Inhibition of adhesion molecules for stem cell therapy

Abstract

An in vitro method for preparing a population of hypoimmune mammalian stem cells, the method comprising: providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule; and modifying the expression of the cell adhesion molecule in the isolated population of mammalian stem cells to reduce or knock out expression of the cell adhesion molecule, to provide the population of hypoimmune mammalian cells. The isolated population of mammalian stem cells may be pluripotent stem cells or embryonic stem cells, and may be human or non-human stem cells.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 380,883, filed October 25, 2022, which is incorporated herein by reference in its entirety. [Technical Field]

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grants HD090256 and HL134764 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Sequence Listing The immediate application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein by reference in its entirety. The XML copy, created on October 22, 2023, is entitled "SEQ_LIST--107668_109" and is 62.1 KB (63,629 bytes) in size. The Sequence Listing does not extend beyond the disclosure in the application as filed. [Background technology]

[0004] One challenge in the field of cell therapy is immune-mediated rejection of transplanted donor cells. Technologies have been developed to "hide" these cells from immune cells and / or to express molecules or factors that prevent immune cells from recognizing them. A careful balance must be struck between preventing immune rejection while not allowing unchecked growth of transplanted cells, which could lead to tumorigenesis or cause all cells to become reservoirs for viral infection and uncontrolled replication.

[0005] In particular, pluripotent stem cell (PSC)-derived cell therapy is a promising remedial treatment for various cardiovascular diseases that kill over 655,000 Americans annually. PSC-derived grafts possess multiple uniquely attractive attributes, such as nearly limitless expandability and the absence of passenger lymphocytes, and may have reduced susceptibility to acute and chronic allograft rejection, which routinely devastates traditional organ transplants. Furthermore, PSCs are amenable to CRISPR / Cas9-based gene editing. In recent short-term studies, multiple research groups have created hypoimmune "universal cells" (e.g., HLA class I and / or HLA class II knockout [KO]) that can evade recognition by T cells, donor-specific antibodies, and / or NK cell-mediated cytotoxicity. Despite these advances, little is known about the potential for long-term tolerance of hypoimmune PSC grafts in patients, including whether drastic interventions such as complete loss of HLA I+II increase the risk of adverse effects (e.g., malignancies, uncontrolled viral replication).

[0006] Therefore, there is a need to develop stem cell (e.g., iPSC or embryonic stem cell) therapies that are functional, curative, and reduce the risk of rejection by the recipient immune system, thereby enabling long-term graft function and improved patient life. Summary of the Invention

[0007] In one aspect, an in vitro method for preparing a population of hypoimmune mammalian stem cells includes providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule; and modifying the expression of the cell adhesion molecule in the isolated population of mammalian stem cells to reduce or knock out the expression of the cell adhesion molecule, thereby providing a population of hypoimmune mammalian stem cells.

[0008] Also included is a population of hypoimmune mammalian stem cells produced by the above method.

[0009] In another aspect, the present invention provides a population of hypoimmune mammalian stem cells in which the expression of cell adhesion molecules is reduced or knocked out by gene editing of the gene for the cell adhesion molecule. The hypoimmune mammalian stem cells can be differentiated into endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or pancreatic islet cells. Also included are transplants containing the differentiated hypoimmune mammalian stem cell population and methods for treating mammalian subjects by introducing the transplants. [Brief explanation of the drawings]

[0010] [Figure 1A-1B] Figures 1A and 1B show RNA sequencing analysis of wild-type and B2M KO H1 PSC-AEC cell culture models (CMs) of allogeneic rejection inflammation. Expression of the adhesion marker ICAM-1 in WT H1 PSC-AECs (1A) before and after 18 hours of coculture with allogeneic peripheral blood mononuclear cells, and in B2M KO H1 PSC-AECs (1B) after 48 hours of stimulation of the cells alone with tumor necrosis factor alpha (TNFα).

[0011] [Figure 2A-2B] Figures 2A and 2B show the transplantation of gene-edited PSC-CVTs into immunodeficient mouse hosts. (2A) Representative flow cytometry AEC phenotypes (CD34+CXCR4+ cells subgated from CD31+CD144+) are shown. (2B) CMs generated from B2M KO H1 PSCs were formed into spheroids and xenografted into the recipient kidney capsule transplant site of NBGSW mice. After 28 days in vivo, animals were anesthetized, and the grafts were macroscopically examined, demonstrating robust contraction and vascularization with human blood vessels.

[0012] [Figure 3]Figure 3 shows the direct and indirect pathways of alloreactivity in first-generation hypoimmune PSC-AECs. H1 WT and B2M-KO PSCs were differentiated into AEC targets and cocultured with HLA-mismatched peripheral blood leukocytes (10:1 E:T ratio) for 6 days. Alloreactivity was assessed by CD4+ and CD8+ cell proliferation (CFSE dye dilution). B2M-KO PSCs showed reduced CD8+ proliferation, but CD8+ proliferation was still present, indicating indirect pathway alloreactivity.

[0013] [Figure 4A-4B] Figures 4A and 4B show evaluation of human T cell phenotype and PSC immunogenicity in NeoThy humanized mice. (4A) Flow cytometry analysis of NeoThy and BLT peripheral blood for the naive T cell marker CD45RA in the CD3+CD4+ population. BLT mice had a significantly higher percentage (p>0.0001) of naive cells, which is less representative of normal patient frequencies. (4B) IHC staining of iPSC-derived CM from an HLA-B / HLA-DR mismatched donor transplanted under the NeoThy kidney capsule, 26 days post-transplant.

[0014] [Figure 5] Figure 5 shows flow cytometry phenotyping of PSC-CMs. Multiple WT and 1st-gen hypoimmune gene-edited PSC lines (H1 B2M KO [n=8], H1 B2M KO+HLA-E dimer [n=2], H1 WT [n=9], iPSC line 4 WT [n=8], and iPSC line 5 WT [n=8], replicate experiments) were differentiated to highly purified and contracted CMs (cTNT+ purity values ​​by flow cytometry).

[0015] [Figure 6] Figure 6 shows a Luminex® assay of human cytokine release in an MLR. PSC-AEC cells were co-cultured with allogeneic PBMCs for 6 days, after which culture supernatants were assessed for 35 human cytokines. N=3 triplicate wells.

[0016] [Figure 7]Figure 7 shows the long-term survival of NeoThy mice. NeoThy mice were generated using an anti-CD2 thymocyte-depleting antibody (n=5) and compared with mice that did not receive depletion (no anti-CD2) (n=4). The study was stopped at 32 weeks.

[0017] [Figure 8] Figure 8 shows bioluminescence imaging (BLI) using the Akaluc reporter. PSC-AECs with a constitutively expressed Akaluc reporter were injected into the hind limbs of NBSGW mice and monitored for retention.

[0018] [Figure 9] Figure 9 shows in vitro maturation of PSC-CMs. 98% cTNT+ PSC-CMs were cultured in vitro, and RNA was collected at two time points: early (14 days, 0 days post-culture) and late (14 days + 28 days post-culture). Differential gene expression is shown for genes associated with CM specification and maturation.

[0019] [Figure 10] Figure 10 shows sequencing-based TCR rearrangement analysis. FACS-sorted CD3+CD8+ T cells from two patients (P1 and P2) were analyzed for baseline ex vivo TCR beta chain clonal diversity using the Adaptive Biotechnologies ImmunoSEQ™ kit.

[0020] [Figure 11A] Figure 11A shows the Crispr gene editing scheme (SEQ ID NO: 5 is the ICAM1 DNA sequence, SEQ ID NO: 6 is the ICAM1 KO ssODN, and SEQ ID NO: 7 is the gRNA). [Figure 11B] FIG. 11B shows the target analysis (SEQ ID NO: 8 is the WT exon and SEQ ID NO: 9 is the edited exon).

[0021] [Figure 12]Figure 12 shows that CD54 KO PSCs can differentiate into highly pure CMs. CMs were assessed for cardiac troponin-T by intracellular flow cytometry on day 17 of differentiation. WT (left) and KO (right) PSCs were similar in purity.

[0022] [Figure 13A] Figure 13A shows a tri-cellular PSC-CVT in culture. The fused spheroids, made from fused PSC-CMs, PSCC-fibers, and ECs, contract in culture (4x magnification).

[0023] [Figure 13B] Figure 13B shows RNAseq data, including the lack of redundancy of other ICAMs (e.g., ICAM2) to compensate for the loss of ICAM1, suggesting that KOs downregulate HLA, which may contribute to reduced immunogenicity / rejection, as well as further insights from the past month of data analysis in KO PSCs and CM. KEGG analysis was performed. H9 WT CM was compared with H9 CD54 KO CM. Multiple KEGG pathways, including extracellular matrix-receptor interactions, focal adhesions, AGE-RAGE signaling, and PI3K-Akt signaling pathways, were enriched in WT versus KO cells. These pathways play important roles in immune cell interactions, suggesting that innate immune cells may be directly affected in KO cells, which may impact adaptive immune responses, i.e., reduced innate and adaptive immune responses to KO cell grafts.

[0024] [Figure 14A] Figure 14A shows a photograph of H9 CD54KO CMs after transplantation: the grafts are evident, with clear evidence of neovascularization and engraftment. [Figure 14B] Figure 14B shows a photograph of a multicellular cardiovascular graft after implantation. The graft contains CMs, endothelial cells, smooth muscle cells, and PSC-derived cardiac fibroblasts.

[0025] [Figures 15A-15B]Figures 15A and 15B show the efficient generation and differentiation of ICAM-1 KO and isogenic WT PSCs. ICAM-1 was ablated via homozygous CRISPR / Cas9 KO. (15A) KO and PSCs were stimulated with IFNγ and TNFα for 48 hours and displayed intact HLA class I, but ICAM-1 was absent in the KO. (15B) Cells were differentiated into highly pure (>85%) (left) cardiac troponin T (cTNT)+ CMs and (right) CD31+CD144+CD184+ AECs. (Unstained cells were used as a comparison control; subgating of additional AEC marker data (not shown)).

[0026] [Figure 16] Figure 16 shows an assay for assessing the direct and indirect pathways of PSC-AEC alloreactivity. H1 WT and B2M-KO PSCs were differentiated into AEC targets and cocultured with HLA-mismatched peripheral blood leukocytes (10:1 E:T ratio) for 6 days. Alloreactivity was assessed by CD4+ and CD8+ cell proliferation (CFSE dye dilution). B2M-KO PSCs showed reduced CD8+ proliferation, but it was still present, indicating indirect pathway alloreactivity. N = 3 biological replicates (BR), error bars = standard deviation (SD), analysis by two-tailed unpaired t-test, Prism 5.04.

[0027] [Figure 17] Figure 17 shows GSH in WT vs. ICAM-1 KO PSCs. GSH levels were assessed in KO vs. WT PSCs at baseline cell culture conditions. N=3 BR, error bars=SD.

[0028] [Figure 18] Figure 18 shows PBMC binding to WT versus ICAM-1 KO PSCs. PSCs were incubated with fluorescently labeled allogeneic PBMCs for 1 hour, washed, and imaged to determine the number of bound immune cells. Counts determined by blinded acquisition of four discontinuous brightfield images prior to fluorescent imaging (10x magnification). Analysis by ImageJ.

[0029] [Figure 19] Figure 19 shows an allogeneic rejection assay using bioluminescence imaging (BLI). ICAM-1 knockout (KO) PSCs (1 x 106) with a constitutively expressed Akaluc reporter were injected into the right hind limb of humanized NeoThy mice with Matrigel®. Isogenic wild-type (WT) PSCs were injected into the left limb. BLI signals were monitored at early, mid, and late / end time points for 32 days. Representative mice are shown, reflecting WT graft loss and KO retention, seen in 3 of 4 mice.

[0030] [Figure 20] Figure 20 shows RNAseq data, including the lack of redundancy of other ICAMs (e.g., ICAM2) to compensate for the loss of ICAM1, suggesting that the KO downregulates HLA, which may contribute to reduced immunogenicity / rejection, and further insights from the past month of data analysis in KO PSCs and CM. EdgeR differential gene expression analysis. H9 WT CM was compared to H9 CD54 KO CM. Multiple genes were differentially upregulated in WT CM versus KO CM. The top 10 most significantly upregulated genes are shown.

[0031] [Figure 21] Figure 21 shows RNAseq data, including the lack of redundancy of other ICAMs (e.g., ICAM2) to compensate for the loss of ICAM1, suggesting that KO downregulates HLA, which may contribute to reduced immunogenicity / rejection, and further insights from the past month of data analysis in KO PSCs and CM. EdgeR differential gene expression analysis focused on IMMPORT genes related to immune response. H9 WT CM was compared with H9 CD54 KO CM. Multiple immune-related genes were differentially upregulated in WT CM versus KO CM. The top 19 most significantly upregulated genes are shown.

[0032] [Figure 22] Figure 22 shows RNAseq data, including the lack of redundancy of other ICAMs (e.g., ICAM2) to compensate for the loss of ICAM1, suggesting that KOs downregulate HLA, which may contribute to reduced immunogenicity / rejection, and further insights from the past month of data analysis on KO PSCs and CM. EdgeR differential gene expression analysis focused on genes associated with immune allorejection (obtained from the GSEA Hallmark Allorejection Gene Set). H9 WT CM was compared with H9 CD54 KO CM. Multiple immune-related genes were differentially upregulated in WT CM versus KO CM. Higher levels of these genes, such as HLA-A and IL11, in WT (and lower levels in KO) may confer immune protection to KO cells.

[0033] [Figures 23A-23B] Figures 23A and 23B show multiplexed Luminex® assays performed on H9 WT CM and CD54 KO CM and validated by RNAseq. (23A) MCP1 (CCL2), an innate immune cell chemoattractant, was significantly higher in WT vs. KO via Luminex® assay, indicating that reduced amounts of KO may result in less recruitment of innate immune cells. n = 3 biological replicates and 2 technical replicates each. (23B) RNAseq was performed. WT had significantly higher mRNA expression levels of MCP1 compared to KO. Two-tailed T-test, p = 0.0011.

[0034] [Figure 24]Figure 24 shows the reduced immunogenicity associated with CD54 knockout. (Top) In vitro mixed lymphocyte reaction (MLR) cultures were established using H9-derived wild-type (WT) and CD54 knockout (KO) cardiomyocytes (>85% cTNT+) cocultured with peripheral blood mononuclear cells from allogeneic (fully HLA-mismatched) donor PBMC3 for 6 days and then assessed by flow cytometry. (Middle) An MLR with a second allogeneic (HLA class II-mismatched) donor was performed, and proliferating T cell subsets were analyzed for differential proliferation. T stem-like central memory cell proliferation is compared between (bottom left) WT H9-derived cells and (bottom right) CD54 KO cells.

[0035] [Figure 25] Figure 25 shows the generation of a CD54 knockout pluripotent stem cell line. CD54 was knocked out by CRISPR / Cas9. The figure shows the KO strategy. (SEQ ID NO: 8 is the WT exon, and SEQ ID NO: 9 is the edited exon).

[0036] [Figures 26A-26B] Figures 26A and 26B show the pluripotency of CD54 KO PSCs. (26A) Cells are positive for SSEA-4 by flow cytometry. (26B) Cells are alkaline phosphatase positive. For both A and B, isogenic WT and KO are shown on the left and right, respectively. These data demonstrate that we successfully generated multiple lineages that maintained pluripotency, i.e., they are bona fide PSCs.

[0037] [Figure 27]Figure 27 shows LFA-1 and MAC-1 staining on immune cells. Both LFA-1 and MAC-1 are ligands for ICAM-1 (CD54) and are present at different levels on many immune cells. We stained peripheral blood lymphocytes (left), monocytes (center), and the monocytic lymphoma line U937 (right) with anti-LFA-1 and anti-MAC-1 antibodies and acquired data by flow cytometry. These data demonstrate that immune cells have a ligand for ICAM-1 and that its binding is disrupted in our KO line.

[0038] [Figure 28] Figure 28 shows that blocking ICAM-1 inhibits immune cell binding. PSC-derived endothelial cells were stimulated with TNFα and IFNg for 48 hours, and then ICAM-1 was blocked by antibody incubation. ECs were then briefly co-cultured with U937 monocytic leukemia cells and peripheral blood mononuclear cells (PBMCs, a mixture of lymphocytes and monocytes). Immune cells were washed away, and all wells were treated equally. Images of fluorescently labeled immune cells co-cultured with ECs were acquired. Bound cells were quantified using Image J software. Five regions of interest were imaged and quantified, and the data were summarized in a plot. These data demonstrate that blocking ICAM-1 inhibits LFA-1 and / or MAC-1 binding, thus reducing the binding of multiple types of immune cells.

[0039] [Figures 29A-29B]Figures 29A and 29B show static immune cell adhesion assays. H9 WT and CD54KO PSCs were differentiated into endothelial cells (ECs) and stimulated with TNFα and IFNg for 48 hours, then briefly cocultured with U937 monocytic leukemia cells. The U937 cells were washed away, and all wells were treated equally. (29A) Image of fluorescently labeled U937 cells cocultured with ECs. Bound cells were quantified using Image J software. Five regions of interest were imaged and quantified, and the data are summarized in (29B). These data are representative of n=3 replicate experiments. Results were consistent with both low- and high-purity PSC-ECs. The significance of these data supports our hypothesis that ICAM-1 KO, in this case, significantly reduces immune cell binding to potential PSC-derived cell therapies. DETAILED DESCRIPTION OF THE INVENTION

[0040] The present disclosure provides improved allogeneic cell tolerance, including methods for generating cells with reduced immunogenicity after grafting, e.g., transplantation, and methods for using those cells. For example, there are specific adhesion proteins on cells that immune cells use to attach / bind to cells, which is one of the first steps for immune cells in rejecting non-self cells. As disclosed herein, knocking down the expression of one or more of these proteins in stem cells, such as pluripotent stem cells, has been found to inhibit or prevent immune cell attachment to differentiated cells resulting from the modified stem cells, which can be used as therapeutic agents. The resulting cells can therefore be used to treat a wide variety of diseases, including, but not limited to, cardiovascular disease, diabetes, neuropathies, liver disease, and viral infections. For example, the cells can be PSC-beta islets (diabetes), PSC-hepatocytes (liver disease), PSC-neuronal and neural subtypes (neurological disease), or PSC-endothelial cells (vascular disease), although any cell type may benefit from knocking out expression, resulting in reduced immune system recognition.

[0041] As used herein, reduced immunogenicity means that the cells are less likely to engage with and / or be rejected by any immune cell(s). Reduced immunogenicity can be demonstrated by reduced binding to immune cells. Reduced immunogenicity can also be demonstrated as a reduced proliferative response from T cells upon encountering ICAM-1 KO cells, such as through a mixed lymphocyte reaction (MLR). Furthermore, in vivo in NeoTHy humanized mice, KO cells persist longer than WT cells. This means that the immune system does not recognize and reject them, as well as WT cells.

[0042] In one aspect, an in vitro method for preparing a population of hypoimmune mammalian stem cells includes providing a population of isolated mammalian stem cells, wherein the isolated mammalian stem cells express a cell adhesion molecule, and modifying the expression of the cell adhesion molecule in the isolated mammalian stem cell population to reduce or knock out the expression of the cell adhesion molecule to provide a population of hypoimmune mammalian cells. In one aspect, the population of hypoimmune mammalian cells is less immunogenic than a corresponding population of isolated mammalian stem cells, as defined above. The isolated population of mammalian stem cells may include pluripotent stem cells or embryonic stem cells, and may be human or non-human stem cells.

[0043] In one embodiment, one or more mammalian stem cells are non-human stem cells, for example, the stem cells of non-human primates, cows, horses, dogs, cats, goats, pigs, mice or sheep.Therefore, knockout cells can be used in xenotransplantation research, for example, in addition to various current gene editing carried out in pigs, CD54 knockout can improve transplantation and / or be less likely to be rejected by human immune cells.

[0044] Thus, in one embodiment, isolated immune-evasive (hypoimmune) stem cells are provided, e.g., human pluripotent stem cells, such as induced human pluripotent stem cells (iPSCs) or human embryonic stem cells, that have reduced or absent expression ("knockdown" or "knockout") of one or more cell adhesion molecules as a result of gene editing of one or more alleles (to produce heterozygotes or homozygotes) or other approaches to inhibit expression, such as inhibitory RNA (e.g., siRNA, shRNA, miRNA, antisense oligonucleotides, etc.). Gene editing can include CRISPR intervention, e.g., using Cas13 or dCas9. In one embodiment, CRISPR gene editing can be used to insert a stop codon within a gene, e.g., within an open reading frame, thus preventing expression of a functional gene product. Populations of hypoimmune mammalian stem cells can be differentiated into any cell type or multiple cell types, e.g., cardiomyocytes or endothelial cells, with the target adhesion protein(s) remaining knocked down or knocked out in the differentiated cells. Exemplary adhesion proteins include, but are not limited to, CD54 (ICAM-1), ICAM-2, ICAM-3, ICAM-4, ICAM-5, VCAM, MADCAM-1, P-selectin, E-selectin, L-selectin, integrins, focal adhesion molecules, extracellular matrix molecules, costimulatory molecules, and other molecules involved in the immune synapse and / or tethering, rolling, and extravasation.

[0045] In one embodiment, isolated cells, e.g., isolated stem cells, e.g., pluripotent stem cells or differentiated cells, e.g., T cells or other immune cells, e.g., hematopoietic stem or progenitor cells, cardiomyocytes, fibroblasts, endothelial cells, lack or reduce expression of CD54 or other adhesion molecule(s), or a combination of adhesion molecules. These cells enable methods of preventing or inhibiting transplant rejection of differentiated cells, including knocking down or eliminating expression of one or more adhesion proteins in these cells. For example, CD54-KO PSC-derived T cells, e.g., CD4 T cells, can be resistant to HIV infection.

[0046] As described herein, gene editing was used on different pluripotent stem cell lineages, such as H9, H1, PED05, and PED04, or their derivatives, and the resulting knockdown or knockout lineages were differentiated into, for example, cardiomyocytes, endothelial cells, or fibroblasts, demonstrating that cells can differentiate without obvious defects after CD54 knockdown (or knockout). For example, cardiomyocytes with CD54 knockout have a normal karyotype and no obvious defects in morphology; for example, they appear precisely normal and behave similarly, for example, with respect to contractility and / or differentiation efficiency. Differentiated cells can be tested to determine their immune cell recognition properties. For example, CD54 knockout PSC-derived T cells can be resistant to HIV infection. Therefore, any PSC immune cell knockout, including T cells (including regulatory T cells, effector T cells, and other subsets), B cells, NK cells, monocytes, macrophages, dendritic cells, etc., can be prepared and used in in vivo methods.

[0047] In one embodiment, the isolated population of mammalian stem cells has reduced or absent expression of one or more different HLA class I molecules, one or more different HLA class II molecules, beta2 microglobulin, or increased expression of CD47, PDL1, secretin, or CTLA4, or any combination or modified version thereof (e.g., class II activator of TNF-α (CIITA) or secreted CTLA4-Ig instead of membrane-bound CTLA-4). The edits herein can improve the function of existing gene edits (e.g., by enhancing existing edits / lineages by increasing the number of immune cells that become unresponsive to those existing edits / lineages, improving their immune evasion capabilities in certain situations, such as when the graft is vascular in nature).

[0048] In one embodiment, the method comprises isolating, expanding, and / or differentiating a population of hypoimmune mammalian stem cells, or any combination thereof. In one embodiment, the expanded population of hypoimmune mammalian stem cells is differentiated. In one embodiment, the isolated cells are differentiated. In one embodiment, the population of hypoimmune mammalian stem cells is differentiated into endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, hematopoietic cells, lymphocytes, or pancreatic islet cells. In one embodiment, the differentiated cells are cardiomyocytes or neurons. Also provided is a population of hypoimmune mammalian stem cells, e.g., hypoimmune cells, produced by the method.

[0049] In one embodiment, a population of hypoimmune mammalian stem cells is provided, which, as a result of genetic modification, have reduced or absent expression of one or more adhesion molecules, for example, compared to mammalian stem cells or differentiated mammalian cells without genetic modification. In one embodiment, the genetic modification is directed to two alleles of a gene encoding an adhesion molecule (homozygous null mutation). In one embodiment, the isolated hypoimmune mammalian cells have reduced or absent expression of ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, VCAM, MADCAM-1, CD54, P-selectin, E-selectin, L-selectin, or a combination thereof. In one embodiment, the parent cells are MHC-1 / 2 knockout lines, thereby providing an additive effect. In one embodiment, the parent cells are hypoimmune mammalian cells with reduced or absent expression of CD54, P-selectin, E-selectin, L-selectin, or a combination thereof, and are subsequently modified to knock out MHC1 and / or MHC2. In one embodiment, the resulting cells or the parent optionally overexpress HLA-E and / or CD47 as a result of genetic modification. The population of hypoimmune mammalian stem cells can be differentiated into endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or pancreatic islet cells.

[0050] Also provided are grafts comprising a plurality of one or more hypoimmune differentiated mammalian cell types, wherein the cells are hypoimmune as a result of reduced or absent expression of one or more adhesion molecules. In one embodiment, the hypoimmune differentiated mammalian cells are hematopoietic cells, lymphoid cells, endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or pancreatic islet cells. In one embodiment, the mammalian cells are human cells. In one embodiment, the graft comprises endothelial cells, fibroblasts, and cardiomyocytes. Methods of using the graft are also provided, for example, by introducing the graft into a mammal in need thereof. In one embodiment, the mammal is human. In one embodiment, the hypoimmune cells are administered as part of a delivery device, such as a patch applied to the heart, a bioengineered blood vessel, or an engineered organ, which may be comprised of multiple PSC-derived cell types.

[0051] In one embodiment, a method for enhancing cell function in a mammal in need thereof is provided, comprising administering to the mammal an effective amount of isolated hypoimmune mammalian stem or differentiated cells. In one embodiment, the mammal is a human. In one embodiment, the mammal is a non-human primate, dog, cat, cow, horse, pig, sheep, or goat. In one embodiment, the differentiated cells comprise one or more endothelial cells, cardiomyocytes, fibroblasts, neurons, hematopoietic cells, lymphoid cells, or pancreatic islet cells.

[0052] Further provided is a method for preventing, inhibiting, or treating neurodegeneration in a mammal, comprising administering to the mammal an effective amount of a composition comprising hypoimmune neural cells that have reduced or absent expression of one or more adhesion molecules as a result of genetic modification. In one embodiment, the composition is administered to the central nervous system. In one embodiment, the composition is injected. In one embodiment, the composition is administered intracerebroventricularly. In one embodiment, the mammal is a human. In one embodiment, the mammal is a human with Alzheimer's disease or has suffered a stroke, and the hypoimmune PSC neurons and / or neural subsets are administered.

[0053] In one embodiment, a method for preventing, inhibiting, or treating diabetes in a mammal is provided, comprising administering to the mammal an effective amount of a composition comprising hypoimmune pancreatic islet cells that have reduced or absent expression of one or more adhesion molecules as a result of genetic modification. In one embodiment, the composition is administered systemically. In one embodiment, the composition is injected. In one embodiment, the composition is administered into the portal vein. In one embodiment, the mammal is a human. In one embodiment, the cells are PCS pancreatic islet cells. In one embodiment, the composition comprises a scaffold for the hypoimmune cells, which are then transplanted into the mammal.

[0054] A method for preventing, inhibiting, or treating vascular disease in a mammal is provided. The method comprises administering to the mammal an effective amount of a composition comprising hypoimmune endothelial cells that have reduced or absent expression of one or more adhesion molecules as a result of genetic modification. In one embodiment, the mammal has peripheral vascular disease. In one embodiment, the mammal has, has had, or is at risk for myocardial infarction. In one embodiment, the composition is injected. In one embodiment, the composition is administered systemically. In one embodiment, the mammal is a human. In one embodiment, the composition is a vascular graft comprising hypoimmune cells.

[0055] Also provided is a method for preventing, inhibiting, or treating myocardial disease in a mammal, comprising administering to the mammal an effective amount of a composition comprising hypoimmune cardiomyocytes that have reduced or absent expression of one or more adhesion molecules as a result of genetic modification. In one embodiment, the composition is injected. In one embodiment, the composition is administered to the heart. In one embodiment, the composition also comprises hypoimmune fibroblasts, hypoimmune endothelial cells, or both. In one embodiment, the mammal is a human. In one embodiment, the composition is a patch comprising the hypoimmune cells.

[0056] In one embodiment, the hypoimmune cells are a bioengineered modified organ or portion thereof, e.g., a decellularized organ or portion thereof is seeded with one or more different types of hypoimmune cells.

[0057] Also provided is a method for controlling or increasing resistance to viral infection or replication in a mammal in need thereof, comprising administering to the mammal an effective amount of a composition comprising hypothyroid immune cells that have reduced or absent expression of one or more adhesion molecules as a result of genetic modification. In one embodiment, the composition is injected. In one embodiment, the mammal is a human. In one embodiment, the virus is a lentivirus such as HIV. In one embodiment, the virus is a rhinovirus or another virus that infects immune cells.

[0058] In one embodiment, an in vitro method for preparing a population of hypoimmune mammalian stem cells can include the use of delivery vectors. Delivery vectors include, for example, plasmids, viral vectors, liposomes and other lipid-containing complexes, such as lipoplexes (DNA and cationic lipids), polyplexes, DNA complexed with cationic polymers such as polyethylene glycol, nanoparticles, such as magnetic inorganic nanoparticles that bind to DNA or are functionalized to bind to DNA, such as Fe3O4 or MnO2 nanoparticles, microparticles formed with polylactide-polygalacid reagents, nanotubes, such as silica nanotubes, and other polymeric complexes that can mediate gene delivery into host cells. Vectors can also contain other components or functionalities that further regulate nucleic acid delivery and / or gene expression or otherwise provide beneficial properties to target cells. Such other components include, for example, components that affect cell binding or targeting (including components that mediate cell-type or tissue-specific binding); components that affect cellular uptake of the vector; components that affect the localization of the transferred gene within the cell after uptake (such as agents that mediate nuclear localization); and components that affect gene expression. Such components may also include markers, such as detectable and / or selectable markers that can be used to detect or select cells that have taken up and are expressing the nucleic acid delivered by the vector. A wide variety of such vectors are known in the art and are generally available.

[0059] Exemplary nucleic acid delivery vehicles deliver, among others, guide RNA, siRNA or vector for its expression, and the coding sequence of recombinase, including CRISPR, TALEN and zinc finger protein, or a combination thereof.Other delivery vehicles can optionally comprise proteins, such as recombinase or antibody or its fragment, or non-protein non-nucleic acid molecules, in combination with nucleic acid.

[0060] Nucleic acid delivery vectors within the scope of the present disclosure include, but are not limited to, isolated nucleic acids, e.g., plasmid-based vectors that can be maintained extrachromosomally, as well as viral vectors, such as recombinant adenoviruses, retroviruses, lentiviruses, herpesviruses, poxviruses, papillomaviruses, or adeno-associated viruses, including viral and non-viral vectors present in liposomes, e.g., DOSPA / DOPE, DOGS / DOPE, or DMRIE / DOPE liposomes, and / or associated with other molecules, such as DNA-anti-DNA antibody-cationic lipid (DOTMA / DOPE) complexes. Exemplary vectors are described below. Nucleic acid delivery vectors can be administered via any route, including, but not limited to, intracranial, intrathecal, intramuscular, buccal, rectal, intravenous, or intracoronary administration, and cellular entry can be enhanced using electroporation and / or iontophoresis, and / or extracellular matrices or scaffolds such as hydrogels, e.g., hydrogel patches.

[0061] CRISPR / Cas System: Type II CRISPR is a well-characterized system that creates double-stranded breaks in target DNA in four sequential steps. First, two non-coding RNAs, the pre-crRNA and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates processing of the pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex targets Cas9 to the target DNA via Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), a further requirement for target recognition. Finally, Cas9 mediates cleavage of the target DNA, creating a double-stranded break within the protospacer. The activity of the CRISPR / Cas system involves three stages: (i) insertion of a foreign DNA sequence into the CRISPR array to prevent future attacks in a process called adaptation; (ii) expression of associated proteins and expression and processing of the array; followed by (iii) RNA-mediated interference with the foreign nucleic acid. Thus, in bacterial cells, several so-called Cas proteins are involved in the natural function of the CRISPR / Cas system. The primary product of the CRISPR locus appears to be a short RNA containing the invader targeting sequence, called a guide RNA.

[0062] "Cas1" polypeptide refers to CRISPR-associated (Cas) protein 1. Cas1 (COG1518 in the Cluster of Proteins Orthologous Group Classification System) is the best marker for CRISPR-associated systems (CASSs). Based on phylogenetic comparisons, seven distinct versions of the CRISPR-associated immune system have been identified (CASSs 1-7). The Cas1 polypeptide used in the methods described herein can be any Cas1 polypeptide present in any prokaryotic organism. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide from an archaeal microorganism. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide from a Euryarchaeota microorganism. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide from a Crenarchaeota microorganism. In certain embodiments, the Cas1 polypeptide is a bacterial Cas1 polypeptide. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide from a Gram-negative or Gram-positive bacterium. In certain embodiments, the Cas1 polypeptide is a Pseudomonas aeruginosa Cas1 polypeptide. In certain embodiments, the Cas1 polypeptide is an Aquifex aeolicus Cas1 polypeptide. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of one of CAS1-7. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of CASS3. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of CASS7. In certain embodiments, the Cas1 polypeptide is a Cas1 polypeptide that is a member of CASS3 or CASS7.

[0063] In some embodiments, the Cas1 polypeptide is identified in GenBank as, for example, Gene ID numbers: 2781520, 1006874, 9001811, 947228, 3169280, 2650014, 1175302, 3993120, 4380485, 906625, 3165126, 905808, 1454460, 1445886, 1485099, 4274010, 888506, 316952 6, 997745, 897836, or 1193018, and / or an amino acid sequence that exhibits homology (e.g., greater than 80%, 90-99%, including 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the amino acid sequences encoded by these polynucleotides, and which polypeptides function as Cas1 polypeptides.

[0064] There are three types of CRISPR / Cas systems, all of which incorporate RNA and Cas proteins: Type I and Type III both have a Cas endonuclease that processes the pre-crRNA and, once fully processed into crRNA, assembles into a multi-Cas protein complex that can cleave nucleic acids complementary to the crRNA.

[0065] In type II CRISPR / Cas systems, crRNA is produced using a different mechanism in which a trans-activating RNA (tracrRNA) complementary to a repeat sequence in the pre-crRNA triggers double-strand-specific RNase III processing in the presence of the Cas9 protein. Cas9 can then cleave target DNA complementary to the mature crRNA; however, cleavage by Cas9 depends on both base pairing between the crRNA and the target DNA and the presence of a short motif in the crRNA called the PAM sequence (protospacer adjacent motif). In addition, the tracrRNA must also be present, base-paired to the crRNA at its 3' end; this association triggers Cas9 activity.

[0066] The Cas9 protein has at least two nuclease domains, one similar to the HNH endonuclease and the other similar to the Ruv endonuclease domain. The HNH-type domain is responsible for cleaving the DNA strand complementary to the crRNA, while the Ruv domain appears to cleave the non-complementary strand.

[0067] The need for a crRNA-tracrRNA complex can be circumvented by the use of an engineered "single guide RNA" (sgRNA) that contains a hairpin normally formed by annealing of the crRNA and tracrRNA. In S. pyrogenes, the engineered tracrRNA:crRNA fusion, or sgRNA, guides Cas9 to cleave the target DNA when a double-stranded RNA:DNA heterodimer forms between the Cas-associated RNA and the target DNA. This system includes the Cas9 protein and an engineered sgRNA.

[0068] "Cas polypeptide" encompasses full-length Cas polypeptides, enzymatically active fragments of Cas polypeptides, and enzymatically active derivatives of Cas polypeptides or fragments thereof. Exemplary derivatives of Cas polypeptides or fragments thereof include, but are not limited to, mutants, fusions, and covalent modifications of Cas proteins or fragments thereof.

[0069] RNA Components of CRISPR / Cas: The Cas9-associated CRISPR / Cas system contains two non-coding RNA components: a tracrRNA and a pre-crRNA array containing nuclease guide sequences (spacers) spaced by identical direct repeats (DRs). To achieve genome engineering using the CRISPR / Cas system, both of these RNA functions must be present. In some embodiments, the tracrRNA and pre-crRNA are provided via separate expression constructs or as separate RNAs. In other embodiments, a chimeric RNA is constructed in which an engineered mature crRNA (which confers target specificity) is fused to the tracrRNA (which provides interaction with Cas9) to create a chimeric cr-RNA-tracrRNA hybrid (also called a single guide RNA).

[0070] Chimeric RNAs, or sgRNAs, can be engineered to contain sequences complementary to any desired target. The RNA contains 22 bases of complementarity to the target and a protospacer adjacent motif (PAM) of the form G[N19] followed by NGG. Thus, in one method, sgRNAs can be designed by (i) aligning the recognition sequence of the ZFN heterodimer with a reference sequence in the relevant genome (human, mouse, or a specific plant species); (ii) identifying the spacer region between the ZFN half-sites; (iii) locating the motif G[N20]GG closest to the spacer region (if two or more such motifs overlap with the spacer, a motif centered relative to the spacer is selected); and (iv) utilizing a known ZFN target in the gene of interest by using that motif as the core of the sgRNA. This method advantageously relies on proven nuclease targets. Alternatively, sgRNAs can be designed to target any region of interest simply by identifying an appropriate target sequence that fits the G[N20]GG formula.

[0071] Donor: As described above, an exogenous sequence (also referred to as a "donor sequence" or "donor" or "transgene" or "gene of interest") is inserted, for example, to correct a mutant gene or increase the expression of a wild-type gene. It should be readily apparent that the donor sequence is typically not identical to the genomic sequence into which it is placed. The donor sequence may contain a non-homologous sequence flanked by two homologous regions to enable efficient HDR at the location of interest. Alternatively, the donor may not have a region of homology to the target location in the DNA and may be integrated by NHEJ-dependent end joining after cleavage at the target site. Furthermore, the donor sequence may comprise a vector molecule containing a sequence that is not homologous to the region of interest in cellular chromatin. The donor molecule may contain several discontinuous regions of homology with cellular chromatin. For example, in the case of targeted insertion of a sequence not normally present in the region of interest, the sequence may be present in the donor nucleic acid molecule and be adjacent to a region of homology to the sequence of the region of interest.

[0072] Donor polynucleotides can be single-stranded and / or double-stranded DNA or RNA, and can be introduced into cells in linear or circular form.When introduced in linear form, the ends of the donor sequence can be protected (for example, from exonuclease degradation) by methods known to those skilled in the art.For example, one or more dideoxynucleotide residues are added to the 3' end of the linear molecule, and / or self-complementary oligonucleotides are linked to one or both ends.Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, adding terminal amino group(s) and using modified internucleotide linkages, such as phosphorothioate, phosphoramidate, and O-methylribose or deoxyribose residues.

[0073] Polynucleotides can be introduced into cells as part of a vector molecule that contains additional sequences such as, for example, an origin of replication, a promoter, and genes encoding antibiotic resistance. Additionally, donor polynucleotides can be introduced as naked nucleic acid, as nucleic acid complexed with agents such as liposomes or poloxamers, or delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus, and integrase-deficient lentivirus (IDLV)).

[0074] The donor is generally inserted such that its expression is driven by the endogenous promoter of the integration site, i.e., the promoter that drives expression of the endogenous gene into which the donor is inserted (e.g., highly expressed albumin, AAVS1, HPRT, etc.). However, it will be apparent that the donor may include a promoter and / or enhancer, e.g., a constitutive promoter or an inducible or tissue-specific promoter.

[0075] The donor molecule can be inserted into an endogenous gene so that all, part, or none of the endogenous gene is expressed. For example, the transgene described herein can be inserted into an albumin or other gene locus, for example, as a fusion with a transgene encoding a lysosomal sequence, so that part (the N-terminus and / or C-terminus of the transgene encoding a lysosomal enzyme) or none of the endogenous albumin sequence is expressed. In other embodiments, the transgene (e.g., with or without additional coding sequences such as albumin) is integrated into any endogenous gene locus, for example, a safe harbor locus. See, for example, U.S. Patent Application Publication Nos. 2008 / 0299580, 2008 / 0159996, and 2010 / 0218264.

[0076] When an endogenous sequence (endogenous or a portion of a transgene) is expressed along with a transgene, the endogenous sequence (e.g., albumin, etc.) can be a full-length sequence (wild-type or mutant) or a partial sequence. The endogenous sequence can be functional. Non-limiting examples of functions of these full-length or partial sequences (e.g., albumin) include increasing the serum half-life of the polypeptide expressed by the transgene (e.g., a therapeutic gene) and / or acting as a carrier.

[0077] Additionally, although not necessary for expression, the exogenous sequence may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides, and / or polyadenylation signals.

[0078] Other Editing Systems: According to another embodiment, in addition to the clustered regularly interspaced short palindromic repeats (CRISPR / Cas) system, the genome editing methods of the present disclosure may include any other form of genome editing, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), as well as RNA editing of precursor mRNAs containing such intronic sequences via CRISPR / Cas12a, CRISPR / Cas13, or other related genome editing approaches.

[0079] In some embodiments, the agent for changing target gene is TALEN system or its equivalent.As used herein, TALEN or " transcription activator-like element nuclease " or " TALE nuclease " refers to the artificial nuclease that comprises a transcription activator, such as a DNA cleavage domain, an effector DNA binding domain to a FokI domain, for example.Several modular assembly schemes have been reported for generating engineered TALE constructs.

[0080] Those skilled in the art will understand that TALE nucleases can be engineered to target virtually any genomic sequence with high specificity, and that such engineered nucleases can be used in embodiments of the present technology to manipulate the genome of a cell, for example, by delivering each TALEN via the methods or strategies disclosed herein under conditions suitable for the TALEN to bind to and cleave its target sequence in the genome of the cell. In some embodiments, the delivered TALEN targets a gene or allele associated with the target circRNA. In some embodiments, delivering TALEN to a subject provides a therapeutic benefit to the subject, such as reducing or eliminating the expression of circRNA in the subject in need thereof.

[0081] In some embodiments, a target gene of a cell, tissue, organ, or organism is altered by a nuclease, such as a CRISPR / cas-9, TALEN, or zinc finger nuclease, or a plurality or combination of such nucleases, delivered to the cell via a strategy or method disclosed herein. In some embodiments, a single- or double-stranded break is introduced by the nuclease at a specific site within the genome, resulting in the disruption of a target genomic sequence, such as an intronic regulatory sequence.

[0082] As used herein, the term "zinc finger" refers to a small nucleic acid-binding protein structural motif characterized by a fold and the coordination of one or more zinc ions that stabilize the fold. Zinc fingers encompass a wide variety of different protein structures. Zinc fingers can be designed to bind to specific sequences of nucleotides, and zinc finger arrays, comprising fusions of a series of zinc fingers, can be designed to bind to virtually any desired target sequence. Such zinc finger arrays, when conjugated to, for example, a nucleic acid cleavage domain, can form the binding domain of a protein, such as a nuclease. Various types of zinc finger motifs are known to those skilled in the art, including, but not limited to, Cys2His2, Gag knuckle, Treble clef, zinc ribbon, Zn2 / Cys6, and TAZ2 domain-like motifs. Typically, a single zinc finger motif binds to three or four nucleotides of a nucleic acid molecule. Thus, a zinc finger domain containing two zinc finger motifs can bind 6-8 nucleotides, a zinc finger domain containing three zinc finger motifs can bind 9-12 nucleotides, a zinc finger domain containing four zinc finger motifs can bind 12-16 nucleotides, etc. Any suitable protein engineering techniques can be used to alter the DNA binding specificity of the zinc fingers and / or to design zinc finger fusions to bind to virtually any desired target sequence between 3 and 30 nucleotides in length.

[0083] Fusions between engineered zinc finger arrays and protein domains that cleave nucleic acids can be used to generate "zinc finger nucleases." Zinc finger nucleases typically contain a zinc finger domain that binds to a specific target site within a nucleic acid molecule and a nucleic acid cleavage domain that cleaves the nucleic acid molecule within or adjacent to the target site bound by the binding domain. Typical engineered zinc finger nucleases contain a binding domain with three to six individual zinc finger motifs and a binding target site ranging in length from 9 to 18 base pairs. Longer target sites are particularly attractive in situations where binding and cleaving unique target sites in a given genome is desired.

[0084] In some embodiments, the agent for altering gene expression is a zinc finger nuclease or other equivalent.As used herein, the term "zinc finger nuclease" refers to a nuclease comprising a nucleic acid cleavage domain conjugated to a binding domain comprising a zinc finger array.In some embodiments, the cleavage domain is the cleavage domain of the type II restriction endonuclease FokI.Zinc finger nucleases can be designed to target virtually any desired sequence within a given nucleic acid molecule for cleavage, and the possibility of designing zinc finger binding domains to bind to unique sites in the context of complex genomes allows for the targeted cleavage of single genomic sites in living cells, for example, to achieve targeted genomic changes of therapeutic value.Targeting double-strand breaks to desired genomic loci can be used to introduce frameshift mutations into the coding sequence of genes due to the error-prone nature of non-homologous DNA repair pathways.

[0085] Zinc finger nucleases can be generated to target sites of interest by methods well known to those skilled in the art.For example, zinc finger binding domains with desired specificity can be designed by combining individual zinc finger motifs of known specificity.The structure of the zinc finger protein Zif268 bound to DNA has informed much of the research in this field, and has described the concept of obtaining zinc fingers for each of the 64 possible base pair triplets, and then mixing and matching these modular zinc fingers to design proteins with any desired sequence specificity.

[0086] In some embodiments, distinct zinc fingers, each recognizing a 3-base pair DNA sequence, are combined to generate 3-, 4-, 5-, or 6-finger arrays that recognize target sites ranging in length from 9 base pairs to 18 base pairs. Longer arrays are contemplated in some embodiments. In other embodiments, two finger modules that recognize 6 to 8 nucleotides are combined to generate 4-, 6-, or 8-zinc finger arrays. In some embodiments, bacterial or phage display is used to express zinc finger domains that recognize desired nucleic acid sequences, e.g., desired nuclease target sites 3 to 30 bp in length.

[0087] In some embodiments, zinc finger nucleases comprise a zinc finger binding domain and a cleavage domain fused or conjugated to each other via a linker, e.g., a polypeptide linker. The length of the linker determines the distance of cleavage from the nucleic acid sequence bound by the zinc finger domain. When a shorter linker is used, the cleavage domain cleaves a nucleic acid closer to the bound nucleic acid sequence, while a longer linker results in a greater distance between the cleaved nucleic acid sequence and the bound nucleic acid sequence. In some embodiments, the cleavage domain of a zinc finger nuclease must dimerize to cleave the bound nucleic acid. In some such embodiments, the dimer is a heterodimer of two monomers, each containing a different zinc finger binding domain. For example, in some embodiments, the dimer may comprise one monomer containing zinc finger domain A conjugated to a FokI cleavage domain and one monomer containing zinc finger domain B conjugated to a FokI cleavage domain. In this non-limiting example, zinc finger domain A binds to a nucleic acid sequence on one side of a target site, zinc finger domain B binds to a nucleic acid sequence on the other side of the target site, and the dimerized FokI domain cleaves the nucleic acid between the zinc finger domain binding sites.

[0088] siRNA: siRNA delivery vectors within the scope of the present disclosure include, but are not limited to, isolated nucleic acids, such as plasmid-based vectors that can be maintained extrachromosomally, as well as viral vectors, including viral and non-viral vectors present in liposomes, such as neutral or cationic liposomes, such as DOSPA / DOPE, DOGS / DOPE, or DMRIE / DOPE liposomes, and / or associated with other molecules, such as DNA-anti-DNA antibody-cationic lipid (DOTMA / DOPE) complexes or natural or synthetic polymers, such as recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papillomavirus, or adeno-associated virus. Exemplary viral gene delivery vectors are described below. Nucleic acid delivery vectors can be administered via any route, including, but not limited to, intracranial, intrathecal, intramuscular, buccal, rectal, intravenous, or intracoronary administration, and cellular transfer can be enhanced using electroporation and / or iontophoresis, and / or extracellular matrices or scaffolds such as hydrogels, e.g., hydrogel patches.

[0089] In one embodiment, vector is a viral vector.Exemplary viral vectors include, for example, retroviral vector, lentiviral vector, herpes simplex virus (HSV)-based vector, parvovirus-based vector, for example, adeno-associated virus (AAV)-based vector, AAV-adenovirus chimeric vector, and adenovirus-based vector.These viral vectors can be prepared using standard recombinant DNA technology, for example, as described in Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, NY (1994).

[0090] Retroviral Vectors: Retroviral vectors exhibit several unique features, including the ability to stably and precisely integrate into the host genome, providing long-term transgene expression. These vectors can be manipulated ex vivo to eliminate infectious gene particles, minimizing the risk of systemic infection and patient-to-patient transmission. Pseudotyped retroviral vectors can alter host cell tropism.

[0091] Lentivirus: Lentiviruses are derived from a family of retroviruses that includes human immunodeficiency virus and feline immunodeficiency virus. However, unlike retroviruses, which infect only dividing cells, lentiviruses can infect both dividing and non-dividing cells. While lentiviruses have specific tropism, pseudotyping of the viral envelope with vesicular stomatitis virus results in a broader spectrum of the virus.

[0092] Adenoviral vectors: Adenoviral vectors can be rendered replication-incompetent by deleting the early (E1A and E1B) genes involved in viral gene expression from the genome, allowing them to stably maintain themselves in host cells in an extrachromosomal form. These vectors have the ability to transfect both replicating and non-replicating cells. Adenoviral vectors have been shown to produce transient expression of therapeutic genes in vivo, peaking on day 7 and persisting for approximately four weeks. Furthermore, adenoviral vectors can be produced at very high titers, enabling efficient gene therapy with small amounts of virus.

[0093] Adeno-associated virus vector: Recombinant adeno-associated virus (rAAV) is derived from a non-pathogenic parvovirus, does not induce cellular immune responses, and produces transgene expression that lasts for several months in most systems.Furthermore, like adenovirus, adeno-associated virus vectors also have the ability to infect replicating and non-replicating cells.AAV vectors include, but are not limited to, AAV1, AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrh10, including chimeric viruses in which the AAV genome is derived from a source different from the capsid.

[0094] Plasmid DNA vectors: Plasmid DNA is often referred to as "naked DNA" to indicate the absence of more complex packaging systems. Direct injection of plasmid DNA into cardiomyocytes in vivo has been achieved. Plasmid-based vectors are relatively non-immunogenic and non-pathogenic and can stably integrate into the cellular genome, resulting in long-term gene expression in postmitotic cells in vivo. Furthermore, plasmid DNA is rapidly degraded in the bloodstream. Therefore, the possibility of transgene expression in distant organ systems is negligible. Plasmid DNA can be delivered to cells as part of a macromolecular complex, such as a liposome or DNA-protein complex, and delivery can be enhanced using techniques including electroporation.

[0095] Hypoimmune Cells: One obstacle preventing the widespread use of differentiated stem cells as therapeutics is immune rejection of transplanted cells. This disclosure describes a platform strategy for modifying stem cells, such as pluripotent stem cell lineages, prior to cell therapy manufacturing. The resulting cells (including differentiated cells therefrom) can be used for biomanufacturing differentiated stem cell therapies because they do not disrupt the immune system balance necessary to prevent transplant rejection while also preventing tumorigenesis.

[0096] For example, gene editing approaches may enable immune evasion of stem cells used for therapy due to the stem cell properties of iPSCs (clonal cell lineages, massively scalable). However, current immune-lowering gene editing approaches are "hammer" approaches that can render transplanted cell grafts susceptible to malignant overgrowth and / or epidemic viral infections. Furthermore, even with HLA-knockout cells, for example, there are redundant mechanisms of allogeneic rejection (indirect pathway) that can result in graft loss in typical clinical situations. The editing described here may circumvent these drawbacks.

[0097] The present disclosure provides an alternative mechanism for circumventing the immune response. Furthermore, this approach may reduce the likelihood of graft loss through two distinct but complementary mechanisms by which grafts are rejected: direct immune cell contact and indirect inflammatory processes.

[0098] In one embodiment, PSCs, such as iPSCs or ESCs, can be genetically modified, e.g., by gene editing or other methods, e.g., in a GMO facility. Cell banks suitable for clinical use are generated. In one embodiment, CD54 knockout (CD54-KO) stem cells (optionally containing additional gene edits such as B2M KO) are differentiated into cell therapies (e.g., cardiomyocytes for myocardial infarction, endothelial cells for vascular disease, neurons for dementia, pancreatic islet cells for diabetes, hepatocytes for liver disease, T cells for HIV infection, retinal pigment epithelial cells for macular degeneration, etc.). These cell therapies can be used incrementally as needed. The cells are transplanted into patients, e.g., as therapeutic, curative, and / or immune-tolerizing cell therapies. As a result, these patients may not need to take immunosuppressive drugs (which have many unobvious adverse effects) or may require fewer immunosuppressive drugs because the cell therapy itself protects against rejection, eliminating the need for systemic and / or multidrug immunosuppression.

[0099] Thus, in one embodiment, by suppressing adaptive and innate immune responses, based on the specific immunobiology of the endothelial cell:immune interface and immune synapse that act as a barrier in allogeneic rejection, treatments such as cardiac therapies could not only improve the quality of life for millions of patients, but also save their lives.

[0100] Exemplary Proteins for Knockdown / Knockout: The present disclosure provides cells with reduced expression of one or more adhesion molecules. Adhesion molecules are generally divided into five groups: members of the immunoglobulin superfamily (IgSF), including integrins, selectins, cadherins, and nectins, and other groups such as mucins.

[0101] Integrins typically bind to the extracellular matrix, while selectins, cadherins and IgSF members are involved in cell-cell adhesion.

[0102] Selectins are further divided into P-, E-, and L-selectins, based on which cell types they were originally found on: platelets, endothelial cells, and leukocytes.

[0103] Members of the IgSF contain at least one immunoglobulin or immunoglobulin-like domain, and most members are type I transmembrane proteins with an extracellular domain (containing one or more Ig domains), a transmembrane domain, and a cytoplasmic tail. The best-known members include major histocompatibility complex (MHC) class I and II molecules and proteins of the T cell receptor (TCR) complex. Intercellular adhesion molecules (ICAMs), vascular cell adhesion molecules (VCAMs), MAdCAM-1, and activated leukocyte cell adhesion molecules (ALCAMs), which are important in leukocyte trafficking events, also belong to this family of adhesion receptors.

[0104] Integrins are large heterodimers consisting of an α-chain and a β-chain that together form an intact receptor in the plasma membrane.

[0105] In one embodiment, the stem cells are human ICAM-1 (CD54), e.g., (SEQ ID NO: 1), a polypeptide having MGSLFPLSLLFFLAAAYPGVGSALGRRTKRAQSPKGSPLAPSGTSVPFWVRMSPEFVAVQPGKSVQLNCSNSCPQPQNSSLRTPLRQGKTLRGPGWVSYQLLDVRAWSSLAHCLVTCAGKTRWATSRITAYSVPGGLLGGDPEAWKPGHLFRKPGALHRPGSGQRDLDLRVCCWTPRLLAARDLPRAPQSRRPGGPQQLGTHYTDARLEPRAHSFGLRFHRCPCRDPPHCGRCVPMQVPSYEVPGVKGDVLCRLSEKKRNMKQSGEMAIHGG (SEQ ID NO: 2), or reduced or absent expression of a polypeptide having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

[0106] In one embodiment, the stem cells express human P-selectin (CD62), e.g., (SEQ ID NO: 3), or reduced or absent expression of a polypeptide having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

[0107] In one embodiment, the stem cells express human E-selectin (CD62E), e.g., (SEQ ID NO: 4), or reduced or absent expression of a polypeptide having at least 80%, 82%, 84%, 85%, 87%, 89%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

[0108] Pharmaceutical compositions: Pharmaceutical compositions of the present disclosure suitable for inoculation, e.g., intranasal, parenteral, or oral administration, e.g., intravenous, intramuscular, intranasal, topical, or subcutaneous, contain one or more low-level immune cell types and optionally further comprise sterile aqueous or non-aqueous solutions, suspensions, and emulsions. The compositions may further comprise adjuvants or excipients as known in the art. The compositions are generally presented in the form of individual doses (unit doses). Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, which may contain adjuvants or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration generally may contain a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups and elixirs containing inert diluents commonly used in the art, such as purified water. In addition to inert diluents, such compositions can also contain adjuvants, wetting agents, emulsifying and suspending agents, or sweeteners, flavoring agents or aromatics.

[0109] In one embodiment, the composition is a patch. The cardiac patch may contain skeletal myoblasts, cardiac stem cells / stromal cells, mesenchymal stem cells (MSCs), and / or human pluripotent stem cells with gene knockdown or knockout. The patch may be formed from synthetic or natural components, such as polymers. Synthetic materials include, but are not limited to, polymers such as poly(vinyl alcohol) (PVA), poly(lactic-co-glycolic) acid (PLGA), poly-(L-lactic) acid (PLLA), or polyurethane (PU). The patch may comprise a hydrogel. Different natural materials, such as collagen, fibrin, alginate, hyaluronic acid, gelatin, and decellularized extracellular matrix (ECM), may also be used.

[0110] When the composition is intended for administration to an individual, the composition may further comprise salts, buffers, adjuvants, or other substances desirable to maintain or improve the effectiveness of the composition.

[0111] The pharmaceutical composition comprises a therapeutically effective amount of cells and a pharmaceutically acceptable carrier. In certain embodiments, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Exemplary pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. These compositions can be formulated as suppositories. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin. Such compositions contain a therapeutically effective amount of the virus, e.g., the virus in purified form, together with an appropriate amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0112] The compositions may be combined with a pharmaceutically acceptable vehicle, such as an inert diluent, and administered systemically, e.g., orally, or locally, e.g., intramuscularly or intramuscularly. For oral administration, the cells may be combined with one or more excipients and used in the form of ingestible capsules, elixirs, suspensions, syrups, wafers, etc. Such compositions should contain at least 0.1% of the active compound. The percentage of compositions and preparations may, of course, be varied and may conveniently be about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such useful compositions is such that an effective dosage level will be obtained.

[0113] The composition can also be administered intravenously or intraperitoneally by infusion or injection.Cell solution can be prepared in water or suitable buffer, and optionally mixed with non-toxic surfactant.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, triacetin, and their mixtures and oils.Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of undesirable microorganisms.

[0114] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders containing the active ingredient, optionally encapsulated in liposomes, suitable for the extemporaneous preparation of sterile injectable or injectable solutions or dispersions. In all cases, the final dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. Liquid carriers or vehicles can be solvents or liquid dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. Fluidity can be maintained, for example, by the formation of liposomes, by maintaining particle size in the case of dispersions, or by the use of surfactants. Prevention of undesirable microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it may be preferable to include isotonic agents, such as sugars, buffers, or sodium chloride.

[0115] Sterile injectable solutions are prepared by incorporating the cells in an appropriate amount in an appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0116] Useful liquid carriers include water, alcohol, or glycol, or water-alcohol / glycol blends, and the virus of the present invention can be dissolved or dispersed at an effective level, optionally using a non-toxic surfactant. Adjuvants such as fragrances and additional antibacterial agents can be added to optimize the properties for a given application. The resulting liquid composition can be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using a pump-type or aerosol sprayer.

[0117] Pharmaceutical purposes: Administration of the composition can be for either "prophylactic" or "therapeutic" purposes. When provided prophylactically, the composition is provided before any symptoms or clinical signs of a disease or disorder become apparent. Prophylactic administration of the composition serves to prevent or attenuate a subsequent disease or disorder or its symptoms. When provided prophylactically, the composition is provided before any symptoms or clinical signs of a disease become apparent. Prophylactic administration of the composition serves to prevent or attenuate one or more symptoms or clinical signs associated with a disease.

[0118] When provided therapeutically, the cells are provided upon detection of a symptom or clinical sign of a disease or disorder. Therapeutic administration of the cell(s) serves to attenuate one or more symptoms of the disease or disorder. When provided therapeutically, a composition comprising the cells is provided upon detection of a symptom or clinical sign of a disease. Therapeutic administration of the cells serves to attenuate the symptom or clinical sign of the disease.

[0119] Thus, the compositions of the present disclosure can be provided before the onset of disease (to prevent or attenuate the disease) or after the detection of the disease. Similarly, the compositions can be provided before any symptoms or clinical signs of a disorder or disease appear, or after one or more symptoms are detected.

[0120] A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient mammal. Such an agent is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. A composition is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient, for example, if it enhances at least one primary or secondary humoral or cellular immune response against at least one strain of virus.

[0121] The "protection" provided need not be absolute, i.e., there is a statistically significant improvement compared to a control population or set of mammals, but it is not necessary to completely prevent or eradicate the disease or its symptoms. Protection may be limited to mitigating the severity or rapidity of onset of disease symptoms or clinical signs. For example, a cardiac patch may improve cardiac ejection fraction, thereby allowing patients to exercise / exercise better, thereby improving the patient's quality of life.

[0122] Pharmaceutical administration: Thus, the present disclosure includes methods for preventing or attenuating a disorder or disease. As used herein, a composition having cells is said to prevent or attenuate a disease if its administration results in a total or partial attenuation (i.e., suppression) of the clinical signs or symptoms of the disease.

[0123] The cell types can be administered by any means that achieves the intended purpose. For example, administration of such compositions can be by various parenteral routes, such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral, or transdermal routes. Parenteral administration can be achieved by bolus injection or gradual perfusion over time.

[0124] In one embodiment, the cells are part of a substrate, such as a patch or hydrogel. For example, the cells can be seeded on a substrate or embedded in a substrate. In one embodiment, the substrate is an artificial vascular graft, such as a vascular graft or intestinal graft. In one embodiment, the cells are part of an organ or a portion thereof. For example, the cells can be seeded on a decellularized organ or a portion thereof.

[0125] Exemplary regimens for preventing, inhibiting, or treating pathologies include administering an effective amount of a composition described herein administered as a single treatment, or multiple doses, for a period of, for example, one week to about 10 years or longer, or any range or value therein.

[0126] According to the present disclosure, an "effective amount" of a composition is an amount sufficient to achieve a desired effect. It is understood that the effective dosage may depend on the recipient's species, age, sex, health condition, and weight, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect. The effective dose ranges provided below are not intended to limit the dosage range.

[0127] Exemplary doses include about 10 4 ~10 8 cells, 10 6 ~10 8 cells, 10 6 ~10 10 cells, or 10 8 ~10 12 cells or more, or about 10 6 ~10 8 cells, 10 8 ~10 10 cells, or 10 10 ~10 12 cells; about 10 4 ~10 8 cells / kg, 10 6 ~10 8 cells / kg, 10 6 ~10 10 cells / kg or 10 8 ~10 12 cells / kg or more, or approximately 10 6 ~10 8 cells / kg, 10 8 ~10 10 cells, or 10 10 ~10 12 Examples of the present invention include, but are not limited to, cells / kg.

[0128] As used herein, the following definitions are provided:

[0129] A "vector" refers to a polymer or assembly of polymers that contains or is associated with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell either in vitro or in vivo. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other nucleic acid delivery vehicles. The polynucleotide to be delivered may also be referred to as a "target polynucleotide" or "transgene," and may contain a coding sequence of interest in gene therapy (such as a gene encoding a therapeutic protein), a coding sequence of interest in vaccine development (e.g., a polynucleotide that expresses a protein, polypeptide, or peptide suitable for eliciting an immune response in a mammal), and / or a selectable or detectable marker.

[0130] As used herein, "transduction," "transfection," "transformation," or "transduction" refers to the process of introducing an exogenous polynucleotide into a host cell, resulting in expression of the polynucleotide, e.g., a transgene, within the cell, and includes the use of recombinant viruses to introduce exogenous polynucleotides into host cells. Transduction, transfection, or transformation of a polynucleotide in a cell can be determined by methods well known in the art, including, but not limited to, measuring protein expression (including steady-state levels), e.g., by ELISA, flow cytometry, and Western blot, and DNA and RNA by hybridization assays, e.g., Northern blot, Southern blot, and gel shift mobility assay. Methods used to introduce an exogenous polynucleotide include well-known techniques such as viral infection or transfection, lipofection, transformation, and electroporation, as well as other non-viral gene delivery techniques. The introduced polynucleotide can be stably or transiently maintained within the host cell.

[0131] "Gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene transfer and may include targeting, binding, uptake, transport, localization, replicon integration and expression.

[0132] "Gene transfer" refers to the introduction of an exogenous polynucleotide into a cell, which may include targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of a gene.

[0133] "Gene expression" or "expression" refers to the processes of transcription, translation, and post-translational modification of a gene.

[0134] An "infectious" virus or virus particle is one that contains a polynucleotide component that the viral species can deliver to a cell for which it is trophic. This term does not necessarily imply the replicative capacity of the virus.

[0135] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form, in linear or cyclic conformations. For purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. These terms can encompass known analogs of natural nucleotides as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). Generally, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of A will base-pair with T.

[0136] An "isolated" polynucleotide, such as a plasmid, virus, polypeptide, cell, or other substance, refers to a preparation of the substance that lacks at least some of the other components that may be present when the substance or similar substance naturally occurs or is initially prepared. Thus, for example, an isolated substance may be prepared by using purification techniques to enrich it from a source mixture. An isolated nucleic acid, peptide, or polypeptide exists in a form or environment different from that found in nature. For example, a given DNA sequence (e.g., a gene) is found on a host cell chromosome in close proximity to neighboring genes. An RNA sequence, such as a particular mRNA sequence encoding a specific protein, is found in a cell as a mixture with many other mRNAs that encode many other proteins. An isolated nucleic acid molecule may exist in single-stranded or double-stranded form. When an isolated nucleic acid molecule is used to express a protein, the molecule contains at least the sense or coding strand (i.e., the molecule may be single-stranded), but may contain both the sense and antisense strands (i.e., the molecule may be double-stranded). Enrichment can be measured in absolute terms, such as weight per volume of solution, or can be measured with respect to a second potential interfering substance present in the source mixture. It is contemplated that embodiments of the present invention may be enriched to an increased degree. Thus, for example, 2-fold enrichment, 10-fold enrichment, 100-fold enrichment, or 1000-fold enrichment.

[0137] "Transcriptional regulatory sequence" refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences used in the present invention generally include at least one transcriptional promoter and may also include one or more enhancers and / or terminators of transcription.

[0138] "Operably linked" refers to the arrangement of two or more components, wherein the components so described are in a relationship permitting them to function in concert. By way of example, a transcriptional regulatory sequence or promoter is operably linked to a coding sequence if the TRS or promoter promotes transcription of the coding sequence. An operably linked TRS is generally linked in cis with the coding sequence, but is not necessarily directly adjacent.

[0139] The terms "operably linked" and "operably linked" (or "operably linked") are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) that permit both components to function normally and allow for the potential for at least one of the components to mediate a function exerted on at least one of the other components. Illustratively, a transcriptional regulatory sequence, such as a promoter, is operably linked to a coding sequence if the transcriptional regulatory sequence controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcriptional regulatory factors. A transcriptional regulatory sequence is generally operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcriptional regulatory sequence operably linked to a coding sequence.

[0140] "Heterologous" means derived from a genotypically distinct entity from the entity to which it is being compared. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (which, when expressed, can encode a heterologous polypeptide). Similarly, a transcriptional regulatory element, such as a promoter, removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcriptional regulatory element.

[0141] "Terminator" refers to a polynucleotide sequence that tends to reduce or prevent read-through transcription (i.e., it reduces or prevents transcription occurring on one side of the terminator from continuing to the other side of the terminator). The degree to which transcription is disrupted is typically a function of the base sequence and / or length of the terminator sequence. In particular, as is well known in many molecular biological systems, certain DNA sequences commonly referred to as "transcription termination sequences" are specific sequences that tend to disrupt read-through transcription by RNA polymerase by terminating and / or disengaging the RNA polymerase molecule from the DNA being transcribed. Typical examples of such sequence-specific terminators include polyadenylation ("polyA") sequences, such as SV40 polyA. In addition to or instead of such sequence-specific terminators, the insertion of a relatively long DNA sequence between the promoter and the coding region also tends to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect is thought to occur because RNA polymerase molecules always tend to disengage from the DNA being transcribed, and increasing the length of the sequence traversed before reaching the coding region generally increases the likelihood that disengagement will occur before transcription of the coding region is completed, or even initiated. Thus, terminators can prevent transcription from only one direction ("unidirectional" terminators) or from both directions ("bidirectional" terminators), and can consist of sequence-specific termination sequences or sequence-nonspecific terminators, or both. A variety of such terminator sequences are known in the art, and exemplary uses of such sequences within the context of the present invention are provided below.

[0142] "Host cell," "cell line," "cell culture," "packaging cell line," and other such terms refer to higher eukaryotic cells useful in this disclosure, e.g., mammalian cells, including human cells. These cells include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or genomic complement) to the original parent cell.

[0143] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction, and / or ligation steps, and other procedures that result in a construct that differs from polynucleotides found in nature. In one embodiment, a recombinant virus is a viral particle that includes a recombinant polynucleotide. These terms include replicas of the original polynucleotide construct and progeny of the original viral construct, respectively.

[0144] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including polynucleotide replication, duplication, transcription, splicing, translation, or degradation. Regulation can affect the frequency, rate, or specificity of a process and can be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, is capable of binding RNA polymerase and initiating transcription of a coding region typically located downstream (3' direction) of the promoter. Promoters include AAV promoters, such as P5, P19, P40, and AAV ITR promoters, as well as heterologous promoters.

[0145] An "expression vector" is a vector containing a region encoding a gene product of interest and is used to express the gene product in the intended target cell. The expression vector also contains control elements operably linked to the coding region to promote expression of the protein in the target. The combination of the control elements and the gene or genes to which they are operably linked for expression is sometimes called an "expression cassette," many of which are known and available in the art or can be easily constructed from components available in the art.

[0146] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The term also encompasses modified amino acid polymers, including, for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component.

[0147] The term "exogenous," when used with reference to a protein, gene, nucleic acid, or polynucleotide in a cell or organism, refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid can be derived from a different organism or cell, or can be one or more additional copies of a nucleic acid that naturally occurs in the organism or cell. As a non-limiting example, an exogenous nucleic acid is at a chromosomal location that is different from that of the native cell, or is flanked by nucleic acid sequences that are different from those found in nature, such as an expression cassette that links a promoter from one gene to an open reading frame for a gene product from a different gene.

[0148] Thus, an "exogenous" molecule is one that is not normally present in a cell but can be introduced into the cell by one or more genetic, biochemical, or other methods. The "normal presence in a cell" is determined with respect to the cell's particular developmental stage and environmental conditions. Thus, for example, a molecule that is present only during muscle embryonic development is exogenous to adult muscle cells. Similarly, a molecule induced by heat shock is exogenous to cells that have not been subjected to heat shock. Exogenous molecules can include, for example, functional versions of dysfunctional endogenous molecules or dysfunctional versions of normally functioning endogenous molecules.

[0149] Exogenous molecules can be small molecules, such as those produced by combinatorial chemical processes, or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complexes containing one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, linear, branched, or circular, and can be of any length. Nucleic acids include those capable of forming duplexes as well as triplex-forming nucleic acids.

[0150] An exogenous molecule can be the same type of molecule as an endogenous molecule, such as an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can include an infectious viral genome, a plasmid or episome introduced into a cell, or a chromosome not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (e.g., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule, but derived from a species different from that from which the cell is derived. For example, a human nucleic acid sequence can be introduced into a cell line originally derived from a mouse or hamster.

[0151] Exogenous molecules can be small molecules, such as those produced by combinatorial chemical processes, or macromolecules, such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complexes containing one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, linear, branched, or circular, and can be of any length. Nucleic acids include those capable of forming duplexes as well as triplex-forming nucleic acids.

[0152] In contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally occurring episomal nucleic acid.

[0153] "Transformed" or "transgenic" are used herein to include any host cell or cell line that has been altered or enhanced by the presence of at least one recombinant DNA sequence. The host cells of the present invention are typically produced by transfection of a DNA sequence in a plasmid expression vector, transfection as an isolated linear DNA sequence, or infection with a recombinant viral vector.

[0154] The term "sequence homology" refers to the percentage of base matches between two nucleic acid sequences or the percentage of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, for example, 50%, the percentage represents the percentage of matches over the length of the selected sequence compared to some other sequence. Gaps (in either of the two sequences) are allowed to maximize matching, and gap lengths of 15 bases or less, 6 bases or less, for example, 2 bases or less are usually used. When oligonucleotides are used as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally 17 or more target base matches (85%) out of 20 possible oligonucleotide base pair matches; 9 or more matches (90%) out of 10 possible base pair matches, or 19 or more matches (95%) out of 20 possible base pair matches.

[0155] Two amino acid sequences are homologous if there is partial or complete identity between them. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum correspondence. Gaps (in either of the two sequences being matched) are allowed when maximizing matching (gap lengths of 5 or less, or 2 or less). Alternatively, two protein sequences (or polypeptide sequences at least 30 amino acids long derived therefrom) are homologous, as this term is used herein, if they have an alignment score of more than 5 (in standard deviation units) using the program ALIGN with a mutation data matrix and a gap penalty of 6 or more. Two sequences or portions thereof are more homologous if their amino acids are 50% or more identical when optimally aligned using the ALIGN program.

[0156] The term "corresponding to" is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion of a reference polypeptide sequence, e.g., they have at least 80%, 85%, 90%, 95% or more, e.g., 99% or 100% sequence identity. The term "complementary" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For purposes of illustration, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA."

[0157] The term "sequence identity" means that two polynucleotide sequences are identical (i.e., nucleotide-by-nucleotide) over the comparison range. The term "sequence identity percentage" means that two polynucleotide sequences are identical (i.e., nucleotide-by-nucleotide) over the comparison range. The term "sequence identity percentage" is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, U, or I) occurs to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the sequence identity percentage. As used herein, the term "substantial identity" refers to a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 85 percent sequence identity, e.g., at least 90-95 percent sequence identity, or at least 99 percent sequence identity, relative to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, where the percentage of sequence identity is calculated by comparing the reference sequence to a polynucleotide sequence that may contain deletions or additions of up to 20 percent of the reference sequence in total over the comparison window.

[0158] "Conservative" amino acid substitutions include, for example, asparagine-glutamic acid as a polar acidic amino acid; lysine / arginine / histidine as a polar basic amino acid; leucine / isoleucine / methionine / valine / alanine / glycine / proline as a nonpolar or hydrophobic amino acid; and serine / threonine as a polar or uncharged hydrophilic amino acid. Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that substituting leucine with isoleucine or valine, substituting aspartic acid with glutamic acid, substituting threonine with serine, or similarly substituting amino acids with structurally related amino acids will not significantly affect the properties of the resulting polypeptide. Whether an amino acid change results in a functional polypeptide can be readily determined by assaying the specific activity of the polypeptide. Naturally occurring residues can be divided into groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that affect chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0159] The present disclosure also contemplates polypeptides with non-conservative substitutions, which involve exchanging a member of one of the above classes for another.

[0160] As used herein, "individual" (as in the subject of treatment) refers to a mammal. Mammals include, for example, humans; non-human primates, such as apes and monkeys; non-primates, such as dogs, cats, rats, mice, cows, horses, sheep and goats. Non-mammals include, for example, fish and birds.

[0161] As used herein, "substantially" means completely or nearly completely. For example, a composition that is "substantially free" of a component either has no such component at all or contains such a small amount that any relevant functional properties of the composition are not affected by the presence of that small amount, or the compound is "substantially pure," with only negligible amounts of impurities present.

[0162] Within the meaning of this specification, "treat" or "treatment" refers to the alleviation of symptoms associated with a disorder or disease, "inhibit" means the inhibition of further progression or worsening of symptoms associated with a disorder or disease, and "prevent" refers to the prevention of symptoms associated with a disorder or disease.

[0163] As used herein, an "effective amount" or "therapeutically effective amount" of an agent refers to an amount of agent that alleviates, in whole or in part, the symptoms associated with a disorder or condition, halts or delays further progression or worsening of those symptoms, or prevents or prevents a disorder or condition, e.g., an amount effective to prevent, inhibit, or treat one or more individual symptoms.

[0164] In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent(s) are outweighed by the therapeutically beneficial effects.

[0165] The term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA, which can be linear, circular or branched, and which can be either single-stranded or double-stranded.

[0166] The term "donor sequence" refers to a nucleotide sequence to be inserted into a genome. The donor sequence can be any length, for example, 2 to 10,000 nucleotides in length (or any integer value therebetween or above), for example, about 100 to 1,000 nucleotides in length (or any integer value therebetween), for example, about 200 to 500 nucleotides in length.

[0167] "Binding" refers to a sequence-specific, non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid). Not all components of a binding interaction need be sequence-specific (e.g., contacts with phosphate residues in a DNA backbone), as long as the interaction as a whole is sequence-specific. "Affinity" refers to the strength of binding; increased binding affinity correlates with a lower Kd.

[0168] A "binding protein" is a protein that can non-covalently bind to another molecule. A binding protein can bind, for example, to a DNA molecule (a DNA-binding protein), an RNA molecule (an RNA-binding protein), and / or a protein molecule (a protein-binding protein). In the case of a protein-binding protein, it can bind to itself (forming a homodimer, homotrimer, etc.) and / or to one or more molecules of one or more different proteins. A binding protein can have more than one type of binding activity.

[0169] A "homologous, non-identical sequence" refers to a first sequence that shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence. For example, a polynucleotide containing the wild-type sequence of a mutant gene is homologous to, but not identical to, the sequence of the mutant gene. In certain embodiments, the degree of homology between the two sequences is sufficient to allow homologous recombination between them, utilizing normal cellular mechanisms. Two homologous, non-identical sequences can be of any length, and their degree of non-homology can be as small as a single nucleotide (e.g., for correction of a genomic point mutation by targeted homologous recombination) or as large as 10 kilobases or more (e.g., for inserting a gene at a predetermined ectopic site in a chromosome). Two polynucleotides containing homologous, non-identical sequences need not be the same length. For example, an exogenous polynucleotide (i.e., donor polynucleotide) of 20 to 10,000 nucleotides or nucleotide pairs can be used.

[0170] A "target site" or "target sequence" is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule will bind, provided sufficient conditions for binding exist. [Example]

[0171] The present invention is further illustrated by the following non-limiting examples.

[0172] Example 1 To date, several groups have knocked out human leukocyte antigen (HLA) genes / associated genes (e.g., beta2-microglobulin) and knocked in other genes (e.g., PDL1, CTLA4-Ig, CD47, and HLA-E). These approaches are effective in suppressing adaptive (T-cell and B-cell mediated) and one innate cell type (NK cell mediated) immune responses.

[0173] However, this approach has several potential drawbacks, including the possibility of compensatory mechanisms (indirect pathways of allorejection) that allow the recipient's immune cells to still reject the transplanted graft and / or the loss of defenses against uncontrolled tumor growth and / or viral infection. This method may be a "gentle" approach that reduces allogeneic rejection while maintaining some degree of antitumor and / or antiviral response. Intercellular adhesion molecule (ICAM-1), E-selectin, and P-selectin, or other adhesion molecules, as well as related inflammatory molecules such as interferon gamma, MCP-1, and other chemoattractants, may be targeted for knockdown or knockout, and, optionally, regulatory cytokines (e.g., IL10) and chemoattractants may be added to the cells (knock-in).

[0174] As described herein, we used a CRISPR / Cas9 editing approach to gene-edit the adhesion molecule CD54 (or ICAM-1) from induced pluripotent stem cells (iPSCs) to generate knockout lines. CD54 is involved in the adhesion of immune cells to parenchymal and antigen-presenting cells. It plays an important role in the formation of the immune synapse during cell attachment / death of many cell types, as well as the tethering, rolling, and extravasation processes by which immune cells interact with endothelial cells. There is also a link between CD54 and inflammation, such that knockout of CD54 can positively affect cellular anti-inflammatory responses. Specifically, loss of CD54 in non-iPSC cell types results in an increase in glutathione (GSH). GSH has an anti-inflammatory effect by helping to remove radical oxygen species that are harmful to cells in inflammatory situations. Knockout of CD54 likely has an anti-rejection effect in the transplant setting by interfering with the ability of the transplant recipient's immune system to counter most cell-mediated and inflammatory immune responses.

[0175] method Experimental procedure: The gRNA sequence used to knock-in the Y324X mutation was the same as that used to induce homozygous mutants in Tsc2 patient iPSC lines. pLenti-CRISPR plasmid delivery was replaced with Cas9-RNP delivery.

[0176] Design of targeting to gene locus: sgRNA identification for the site of interest was achieved using the CRISPOR design tool described in the art. The sgRNA used was 1.5 nmol of synthetic sgRNA with 2'-O-methyl 3' phosphorothioate modifications in the first and last three nucleotides.

[0177] Electroporation, selection, and expansion: iPSCs were cultured in mTeSR™ PLUS medium (StemCell Technologies) on Matrigel® according to standard cell culture protocols until approximately 80% confluent. 24 hours prior to electroporation, cells were treated with CloneR™ (StemCell Technologies) according to the manufacturer's protocol. Prior to electroporation, the sgRNA construct was reconstituted to a concentration of 150 pmole / µL according to the manufacturer's protocol. 1 µL of reconstituted sgRNA was pooled with 4 µg of Cas9 nuclease protein (TrueCut™ Cas9 Protein V2, Thermo Fisher Scientific) and 5 µL of Neon™ Buffer R (Invitrogen) to promote Cas9-RNP complex formation. After 15 minutes, 1.0 μL of ssODN primer (designed to have at least 40 base pair homology overhangs, reconstituted to a concentration of 1 μg / μL) was added to the Cas9-RNP mix.

[0178] Cells were isolated and lifted in a 1:1 mixture of 0.5 mM EDTA:Accutase® for 3-4 minutes, resuspended in 1 mL of PBS, and pelleted. Approximately 400,000 cells were resuspended in 35 μL of Neon™ Buffer R and mixed with 8 μL of pre-prepared Cas9-RNP complex containing repaired ssODN. Cells were electroporated in 10 μL of the NEON electroporation format using a 1200 V, 30 msec, 1x pulse setting. After four electroporations, cells were pooled and serially diluted and seeded into TeSR™ PLUS medium containing CloneR™ supplement at the manufacturer's recommended concentration to promote single-cell clonal expansion. After 10-14 days of growth, clones were identified and selected using standard techniques.

[0179] Genotyping: Bulk gDNA was collected from dissociated cells using QuickExtract™ DNA Extraction Solution 1.0 (Epicentre) to confirm editing efficiency prior to clone selection. Single-cell clones were manually selected and mechanically disaggregated. Genomic DNA was isolated from some of these clones using QuickExtract™ DNA Extraction Solution 1.0 (Epicentre). Genotyping primers were designed flanking the mutation site, allowing amplification of this region using Q5® polymerase-based PCR (NEB). PCR products were identified by agarose gel and purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research). Clones were submitted to Quintara Biosciences for Sanger sequencing to identify clones with the appropriate gene modifications.

[0180] Off-target analysis: To determine whether the CRISPR-Cas9 system resulted in any nonspecific genome editing, suspected off-target sites for genome modification were analyzed. Using the five most likely off-target sites for each sgRNA predicted by the CRISPOR algorithm, genotyping primers were prepared to amplify these regions via Q5®-polymerase PCR. PCR products were identified by agarose gel, purified using the Zymoclean™ Gel DNA Recovery Kit, and submitted to Quintara Biosciences for Sanger sequencing.

[0181] Results: One example of cell therapy is PSC-based cardiovascular therapy (PSC-CVT), which is immune tolerant and significantly improves patient health and quality of life with little or no ongoing need for immunosuppression. Different differentiated cells can be used in this type of therapy. For example, endothelial cells, a major interface between transplant tolerance and rejection, are targeted by genetically disrupting the ability of allogeneic adaptive and innate immune cells to adhere to, infiltrate, and destroy the graft. Hypoimmune grafts, e.g., hypoimmune PSC-CVT grafts, optionally bearing a combination of different cell types, each derived from one or more adhesion molecule knockout stem cells / stem cell lineages, are evaluated for alloimmunogenicity in humanized mouse models, e.g., the NeoThy model, a more robust (non-fetal) alternative to the gold-standard, but suboptimal, bone marrow-liver-thymus (BLT) fetal model. Because there is a rapid upregulation of adhesion molecules (e.g., ICAM-1) on PSC-derived arterial endothelial cells (AECs) after encounter with HLA-mismatched peripheral blood leukocytes (Figure 1), targeted disruption of leukocyte adhesion promotes immune tolerance of angiogenic tri-cellular PSC-CVTs generated from gene-edited PSC-AECs, cardiomyocytes (CMs), and cardiac fibroblasts (C-fibs), and these grafts are tested for functionality and immunogenicity.

[0182] We evaluated the in vitro phenotype, function, and immunogenicity of adhesive gene-edited PSC-CVT cell subtypes. When comparing flow cytometry phenotype, gene expression, and cell type-specific functional assays, CRISPR / Cas9 KO of the leukocyte adhesion (AD) molecule ICAM-1 in PSC-derived AECs, CMs, and C-fib (PSC-AD-CVT cells) showed normal identity and function relative to isogenic controls, and were more immunotolerant in immune cell proliferation and cytotoxicity assays compared to WT, enhancing the tolerogenic potential of first-generation hypoimmune (HLA I+II KO, HLA-E dimer) PSC-derived cells.

[0183] We generate angiogenic next-generation PSC-AD-CVT grafts that are immune-tolerant in vivo in the NeoThy humanized mouse model. PSC-AD-CVT grafts composed of hypoimmune PSC-AECs, -CMs, and -C-Fibs (e.g., 1 x 105 at a 1:1:1 ratio, followed by doubling each type one at a time) form vascularized cardiac organoids with contractile function that are immune-tolerant in the NeoThy model (e.g., reduced graft immune cell infiltration). These data clearly demonstrate the benefits of validating grafts in heterotopic kidney capsule transplantation sites.

[0184] We evaluated the repair capacity and immune tolerance of gene-edited PSC-AD-CVT grafts in the NeoThy humanized mouse model of inflammatory post-myocardial infarction (MI). After MI, orthotopic transplantation of hypoimmunized PSC-CVT resulted in vascularized, persistently tolerated grafts that improved cardiac function, reduced infarct size, and increased ventricular wall thickness. PSC-CVT possesses adaptive and innate immune tolerance, safely improving cardiac function in a robust in vivo model of human cardiomyopathy.

[0185] Example 2 After a first myocardial infarction (MI), the adult heart recovers via scar formation, with 36% of male and 47% of female patients dying within five years, demonstrating the serious inadequacy of current treatments. Pluripotent stem cell (PSC)-based cardiovascular therapy (PSC-CVT) has the potential to dramatically reduce MI mortality and improve patients' quality of life. Artificial PSCs (iPSCs), created by reprogramming primary cells such as peripheral blood T cells from individual patients and then differentiating them into autologous replacement cells for transplantation (e.g., cardiomyocytes [CMs], endothelial cells [ECs]), hold particular promise. These therapies could theoretically be accepted as "autologous" by the patient's immune system without the need for immunosuppressive drugs and their associated adverse effects, thereby avoiding the detrimental immune disorder of transplant allo-rejection. While several autologous iPSC therapies are being investigated in clinical trials for diseases such as macular degeneration, clinical-grade patient-specific therapies are currently very expensive to manufacture and, without new technological advances, present quality control and other regulatory obstacles that make them suboptimal for widespread clinical use. Furthermore, autologous cells are of limited use in the context of autoimmune disease and time-sensitive situations (e.g., subacute MI) where patient-specific iPSCs cannot be generated in time for administration to the critically ill recipient.

[0186] Highly pure PSC-derived grafts possess many uniquely attractive attributes, such as near-infinite expandability and the absence of passenger lymphocytes, which may reduce susceptibility to the acute and chronic allograft rejection that routinely afflicts transplanted organs, especially in HLA-matched settings. Therefore, researchers in the PSC field have focused on two strategies for developing allogeneic therapies to achieve tolerance in patients: 1) banking HLA-matched PSC lines from a broad range of donors representing large populations; and 2) gene editing to modulate the immunogenicity of cells and shield them from immune-mediated destruction. While both of these approaches have advantages and disadvantages, HLA banking in the United States faces several hurdles (e.g., ensuring representation of genetically diverse racial and ethnic minorities). Research is now shifting toward developing universal, immuno-hypotensive PSCs, whereby a single cell line could be used for most, if not all, patients in the country, including those with rare HLA types.

[0187] Methods: A hypoimmunized PSC-CVT for MI is provided for use in any form of cardiac disease requiring replacement of damaged and / or diseased cardiac tissue with vascularized, functional cells that are tolerated by the recipient's immune system. This approach is designed to tolerate both adaptive immune cells (e.g., T cells) and innate immune cells (e.g., monocytes), the latter of which are not directly targeted by current hypoimmunized PSC therapies. The graft provides a treatment for cardiac disease that kills and / or reduces the quality of life of millions of people worldwide.

[0188] Next-generation low-immunity PSC-CVT design: 1st generation (1 st(HLA-gen) hypoimmune gene-edited cells (e.g., knockout [KO] of MHC class I+II) can avoid recognition by T cells, donor-specific antibodies, and / or NK cell-mediated cytotoxicity in short-term studies. These gene-edited cells have been shown to maintain phenotypic and functional attributes and have been used to differentiate into multiple PSC-derived cell types, including CMs, which, in purified form, do not develop teratomas after gene editing. Conventional wisdom in the PSC field is that the potential benefits of HLA ablation outweigh the risks of MHC absence or downregulation, a key immune evasion mechanism used by 40–90% of human tumors and multiple viral pathogens. However, the potential for tumor propensity and / or viral reservoirs within HLA I+II KO PSC-derived cells has not been adequately investigated. Furthermore, HLA ablation and related published strategies are intended to reduce adaptive immunity (T cells, donor-specific antibodies) and NK cells, ignoring other innate immune cells (e.g., monocytes) that play a key role in allogeneic rejection. One approach to gene editing described herein is to focus on targeted gene editing that is important for cell biology and immune cell interactions of individual cell types (e.g., adhesion of adaptive and innate immune cells to arterial endothelial cells [AECs]), which are also important for effector immune synapse formation with other parenchymal cells (e.g., CMs).

[0189] The endothelial:immune cell interface in allorejection and tolerance: One strategy focuses on the inherent biology and interactions of specific cell types intimately involved in allorejection and tolerance, with the goal of enhancing both therapeutic efficacy and safety. Furthermore, there may be significant advantages to designing PSC therapy based on targeted consideration of the inherent biology of specific cells versus strict global HLA ablation. ECs are at the primary interface where transplant rejection begins and / or immune tolerance is maintained. The PSC-CVT described herein contains AECs that form a luminal barrier in larger cardiac vessels (e.g., coronary arteries) and, crucially, have a lower baseline propensity for leukocyte adhesion than vascular or other EC subtypes. Targeted destruction of ICAM-1-mediated leukocytes: Endothelial adhesion may confer immune tolerance to AECs, as well as CMs and cardiac fibroblasts (C-fib) within the PSC-CVT graft by preventing initial leukocyte adhesion and immune synapse formation.

[0190] KO of donor adhesion molecules resulted in a reduction in allogeneic T cell responses and nearly doubled the survival time of allogeneic matched hearts compared to wild-type (WT) grafts. Importantly, KO did not cause major pathogenic vascular defects and / or embryonic lethality. Furthermore, because ECs function as semi-professional antigen-presenting cells, the gene editing approach described herein to prevent stable leukocyte:EC interactions has the potential to disrupt effector memory T cell function. Figures 1A and 1B show the effects of beta2 microglobulin (B2M) KO 1 cells lacking surface MHC class I. stSimilar to published reports on immortalized mouse and primary human ECs, we show that gene expression of ICAM-1 increases during activation / inflammation and after the addition of inflammatory stimuli (e.g., TNFα) in B2M-gen PSC-AECs. This further validates the strategy of focusing on molecules that play a role in allorejection and suggests that adhesion molecules (and the innate immune cells that bind to them) may still contribute to allorejection in B2M KO PSC grafts. Because ICAM-1 plays a role in immune synapse formation during parenchymal cell cytolysis, the KO approach likely confers additional adaptive and innate immune protection to PSC-CMs, AECs, and B2M-fibs within the graft.

[0191] Designing PSC-CVT grafts with durable function and immune tolerance: Repair function and immunogenicity are closely related. PSC-CVTs must maintain their in vivo function while avoiding destruction through the individual and coordinated actions of the recipient's adaptive and innate immune cells. 1 st We derived AECs and CMs from C-fibs-based HLA class I KO PSCs and confirmed that the differentiation protocol maintained high yield and efficiency for both WT and gene-edited cells, and that the cells maintained characteristic in vivo functions, such as robust engraftment in immunodeficient mouse hosts (Figure 2A and Figure 2B). Prior to in vivo studies, we will determine the functional attributes of next-generation gene-edited AECs, CMs, and C-fibs, in addition to assessing their immunogenicity in vitro.

[0192] In addition to incorporating immune-evading cells, we have prepared and tested advanced tricellular PSC-CVTs (containing a ratio of three different PSC-derived cell types: 1) CMs, 2) C-fibs, and 3) AECs. Previous transplantation studies have typically utilized grafts composed primarily of CMs, and their methods have not necessarily identified the purity of the preparation and / or the effects of unintended non-CM populations within the graft. These grafts are prone to post-transplant arrhythmias, potentially related to the immature developmental state of the PSC-CMs used, cellular impurities, and / or the lack of homeostatic cues from accessory cell types (e.g., C-fib) present in the normal heart but lacking in the relatively homogenous population of PSC-CMs. The PSC-CVT graft design disclosed herein utilizes an advanced protocol to intentionally mix highly purified CMs (to avoid teratomas) with C-fib (to provide homeostatic cues and aid maturation). A third specialized purified cell population, AECs, is then added to promote neovascularization, CM maturation, and homeostatic crosstalk. Tricellular hypoimmune grafts are relatively mature and have high potential for clinical translation with long-term function and immune tolerance. The input ratios of these individual cell types (CMs, C-Fibs, and AECs) can be varied in in vivo studies. Figures 2A and 2B show robust, leukocyte-adherent gene-edited PSC-AD-CVT grafts demonstrating identity, function (e.g., contractility), and angiogenesis upon transplantation into humanized mice. Both the repair and immune tolerance capacities of PSC-AD-CVT are evaluated in the inflamed, physiologically complex orthotopic site of the heart in a humanized mouse model of myocardial infarction. Grafts are tested in vitro, in hypoinflammatory in vivo, and in inflammatory post-infarction in vivo environments.

[0193] Accurate assessment of human immune responses to PSC-CVTs: Rigorous immunogenicity studies must be performed using high-fidelity in vitro assays and in vivo models that accurately recapitulate human immunology. This gap is the result of 1) the disparate methodologies and T cell / NK cell bias of assays used by various groups in previous studies (ignoring monocytes and other non-NK innate cells); and 2) the lack of adequate preclinical in vivo models of human immune responses to PSC grafts. As described herein, a robust in vitro mixed lymphocyte reaction (MLR) assay is used, informed by historical transplant immunology lessons and stem cell biology expertise, which purposefully examines both adaptive and innate immunity. The MLR data in Figure 3 demonstrate that 1 st These results demonstrate that B2M KO PSC AECs are capable of eliciting CD8+ T cell proliferative responses in the presence of antigen-presenting cells, as shown by a significant decrease in proliferation in B2M KO compared with WT cells. However, B2M KO may protect against direct pathway allorejection, as shown by a significant decrease in proliferation in B2M KO compared with WT cells. st This indicates that gen-gene-edited PSC therapy may still be prone to the indirect pathway-mediated chronic rejection seen in solid organ transplant patients, i.e., the number of expanded T CD8 cells increased compared to unstimulated controls. + A large population of T cells still exists. This finding reinforces the importance of creating PSC-CVTs that avoid both direct and indirect allorecognition to increase the likelihood of successful clinical translation. Innate immune cells such as dendritic cells and monocytes play a key role in allorejection, and this study, using a controlled, highly purified target cell population, is relevant to the successful clinical translation of PSC-CVTs and also provides insight into the mechanisms of conventional solid organ allorejection.

[0194] We used a humanized mouse model, NeoThy. Humanized mice are a powerful research tool for modeling in vivo human immune responses to PSC transplantation. The existing gold-standard humanized mouse model, fetal tissue-based "BLT," has been reported to be suboptimal for allogeneic rejection testing due to naive and regulatory T cell bias. In contrast, NeoThy maintains naive CD4 T cells, which are more similar to adult patients than the BLT model. + It has a T cell compartment (Figure 4A) and is a high-fidelity model of allorejection (e.g., extensive immune infiltration) of transplanted PSC-CM grafts (Figure 4B).

[0195] Hypoimmune PSC-AD-CVT grafts with a tri-cellular composition lacking alloreactivity in vitro and in vivo can be used to restore function after MI in the absence of alloreactivity in vivo, as tested, for example, in the advanced NeoThy humanized mouse MI model, a testing platform for hypoimmune PSC therapy, which may be useful for many patients with MI and other pathologies characterized by cellular dysfunction in immunocompetent anatomical sites.

[0196] Example 3 MI is a devastating condition, resulting in death or reduced quality of life for millions of patients. While existing treatments are suboptimal, PSC-based cell therapy holds great potential for improving and saving lives, especially in the presence of sustained immune tolerance. Scalability and pluripotent differentiation potential are two key advantages of PSCs over other primary cell-based CVTs. The current state of the art for gene-edited PSC therapy is the use of HLA I+II KO lines, a promising but generalized approach that ignores subtle cell-type-specific interactions with the immune system and cooperates with monocytes and other innate immune cells, rendering the graft vulnerable to indirect pathway-mediated rejection. In one embodiment, a gene-editing strategy is used to focus on preventing immune cell adhesion at the endothelial cell:immune interface and at the adaptive and innate immune synapses, which are critical for parenchymal cell rejection / tolerance. In one embodiment, a tricellular PSC-AD-CVT graft is prepared for superior repair function and low immunogenicity. In one embodiment, human immune responses are investigated using a multifaceted approach that utilizes classical transplant immunology techniques and stem cell biology, leveraging an advanced in vivo humanized mouse model. The data allow for a better understanding of the mechanisms of leukocyte adhesion to AECs and the mechanisms mediating activation and inflammation during the allogeneic rejection process. The NeoThy model provides the regenerative medicine community with a tool for long-term cardiomyopathy research in the context of the human immune system, which is not feasible with other (e.g., BLT-type) humanized mice. PSC differentiation protocols, combined with robust assessment of human immunogenicity in vitro and in vivo, generate tricellular PSC-AD-CVT grafts that are reparative and sustainably tolerated, enabling further clinical use of PSC-CVT.

[0197] Approach: Experiments involve paying careful attention to mouse strain, genotyping, and age, tracking cell line identity and gender, and disaggregating data to allow for analysis of gender-based differences. Experiments use technical replicates and a minimum of n = 3 biological replicates, and >90% pure cells. Statistical power calculations can be used to estimate sample size.

[0198] Evaluating the in vitro phenotype, function, and immunogenicity of adhesive gene-edited PSC-CVT cell subtypes: Attributes: ICAM-1 KO PSC-AEC, CM, and C-fibs (PSC-AD-CVT) have normal phenotypic identity. PSC-AD-CVT has normal cell type-specific functions. PSC-AD-CVT is immune tolerant compared with WT. · Addition of ICAM-1 KO to first-generation hypoimmune PSC-CVT improves immune tolerance.

[0199] The hypoimmune tricellular PSC-AD-CVT incorporates genetic ablation of the key cell adhesion molecule ICAM-1, which is used by multiple adaptive and innate immune cells during a critical phase of allogeneic rejection. Therapeutic design and immunogenicity assays address and experimentally examine innate and adaptive immune responses. For each of the three tested PSC-AD-CVT cell types, immunomodulatory gene editing does not significantly disrupt cell phenotype and function (e.g., reduce repair capacity) for their use in clinically relevant hypoimmune PSC therapies. Therefore, after preparing gene-edited PSCs, we differentiate them into the three PSC-CVT cell types. We then conduct phenotypic, functional, and immunogenic studies on each individual gene-edited cell type to characterize adhesion molecules in the interactions between immune cells and AECs, CMs, and C-fib, and to determine how ICAM-1 ablation affects independent cell biological functions. This may enable sustainably tolerated PSC-CVT, which could improve the health and well-being of millions of MI patients worldwide.

[0200] ICAM-1 deletion does not significantly alter the phenotype or impair the function of PSC-AECs, -CMs, or -C-fibs. Furthermore, there is a decrease in immune cell interactions with each of the three cell types, and a concomitant decrease in in vitro measures of allorejection. These effects are evident with ICAM-1 KO alone, but not with 1 stThis is enhanced when combined with the -gen gene editing lineage (i.e., removal of MHC class I+II, addition of HLA-E dimers).

[0201] ICAM-1 KO PSC-AECs, CMs, and C-fibs (PSC-AD-CVTs) have normal phenotypic identity: CRISPR / Cas9-based gene editing is efficient. To minimize off-target effects, we use a high-fidelity Cas9 mutant to knock out ICAM-1 and introduce a constitutively expressed Akaluc luminescence reporter for use in downstream bioluminescence imaging (BLI) studies. In one experiment, we generated the H9 B2M KO / CIITA KO / HLA-E dimer (H9 KO E dimer) line. st The ICAM-1 KO line can be used as a gen control line. An ICAM-1 KO line is edited to confirm that it was selected for KO via IFNγ-based upregulation of ICAM-1 in non-target cells. Multiple KO clones are pooled, karyotyped, and screened for off-target activity. Four replicates of PSCs are used for downstream experiments: 1) ICAM-1 KO only; 2) 1 st -gen KO E dimer only, 3)1 st 1) ICAM-1 KO added to the -gen KO E dimer, and 2) WT (unedited) H9. st Studies on ICAM-1-hyperimmune PSC lines showed that they could differentiate into phenotypically normal and functional AECs and CMs (Figure 5). To verify that KO of ICAM-1 does not adversely affect differentiation potential, we differentiated four PSC replicates into AECs, CMs, and C-fibs and assessed their phenotypes by flow cytometry and bulk RNA sequencing.

[0202] Other PSC lines (or H9) with KO of other adhesion molecules, for example, double KO of E-selectin and P-selectin, can also be prepared.

[0203] PSC-AD-CVTs have normal cell type-specific functions: In addition to cellular phenotype and morphology, normal cell type-specific functions after KO are demonstrated. Previously described methods are used to validate gene-edited PSC-AECs, PSC-CMs, and PSC-C-fibs. Briefly, normal AEC function is verified by assessing oxygen consumption rate, nitric oxide production levels, and shear stress response (CMs by macroscopic contractility; and C-fibs by immunolabeling to demonstrate extracellular matrix formation via staining for collagen I and fibronectin).

[0204] PSC-AD-CVT is immunotolerant compared to WT: In vitro assessment of immunogenicity is one step in assessing the tolerogenic potential of the disclosed PSC-AD-CVT. Adaptive immune cells (e.g., T cells) and innate immune cells (e.g., monocytes) adhere largely via ICAM-1, and therefore these cells as well as innate NK cells (in solid organ transplants and 1 stWe will evaluate ICAM-1 (a key mediator of missing-self cytotoxicity in gene-hypoimmune PSC therapy). A 6-day MLR will be performed based on a previously published protocol targeting allogeneic peripheral blood mononuclear cells (PBMCs) (containing the above three cell populations) and the individual gene-edited PSC-derived cells described above, as shown in Figure 3. To assess alloreactivity, we will measure proliferation by flow cytometry for gated cell subtypes (e.g., effector memory T cells), and assess LAMP1 and T cell / NK cytotoxic activity by flow cytometry and CytoTox96® assay, respectively. Furthermore, we will measure PD1, LAG3, and other markers of T cell exhaustion to assess the effect of ICAM-1 ablation on effector function. Cell culture supernatants will be used to detect cell type-specific cytokine release using a Luminex® 35-plex human panel. The data in Figure 6 demonstrate that monocyte chemoattractant protein 1 (MCP-1) is significantly produced in MLR cocultures of WT PSC-AECs and allogeneic PBMCs, thus illustrating the role of innate cells / monocytes in the natural allogeneic response to unedited PSC-derived cells. These experiments also evaluate the differential immunogenicity between WT versions of the three cell types, as well as between edited versions, to determine whether edited versions of certain cells have a greater tolerance-promoting effect.

[0205] Decreased proliferation and cytotoxic (allo-reactive) T cell and NK responses are observed in the PSC-AD-CVT cell type compared to WT cells. Furthermore, there is a reduction in inflammatory cytokines involved in adaptive and innate alloreactions. Edited AECs may be the most hypoimmune of the three cell types due to their role as semi-professional antigen-presenting cells in allorejection, which is disrupted by ICAM-1 knockout.

[0206] Addition of ICAM-1 KO to first-generation hypoimmune PSC-CVT improves immune tolerance: Figure 3 shows that 1 stWe demonstrate that B2M-gen (B2M KO) hypoimmune PSC-AECs are less immunogenic than WT cells but still have a proliferative response in the MLR, suggesting that an indirect pathway of allogeneic rejection may be involved. st The -gen KO E-dimer PSC line was also edited to incorporate ICAM-1 KO, with the intention of further reducing rejection by preventing effective adhesion. In ICAM-1 KO cells, reduced adhesion of T cells, B cells, NK cells, and monocytes was observed in immunofluorescence microscopy and flow cytometry-based studies, and reduced immune responses were observed in MLR assays. The increased frequency of T cells with an exhausted phenotype mediates reduced direct and indirect pathway responses.

[0207] We will prepare vascularized next-generation PSC-AD-CVT grafts that are immune-tolerized in vivo in the NeoThy humanized mouse model. PSC-AD-CVT grafts composed of a ratio of hypoimmune PSC-AECs, -CMs, and -C-Fibs form vascularized cardiac organoids with contractile function in ectopic kidney capsule sites in immunodeficient mice. PSC-AD-CVT grafts are immunotolerant in the short- and long-term in the minimally inflammatory environment of the NeoThy model.

[0208] The inclusion of hypoimmune AECs alone may prevent immune cells from breaching the graft's endothelial barrier, and when included in PSC-CVT, it may protect WT PSC-CM and PSC-C-fib from rejection. In vivo studies will then be used to validate the clinical utility of the disclosed PSC-AD-CVT. To this end, we will use the NeoThy humanized mouse model. NeoThy overcomes a weakness of other humanized mice: graft-versus-host disease (GVHD). GVHD causes systemic inflammation, which prevents the identification of model-specific immune effects versus experimental design, and shortens the experimental window by causing early mouse death. Mouse host irradiation and anti-host passenger thymocytes within transplanted human thymic fragments are thought to be mediators of GVHD. NBSGW mice, which do not require irradiation for humanization, along with anti-CD2 passenger thymocyte depletion, have a significantly reduced premature death rate and a correspondingly larger experimental window for long-term transplantation studies when used to generate NeoThy, as demonstrated in Figure 7. The use of NBSGW mice allows for the control of the inflammatory environment and for long-term studies to be performed (few published PSC transplantation studies have time points beyond 30 days, diminishing their relevance for chronic rejection assessment).

[0209] The input cell ratio of the three-cell PSC-AD-CVT was varied in non-humanized NBSGW mice to generate contractile vascularized grafts, as shown in Figure 2. The immunogenicity of the grafts was evaluated in the NeoThy model. To examine graft function and the interaction between the graft and human immune cells, PSC-AD-CVTs were transplanted into the xenogeneic kidney capsule (Figure 2), a blood-rich location. Graft composition and immunogenicity are interrelated. Hypoimmune grafts were functional in non-humanized and NeoThy mice, and in NeoThy mice, the grafts were tolerated by the allogeneic human immune system.

[0210] PSC-AD-CVT grafts composed of a ratio of hypoimmune PSC-AECs, -CM, and -C-Fibs form vascularized cardiac organoids with contractile function in ectopic kidney capsule sites in immunodeficient mice.

[0211] The human heart is composed of multiple cell types present at varying frequencies, which synergistically maintain homeostasis and proper biological function within the cells themselves and the organ as a whole. The tricellular PSC-AD-CVT utilizes highly purified PSC-derived AECs, CMs, and C-fibs such that the proportions of these cells are controlled (and undefined populations with unknown function and / or teratoma potential are minimized) to achieve engraftment and repair function. The input ratio of the three graft cell types was varied, and the grafts were then transplanted in spheroid form (Figure 3). Engraftment into the kidney capsule (ectopic) and heart (orthotopic) of immunodeficient mouse hosts was demonstrated, respectively. Matrices were used to vary cell number and spheroid size prior to transplantation. PSC-AD-CVT spheroid preparations were evaluated for macroscopic contractility before transplantation under the kidney capsule of 8-week-old NBSGW immunodeficient mice. Graft integrity / engraftment is monitored by weekly BLI tracking via the Akaluc reporter co-engineered into hypoimmune PSCs (Figure 8). Grafts positive by BLI are anesthetized at 1 month and undergo survival surgery to observe graft size, vascularization, and contraction rate. At 3 months, this procedure is repeated, and then observations are recorded before the animals are sacrificed and tissues are collected. Tissues are evaluated by histology (for evidence of immune infiltration and teratoma) and RNA sequencing for CM, AEC, and C-fib markers (e.g., cardiac troponin T [cTNT], CD31), and maturation-related genes, compared with d0 grafts retained as controls. Small graft samples from all transplantation experiments are retained for further studies to analyze viral infection trends. PSC-AD-CVTs are compared with WT tri-cellular PSC-CVTs and PSC-CMs as controls. Robust graft engraftment was observed in PSC-AD-CVT and WT PSC-CVT, with both tri-cellular grafts exhibiting greater vascularization and contraction than PSC-CM-only grafts. Grafts mature in vivo when compared to d0-3 month timepoint gene expression signatures of cardiac maturation-related genes (e.g., SCN5A, GJA1, KCNJ4), similar to in vitro studies demonstrating time-related maturation changes (Figure 9).

[0212] PSC-AD-CVT grafts are immune tolerant in the short and long term in the minimally inflammatory environment of the NeoThy model: repeat transplantation studies are performed using graft candidate(s) (determined by graft size, vascularization, contractility, and maturity) in NeoThy humanized mice humanized with an allogeneic immune system (fully HLA-mismatched). As shown in Figure 7 above, an advantage of NeoThy is that long-term studies (e.g., over 3 months) can be performed compared to traditional fetal tissue-based models, which often result in animal loss and loss of experimental power due to early GVHD-related mouse death. Using the NeoThy model, we evaluated short-term time points (1 month) commonly used in the humanized mouse and / or gene-edited PSC literature, as well as long-term time points (3 months) that allow for the determination of susceptibility to chronic allogeneic rejection. Systemic inflammation was assessed by Luminex (Figure 6) before and after transplantation. Effector memory T cells were quantified by flow cytometry, including analysis for markers of T cell activation and exhaustion. Transplanted PSC-AD-CVTs were evaluated for infiltration of human CD4+, CD8+, and FoxP3+ T cells and human monocytes / macrophages. Tissue fibrosis and anatomical integrity were assessed by histopathology. Compared to WT controls, animals receiving PSC-AD-CVT had reduced inflammatory cytokines, reduced graft immune infiltration, fewer circulating effector memory T cells, increased exhausted and regulatory phenotypes, and normal graft structural integrity. Day 0 blood samples from individual animals were used to establish baseline inflammatory and immune cell levels for these comparisons. BLI was performed at 2 months, and blood samples were retained to track increased inflammatory cytokines and / or increased effector immune phenotypes, which may indicate an impending allogeneic response that could lead to intervention, including the possible administration of immunosuppressive drugs.

[0213] The inclusion of hypoimmune AECs alone may prevent immune cells from breaching the graft's endothelial barrier, and when included in PSC-CVT, may protect WT PSC-CM and PSC-C-fib from rejection. One experiment included the same ratio of cells as above, but added only ICAM-1 KO PSC-AECs to WT PSC-CM and PSC-C-fib. The protective role of gene-edited AECs may occur, as evidenced by the lack of immune cell infiltration into the WT parenchyma (CM and C-fib) of the graft. If there was evidence of immune infiltration, its extent was quantified by image analysis and compared to all three cell types in the edited PSC-AD-CVT. The local infiltrate was analyzed to determine whether CM or C-fib were targeted and whether it was reduced relative to WT. Additional gene editing, such as P / E-selectin and / or molecules specific to the biology of the affected cell(s), was optionally performed.

[0214] Evaluating the repair capacity and immune tolerance of gene-edited PSC-AD-CVT grafts in the inflammatory post-myocardial infarction (MI) NeoThy humanized mouse model: Induction of MI in the NeoThy model releases systemic and local mediators of inflammation, leading to local monocyte recruitment and differentiation into tissue macrophages in the infarct and a higher frequency of activated oligoclonal effector memory T cell phenotypes in the periphery. After MI, orthotopic transplantation of gene-edited PSC-AD-CVT results in a vascularized graft that improves cardiac function, reduces infarct size, and increases ventricular wall thickness. Gene-edited PSC-AD-CVT is persistently tolerated by allogeneic human adaptive and innate immune cells in inflamed humanized mouse hearts after MI.

[0215] We tested PSC-AD-CVTs for repair function and immune tolerance potential in a high-fidelity model that mimics the inflamed, complex physiological environment found in the heart of an MI patient. Specifically, we tested whether hypoimmune PSC-AD-CVTs are immune tolerant in a robust, clinically relevant in vivo environment. Demonstration of sustained repair function when encountered with a human immune response after a complex MI is a clear indicator of translational potential. Although immunodeficient animals are receptive to transplanted xenogeneic tissue, their response to injury differs from that of immunocompetent WT animals. This model reconstitutes a functional human immune system and more closely models the role of normal human immunity in cardiac injury and transplantation. Tolerant PSC-AD-CVTs have been used in numerous patient populations and require little or no immunosuppressive medications.

[0216] Induction of MI in the NeoThy model releases systemic and local mediators of inflammation, leading to local monocyte recruitment and differentiation into tissue macrophages in the infarct and a higher frequency of activated oligoclonal effector memory T cell phenotypes in the periphery. Using the NeoThy MI model, MI is induced in human-chimeric animals (16 weeks post-humanization) by left anterior descending artery (LAD) ligation with non-absorbable sutures using IACUC-approved surgical techniques and pain relief measures. Baseline inflammation was assessed by Luminex® assay of peripheral blood compared with non-MI (sham-operated without the ligation procedure) NeoThy mice of similar age and time post-humanization, as well as naive NBSGW controls. Echocardiographic imaging was performed, and images were analyzed for MI by calculating left ventricular ejection fraction, left ventricular fractional shortening, end-diastolic volume, and end-systolic volume. Furthermore, after 4 weeks, immunohistochemistry of mouse hearts was performed to evaluate infarct size, reduction in ventricular wall thickness, and apoptosis (TUNEL assay). MI was associated with an increase in human inflammatory cytokines (e.g., IFNγ, IL1β, TNFα) and human CD11b. + CD33 + CD16 +This resulted in increased infiltration of macrophages. As shown in Figure 10, activated effector memory CD4 + and CD8 + T cell as well as inflammation-induced clonal expansion was detectable by adaptive bioengineered TCR β chain rearrangement sequencing.

[0217] Orthotopic transplantation of gene-edited PSC-AD-CVT after MI results in vascularized grafts that improve cardiac function, reduce infarct size, and increase ventricular wall thickness. PSC-AD-CVT were transplanted into MI-treated nonhumanized NBSGW mice and humanized NeoThy mice, and MI was induced using the same method as described above. Immediately after LAD ligation, 5 to 10 spheroids were suspended in a fibrin matrix patch placed over the infarct site. Animals were monitored weekly for engraftment by BLI monitoring. One cohort of animals was analyzed at 1 month and the other at 3 months, first for cardiac function and engraftment (BLI) as described above, and then for gross vascularization and graft contraction under anesthesia before sacrifice. After sacrifice, infarct size and ventricular wall thickness were assessed in these mice versus sham-treated animals. Improved cardiac function, reduced infarct size, and increased ventricular wall thickness were observed in nonhumanized NBSGW mice and MI-treated NeoThy mice.

[0218] Gene-edited PSC-AD-CVT are persistently tolerated by allogeneic human adaptive and innate immune cells in inflamed humanized mouse hearts after MI: In addition to their engraftment and repair function in non-humanized NBSGW mice, hypoimmunized PSC-AD-CVTs also produce the same engraftment and repair outcomes in NeoThy mice, but not as well as WT PSC-CVTs and 1 st The -gen gene-edited PSC-CVTs are rejected by the allogeneic human immune system, although to a lesser extent. Rejection is evidenced by a decrease in BLI signal over time, as well as the presence of human Ku80 in immunohistochemical analysis of mouse hearts. +This was evident by a decrease in cells and an increase in T cell and macrophage infiltration in the grafts compared with the tolerated grafts. Improved cardiac function, durable (3-month) tolerance of PSC-AD-CVT, a >10% reduction in infarct size, and a decrease in cardiac fibrotic tissue were also observed in the hypoimmune grafts.

[0219] Statistical and Gender-Based Considerations: Using data from previous studies, two-tailed, two-sample t-tests were performed at a significance level of 0.05. To have sufficient (80%) power to find significant differences between groups, a minimum of five animals per group (one additional mouse to account for any unexpected mouse deaths) was used in all humanized mouse studies. Male and female mice (n=5 each) were used. Data were disaggregated to allow for analysis of any gender-based differences in the experimental metrics listed above.

[0220] Example 4 Pluripotent stem cell (PSC)-derived cell therapies are promising restorative treatments for various cardiovascular diseases that kill over 655,000 Americans annually, and preventing their immune rejection after transplantation is crucial for effective clinical translation. Due to their scalability, which allows for large-scale cell banking, PSCs are an ideal cell source for gene-editing approaches to improve transplant outcomes and achieve immune tolerance. Recently, multiple research groups have created gene-edited hypoimmune PSCs (e.g., human leukocyte antigen [HLA] knockout [KO]) that can circumvent allogeneic rejection by T cells, donor-specific antibodies, and / or natural killer cell-mediated cytotoxicity in short-term studies. Despite these advances, little is known about the potential for long-term tolerance of HLA-KO PSC grafts in patients, including whether drastic interventions such as the complete loss of HLA class I and / or HLA class II increase the long-term risk of adverse effects (e.g., malignancies). There remains a critical need to develop strategies for immune tolerance induction and gene editing approaches informed by the cell-specific biology of the graft: immune cell interactions hold great promise for meeting this challenge. Furthermore, to determine potential clinical utility and efficacy, rigorous translational studies require high-fidelity in vitro and in vivo models relevant to human immunology.

[0221] Described herein are PSC-based cell therapies that are immune-tolerant, require little or no sustained immunosuppression, and significantly improve patient health and quality of life. For example, 1) we use a CRISPR / Cas9 gene editing approach to target adhesion molecules (AMs) (e.g., ICAM-1) in human PSC-derived cardiovascular therapies (CVTs) to disrupt the adhesion, infiltration, and destruction of vascularized grafts by allogeneic immune cells; and 2) we investigate the cellular composition and immunogenicity profile of next-generation immune-hypothesized PSC-CVT grafts for their repair potential in the inflammatory setting of myocardial infarction (MI).

[0222] Targeted deletion of AM genes promotes immune tolerance of PSC-CVTs through two mechanisms: 1) reduced immune cell contact-mediated destruction; and 2) anti-inflammatory effects (e.g., altered secreted factors and gene expression) directly related to genetic disruption of AM function. Hypoimmune PSCs are a clinical platform for immune tolerance of PSC grafts, for example.

[0223] We define the effect of ICAM-1 ablation on immune cell contact-mediated PSC-CVT graft destruction. Preliminary data demonstrated reduced leukocyte binding to ICAM-1 KO PSCs in vitro and a normal PSC phenotype. AM KO can prevent allogeneic destruction of tricellular PSC-CVT grafts (composed of PSC-derived arterial endothelial cells [AECs], cardiomyocytes [CMs], and cardiac fibroblasts [Cfib]). ICAM-1 is directly involved in immune cell tethering, rolling, extravasation, and cytotoxic immune synapse formation. Targeting this AM gene prevents leukocyte binding to each of the individual PSC-CVT cell subtypes, reducing KO graft cell loss in vitro and in vivo.

[0224] Immune cell-initiated inflammatory responses define PSC-CVT graft interactions. Preliminary data demonstrate a direct relationship between inflammatory stimuli (TNFα) and ICAM-1 function in PSCs and indicate that ICAM-1 deletion increases PSC free radical scavenging capacity. Adaptive and innate immune cells may release lower amounts of pro-inflammatory cytokines and chemokines and more anti-inflammatory factors upon in vitro interaction with ICAM-1 KO cells. KO cells may inherently be more resistant to inflammatory cues (e.g., oxidative stress) compared to WT cells. MI can induce systemic inflammatory cytokines in the humanized NeoThy mouse model.

[0225] Gene-edited PSC-CVTs can show clear evidence of immune tolerance. The results contribute to data on the mechanisms of PSC immunogenicity and transplant tolerance and validate a gene-editing approach that directly targets grafts: immune cell adhesion and related inflammatory pathways.

[0226] After a first myocardial infarction (MI), the adult heart recovers through scar formation. The fact that 36% of male and 47% of female patients die within 5 years indicates that current treatments are grossly inadequate. Pluripotent stem cell (PSC)-based cardiovascular therapy (PSC-CVT) has the potential to dramatically reduce MI-related mortality and improve patients' quality of life. Artificial PSCs (iPSCs), created by reprogramming primary cells such as peripheral blood T cells from an individual patient and then differentiating them into autologous replacement cells for transplantation (e.g., cardiomyocytes [CMs], endothelial cells [ECs]), hold particular promise. These tissues could theoretically be accepted as "self" by the patient's immune system without the need for immunosuppressive drugs and their associated adverse effects, thereby avoiding the detrimental immune disorder of transplant allo-rejection. Although several autologous iPSC therapies are being investigated in clinical trials for diseases such as macular degeneration, clinical-grade patient-specific therapies are currently very expensive to manufacture and, without new technological advances, present quality control and other regulatory obstacles that make them suboptimal for widespread clinical use. Furthermore, autologous cells have limited use in the context of autoimmune diseases and time-sensitive situations (e.g., subacute MI) where patient-specific iPSCs cannot be generated in time for administration to critically ill recipients. For this reason, there is an urgent need for "off-the-shelf" allogeneic iPSC-CVTs that can be rapidly utilized in the clinic.

[0227] Therefore, researchers in the PSC field have focused on two strategies for developing allogeneic therapies to achieve tolerance in patients: 1) banking human leukocyte antigen (HLA)-matched PSC lines from a broad range of donors representing large populations; and 2) gene editing to modulate the cells' immunogenicity and shield them from immune-mediated destruction. While both of these approaches have advantages and disadvantages, in the United States, HLA banking faces several hurdles (e.g., ensuring representation of genetically diverse racial and ethnic minorities). Research is shifting toward developing gene-edited, universally donor, immuno-hypothesized PSCs, so that a single cell line can be used for most, if not all, patients in the country. Importantly, this includes racial and ethnic minorities and / or individuals with rare HLA types, who have traditionally been underrepresented in clinical trials and PSC banks.

[0228] Disclosed herein is a gene-edited PSC-CVT for MI, which has the potential for future use in any form of cardiac disease requiring the replacement of damaged and / or diseased cardiac tissue with vascularized, functional cells that can be tolerated by the recipient's immune system. This approach targets adhesion molecules (AMs), specifically ICAM-1, a key AM involved in immune cell binding and / or inflammation. This approach is designed for tolerance by both adaptive immune cells (e.g., T cells) and innate immune cells (e.g., monocytes), the latter of which are not directly targeted by currently available hypoimmune PSC therapies (e.g., HLA knockout [KO]). In addition to validating an entirely new targeted approach for hypoimmune gene editing, gene-edited PSC-CVT may be useful in treating cardiac diseases that kill and / or reduce the quality of life of millions of people worldwide.

[0229] Next-generation gene-edited hypoimmune PSC-CVT: Recently, first-generation (1st-gen) gene-edited hypoimmune cells (e.g., HLA class I+II KO) capable of evading recognition by T cells, donor-specific antibodies, and / or natural killer (NK) cell-mediated cytotoxicity in short-term studies have been described. These gene-edited cells have been used to differentiate into multiple PSC-derived cell types, including CMs. These cells maintain their phenotypic and functional attributes and, in purified form, do not develop teratomas after gene editing. Conventional wisdom in the PSC field is that the potential benefits of HLA elimination outweigh the risks of HLA absence or downregulation, a key immune evasion mechanism used by 40–90% of human tumors and multiple viral pathogens. However, no publications to date have explicitly examined the potential for tumor propensity and / or viral reservoirs within HLA I+II KO PSC-derived cells. Furthermore, HLA depletion and related published strategies are intended to reduce adaptive immunity (T cells, donor-specific antibodies) and NK cells, while ignoring other innate immune cells (e.g., monocytes) that play a key role in allogeneic rejection. Adverse events may jeopardize the 1st-gen platform, and innate immune-mediated rejection may play a clinically important role in PSC graft failure. Therefore, an alternative strategy for hypoimmune PSC engineering is provided herein. The gene editing approach focuses on targeting the ICAM-1 gene, an AM gene closely involved in immune cell adhesion and inflammatory responses.

[0230] Designing PSC-CVT grafts with durable function and immune tolerance: Repair function and immunogenicity are closely related. PSC-CVTs must maintain their in vivo function while avoiding destruction through the individual and coordinated actions of the recipient's adaptive and innate immune cells. The differentiation protocol used yields cells with strong repair potential by maintaining their cell type-specific in vivo functions (e.g., contractility) and angiogenesis in immunodeficient mouse hosts, as shown by first-generation gene-edited (beta2 microglobulin [B2M] KO) PSC-CMs in Figure 2B.

[0231] To overcome the non-immunological limitations associated with previously published PSC-CM grafts, we used an advanced tricellular PSC-CVT containing three distinct PSC-derived cell types (CMs, Cfib, and arterial ECs [AECs]). Previous transplantation studies typically utilized grafts composed primarily of CMs, and their methods did not necessarily identify the purity of the preparation and / or the effects of unintended non-CM populations within the graft. These grafts are prone to post-transplant arrhythmias, potentially related to the immature developmental state of the PSC-CMs used, potentially related to cellular impurities, and / or the lack of homeostatic cues from accessory cell types (e.g., C-Fib) that are present in the normal heart but lacking in the relatively homogenous population of PSC-CMs. Our PSC-CVT graft design utilizes an advanced protocol to intentionally mix highly purified CMs (to avoid teratomas) with C-fib (to provide homeostatic cues and aid maturation). A third specialized purified cell population, AECs, was then added to promote neovascularization, CM maturation, and homeostatic crosstalk. The hypoimmune tricellular grafts described herein may be more mature than CM alone and have high potential in the clinic due to their long-term functional and immune-tolerant capabilities.

[0232] Targeting immune cell adhesion in allogeneic rejection: A strategy to achieve PSC-CVT resistance combines cutting-edge CRISPR / Cas9 gene editing technology with over 50 years of transplant immunology lessons. Importantly, specific focus is placed on the unique biology of AMs as an alternative target to global HLA ablation, which may be too extreme (e.g., it can render cells susceptible to malignancy). In one aspect, the present disclosure provides a strategy that attenuates, but intentionally does not completely eliminate, immune cell adhesion and pathological inflammatory responses. Tricellular PSC-CVT grafts contain AECs, which form a luminal barrier in larger cardiac vessels (e.g., coronary arteries), promoting post-transplant angiogenesis. The vasculature is a major interface for transplant rejection, and crucially, AECs have a lower baseline propensity for leukocyte adhesion than vasculature or other EC subtypes.

[0233] The first part of the hypothesis is that targeted attenuation of immune cell adhesion to cellular targets via ICAM-1 knockout confers immune tolerance to PSC-CVT graft vasculature and parenchymal cells. ICAM-1 is involved in post-transplant immune interactions with the vasculature (tethering, rolling, extravasation) and cytolysis during immune cell killing (immune synapse formation). LFA-1 is the primary ligand for ICAM-1 and is expressed on adaptive (e.g., T cells) and innate (e.g., monocytes and dendritic cells) immune cells, all of which play important and coordinated roles in allogeneic rejection. Previous mouse studies have shown that ICAM-1 knockout results in a reduction in allogeneic T cell responses and nearly doubles the survival time of allogeneic hearts compared to WT grafts. Importantly, this knockout does not cause major pathogenic vascular defects and / or embryonic lethality. Furthermore, because ECs function as semi-professional antigen-presenting cells, gene editing approaches to prevent stable leukocyte:graft interactions could disrupt effector memory T cell function. Importantly, preliminary data show that karyotypically normal human ICAM-1 KO PSCs can be readily differentiated into CMs and AECs comparable to isogenic WT PSCs (Figure 15). This provides strong evidence that this project is feasible and that our strategy is compatible with existing PSC differentiation protocols.

[0234] Accurate assessment of human immune responses to PSC grafts: Data, as well as multiple recent publications from other groups, highlight the importance and potential of new discoveries related to gene-edited hypoimmune PSC grafts. A gap exists in existing knowledge: the need to conduct rigorous immunogenicity studies using high-fidelity in vitro assays and in vivo models that accurately recapitulate human immunology. This gap is the result of 1) the disparate methodologies and T cell / NK cell bias (ignoring monocytes and other non-NK innate cells) of assays used by various groups in previous studies; and 2) the lack of an appropriate preclinical in vivo model of the human immune response to PSC grafts. To close the gap, we employ robust assays. For example, Figure 16 shows a mixed lymphocyte reaction (MLR) assay that can be used to experimentally test immunogenicity. This approach is informed by past lessons from transplant immunology and stem cell biology expertise, purposefully examining both adaptive and innate immunity. The MLR data demonstrate that 1st-gen B2M KO PSC-AECs express CD8 in the presence of antigen-presenting cells. + Our results demonstrate that B2M KO can induce T cell proliferative responses, and although B2M KO may protect against direct pathway allorejection (as indicated by a significant decrease in proliferation in B2M KO vs. WT cells), such first-gen gene-edited PSC therapy may still be prone to indirect pathway-mediated chronic rejection, as seen in solid organ transplant patients. Notably, a large population of proliferating CD8+ T cells still exists compared to unstimulated controls. This finding enhances the potential importance of our proposal, as creating a PSC-CVT that circumvents both direct and indirect allorecognition by knockout of AMs involved in both of these rejection pathways would have a higher chance of successful clinical conversion.

[0235] Controlled and highly pure WT and KO target cell populations will enable successful clinical translation of PSC-CVT and also provide insight into traditional solid organ allogeneic rejection mechanisms using high-fidelity in vitro assays and in vivo models relevant to human immunology, including humanized mouse models.

[0236] Beyond immune cell adhesion: Effects of ICAM-1 KO on inflammation: Pathological inflammation plays a key role in solid organ allo-rejection, both in the parenchymal tissue and the graft vasculature. Immune cell-derived inflammatory cytokines and / or oxidative stress can initiate an inflammatory response in endothelial cells and other graft cells, which feeds back (in association with ICAM) via further release of cytokines and radical oxygen species, which may further reduce graft cell viability. To develop effective mitigation strategies, the deleterious effects of inflammatory stimuli on PSC-derived cell therapy must be defined. Data in Figure 1 show that ICAM-1 gene expression (by RNA sequencing [RNAseq]) increases during immune cell-mediated activation / inflammation as well as after the addition of inflammatory stimuli (e.g., TNFα) in the absence of adherent cells. This also occurs in 1st-gen B2M KO PSC-AECs, which lack surface major histocompatibility complex (MHC) class I, similar to published reports on immortalized mouse and primary human ECs. This validates the strategy of focusing on molecules that play a key role in allorejection and suggests that AMs (and the innate immune cells that bind to them) may still contribute to allorejection in 1st-gen KO PSC grafts.

[0237] Furthermore, AM plays an important role in the response to oxidative stress during inflammatory responses. Using mouse ICAM-1 KO aortic ECs, an inverse relationship between ICAM-1 and glutathione (GSH) production was demonstrated. GSH is a major source of intracellular GSH and plays a key role in the anti-inflammatory response to oxidative stress. Data show increased ThiolTracker™ dye levels in ICAM-1 KO PSCs compared to WT, indicating increased GSH and a concomitant increase in free radical scavenging capacity within PSC-CVTs.

[0238] Investigating two mechanisms of immune rejection in SA1 (via immune cell adhesion) and SA2 (via inflammation) enhances the generalizable significance of our AM gene editing approach and its potential to improve transplant outcomes and patient health.

[0239] Evaluating PSC-CVT engraftment in a novel human immune model: using the humanized mouse, NeoThy. Humanized mice are powerful research tools for modeling the in vivo human immune response to PSC transplantation. NeoThy contrasts with the existing gold-standard humanized mouse (fetal tissue-based "BLT"), which has been reported to be suboptimal for allogeneic rejection studies due to naive T cell and regulatory T cell bias. NeoThy possesses naive CD4 T cells, which are more similar to adult patients than the BLT model. + The NeoThy model possesses a T cell compartment and is a high-fidelity model of allorejection (e.g., extensive immune infiltration) of transplanted PSC-CM grafts. The NeoThy model may be a more reproducible and reproducible system for broader applications, and such technology would benefit from investments to accelerate research in these fields. Testing the alloimmune response to gene-edited PSC-CVT in NeoThy, as well as measuring local and systemic inflammation pre- and post-MI in the model, will generate important data for evaluating in vivo orthotopic engraftment and repair function in an inflammatory environment in the context of human immune responses. The result is a hypoimmunized PSC-CVT graft with a tricellular composition lacking alloreactivity in vitro and in vivo. This hypoimmunized graft strategy is appropriate for MI patients.

[0240] MI is a devastating condition that results in death or reduced quality of life for millions of patients. While existing treatments are suboptimal, PSC-based cell therapy holds great potential for improving and saving lives, especially in the presence of sustained immune tolerance. Scalability and pluripotent differentiation potential are two advantages of PSCs over other primary cell-based CVTs. The current state of the art in gene-edited PSC therapy is the use of HLA I+II KO lines, a promising but biologically invasive approach that ignores subtle cell-type-specific interactions with the immune system and renders the graft vulnerable to indirect pathway-mediated rejection in concert with monocytes and other innate immune cells. The graft utilizes a gene-editing strategy focused on preventing immune cell-mediated allorejection, and the disclosed tricellular graft allows for superior repair function and low immunogenicity. The human immune response can be investigated with a multifaceted approach that utilizes classical transplant immunology techniques and stem cell biology, as well as advanced in vivo humanized mouse models, allowing for understanding the mechanisms of leukocyte adhesion to vasculature and parenchymal cells, and mediating activation and inflammation during the allorejection process.

[0241] We defined the effect of ICAM-1 ablation on immune cell contact-mediated PSC-CVT graft destruction. ICAM-1 KO prevented allogeneic destruction of engraftable tricellular PSC-CVTs. Hypoimmune tricellular PSC-CVTs incorporate genetic loss of a key cellular AM, used by multiple adaptive and innate immune cells during critical stages of allogeneic rejection. Addressing and experimentally examining innate and adaptive immune responses in therapeutic design and immunogenicity assays will enable improved graft materials. ICAM-1 KO PSCs were generated with normal karyotypes and differentiation yields typical of phenotypically normal PSC-CVT cell types. Tricellular spheroids exhibiting in vitro contractility were generated (Figure 13A). Multiple gene-edited PSC lines were generated and then differentiated into the three PSC-CVT cell types. The immunogenicity of each gene-edited cell type was then assessed in multiple assays. The mechanistic role played by AM in the interactions between immune cells and AECs, CMs, and C-fib, as well as how ICAM-1 ablation affects immune cell adhesion and downstream immunogenic responses, will be defined to develop sustainably tolerated PSC-CVTs that will improve the health and well-being of millions of patients worldwide. ICAM-1 knockout results in reduced immune cell interactions and cell loss with each of the three cell types. These effects of ICAM-1 knockout alone can be enhanced when combined with first-generation gene editing (i.e., removal of MHC class I+II, addition of HLA-E dimers).

[0242] The gene-edited PSC-CVT cell line will have normal phenotypic identity and function: CRISPR / Cas9-based gene editing is efficient and cost-effective. To minimize off-target effects, we used a high-fidelity Cas9 mutant to KO ICAM-1 and introduce a constitutively expressed Akaluc luminescent reporter for use in downstream bioluminescence imaging (BLI) studies. The ICAM-1 KO described in Figure 15 and the B2M KO / CIITA KO / HLA-E dimer (KO E dimer) line used as the first-gen control line for these experiments were used. ICAM-1 KO lines were generated from multiple existing PSC lines, and the KO was then validated via IFNγ- and / or TNFα-based upregulation of ICAM-1 (see Figure 15). Multiple KO clones were pooled, karyotyped, and screened for off-target activity. Multiple 1 st The 1st-gen hypoimmune PSC line was able to differentiate into phenotypically normal, functional AECs and CMs (Figure 15 and additional data not shown). Upon verifying a normal karyotype by g-banding, all of our PSC replicates were differentiated into AECs, CMs, and C-fibs to assess their phenotypes. Five PSC replicates were used: 1) ICAM-1 KO only, 2) 1st-gen KOE dimer only, 3) ICAM-1 KO added to 1st-gen KOE dimer, 4) ICAM-1 KO with additional KO of both E- and P-selectin, and 5) WT. All lines contained Akaluc. E- and P-selectin have compensatory / redundant functions and are therefore ablated together.

[0243] Using flow cytometry and bulk RNA sequencing, we verified that knockout of ICAM-1, E- and P-selectin, and / or 1st-gen gene targets did not result in off-target gene and protein expression effects or adversely affect the cells. Normal cell type-specific function was demonstrated after knockout, validating gene-edited PSC-AECs, PSC-CMs, and PSC-C-fibs. Briefly, we verified normal AEC function by assessing oxygen consumption rate, CMs by contractility, and C-fibs by immunolabeling to demonstrate extracellular matrix formation via staining for collagen I and fibronectin.

[0244] ICAM-1 KO PSC-CVT cell lines induce reduced allogeneic immune cell binding and proliferation compared to WT cells: In vitro assessment of immunogenicity is the first step in the methodological evaluation of the tolerogenic potential of PSC-CVT cells. ICAM-1 plays a key role in adaptive (e.g., T cells) and innate immune cell (e.g., monocyte) adhesion to cellular targets via LFA-1 binding; therefore, we evaluated these cells as well as innate NK cells (mediators of autologous cytotoxicity in solid organ transplantation and first-generation hypoimmune PSC therapy). Data show reduced peripheral blood mononuclear cell (PBMC) binding to ICAM-1 KO PSCs compared to WT cells (Figure 18). AECs, CMs, and C-fib differentiated from KO PSCs also demonstrated attenuated immune binding and less target loss. As shown in Figure 3, a 6-day MLR is performed based on a previously published protocol using allogeneic PBMCs (containing the above three cell populations) and the above individual gene-edited PSC-derived cells as targets.

[0245] To assess alloreactivity, proliferation was measured by flow cytometry (CFSE dilution method) for gated cell subtypes (e.g., effector memory T cells), and target cell loss was measured microscopically via changes in morphology and the degree of monolayer integrity. Additionally, monocyte / macrophage inflammatory and regulatory phenotypes, markers of T cell exhaustion (e.g., PD1, LAG3) were measured to assess the effect of ICAM-1 ablation on effector function, and finally, regulatory (CD3) responses were assessed. + CD4 + CD25 + CD127 lo FoxP3 + ) T cell proliferation was assessed. Cell culture supernatants were collected and frozen to assess inflammatory cytokines in SA2. These experiments also assessed the differential immunogenicity between wild-type and gene-edited versions of the three cell types to determine whether ICAM-1 knockout in specific cells has a more profound tolerance-promoting effect.

[0246] The hypoimmune KO PSC-CVT cell type demonstrated reduced proliferative T cell, NK cell, and monocyte / macrophage responses compared to WT cells, comparable to or superior to cell types derived from the first-gen KO PSC line. Furthermore, reductions in inflammatory cytokines involved in adaptive and innate alloreactions were determined by Luminex® evaluation of cell culture supernatants and intracellular cytokine flow cytometry (e.g., IFNγ), as described below. Gene-edited AECs are the most hypoimmune of the three cell types due to their role as semi-professional antigen-presenting cells in allogeneic rejection, which is abolished by ICAM-1 KO. Other AMs, such as the double KO of E-selectin and P-selectin in the first-gen line generated above, may also be incorporated.

[0247] Gene-edited PSC-CVT yield vascularized grafts after transplantation into immunodeficient mouse hosts: Different ratios of three PSC-CVT cells in each of the five categories were used with different cell numbers (see Figure 2B and Figure 13; e.g., 1 x 10 cells at a 1:1:1 ratio). 5 These (non-humanized) host strains were combined in a four-level matrix (100x, then doubled one of each type) and transplanted into immunodeficient mice via kidney capsules to obtain the most achievable contractile and vascularized grafts. These (non-humanized) host strains were used for humanization experiments, and the kidney capsule site is the recipient niche, an anatomical site for assessing baseline engraftment and development in the absence of immune cells. Positive engraftment in the kidney indicates the likelihood of positive engraftment in the heart. Grafts were monitored by live animal imaging (BLI) tracking of the grafts (Figure 19) as well as histology 30 days post-transplant at the end of the experiment (described in more detail below).

[0248] Individually injected gene-edited PSC-CVT cell lines are tolerated in humanized NeoThy mouse xenografts. The NeoThy humanized mouse model overcomes a weakness of existing humanized mice: graft-versus-host disease (GVHD). GVHD causes systemic inflammation, which prevents the identification of model-specific immune effects versus experimental design, and shortens the experimental window by causing early mouse death. Mouse host irradiation and anti-host passenger thymocytes within transplanted human thymic fragments are mediators of GVHD in humanized mice. NBSGW mice do not require irradiation for humanization. Furthermore, when used to generate NeoThy mice with anti-CD2 passenger thymocyte depletion, NBSGW mice significantly reduce early death and have a correspondingly larger experimental window for long-term transplantation studies.

[0249] We evaluated the in vivo immune response to ICAM-1 KO PSC-CVT in a non-MI setting (to assess baseline alloimmune responses). We assessed post-MI inflammatory dynamics in NeoThy mice in the absence of PSC-CVT, mimicking clinical experience. Genetically immunodeficient mice are receptive to transplanted xenogeneic tissue, but their response to injury differs from that of immunocompetent WT animals. The NeoThy model reconstitutes a functional human immune system and more closely models the role of normal human immunity in cardiac injury and transplantation.

[0250] Similar to the pilot experiment shown in Figure 21, highly purified populations of ICAM-1 KO PSC-derived CM, AECs, and c-Fibs from one donor were individually injected with Matrigel® into the hind limbs of humanized NeoThy mice (minimum 10% human CD45, 10% human CD3 T cells) and WT controls were placed in the other limb. BLI signals were monitored for 30 days at three time points: immediately after injection (d1-6), midway (approximately d15), and at the final endpoint (approximately d30). KO cells of the three PSC-CVT cell types were retained / tolerated in the animals, whereas WT cells were rejected (Figure 8, n = 4 animals). Three of the four animals showed loss of WT but not KO grafts, indicating immune tolerance of the KO and rejection of the WT. The fourth animal showed no loss of any cell type. Consult a biostatistician to test n = 19 mice per PSC cell type (CM, AEC, c-Fib) to have sufficient statistical power to assess the likelihood of rejection between WT and KO. This is based on the 75% response and Fisher's exact test in Figure 21, performed assuming 80% power at a significance level of 0.05. A non-humanized control (n = 5) was included to verify that there was no significant loss of either WT or KO PSC-derived cells in the absence of human immune cells.

[0251] Thus, NeoThy is useful for assessing allogeneic rejection and tolerance of heterotopically transplanted PSC-CVT cells, providing data on hypoimmune states, and mechanistic immunogenicity studies of orthotopically transplanted intact PSC-CVTs after MI.

[0252] Immune cells: Define the inflammatory response initiated during PSC-CVT graft interactions in vitro and in NeoThy mice after MI in vivo: adaptive and innate immune cells release lower amounts of pro-inflammatory cytokines and chemokines and more anti-inflammatory factors upon in vitro interaction with ICAM-1 KO cells, KO cells are more resistant to inflammatory cues than WT cells, and MI induces systemic inflammatory cytokines in a humanized NeoThy mouse model.

[0253] Pathological inflammation plays a driving role in allogeneic rejection. As shown by TNFα-induced ICAM-1 upregulation in Figure 1, AM cell biology and inflammatory stimuli are closely linked. To focus on the cell adhesion component of the immune response (e.g., can lysis be prevented by removing critical AMs required for T cell immune synapse formation?), we investigated rejection mechanisms related to direct cell binding. Because the literature has demonstrated an inverse relationship between ICAM-1 expression in multiple cell types and anti-inflammatory responses to oxidative stress, we measured pro-inflammatory cytokines to determine the role ICAM-1 KO plays in the response to these cues. KO of ICAM-1 conferred an anti-inflammatory benefit to PSC-CVT that is specific to the graft's cells and also influences the effector immune response.

[0254] Adaptive and innate immune cell subtypes exhibit reduced inflammatory cytokine responses to KO PSC-CVT cells, and KO PSC-CVT cells exhibit reduced inflammatory cytokine (protein and gene expression) responses after immune cell encounter. We prepared 18-hour MLR cocultures between whole PBMCs and ICAM-1 KO, 1st-gen KO, ICAM-1 KO + 1st-gen KO-derived cells, ICAM-1 KO + E- / P-selectin, and WT PSC-CVT cell types. These short-term MLRs allow for the preservation of intact target cells, which is not possible in 6-day MLRs due to advanced target cell destruction. Target cells were collected by enzymatic dissociation, and effector immune cells were removed via CD45 magnetic beads and stained for intracellular cytokine production (e.g., IFNγ) by flow cytometry. PSC-CVT cell types (which also produce cytokines in response to inflammatory stress) were analyzed by bulk RNA-seq versus control cells without immune cell exposure. Identifying expression patterns in inflammation-resistant cells allows for the analysis of subpopulations via single-cell RNAseq, enabling the evaluation of immune evasion mechanisms and the design of hypoimmune therapies.

[0255] Cell culture supernatants were collected for detection of cell-type-specific cytokine release using a Luminex® 35-plex human panel. Figure 6 shows an example of one cytokine, monocyte chemoattractant protein 1 (MCP-1), produced in large amounts in 18-hour MLR cocultures of WT PSC-AECs and allogeneic PBMCs. This finding demonstrates the role of innate cells / monocytes in the natural allogeneic response to unedited WT PSC-derived cells, which is reduced as a direct result of our AM gene editing. Analysis of immune cell intracellular cytokine flow and gene expression of targeted KO PSC-CVT therapy allows for identification of the source of supernatant cytokines.

[0256] Upon encounter between KO PSC cell types and PBMCs, T cells and monocytes secrete anti-inflammatory cytokines (e.g., IL10), whereas WT cells induce relatively high levels of pro-inflammatory cytokines (e.g., IFNγ, IL1β, TNFα). These differences relate to subcell types across the bulk of specific cell types (e.g., upregulated in CMs versus AECs) and are specific to the type of gene editing (e.g., maximal increase in MCP-1 in monocyte-only cocultures versus T-cell-only for ICAM-1 KO cells, and 1 st gen HLA KO and vice versa).

[0257] KO cells would inherently be more resistant to inflammatory cytokines than WT cells: To assess the direct effects of inflammatory cytokines without confounding effects associated with the presence of immune cells in culture (e.g., nutrient depletion in the culture medium), purified PSC-CVT cells alone were cultured in the presence of a panel of 20 individual cytokines, chemokines, and factors associated with the inflammatory immune response of multiple immune cell types (e.g., IFNγ, IL1β, TNFα, IL17, TGFβ, IL15, MIP-1α, and RANTES). After 48 hours, cells were washed and RNA was collected for bulk RNA-seq to analyze differences in cytokine / chemokine gene expression, inflammatory gene pathways, cell identity-related genes to monitor for potential dedifferentiation, and other mechanisms of apoptosis / cell death (e.g., NF-κB). Based on scRNAseq analysis of inflammatory cues for heterogeneous subpopulations within the PSC-CVT cell type, we assessed cytokines / factors resulting in bulk RNAseq responses indicative of enhanced anti-inflammatory effects associated with ICAM-1 KO (i.e., "hit"), as well as whether compensatory mechanisms play a role for ICAM-1 in the KO lines by looking for altered expression of other AMs in bulk RNAseq analysis of naive KO PSC-derived cells.

[0258] KO cells are more resistant to oxidative stress compared to WT cells, as evidenced by increased GSH levels. Purified cultures of KO and WT PSC-CVT cell types were used to determine GSH levels using ThiolTracker™ dye (shown in Figure 17 for PSCs). High levels similar to those observed in undifferentiated PSCs were observed in KO PSC-derived cells (e.g., CMs). Compounds known to cause oxidative stress (e.g., hydrogen peroxide and nitric oxide) and compounds known to damage associated molecular patterns (DAMPs) may be used to determine whether KO confers better cell viability. Furthermore, cell apoptosis is assessed via caspase 3 / 7 and annexin V / 7-AAD. Decreased apoptosis in response to oxidative stress in KO cells is observed compared to baseline in WT cells.

[0259] Induction of MI in the NeoThy mouse model releases systemic and local mediators of inflammation (at baseline, in the absence of PSC-CVT transplantation) and leads to local monocyte recruitment and differentiation into tissue macrophages in the infarct. MI was induced in chimeric NeoThy animals (16 weeks post-humanization) by left anterior descending artery (LAD) ligation with non-absorbable sutures using IACUC-approved surgical techniques and pain relief measures. Systemic inflammation was assessed by Luminex® assay of peripheral blood pre- and post-MI in the same animals. All mice were of comparable human chimerism, age, and time post-humanization. Echocardiographic imaging was performed, and images were analyzed for MI by calculation of left ventricular ejection fraction, left ventricular fractional shortening, end-diastolic volume, and end-systolic volume. Furthermore, at the end of 30 days, in addition to collecting serum for inflammatory factor analysis, immunohistochemistry of mouse hearts was performed to evaluate infarct size, reduction in ventricular wall thickness, and apoptosis (TUNEL assay). MI was associated with an increase in human inflammatory cytokines (e.g., IFNγ, IL1β, TNFα) in serum and human CD11b in the heart. + CD33 + CD16 +This resulted in the infiltration of macrophages. Activated effector memory CD4 + and CD8 + T cells are observed in the periphery and are detected by flow cytometry analysis.

[0260] Additional statistical and gender-based considerations: For MI studies, a two-tailed, two-sample test at a significance level of 0.05 was performed and it was found that only four animals per group were needed to have sufficient (80%) power to find significant differences in inflammation between pre- and post-MI groups. For all animal studies, equal groups of male and female mice were used and no gender-based differences were assumed.

[0261] The use of the terms "a," "an," and "the," and similar referents (particularly in the context of the claims below), should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. As used herein, the terms "first," "second," etc., are not meant to denote a particular order, but are merely meant for convenience to denote a plurality, e.g., layers. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated. The recitation of ranges of values ​​is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein as if it were individually recited herein. The endpoints of all ranges are included within the range and are independently combinable. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") is intended merely to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention as used herein.

[0262] While the present invention has been described with reference to exemplary embodiments, those skilled in the art will recognize that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope of the invention. Therefore, it is not intended that the invention be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but the invention is intended to include all embodiments falling within the scope of the appended claims. Any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0263] [Table 1]

[0264] [Table 2]

Claims

1. 1. An in vitro method for preparing a population of hypoimmune mammalian stem cells, comprising: providing an isolated population of mammalian stem cells, wherein said isolated mammalian stem cells express a cell adhesion molecule; modifying the expression of the cell adhesion molecule in the isolated population of mammalian stem cells to reduce or knock out expression of the cell adhesion molecule, thereby providing the population of hypoimmune mammalian stem cells; A method comprising:

2. The method of claim 1 , wherein the population of hypoimmune mammalian cells is less immunogenic than a corresponding population of isolated mammalian stem cells.

3. The method of claim 1 or 2, wherein the one or more mammalian stem cells are induced pluripotent stem cells or embryonic stem cells.

4. 10. The method of any preceding claim, wherein the one or more mammalian stem cells are human stem cells or non-human stem cells.

5. 10. The method of any preceding claim, wherein the expression of the cell adhesion molecule is altered by gene editing of the gene for the cell adhesion molecule or by inhibiting expression with an inhibitory RNA.

6. 10. The method of any preceding claim, wherein the cell adhesion molecule comprises ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, VCAM, MADCAM-1, P-selectin, L-selectin, E-selectin, or a combination thereof.

7. 10. The method of any of the preceding claims, wherein the isolated mammalian stem cell population has reduced or absent expression of HLA class I molecules, reduced or absent expression of HLA class II molecules, reduced or absent expression of beta2 microglobulin, increased expression of CD47, increased expression of PDL1, increased expression of secretin, increased expression of CTLA4, or a combination thereof.

8. 10. The method of any preceding claim, further comprising isolating, expanding and / or differentiating said population of hypoimmune mammalian stem cells, or any combination thereof.

9. 9. The method of claim 8, wherein the population of hypoimmune mammalian stem cells are differentiated into endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells, or pancreatic islet cells.

10. The method of claim 9 , wherein the differentiated cells are cardiomyocytes or neurons.

11. A population of hypoimmune mammalian stem cells produced by the method of any one of claims 1 to 10.

12. A population of hypoimmune mammalian stem cells in which the expression of a cell adhesion molecule is reduced or knocked out by modifying the gene for said cell adhesion molecule by gene editing.

13. 13. The population of hypoimmune mammalian stem cells of claim 12, wherein the cell adhesion molecules include ICAM-1, ICAM-2, ICAM-3, ICAM-4, ICAM-5, VCAM, MADCAM-1, P-selectin, L-selectin, E-selectin, or a combination thereof.

14. The population of hypoimmune mammalian stem cells according to any one of claims 11 to 13, wherein the hypoimmune mammalian stem cells are differentiated into endothelial cells, cardiac cells, fibroblasts, pancreatic cells, neural cells or pancreatic islet cells.

14. A graft comprising a population of differentiated hypoimmune mammalian stem cells according to claim 14.

15. 15. A method of treating a mammalian subject comprising introducing the implant of claim 14 into a mammalian subject in need thereof.

16. 16. The method of claim 15, wherein the mammal is a human, dog, cat, cow, horse, pig, sheep, or goat.

17. 17. The method of claim 15 or 16, wherein the mammalian subject is in need of treatment for neurodegeneration, diabetes, vascular disease, myocardial disease, or a combination thereof.

18. 18. The method of claim 17, wherein the mammalian subject is in need of treatment for neurodegeneration, the graft is administered to the central nervous system, and the mammalian subject has Parkinson's disease, Alzheimer's disease, stroke, or ALS.

19. 18. The method of claim 17, wherein the mammalian subject is in need of treatment for diabetes and the composition is administered systemically.

20. 18. The method of claim 17, wherein the mammalian subject is in need of treatment for a vascular disease, and the mammalian subject has peripheral vascular disease, ischemia, or traumatic injury.

21. 18. The method of claim 17, wherein the mammalian subject is in need of treatment for a myocardial disease and the composition is administered to the heart.

22. 22. The method of claim 21, further comprising administering hypoimmune fibroblasts, hypoimmune endothelial cells, or both.

23. 22. The method of claim 21, wherein the mammalian subject is at risk for or has a myocardial infarction, congenital heart disease, heart disease or cardiac malformation.

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