein, e.g., a population of hypoimmunogenic cells, can be transplanted, e.g., the population of hypoimmunogenic cells can be administered to a subject. In some embodiments, SC islets comprising hypoimmunogenic cells (e.g., hypoimmunogenic SC-β cells) can be transplanted, e.g., the SC islets can be administered to a subject. In some embodiments, the subject receiving the population of hypoimmunogenic cells is the same subject from which the pluripotent stem cells used to differentiate into the hypoimmunogenic cells were obtained (e.g., for autologous cell therapy). In some embodiments, the subject is a different subject. In some embodiments, the subject is either suffering from diabetes, such as insulin-dependent diabetes, or is a normal subject. For example, the transplant cells (e.g., a composition comprising a population of hypoimmunogenic cells, or SC islets comprising a population of hypoimmunogenic cells) can be in a form suitable for transplantation, e.g., organ transplantation.

[0119] The method may further include administering the cells to a subject in need thereof, e.g., a mammalian subject, e.g., a human subject. The source of the cells may be a mammal, preferably a human. The source or recipient of the cells may also be a non-human subject, e.g., an animal model. The term "mammal" includes organisms including mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Similarly, transplantable cells can be obtained from any of these organisms, including non-human transgenic organisms. In one embodiment, the transplantable cells are genetically engineered, e.g., the cells are genetically engineered to contain an exogenous gene or to inactivate or modify an endogenous gene.

[0120] Compositions comprising a population of hypoimmunogenic cells (e.g., hypoimmunogenic pancreatic stem cell-derived cells such as SC-β cells) can be administered to a subject using an implantable device. Implantable devices and related technology are known in the art and are useful as delivery systems when continuous or timed-release delivery of the compounds or compositions described herein is desired. Furthermore, implantable device delivery systems are useful for targeting specific points of compound or composition delivery (e.g., localized sites, organs) (Negrin et al., Biomaterials, 22(6):563(2001)). Timed-release technologies involving alternative delivery methods can also be used in the present invention. For example, timed-release formulations based on polymeric, sustained-release, and encapsulation technologies (e.g., polymers, liposomes) can also be used for delivery of the compounds and compositions described herein.

[0121] For administration to a subject, a cell population, e.g., a population of hypoimmunogenic cells (e.g., hypoimmunogenic β cells) or SC islets comprising hypoimmunogenic cells, produced by the methods disclosed herein can be administered to a subject, for example, in a pharmaceutically acceptable composition. These pharmaceutically acceptable compositions comprise a therapeutically effective amount of the hypoimmunogenic cell population described above formulated together with one or more pharmaceutically acceptable carriers (excipients) and / or diluents.

[0122] As described in detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., buccal, sublingual, and those targeted for systemic absorption), boluses, powders, granules, or pastes for application to the tongue; (2) parenteral administration, e.g., as a sterile solution or suspension, or as a sustained-release formulation, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection; (3) topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; (5) sublingual administration; (6) ophthalmic administration; (7) transdermal administration; (8) transmucosal administration; or (9) nasal administration. Additionally, the compounds can be implanted into a patient or injected using a drug delivery system. See, e.g., Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24: 199-236 (1984), Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981), U.S. Pat. No. 3,773,919, and U.S. Pat. No. 353,270,960.

[0123] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0124] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or steric acid), or solvent encapsulating material that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Examples of substances that can function as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol Cholesterol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C12 alcohols, such as ethanol; and (25) other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweeteners, flavoring agents, perfumes, preservatives, and antioxidants may also be present in the formulation.The terms "excipient," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably herein.

[0125] As used herein with respect to a cell population, the phrase "therapeutically effective amount" refers to an amount of a cell population, e.g., hypoimmunogenic cells, or relevant cells in a composition comprising the hypoimmunogenic cells of the present invention, effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment. For example, the amount of a population of hypoimmunogenic cells administered to a subject is sufficient to produce a statistically significant and measurable change in at least one symptom of type 1, type 1.5, or type 2 diabetes, such as glycosylated hemoglobin levels, fasting blood glucose levels, hypoinsulinemia, etc. Determining a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. Generally, a therapeutically effective amount may vary depending on the subject's medical history, age, condition, and sex, as well as the severity and type of the subject's condition and the administration of other pharmaceutically active agents.

[0126] As used herein, the term "administering" refers to placing a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site so that a desired effect occurs. The compounds or compositions described herein may be administered by any suitable route known in the art, including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, respiratory (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration.

[0127] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or oral ingestion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In a preferred embodiment, the composition is administered by intravenous infusion or injection.

[0128] "Treating," "preventing," or "ameliorating" a disease or disorder means delaying or preventing the onset of such disease or disorder, reversing, alleviating, ameliorating, inhibiting, slowing or halting the progression, severity, or worsening of the conditions associated with such disease or disorder. In one embodiment, symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0129] Treatment for diabetes is determined by standard medical methods. The goal of diabetes treatment is to lower blood sugar levels as close to normal as safely possible. Commonly established targets are 80-120 milligrams per deciliter (mg / dl) before meals and 100-140 mg / dl at bedtime. A particular physician may set different goals for a patient depending on other factors, such as the frequency of the patient's hypoglycemic reactions. Useful medical tests include testing the patient's blood and urine to measure blood glucose levels, glycosylated hemoglobin levels (HbA1c; a measure of average blood glucose levels over the past 2-3 months; normal range is 4-6%), cholesterol and fat levels, and urine protein levels. Such tests are standard tests known to those skilled in the art (see, e.g., American Diabetes Association, 1998). A successful treatment program can also be determined by the low number of patients undergoing the program who have diabetes-related complications, such as eye, kidney, or nerve problems.

[0130] Delaying the onset of diabetes in a subject refers to delaying the onset of at least one symptom of diabetes, e.g., hyperglycemia, hypoinsulinemia, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, or a combination thereof, by at least 1 week, at least 2 weeks, at least 1 month, at least 2 months, at least 6 months, at least 1 year, at least 2 years, at least 5 years, at least 10 years, at least 20 years, at least 30 years, at least 40 years or more, including the entire lifespan of the subject.

[0131] In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient" and "subject" are used interchangeably herein. Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects representing animal models of type 1 diabetes, type 2 diabetes, or a pre-diabetic state. Furthermore, the methods described herein can be used to treat livestock animals and / or pets. The subject may be male or female. The subject may be a person who has been previously diagnosed with or identified as having diabetes (e.g., type 1 or type 2), one or more complications associated with diabetes, or a pre-diabetic state, and optionally need not have already been treated for diabetes, one or more complications associated with diabetes, or a pre-diabetic state. The subject may also be a person who does not have diabetes or a pre-diabetic state. The subject may also be a person who has been diagnosed with or identified as having diabetes, one or more complications related to diabetes, or a prediabetic condition, but who has received one or more treatments for diabetes, one or more complications related to diabetes, or a prediabetic condition and has shown improvement in known diabetes risk factors. Alternatively, the subject may not have been previously diagnosed with diabetes, one or more complications related to diabetes, or a prediabetic condition. For example, the subject may exhibit one or more risk factors for diabetes, one or more complications related to diabetes, or a prediabetic condition, or a subject who does not exhibit diabetes risk factors, or a subject who is asymptomatic for diabetes, one or more complications related to diabetes, or a prediabetic condition. The subject may also be a person who has or is at risk of developing diabetes or a prediabetic condition. The subject may also be a person who has been diagnosed with or identified as having one or more complications related to diabetes or a prediabetic condition as defined herein, or a subject who has not previously been diagnosed with or identified as having one or more complications related to diabetes or a prediabetic condition.

[0132] As used herein, the phrase "subject in need of hypoimmunogenic beta cells" refers to a subject who has been diagnosed with, is suffering from, has, or is identified as being at risk of developing diabetes (e.g., type 1, type 1.5, or type 2), one or more complications associated with diabetes, or a pre-diabetic condition.

[0133] Subjects who require a population of hypoimmunogenic beta cells can be identified using any method used to diagnose diabetes. For example, type 1 diabetes can be diagnosed using glycosylated hemoglobin (A1C) testing, random blood glucose testing, and / or fasting blood glucose testing. Parameters for diagnosing diabetes are known in the art and can be obtained by those skilled in the art without much effort.

[0134] In some embodiments, the methods of the present invention further include selecting a subject identified as needing additional hypoimmunogenic beta cells. Subjects in need of a hypoimmunogenic beta cell population can be selected based on the symptoms they exhibit, such as symptoms of type 1, type 1.5, or type 2 diabetes. Exemplary symptoms of diabetes include, but are not limited to, excessive thirst (polydipsia), frequent urination (polyuria), extreme hunger (hyperphagia), extreme fatigue, weight loss, hyperglycemia, low levels of insulin, hyperglycemia (e.g., glucose levels greater than 250 mg, greater than 300 mg), the presence of ketones in the urine, fatigue, dry and / or itchy skin, blurred vision, slow-healing cuts or sores, more infections than usual, numbness and tingling in the feet, diabetic retinopathy, diabetic nephropathy, blindness, memory loss, renal failure, cardiovascular disease (including coronary artery disease, peripheral artery disease, cerebrovascular disease, atherosclerosis, and hypertension), neuropathy, autonomic dysfunction, hyperglycemic hyperosmolar coma, and combinations thereof.

[0135] In some embodiments, a composition comprising a population of hypoimmunogenic beta cells for administration to a subject may further comprise a pharmaceutically active agent, such as an agent known in the art for the treatment of diabetes and / or for having antihyperglycemic activity (e.g., dipeptidyl peptidase 4 (DPP-4) inhibitors (e.g., alogliptin, linagliptin, saxagliptin, sitagliptin, vildagliptin, and berberine), biguanides (e.g., metformin, buformin, and phenformin), peroxisome proliferator-activated receptor (PPAR) modulators such as thiazolidinediones (TZDs) (e.g., pioglitazone, rivoglitazone, rosiglitazone, and troglitazone), dual PPAR agonists (e.g., aleglitazar, muraglitazar, and tesaglitazar), sulfonylureas (e.g., acetophenone), and the like. cyclosporine, ... T2) inhibitors (e.g., dapagliflozin, remogliflozin, and sergliflozin), and others (e.g., benfluorex and trestat).

[0136] In some embodiments, the composition comprising a population of hypoimmunogenic beta cells for administration to a subject may further comprise a pharmaceutically active agent, e.g., an agent known in the art for suppressing the immune system, i.e., an immunosuppressant. Non-limiting examples of immunosuppressants include biologics (e.g., adalimumab and infliximab), calcineurin inhibitors (e.g., tacrolimus and cyclosporine), corticosteroids (e.g., prednisone), inosine monophosphate dehydrogenase (IMDH) inhibitors (e.g., mycophenolate mofetil), Janus kinase inhibitors (e.g., tofacitinib), mechanistic target of rapamycin (mTOR) inhibitors (e.g., sirolimus), and monoclonal antibodies (e.g., basiliximab).

[0137] The composition comprising hypoimmunogenic cells can be administered to a subject simultaneously with or at different times than the administration of a pharmaceutically active agent or composition comprising the same. When administered at different times, the composition comprising a population of hypoimmunogenic cells and / or the composition comprising a pharmaceutically active agent for administration to a subject can be administered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, or 24 hours of the other. When the composition comprising a population of hypoimmunogenic cells and the composition comprising a pharmaceutically active agent are administered as different pharmaceutical compositions, the routes of administration may be different. In some embodiments, a subject is administered a composition comprising hypoimmunogenic cells. In other embodiments, a subject is administered a composition comprising a pharmaceutically active agent. In another embodiment, a subject is administered a composition comprising a population of hypoimmunogenic cells mixed with a pharmaceutically active agent. In another embodiment, a subject is administered a composition comprising a population of hypoimmunogenic cells and a composition comprising a pharmaceutically active agent, the administrations being substantially simultaneous or subsequent to each other.

[0138] Toxicity and therapeutic efficacy of administration of a composition comprising a population of hypoimmunogenic cells can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). Compositions comprising a population of hypoimmunogenic cells that exhibit large therapeutic indices are preferred.

[0139] The amount of a composition containing a population of low immunogenic cells can be tested using several well-established animal models.

[0140] Non-obese diabetic (NOD) mice have a genetic defect that results in insulitis that manifests within a few weeks of age (Yoshida et al., Rev. Immunogenet. 2:140, 2000). 60-90% of female mice develop overt diabetes by 20-30 weeks of age. The immune-mediated pathology appears to be similar to that of human type 1 diabetes. Other models of type 1 diabetes are mice with transgene and knockout mutations (Wong et al., Immunol. Rev. 169:93, 1999). Recently, Lenzen et al. reported a rat model of spontaneous type 1 diabetes (Diabetologia 44:1189, 2001). Hyperglycemia can also be induced in mice (glucose >500 mg / dL) by a single intraperitoneal injection of streptozotocin (Soria et al., Diabetes 49:157, 2000) or by sequential low doses of streptozotocin (Ito et al., Environ. Toxicol. Pharmacol. 9:71, 2001). To assess the efficacy of transplanted islet cells, mice are monitored for the return of glucose to normal levels (<200 mg / dL).

[0141] Large animals provide a good model for tracking the sequelae of chronic hyperglycemia. Dogs can be made insulin-dependent by removing their pancreas (J. Endocrinol. 158:49, 2001) or by feeding them galactose (Kador et al., Arch. Opthalmol. 113:352, 1995). The Keeshond is a genetic model of type 1 diabetes (Am. J. Pathol. 105:194, 1981). Early studies using the canine model (Banting et al., Can. Med. Assoc. J. 22:141, 1922) led to the discovery of a group of two or three Canadians who embarked on a long sea voyage to Stockholm in February 1925.

[0142] In some embodiments, data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized.

[0143] A therapeutically effective amount of a composition comprising a population of hypoimmunogenic cells can be estimated initially from cell culture assays, or the effect of any particular dosage can be monitored by a suitable bioassay.

[0144] Regarding the duration and frequency of treatment, a skilled clinician typically monitors the subject to determine if the treatment is providing therapeutic benefit and decides whether to increase or decrease the dosage, increase or decrease the frequency of administration, discontinue treatment, resume treatment, or make other changes to the treatment regimen. The administration schedule can vary from once a week to daily, depending on various clinical factors, such as the subject's sensitivity to stem cell-derived cells. The desired dose can be administered all at once or divided into subdoses, e.g., two to four subdoses, administered over a period of time, e.g., at appropriate intervals throughout the day, or according to any other suitable schedule. Such subdoses can be administered as a unit dosage form. In some embodiments, administration is chronic, e.g., one or more doses per day over a period of several weeks or months. Exemplary administration schedules include daily, twice-daily, three-daily, or four-daily or more doses for periods of one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, or six months or longer.

[0145] In another aspect of the present invention, methods provide for the use of the isolated population of hypoimmunogenic cells disclosed herein. In one embodiment of the present invention, the isolated population of hypoimmunogenic cells disclosed herein can be used to generate a pharmaceutical composition for use in transplantation into a subject in need of treatment, for example, a subject with diabetes or at risk of developing diabetes, including, but not limited to, subjects with congenital and acquired diabetes. In one embodiment, the isolated population of hypoimmunogenic cells can be genetically modified. In another aspect, the subject can have or be at risk for diabetes and / or metabolic disorders. In some embodiments, the isolated population of hypoimmunogenic cells disclosed herein can be autologous and / or allogeneic. In some embodiments, the subject is a mammal, and in other embodiments, the mammal is a human.

[0146] The use of the isolated population of hypoimmunogenic cells disclosed herein offers advantages over existing methods because the population of hypoimmunogenic cells can be differentiated from endocrine precursor cells or their precursors derived from stem cells (e.g., iPS cells obtained or harvested from a subject administered the isolated population of hypoimmunogenic cells). This is highly advantageous because it provides a renewable source of hypoimmunogenic cells that can be differentiated from stem cells into endocrine precursor cells by methods generally known to those of skill in the art and further differentiated into pancreatic β-like cells, particularly SC islets comprising hypoimmunogenic pancreatic endocrine cells, for transplantation into a subject by the methods described herein, without the risks and limitations of cells derived from other lineages.

[0147] One embodiment of the present invention relates to a method of treating diabetes or a metabolic disorder in a subject, comprising administering to a subject having diabetes and / or a metabolic disorder an effective amount of a composition comprising a population of hypoimmunogenic cells (e.g., hypoimmunogenic beta cells) disclosed herein. In a further embodiment, the present invention provides a method of treating diabetes, comprising administering to a subject having or at high risk of developing diabetes a composition comprising a population of hypoimmunogenic cells disclosed herein.

[0148] In one embodiment of the above method, the subject is a human and the population of hypoimmunogenic cells disclosed herein are human cells. In some embodiments, the present invention contemplates that the population of hypoimmunogenic cells disclosed herein is administered directly to the subject's pancreas or administered systemically. In some embodiments, the population of hypoimmunogenic cells disclosed herein can be administered to any suitable location in the subject, for example, into a blood vessel or into the liver capsule.

[0149] The present invention also relates to methods for treating subjects with diabetes or metabolic disorders resulting from genetic defects, physical injury, environmental insults or conditions, poor health, obesity, and other diabetes risk factors commonly known to those skilled in the art. The effectiveness of treatment of a subject administered a composition comprising a population of hypoimmunogenic cells (e.g., hypoimmunogenic beta cells) can be monitored by clinically accepted standards and tests, including, for example, (i) a glycated hemoglobin (A1C) test (which measures the percentage of blood glucose bound to hemoglobin, the oxygen-carrying protein in red blood cells, thereby indicating the subject's average blood glucose level over the past two to three months). The higher the blood glucose level, the higher the percentage of hemoglobin bound to glucose. An A1C level of 6.5% or higher on two separate tests indicates that the subject has diabetes. A test value of 6 to 6.5% suggests that the subject has prediabetes. (ii) random blood glucose testing. A blood sample is drawn from the subject at random times. A random blood glucose level of 200 milligrams per deciliter (mg / dL), or 11.1 millimoles per liter (mmol / L), or higher indicates that the subject has diabetes. (iii) Fasting Blood Glucose Test. A blood sample is drawn from the subject after an overnight fast. A fasting blood glucose level of 70-99 mg / dL (3.9-5.5 mmol / L) is normal. A fasting blood glucose level of 126 mg / dL (7 mmol / L) or higher on two separate occasions indicates that the subject has diabetes. A blood glucose level of 100-125 mg / dL (5.6-6.9 mmol / L) indicates that the subject has prediabetes. (iv) Oral Glucose Tolerance Test. A blood sample is drawn from the subject after they have fasted for at least 8 hours or overnight, and then they ingest a sugary solution, and their blood glucose level is measured two hours later. A blood glucose level below 140 mg / dL (7.8 mmol / L) is normal. A blood glucose level between 140 and 199 mg / dL (7.8 and 11 mmol / L) is considered prediabetic. This is sometimes called impaired glucose tolerance (IGT). A blood glucose level of 200 mg / dL (11.1 mmol / L) or higher may indicate diabetes.

[0150] In some embodiments, the effects of administering a hypoimmunogenic cell population (e.g., hypoimmunogenic beta cells) disclosed herein to a subject in need of treatment are associated with improved exercise tolerance or other quality of life measures, and reduced mortality. The effects of hypoimmunogenic cell population cell therapy may become apparent over the course of days to weeks following the procedure. However, beneficial effects may be observed as early as hours after the procedure and may persist for several years. In some embodiments, the effects of hypoimmunogenic cell population cell therapy are apparent within two weeks following the procedure.

[0151] In some embodiments, the hypoimmunogenic cell populations (e.g., hypoimmunogenic β cells) disclosed herein can be used for tissue reconstruction or regeneration in human patients or other subjects in need of such treatment. In some embodiments, compositions of hypoimmunogenic cell populations can be administered in a manner that allows them to engraft or migrate to the intended tissue site and reconstruct or regenerate functionally deficient areas. Specialized devices are available that are adapted to administer cells capable of reconstructing a population of hypoimmunogenic pancreatic cells (e.g., β cells) to the pancreas or an alternative desired location. Thus, the hypoimmunogenic cells can be administered by injection into the recipient subject's pancreas or intramuscularly. [Example]

[0152] Sustained transgene expression is essential for maintaining a desirable, low-immunogenic phenotype both throughout differentiation and after transplantation. Previously, transgene silencing has been observed when tolerogenic molecules were targeted to the AAVS1 safe harbor locus in hESCs and subsequently differentiated into SC-β cells. Because housekeeping genes such as GAPDH are constitutively expressed in all cells of human pancreatic islets, it was hypothesized that targeting tolerogenic molecule expression to the GAPDH locus in hESCs would result in strong and sustained transgene expression after differentiation into SC islet cells and after transplantation. Table 1 provides a non-exhaustive list of other housekeeping genes suitable for use in the present invention. [Table 1]

[0153] Generation of low-immunogenic SC islet cells

[0154] To generate hypoimmunogenic SC islet cells, we used a targeted approach that minimizes the number of editing steps. First, we disrupted the antigen-presenting ability of stem cells and their differentiated progeny by knocking out the B2M gene in hESCs to eliminate HLA class I expression. In doing so, we reduced the ability of T cells to bind to and destroy allogeneic SC islet cells. We also targeted the expression of the immunoregulatory molecule PDL1 to the GAPDH locus to provide additional inhibitory signaling to T cells. Other candidate molecules for expression from the GAPDH locus include soluble PDL1-Ig and CTLA4-Ig, CD47 (macrophage and NK cell inhibitor), HLA-G single-chain fusion (NK cell inhibitor), SERPINB9 (granzyme inhibitor), CCL21 (macrophage cytokine inhibitor), FASL (T cell apoptosis inhibitor), CD200 (myeloid cell inhibitor), CD55, and CD46 (complement inactivation). For multiple transgene expression, polycistronic RNA is transcribed from the GADPH locus encoding GAPDH, and the tolerogenic molecules are separated by a self-cleaving peptide. Because HLA class II expression has not been observed in our SC islet cells, we chose not to interfere with HLA class II expression.

[0155] Because HLA class I-deficient cells are susceptible to NK cell cytotoxicity, we next introduced an HLA-E single-chain fusion gene into the GAPDH locus so that SC islet cells express a single HLA molecule involved in NK cell inhibition. Furthermore, we selectively loaded the peptide VMAPRTLLL, which has been shown to mediate preferential interaction between HLA-E and its cognate inhibitory receptor, NKG2A, on NK cells. In comparison, another HLA-E single-chain fusion loaded with a peptide derived from the leader sequence of HLA-G (VMAPRTLFL)3 has been shown to activate NK cells through interaction with HLA-E's cognate activating receptor, NKG2C. Other potential peptide candidates for loading into HLA-E fusions that do not activate NK cells via NKG2C include VMAPRTLVL, VMAPRTLIL, IMAPRTLVL, VMPPRTLLL, VMAPRTVLL, VTAPRTLLL, VTAPRTVLL, VMAPRTLTL, and VMAPRALLL. After generating hypoimmunogenic hESCs, these cells are differentiated into SC-β cells that do not express classical HLA and constitutively express tolerogenic molecules that can inhibit T cell and NK cell responses in allogeneic settings.

[0156] SC islet cells as mediators of localized immune tolerance

[0157] We also attempted to use our engineering strategy to confer the ability of SC islet cells to regulate their local immune microenvironment by secreting immunomodulatory cytokines. We hypothesized that the immunomodulatory cytokines secreted by islet cells could induce localized immune tolerance and thereby protect allografts from immune destruction. We engineered a GAPDH locus targeting construct to contain the tolerogenic cytokines IL-10, TGF-β, and modified IL-2, and introduced these cytokines into the GAPDH locus of hESCs. The cytokine-secreting hESCs were then differentiated into SC islet cells and transplanted into immunocompetent mice.

[0158] method

[0159] mouse

[0160] Male B6 / albino, Scid / beige, and NSG-MHC class I / II knockout (KO) mice (6–8 weeks old) were purchased from Jackson Labs. Mice were housed under specific pathogen-free conditions at Harvard University. All animal experiments were performed with approval from the Harvard IACUC.

[0161] cell culture

[0162] Maintenance and differentiation of human ES cells were performed as previously described (Millman et al., 2016; Pagliuca et al., 2014). SC islet cell differentiation was initiated 72 hours after the first passage by aspirating mTeSR1 and supplementing with stage- and day-specific medium supplemented with appropriate small molecules or growth factors, as previously described (Millman et al., 2016; Pagliuca et al., 2014; Veres et al., 2019). All cell lines were routinely tested for and found to be mycoplasma-free. All experiments involving human cells were approved by the Harvard University IRB and ESCRO committees.

[0163] Generation of immune-evasive hESCs

[0164] The existing GAPluc (WT) hESC line was used as the starting material for generating HLA-deficient hESCs (Gerace et al., 2021). To generate HLA-deficient hESCs, 1 x 10 WT hESCs were nucleofected with 120 pmol of B2M gRNA (5'-GCTACTCTCTCTTTCTGGCC'3, SEQ ID NO: 1) (Mandal et al., 2014) (IDT) and 10 pmol of RNP complexed with Alt-R® Sp HiFi Cas9 Nuclease V3 (IDT) using a 4D-Nucleofector (Lonza) according to the manufacturer's instructions. Nucleofected cells were resuspended in mTesR1 (STEMCELL Technologies, 85850) + 10 μM Y27632 (DNSK International, DNSK-KI-15-02) and seeded onto Matrigel-coated tissue culture plates. After 48 hours, cells were treated with 10 ng / ml IFN-γ (R&D, 285-IF-100) for 24 hours and then stained with APC anti-human HLA-ABC (W6 / 32, 1:100) (Biolegend, 311409). HLA-ABC- / - cells were sorted using a FACS Aria II (BD Biosciences) and seeded onto Matrigel-coated tissue culture plates containing mTesR1 + CloneR (STEMCELL Technologies, 05888). Single colonies were picked and expanded. The resulting HLA-deficient line was designated B2M- / -.

[0165] To generate GAP-PD and GAP-BEC hESCs, human PD-L1 and peptide::B2M::HLA-E fusion sequences were synthesized as gBlock (Genscript) and cloned downstream of the Luc2 gene in our existing GAPluc targeting plasmid. The covalently linked peptide sequence (VMAPRTLLL (SEQ ID NO: 2)) in the peptide::B2M::HLA-E fusion is derived from the HLA-Cw7 molecule (Kaiser et al., 2005). hESCs were nucleofected with the GAP-PD or GAP-BEC targeting plasmid and GAPDH-targeting RNP as previously described (Gerace et al., 2021). For GAP-B2P and GAP-BEC lines, polyclonal populations of puromycin-selected cells were then nucleofected with B2M-targeting RNP, and single colonies were picked and expanded after FACS sorting as described above. The gating strategy for sorting HLA-ABC − / − hESCs is shown in Figure 5G.

[0166] Flow cytometry

[0167] Differentiated WT, B2M- / -, BEC, PD-L1, and B2P SC islet cells were treated with or without 10 ng / ml IFN-γ for 24 hours before staining. Cells were dissociated with Accutase (STEMCELL Technologies, 07920), washed twice with PBS + 0.1% BSA (Gibco, A10008-01), and blocked with PBS + 5% donkey serum (Jackson Labs, 100181-234) on ice for 30 minutes. Cells were then stained for 30 minutes on ice in blocking buffer containing PE or APC anti-human HLA-ABC (Biolegend, 311405), PE anti-human HLA-E (Biolegend, 342603), and PE anti-human PD-L1 (Biolegend, 393607). Cells were then washed three times and fixed in 4% PFA (EMS, 15710) for 15 minutes at 4°C. Fixed cells were then incubated in blocking buffer containing rat anti-human C-peptide (DHSB, GN-ID4) and mouse anti-human Nkx6.1 (DHSB, F55A12) (overnight at 4°C), washed three times with blocking buffer, and incubated with goat anti-rat 647 (Life Technologies, A-21247, 1:300) and goat anti-mouse 405 (Life Technologies, A-31553, 1:300) in blocking solution (1 hour at room temperature), washed three times, and resuspended in PBS + 0.1% BSA. Samples were acquired on an LSR II (BD) flow cytometer and analyzed using FlowJo 10.7.1 (BD). Unless otherwise noted, all antibodies were used at 1:100. The gating strategy for identifying SC-β cells is shown in Figure 5H. PE mouse IgG2a (Biolegend, 400213) and PE mouse IgG1 (Biolegend, 400111) were used as isotype controls.

[0168] Magnetic enrichment and reaggregation using CD49a

[0169] WT and BEC SC-β cells were magnetically enriched from SC islet clusters as previously described (Veres et al., 2019). Enriched cells were resuspended in S6 medium and seeded at 5 × 10 cells / well in low-attachment 96-well v-bottom tissue culture plates (Thermo Scientific, 277143). They were centrifuged at 300 g for 1 minute and incubated at 37°C for 4–7 days. CD49a-enriched SC-β cell clusters were fed with fresh S6 medium every 2 days.

[0170] SC-β cell immunohistochemistry

[0171] CD49a-enriched SC-β cell clusters were fixed in 4% PFA for 1 hour at room temperature, washed, frozen in OCT (Tissue-Tek, 4583), and sectioned at 14 μm. Prior to staining, paraffin-embedded samples were treated with Histo-Clear (EMS, 64110-01) to remove paraffin. For staining, slides were incubated in blocking buffer (PBS + 0.1% Triton®-X + 5% donkey serum) for 1 h at room temperature, then incubated in PBS + 5% donkey serum containing rat anti-human C-peptide (1:300), mouse anti-human Nkx6.1 (1:100), and rabbit anti-human HLA-E (Sigma-Aldrich, HPA031454, 1:100) for 1 h at room temperature, washed three times, and then incubated in goat anti-mouse 594 (Life Technologies, A-11032, 1:500), goat anti-rat 488 (Life Technologies, A-11006, 1:500), and goat anti-rabbit 647 (Life technologies, A-21244, 1:500) for 2 h at room temperature, washed, and then stained with Vectashield (Vector) containing DAPI. The sections were mounted in a 500 ml PBS (DeepLab Laboratories, H-1200), coverslipped, and sealed with clear nail polish. Representative fields were imaged using a Zeiss Z2 with an Apotome microscope.

[0172] Immune cell isolation

[0173] PBMCs, CD8 and CD4 T cells, and NK cells were isolated from apheresis leukapheresis collars (n = 5 donors) obtained from Brigham and Women's Hospital under IRB approval. PBMCs were isolated by density gradient centrifugation using Lymphoprep (STEMCELL Technologies, 07801) and SepMate™-50 (IVD) tubes (STEMCELL Technologies, 85450) according to the manufacturer's instructions. After adding RosetteSep human CD4+ T cell enrichment cocktail (STEMCELL Technologies, 15062), CD8+ T cell enrichment cocktail (STEMCELL Technologies, 15063), and NK cell enrichment cocktail (STEMCELL Technologies, 15065), respectively, CD4, CD8, and NK cells were isolated by density gradient centrifugation using Lymphoprep and SepMate™-50 (IVD) tubes. Each enriched immune cell was cryopreserved in CryoStor CS10 (STEMCELL Technologies, 07930) at a concentration of 10 M cells / vial.

[0174] Flow cytometry analysis of enriched immune cell subpopulations was performed by staining cells in blocking buffer containing APC anti-human CD3 (Biolegend, 300311), PE anti-human CD4 (Biolegend, 357403), Pacific Blue anti-human CD8 (Biolegend, 344717), and Pacific Blue anti-human CD56 (Biolegend, 362519). Isotype controls were Pacific Blue mouse IgG1 (Biolegend, 400131), APC mouse IgG2a (Biolegend, 400221), and PE rat IgG2b (Biolegend, 400607). All antibodies were used at 1:100 unless otherwise noted. Representative gating strategies are shown in Figures 6G-6I.

[0175] In vitro T cell cytotoxicity assay

[0176] WT, B2M- / -, and BEC SC islet cells were seeded in S3 medium at 5 x 104 cells per well in Matrigel-coated 96-well black flat-bottom plates (Corning, 3916) in the presence or absence of IFN-γ (10 ng / ml). After 24 hours, the medium was switched to T cell medium for immune cell coculture (ImmunoCult™-XF T Cell Expansion Medium (STEMCELL Technologies, 10981) + 100 U / ml rhIL-2). Primary human PBMC, CD4, or CD8 cells (n = 5 donors) cultured in T cell medium were added to the SC islet cells at effector:target ratios of 1:1 and 3:1 with or without ImmunoCult™ human CD3 / CD28 T cell activator (STEMCELL Technologies, 10991). All T cell cytotoxicity assays were performed after 72 hours of co-culture. After the addition of 150 μg / ml D-luciferin (Gold Biotechnology, LUCK-2G), luminescence was measured using a CLARIOstar microplate reader (BMG LABTECH). The viability of SC islet cells was calculated as a percentage (%) of luminescence in the absence of T cells (luminescence of test SC islet cells / luminescence of SC islet cells without T cells × 100).

[0177] In vitro NK cell cytotoxicity assay

[0178] WT, B2M- / -, and BEC SC-islet cells were seeded at 2 x 104 cells per well in 96-well black round-bottom ultra-low attachment plates (Corning, 4591) in NK cell medium (NK MACS medium (Milteny Biotec, 130-114-429) + 5% human AB serum (Valley Biomed, HP1022HI) + 5% HyClone FBS (GE Healthcare, SH30070.03) + 0.5 ng / ml rhIL-2 (Peprotech, 200-02)). Primary NK cells (n = 5 donors) cultured for 5 days in NK cell medium were added to SC islet cells at effector:target ratios of 1:1 and 10:1. K562-Luc2 (Biocytogen, BCG-PS-015-luc) and Raji-GFP-Luc2 (Biocytogen, BCG-PS-087-luc) cells were used as positive and negative controls, respectively. All NK cell cytotoxicity assays were performed after 5 h of co-culture, and luminescence and SC islet cell viability were measured as described above.

[0179] In vivo NK cell cytotoxicity

[0180] 2.5 × 10 wild-type and B2M- / - SC islet cell clusters were resuspended in phenol-free Matrigel (Corning, 356237) alone or with 7.5 × 10 primary human NK cells (pretreated with 0.5 ng / ml rhIL-2 for 12 hours) and subcutaneously implanted into Scid / beige mice (n = 5). Bioluminescence was measured 10 minutes after intraperitoneal injection of 10 μL / g D-luciferin (15 mg / ml) on days 1 and 5 after cell implantation, as previously described (Gerace et al., 2021) using an IVIS Spectrum (PerkinElmer, 124262).

[0181] Endothelial cell differentiation

[0182] WT and B2M- / - hESCs were plated in mTesR1 plus 10 μM Y27632 at 7.5 × 10 per well of a Matrigel-coated 6-well tissue culture plate (Corning, 3516). 4 Cells were seeded at a concentration of 1:100. The cells were then differentiated into endothelial cells using the STEMdiff™ Endothelial Differentiation Kit (STEMCELL Technologies, 08005) according to the manufacturer's instructions. The endothelial cells were stained with Pacific Blue anti-human CD31 (Biologend, 303113) and then the differentiation efficiency was quantified by FACS analysis. Pacific Blue mouse IgG1 (Biolegend, 400131) was used as an isotype control. Unless otherwise noted, all antibodies were used at 1:100.

[0183] Bulk RNA sequencing

[0184] Magnetically enriched CD49a+ SC-β cells and SC endothelial cells in 6-well plates at 1 x 10 6 Cells were seeded in triplicate at 1000 kJ / well and treated with or without 10 ng / ml IFN-γ for 24 hours. Cells were then harvested using Accutase, and pellets were flash-frozen on dry ice. RNA extraction, library preparation, and sequencing reactions were performed at GENEWIZ, LLC (South Plainfield, NJ, USA). Total RNA was extracted from cell pellet samples using the RNeasy Plus Mini Kit (Qiagen, Germantown, MD, USA) and quantified using a Qubit Fluorometer (Life Technologies, Carlsbad, CA, USA). Full-length cDNA synthesis and amplification were performed using the SMART-Seq v4 Ultra Low Input Kit for Sequencing (Clontech, Mountain View, CA), and sequencing libraries were prepared using Illumina Nextera XT libraries.

[0185] The multiplexed sequencing library was loaded onto an Illumina HiSeq instrument and sequenced using the 2x150 Paired End (PE) configuration. Image analysis and base calling were performed using HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from the Illumina HiSeq were converted to FASTQ files and demultiplexed using Illumina's bcl2fastq 2.17 software. One mismatch was allowed to identify the index sequence.

[0186] After inspecting the quality of the raw data, sequence reads were trimmed using Trimmomatic v.0.36 to remove potential adapter sequences and low-quality nucleotides. Trimmed reads were mapped to the Homo sapiens reference genome available in ENSEMBL using STAR Aligner v.2.5.2b. STAR Aligner is a splice aligner that detects splice junctions and helps align the entire read sequence by incorporating them. This step resulted in a BAM file. Unique gene hit counts were calculated using the feature Counts in the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted.

[0187] After extracting gene hit counts, the gene hit count table was used for downstream differential expression analysis. DESeq2 was used to compare gene expression levels between sample groups. Wald tests were used to generate p-values ​​and Log2 fold changes. Genes with an adjusted p-value <0.05 and an absolute log2 fold change >1 were designated as differentially expressed for each comparison. Gene ontology analysis was performed on the statistically significant set of genes by running the software g:Profiler (Raudvere et al., 2019). The gene accession number for the unique RNAseq dataset generated in this study is GSE200021.

[0188] Analysis of NK cell ligands

[0189] WT and B2M- / - SC islets and SC endothelial cells were washed twice with washing buffer and blocked with blocking buffer for 30 minutes on ice. Then, cells were stained with APC anti-human CD47 (Biolegend, 323123), APC anti-human CD324 (E-cadherin) (Biolegend, 324107), PE anti-human CD325 (N-cadherin) (Biolegend, 350806), PE anti-human CD112 (Nectin-2) (Biolegend, 337409), APC anti-human CD155 (PVR) (Biolegend, 337617), PE anti-human CD111 (Nectin-1) (Biolegend, 340404), APC anti-human MICA / MICB (Biolegend, 320907), and AF647 anti-human PCNA (Biolegend, 320907). The cells were stained for 30 minutes on ice using blocking buffer containing: APC anti-human CD48 (Biolegend, 336713), PE anti-human CD70 (Biolegend, 355103), mouse anti-human CD113 (nectin-3) (Millipore-Sigma, MABT63), APC anti-human ULBP1 (R&D, FAB1380A), PE anti-human ULBP2 / 5 / 6 (R&D, FAB1298P), APC anti-human CLEC2D (R&D, FAB3480A), and PE anti-human CD72 (Biolegend, 316207). PE mouse IgG1 (Biolegend, 400111), APC mouse IgG1 (Biolegend, 400121), APC mouse IgG2a (Biolegend, 400221), PE mouse IgG2a (Biolegend, 400213), APC rat IgG1 (Biolegend, 401903), PE rabbit IgG (Cell Signaling Technology, 5742S), and goat anti-mouse 488 (Thermofisher, A-11001, 1:300) were used as isotype controls. Unless otherwise noted, all antibodies were used at 1:100.

[0190] Transplantation of HLA-deficient SC islet cells

[0191] Diabetes was induced in NSG-MHC class I / II KO mice by multiple intraperitoneal injections of low-dose (40 mg / kg) streptozotocin (STZ) as previously described (Furman, 2021). Once the animals reached a blood glucose level of >500 mg / dL, they were injected with 5 × 10 6 WT or B2M- / - SC islets were transplanted under the kidney capsule (n = 10) as previously described (Millman et al., 2016; Pagliuca et al., 2014). Blood glucose and body weight were measured twice weekly after transplantation.

[0192] Ten weeks after transplantation (before PBMC injection) and seven weeks after PBMC injection, the function of the transplanted cells was assessed by performing an in vivo peritoneal glucose-stimulated insulin secretion (IPGTT) as previously described (Millman et al., 2016; Pagliuca et al., 2014). Insulin secretion was quantified using the Human Ultrasensitive Insulin ELISA (ALPCO Diagnostics; 80-INSHUU-E01.1).

[0193] Generation of immune-tolerized SC islet cells

[0194] Human IL-2, TGF-β, and IL-10 cDNAs were synthesized as polycistronic gBlocks (Genscript) and cloned into an existing GAPluc targeting plasmid as described above to generate the GAP-2B10 plasmid. The human IL-2 sequence was modified by a single amino acid substitution (N88D) as previously described (Peterson et al., 2018). GAP-2B10 hESCs were generated by co-nucleofection of the GAP-2B10 targeting plasmid and GAPDH-targeting RNP. GAP-2B10 SC islet cells were differentiated as previously described (Millman et al., 2016; Pagliuca et al., 2014).

[0195] In vitro glucose-stimulated insulin secretion

[0196] In vitro function was assessed by measuring glucose-stimulated insulin secretion (GSIS) as previously described (Millman et al., 2016; Pagliuca et al., 2014). SC islet clusters were washed twice in Krebs buffer (KRB) and preincubated for 1 hour at 37°C in KRB containing 2 mM glucose (low glucose). Clusters were then challenged with three sequential treatments alternating low-high-low KRB containing glucose (high, 20 mM), followed by depolarization with low KRB containing 30 mM KCl. Each treatment lasted 30 minutes, after which 100 μl of supernatant was collected and human insulin quantified using the Human Ultrasensitive Insulin ELISA. Human insulin measurements were obtained by dispersing clusters with TrypLE Express (Thermofisher, 12604013) and normalized to the number of viable cells counted using a ViCell (Beckman Coulter) kit.

[0197] In vitro SC islet cell cytokine secretion and PBMC cytotoxicity assays

[0198] GAP-2B10 SC islet cell clusters were dispersed with TrypLE Express and plated onto Matrigel-coated 96-well plates at linear concentrations (1, 2, 4, 6, 8 × 10 4 , and 1 × 10 5Cells were seeded in duplicate in S3 medium at 1000 x g (cells / well). After 24 h, supernatants were collected and centrifuged at 3000 x g for 5 min. The cytokines IL-2, TGF-β, and IL-10 were quantified using a Legend Max ELISA according to the manufacturer's instructions (IL-2, Biolegend, 431807; TGF-β, Biolegend, 436707; IL-10, Biolegend, 430607). For cytotoxicity assays, WT and 2B10 SC islet cells were cocultured with human PBMCs as described above.

[0199] Xenotransplantation of GAP-2B10 SC islet cells

[0200] Clusters of WT and GAP-2B10 SC islet cells (5 × 10 6 SC islet grafts were transplanted under the kidney capsule of B6 / albino mice (n = 3 / group), and graft viability was monitored weekly for 9 weeks by bioluminescence following intraperitoneal injection of D-luciferin, as described above. Five weeks after transplantation, SC islet grafts were removed for immunohistochemical analysis of surviving INS+ cells and CD8+ T and FOXP3+ Treg cells.

[0201] Immunohistochemistry of SC islet grafts

[0202] Kidneys from B6 / albino mice transplanted with WT and 2B10 SC islets were excised, fixed in 4% paraformaldehyde (PFA) overnight at room temperature, and embedded in paraffin. Sections were pre-cleared with Histo-Clear, rewetted using an ethanol gradient, and antigens were fixed by incubation in boiling antigen retrieval reagent (10 mM sodium citrate, pH 6.0) for 50 minutes. Slides were then blocked in 5% donkey serum for 1 hour and stained overnight at 4°C with guinea pig anti-human insulin (DAKO, A0564), rat anti-mouse CD8α (Biolegend, 100702), and mouse anti-mouse FOXP3 (Biolegend, 320002). Slides were then washed three times and incubated in secondary antibodies goat anti-mouse 594 (Life Technologies, A-11032), goat anti-rat 488 (Life Technologies, A-11006), and goat anti-guinea pig 647 (Life Technologies, A-21450) for 2 hours at room temperature. They were then washed, mounted in Vectashield with DAPI (Vector Laboratories, H-1200), coverslipped, and sealed with clear nail polish. Representative fields were imaged using a Zeiss Z2 with an Apotome microscope. All primary and secondary antibodies were used at dilutions of 1:200 and 1:500, respectively.

[0203] statistics

[0204] All data are presented as mean ± SD and analyzed using GraphPad Prism 9 (GraphPad Software). Statistical significance was determined by one-way or two-way analysis of variance with Tukey and Sidak post hoc tests for multiple comparisons and two-tailed t-tests for pairwise comparisons. In the figures, p values ​​are indicated as *p<0.05, **p<0.01, ***p<0.005, and ****p<0.001.

[0205] result

[0206] Engineering low-immunogenic SC islet cells

[0207] Because GAPDH is constitutively expressed in all cells of human pancreatic islets (Figures 5A-5B), we targeted expression of PD-L1 and HLA-E long fusions to the GAPDH locus in hESCs and used luminescence as a reporter of cell viability. We chose to overexpress PD-L1 because PD-L1 has previously been shown to protect SC islet cells from xenorejection (Yoshihara et al., 2020), while HLA-E long fusions inhibit NK cells in the context of HLA deficiency (Gornalusse et al., 2017; Mattapally et al., 2018; Riolobos et al., 2013; Wang et al., 2015). The GAPDH-targeting plasmid was modified to contain PD-L1 or HLA-E (Figures 5C-5E) and used to generate five hESC lines (Figure 1A): wild-type (WT), HLA-deficient (B2M- / -), PD-L1-expressing (PD-L1), HLA-deficient / PD-L1-expressing (B2P), and HLA-deficient / HLA-E-expressing (BEC) lines. All five genetically modified hESC lines successfully differentiated into SC-β cells with similar efficiency, as assessed by C-peptide and Nkx6.1 expression (Figure 1B). Knockout of the beta2-microglobulin (B2M) gene effectively abolished HLA class I expression on B2M- / -, B2P, and BEC SC islet cells. After stimulation with IFN-γ, HLA class I and PD-L1 were upregulated in WT SC islet cells (Castro-Gutierrez et al., 2021), whereas B2P SC islet cells constitutively overexpressed PD-L1 and lacked HLA class I expression (Figure 1C). Similar expression profiles of HLA-E and HLA class I expression were observed in WT and BEC SC islet cells (Figure 1D) (Gornalusse et al., 2017). Immunohistochemistry of magnetically enriched SC-β cells showed localization of HLA-E long chain fusions to the membrane of BEC SC-β cells (Figure 1E) (Veres et al., 2019).

[0208] To confirm that human PD-L1 expressed on the surface of SC islet cells binds to PD-1, we assessed the binding of fluorescently labeled soluble human and mouse PD-1-Fc to WT and B2P SC islet cells (Figure 5F). While both soluble human and mouse PD-1 bind to membrane-bound PD-L1 on B2P SC islet cells, WT SC islet cells do not endogenously express sufficient levels of PD-L1 to detectably bind to human or mouse PD-1. We also observed a reduced binding affinity of soluble mouse PD-1 to human PD-L1 (Freeman et al., 2000). When WT, PD-L1, and B2P SC islet cells were transplanted under the kidney capsule of B6 / albino mice (Figure 1F), all genetically modified SC islet cells were rejected within 10 days of transplantation (Figure 1G). These results suggest that overexpression of PD-L1 is not sufficient to protect HUES8-derived SC islet cells from xenorejection and that loss of HLA expression does not improve xenograft survival in our animal model of xenorejection.

[0209] HLA-deficient SC islet cells are resistant to allogeneic immune cell destruction in vitro

[0210] Next, we evaluated the in vitro viability of gene-modified SC islet cells in coculture with allogeneic human immune cells (Figure 2A). Consistent with previous studies, NK cells and CD4+ and CD8+ T cells accounted for approximately 8, 35, and 10% of enriched PBMCs, respectively (Figures 6A-6B) (Kleiveland, 2015). When SC islet cells were pretreated with IFN-γ and cocultured with human PBMCs at multiple effector:target ratios, B2M- / - and BEC SC islet cells showed significantly improved viability compared to WT cells (Figures 2B-2C). HLA-E overexpression did not confer any additional protective benefit. Furthermore, a similar viability pattern was observed when SC islet cells were cocultured with purified CD8+ and CD4+ T cells (Figures 6C-6F). In all coculture assays, there was no significant difference in the viability of all gene-modified SC islet cells cultured with CD3 / CD28-activated cells.

[0211] Because the expression of T cell co-activating and co-inhibitory ligands determines T cell function and is regulated by various stimuli, including IFN-γ (Chen and Flies, 2013), we performed bulk RNA sequencing on WT CD49a+ SC-β cells to assess T cell ligand expression after IFN-γ stimulation (Figure 2D). As expected, IFN-γ stimulation upregulated the T cell co-inhibitory ligands PD-L1 and LGALS9 (galectin 9) in SC-β cells (Figure 2E) (Garcia-Diaz et al., 2017; Imaizumi et al., 2002). Conversely, although we did not detect transcripts of many T cell coactivation ligands other than HLA genes, we found that IFN-γ stimulation upregulated the TNF receptor superfamily members HVEM (TNF receptor superfamily member 14) and CD40 (tumor necrosis factor receptor superfamily member 5) in SC-β cells (Figure 2F) (Benci et al., 2016; Wagner et al., 2002). Interestingly, BNT3A1 (butyrophilin subfamily 3 member A1), an MHC-related gene that regulates T cell activation and proliferation, was also upregulated (Rigau et al., 2020). These results suggest that other coactivator and coinhibitory T cell ligands that may affect T cell function are expressed in SC-β cells in the absence of classical HLA-TCR signaling due to HLA knockout.

[0212] HLA-deficient SC islet cells are resistant to destruction by allogeneic NK cells in vitro and in vivo

[0213] We next evaluated the effects of HLA-deficiency and HLA-E overexpression on NK cell function against SC islet cells. When cocultured with NK92mi cells, BEC SC islet cells showed no significant difference in viability compared to WT SC islet cells, whereas HLA-deficient SC islet cells were sensitive to NK92mi cytotoxicity (Figure 7A) (Gornalusse et al., 2017). The strong protective effect of HLA-E on NK92mi cells is likely due to the high percentage (approximately 96.1%) of NKG2A+ / NKG2C- cells (Figure 7B), which biases NK cell inhibition.

[0214] NK cell lines are useful tools for assessing NK cell cytotoxicity, but they do not accurately recapitulate the receptor expression and function of primary NK cells. Previous studies have shown that human NK cells do not destroy HLA-deficient endothelial cells and platelets in the absence of IL-2 activation (Deuse et al., 2021; Suzuki et al., 2020). Therefore, we pre-activated human NK cells with IL-2 for 5 days prior to co-culture with genetically modified SC islet cells, as previously described (Deuse et al., 2021). The enriched NK cells consisted of approximately 80% CD56-low NK cells and approximately 5% CD56-high NK cells (Figures 7C-7D). Surprisingly, despite pre-activation with IL-2, no significant differences in viability were observed between genetically modified SC islet cells at multiple effector:target ratios (Figure 2G). Because overexpression of HLA-E does not confer any additional protective benefit to HLA-deficient SC islet cells against NK cell cytotoxicity, we chose to continue examining WT and B2M- / - SC islet cells. We evaluated the survival of WT and B2M- / - SC islet cells after transplantation with IL-2-preactivated NK cells in Scid / beige mice (Figure 2H). Again, no significant difference was observed between the survival rates of WT and B2M- / - SC islet cells in vivo (Figures 2I and 7E). Collectively, these results suggest that HLA-deficient SC islet cells are intrinsically resistant to NK cell cytotoxicity and that their in vitro survival is recapitulated in vivo.

[0215] Predominantly, SC-β cells inherently possess and maintain an NK cell-evasive ligand profile following inflammatory stimuli.

[0216] Because NK cell function is determined by the balance between inhibitory and activating signals, we compared the ligand profile of CD49a+ SC-β cells with that of stem cell-derived endothelial (SC-endo) cells. Because HLA-deficient endothelial cells have previously been shown to be sensitive to pre-activated NK cells (Deuse et al., 2021), we chose endothelial cells as a comparative cell type. Differentiation of endothelial cells yielded over 95% CD31+ SC-Endo cells derived from both WT and B2M- / - hESCs (Figures 7F-7G). Bulk RNA sequencing and transcript analysis of NK cell ligands in SC-β and SC-endo cells confirmed that SC-endo cells possessed an activating NK cell ligand profile characterized by the expression of MICA, MICB, ULBP1, ULBP2, and RAET1G, whereas transcripts of these genes were undetectable in SC-β cells (Figures 3A-3B). Importantly, we found that the expression of most NK cell ligands, with the exception of HLA molecules, was not regulated by IFN-γ. Thus, within the context of HLA deficiency, expression of non-HLA NK cell ligands remained stable after inflammatory stimulation. We confirmed the bulk RNA-sequencing analysis of NK cell ligand expression by flow cytometry, which showed that MIC and ULBP proteins were absent or poorly expressed on SC-β cells (Figures 3C-3D). Together, these data suggest that SC-β cells intrinsically possess and maintain a reduced NK cell-activating ligand profile after inflammatory stimulation, and that this ligand phenotype may explain their resistance to the cytotoxicity of IL-2-preactivated NK cells.

[0217] HLA-deficient SC islet cells reverse diabetes and delay graft rejection in humanized mice

[0218] Based on our findings that HLA-deficient SC islet cells are resistant to PBMC cytotoxicity in vitro and to NK cell cytotoxicity in vitro and in vivo, we evaluated the viability of HLA-deficient SC islet cells in diabetic NSG-DKO mice before PBMC injection (Figure 3E). By 10 weeks after transplantation, there was no significant difference in blood glucose levels between mice transplanted with WT and B2M- / - SC islet cells (Figure 3F). Once blood glucose levels were normalized by SC islet transplantation, we injected HLA-A2- (mismatched) PBMCs and assessed allograft rejection by monitoring blood glucose levels. While WT SC islet cells were destroyed within 2 weeks, rejection of B2M- / - SC islet cells was delayed. Seven weeks after PBMC injection, in vivo GSIS assays demonstrated that animals transplanted with WT SC islets lost in vivo graft function, whereas animals transplanted with B2M- / - SC islets maintained some graft function (Figure 3G). These results suggest that B2M- / - SC islets reverse diabetes and delay allograft rejection.

[0219] Cytokine-secreting SC-β cells induce a local tolerogenic microenvironment that confers protection from xenorejection

[0220] As a result of the limited success of immune evasion engineering to protect SC islet cells, we explored a complementary strategy by engineering SC islet cells to secrete the cytokines IL-2 mutein, TGF-β, and IL-10 as vehicles for localized immunity (Figure 4A). IL-2 mutein (N88D) has low affinity for the IL-2Rβγ receptor, resulting in the generation of a Treg-selective molecule that preferentially expands Tregs while minimizing the impact on CD4+ and CD8+ memory T cells (Khoryati et al., 2020; Peterson et al., 2018). Because IL-10 and TGF-β are required for the immunosuppressive phenotype of Tregs, we included these two cytokines in our immune tolerization construct (Horwitz et al., 2008).

[0221] Differentiation of this genetically engineered cell line, designated 2B10, yielded approximately 30% Nkx6.1+ / C-peptide+ SC-β cells (Figure 4B), which possessed physiological function as assessed in vitro by GSIS (Figure 4C). We also confirmed that 2B10 SC islet cells secreted engineered IL-2, TGF-β, and IL-10 in a cell concentration-dependent manner (Figure 4D). When WT and 2B10 SC islet cells were cocultured with human PBMCs in vitro, 2B10 SC islet cells exhibited significantly improved survival (Figure 4E). Notably, 2B10 SC islet cells transplanted under the kidney capsule of B6 / albino mice survived for up to 9 weeks after transplantation, whereas WT SC islet cells were destroyed within 2 weeks (Figures 4F-4G). Furthermore, we found that WT grafts contained few or no residual SC islet cells, whereas 2B10 grafts contained viable insulin-producing cells and Tregs localized within the graft (Figure 4H). Collectively, these data suggest that SC islet cells can be engineered to co-secrete immunomodulatory cytokines that induce a tolerogenic local microenvironment characterized by infiltration of Treg cells that sustains xenograft survival.

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Claims

1. Cells manipulated to express one or more exogenous genes encoding one or more immunomodulators, wherein the one or more immunomodulators are selected from IL-10, TGF-β, IL-2, or modified IL-2.

2. The cell according to claim 1, wherein the cell is a stem cell.

3. The cell according to claim 1, wherein the cell is an induced pluripotent cell.

4. The cell according to claim 1, wherein the cell is a β-cell progenitor cell.

5. The cell according to claim 1, wherein the cell is a β-cell.

6. The cell according to claim 1, wherein the cell is an α-cell.

7. The cell according to claim 1, wherein the exogenous gene is expressed from the locus of a housekeeping gene.

8. The cell according to claim 7, wherein the housekeeping gene is selected from the group consisting of actin, ubiquitin, and GAPDH.

9. The cell according to claim 1, wherein the one or more immunomodulators are IL-10.

10. The cell according to claim 1, wherein the one or more immunomodulators are TGF-β.

11. The cell according to claim 1, wherein the one or more immunomodulators are IL-2.

12. The cell according to claim 1, wherein the one or more immunomodulators are modified IL-2.

13. The cell according to claim 12, wherein the modified IL-2 is IL-2 containing the N88D mutation.

14. The cell according to claim 1, wherein the one or more immunomodulators are IL-2 (or modified IL-2 including the N88D mutation) and IL-10.

15. The cell according to claim 1, wherein the one or more immunomodulators are IL-2 (or modified IL-2 including the N88D mutation) and TGF-β.

16. The cell according to claim 1, wherein the one or more immunomodulators are IL-2 (or modified IL-2 including the N88D mutation), IL-10, and TGF-β.

17. The cell according to claim 1, wherein the cell is a human pancreatic islet cell.

18. A composition for use in the treatment of diabetes requiring treatment, comprising the cells described in any one of claims 1 to 17.

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