Generation Of Hypoimmunogenic Human Embryonic Stem Cells Expressing ?2m-HLA-G Via Genetic Engineering

Human pluripotent stem cells(hPSCs)are capable of proliferating indefinitely and differentiating into all types of cells from three germ layers.Thus,these cells could theoretically serve as a potentially inexhaustible source of all cell types in the body for regenerative medicine.However,hPSCs and their derivations are immunogenic,which means,transplantation of cells/tissues differentiated from hPSCs will induce an allogeneic immune rejection in recipient patients,resulting in the necrosis of the graft.Therefore,the construction of hypoimmunogenic hPSCs has become an important part of promoting the application of hPSCs in regenerative medicine.The most robust and ubiquitous example of immune accommodation observed in mammalian is the maternal-fetal barrier.The extravillous trophoblasts(EVTs)are directly contact with the maternal uterine decidua,which effectively protect the fetus from attack by maternal immune system and ensure the normal development of the fetus in the womb.EVTs do not express HLA-A,-B.The expression level of HLA-C in EVTs is low,while expression level of HLA-G is high.HLA-G protein is a non-classical HLA I molecule.It has been reported that the HLA-G molecule can accommodate activation of NK cells,T cells,DC cells,etc.In order to construct human embryonic stem cells(hESCs)with HLA I expression pattern similar to EVTs,we establish and optimize an efficient gene editing technology.With this technology,we are able to knockout B2M gene.B2M is responsible for membrane localization of HLA I molecules.Knock out of B2M in hPSCs will lead to absence of HLA I expression on cell membrane.First,we constructed an efficient,predictable,donor-free double gRNAs knockout strategy.By electroporating a pair of adjacent gRNAs(paired-KO)with CRISPR/Cas9 plasmid into hPSCs,an expected DNA fragment is removed.More importantly,DN A is repaired mostly through direct end joining without insertions/deletions during paired-KO,producing predictable lesion product.Through paired-KO,we succeed in targeting more than a dozen of genes including SMAD3 and?-Catenin.In addition,we attempted to introduce gRNAs designed for multiple genes into hPSCs with CRISPR/Cas9 plasmid,the results showed that paired-KO can simultaneously knock out multiple genes with extremely high homozygous-targeting efficacy.For knock-out of encoded microRNAs(miRNAs)and long non-coding RNAs(lncRNAs),we succeed in knocking out miRNA MIR1193 by removing whole transcription DNA sequence through paired-KO,and we carried out a specific knockout of a short fragment of the core promoter region to eradicate downstream MALAT1 gene transcription.The paired-KO strategy we designed is highly efficient,suitable for knocking out both coding and non-coding genes,able to knock out multiple genes simultaneously.The gene product after paired-KO can be accurately predicted.Based on this principle,we used paired-KO system to construct hypoimmunogenic hESCs with endogenous B2M knockout.In order to generate hypoimmunogenic hESCs which express non-classical HLA-G,while lack membrane expression of classical HLAI molecules,we replaced the stop codon of endogenous B2M with HLA-G1 heavy chain reading frame and flexible(G4S)4 linker coding sequence by homologous recombination.Then,a DNA sequence encoding a B2M-(G4S)3-HLA-G5 fusion protein was introduced into the cell based on the engineered cell line using a lentiviral vector.Results have shown that B2M knockout hPSCs lacked expression of ?2m and HLA I molecules on the cell membrane.However,HLA-G knock-in hESCs successfully expressed ?2m-HLA-G1 and ?2m-HLA-G5.We found that ?2m-HLA-G1 could reach the cell membrane and the expression of this protein was regulated by the endogenous B2M gene promoter,which could respond to IFN-y stimulation.Western blot analysis confirmed ?2m-HLA-G5 was secreted into the extracellular culture environment.At the same time,the classical HLA I protein complex could not be assembled,resulting in the lack of HLA-A,-B,-C protein distribution on the surface of the cell membrane after genetic modification,due to the deletion of the free endogenous ?2m protein.Both in vitro and in vivo allogenic immune analysis demonstrated that HLA-G expressing cell lines were hypoimmunogenic compared with wild-type hESCs.HLA-G expressing cell lines were more effective in inhibiting NK cell activation than B2M gene-deficient cell lines.In addition,?2m-HLA-G5 secreted protein could significantly inhibit activation of NK cells co-cultured with wild-type hESCs,and safeguard low immunogenic environments in allografts.In summary,we have successfully constructed a paired-KO gene editing system,enabling efficient and predictable gene knockout in human embryonic stem cells.We also generated hypoimmunogenic hESCs expressing membrane-bound and secreted HLA-G,while with no distribution of classical HLAI molecules on cell membrane.This cell line can effectively avoid T and NK cell-mediated immune rejection and create a non-anomalous immune environment,which might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts,tissues or organs in the future.