Molecular Design And Mechanism Of Recombinant CD47 That Inhibits Graft Damage

It is clear that allograft of human cells,tissues,or organs is crucial for treating serious diseases and injuries.However,the shortage of organ donors remains a significant problem.Xenotransplantation,particularly using pigs as a source of organs,is seen as a promising solution to this issue.Pigs are attractive for xenotransplantation due to their physiological similarities to humans and their rapid reproduction.Despite the potential of pig organ xenotransplantation,rejection caused by the incompatibility between species has been a major obstacle.However,advancements in gene editing technology have shown promise in addressing these challenges.The expression of human CD47 on pig cells,for example,can inhibit rejection caused by macrophages during xenograft.In conclusion,while the shortage of organ donors continues to be a critical issue,xenotransplantation using pig organs holds promise for addressing this problem.Advances in gene editing technology are making it increasingly feasible to overcome the rejection issues associated with xenotransplantation,offering hope for the future of organ transplantation.CD47 is a five-pass transmembrane protein that is widely expressed on the surface of normal cells and belongs to the immunoglobulin superfamily.It has a molecular weight of approximately 50 k D.The protein comprises an extracellular N-terminal Ig V globular domain,five transmembrane helices,two intracellular loops,two extracellular loops,and a short tail with variable splicing at the intracellular C-terminal.Notably,the second extracellular loop has the capability to form a stable disulfide bond with the Ig V domain.SIRPα,a member of the signal regulatory protein family,serves as one of the receptors for CD47.The CD47/SIRPα signal acts as a marker for the body to distinguish between “self” and “non-self.” CD47 can activate SIRPα to transmit a “don’t eat me”signal.The specific mechanism involves CD47 activation of SIRPα,leading to the phosphorylation of the SIRPα intracellular immune receptor tyrosine inhibitory motif(ITIM),which in turn transmits immunosuppressive signals,ultimately inhibiting the phagocytic function of macrophages.It is important to note that the recognition and activation of CD47 and SIRPα are species-specific.For instance,pig CD47 is unable to effectively activate human SIRPα to inhibit macrophage-mediated rejection.Therefore,the accurate transmission of CD47/SIRPα signals is a crucial factor for the success of xenograft procedures.CD47 can be activated by TSP-1 to initiate a series of survival suppression signals.TSP-1,a member of the platelet thrombospondin family,is composed of three identical subunits covalently bound to form a trimeric protein.The TSP-1/CD47 signaling axis can impact the normal physiological function of cells by inhibiting the NO/c GMP survival-promoting signaling pathway,inducing ROS production,antagonizing the VEGF signaling pathway,inhibiting angiogenesis,and even mediating apoptosis or cell death.Studies have shown that blocking the interaction between CD47 and TSP-1 can significantly alleviate graft damage caused by ischemia and reperfusion,ultimately improving the survival rate of grafts.In summary,CD47 demonstrates two distinct effects in transplantation.Firstly,it serves as a “self-marker” recognized by the body’s immune system.By activating SIRPα expressed on the surface of myeloid cells,it inhibits macrophage phagocytosis,dampens dendritic cell activation and antigen presentation,and suppresses the recruitment and activation of NK cells and T cells,thereby mitigating immune rejection mediated by myeloid cells.Secondly,during the transplantation process,the body’s TSP-1 levels increase due to ischemia-reperfusion injury and other factors.As an important ligand of CD47,TSP-1 activates CD47,transmitting a variety of survival inhibition signals to the cell interior,inducing apoptosis or cell death,and also hindering blood flow and angiogenesis.Ultimately,this exacerbates transplant rejection damage to cells and organ grafts.In response to the dual challenges posed by the CD47 molecule in xenograft,there is an urgent need for solutions.As a response,we have undertaken the modification of the CD47 protein.The objective is to preserve its anti-phagocytosis ability,while also integrating the benefits of TSP-1/CD47 signal blockade to enhance xenograft survival.This approach seeks to address the complexities associated with CD47’s role in transplantation and aims to optimize its functionality for improved outcomes.Method1.Design and construct stable expression cell lines of human and mouse CD47-Ig V mutants: that is,CRISPR/Cas 9 is used to knock out endogenous CD47 of Jurkat,EA.hy926 and A20 cells,construct CD47-Ig V and CD47-iso2(the most common isoform of CD47)lentiviral expression vectors,package lentiviruses and infect CD47 knock-out cell lines,and obtain stably expressed cell lines through flow sorting;flow detection of CD47-Ig V and CD47-iso2 expression.2.In order to ascertain whether CD47-Ig V transmits death signals,apoptosis induction experiments were conducted in vitro using Jurkat cell lines expressing human CD47-Ig V,CD47-iso2,and CD47 KO.The cells were treated with CD47 agonist antibodies,and subsequent analysis revealed the induction of apoptosis.This investigation aimed to determine the potential of CD47-Ig V in transmitting death signals.3.Through in vitro tube formation assay and Matrigel plug assay,the angiogenic capabilities of human CD47-Ig V,CD47-iso2,CD47 KO,and WT endothelial cell lines were assessed.The objective was to determine the impact of CD47-Ig V on angiogenesis.4.Through in vivo and in vitro phagocytosis experiments,the resistance of Jurkat cells expressing human CD47-Ig V,CD47-iso2,and CD47 KO,as well as A20 cells expressing mouse CD47-Ig V,CD47-iso2,and CD47 KO to macrophage phagocytosis was evaluated.The aim was to ascertain whether human/mouse CD47-Ig V can protect its expressing cells from macrophage phagocytosis.5.In the syngeneic stem cells transplantation experiment,the chimerism level and lineage distribution of donor-derived cells in two groups of hematopoietic stem cell transplanted mice with stable expression of CD47-Ig V and CD47-iso2 were compared.The objective was to evaluate the ability of the corresponding cells to engraft and differentiate in the recipient mice,and to determine whether the overexpression of CD47-Ig V affects the function of normal cells.Result1.The GPI structure was effectively linked to the CD47 extracellular Ig V domain to generate CD47 mutants.CD47-Ig V was able to be expressed at high levels on the surface of various cells,and its expression was comparable to that of CD47-iso2.Additionally,it was observed that CD47-Ig V mutants did not exhibit intracellular retention.2.CD47-Ig V was found to not transmit death signals induced by CD47-activated antibodies.Additionally,the expression of CD47-Ig V on endothelial cells did not inhibit the angiogenic ability of these cells.In fact,compared to the expression of wild-type CD47,the expression of CD47-Ig V was observed to improve the decline in angiogenesis ability caused by the aging of endothelial cells.Moreover,in a mouse in vivo angiogenesis model,cells with stable expression of CD47-Ig V continued to display superior angiogenic ability compared to cells with stable expression of wild-type CD47.3.CD47-Ig V was found to demonstrate a similar level of anti-phagocytosis ability as CD47-iso2 in both human and mouse cells.Furthermore,due to its inability to transmit death signals,tumor cells that overexpress the mutant exhibited stronger tumor-forming capabilities than CD47-iso2-expressing cells in in vivo experiments.4.In the syngeneic stem cells transplantation experiment,the expression of CD47-Ig V in donor hematopoietic stem cells was found to have no impact on the proportion and function of mature cells derived from donor differentiation in peripheral blood,tissue,and bone marrow.Furthermore,CD47-Ig V did not affect the proportion and function of donor-derived stem progenitor cells in the recipient bone marrow.Additionally,CD47 KO hematopoietic stem cells expressing m CD47-Ig V were observed to persist in recipient mice for an extended period,providing further evidence of CD47-Ig V’s ability to inhibit phagocytosis.ConclusionCD47-Ig V has demonstrated successful high expression on the cell membrane.Compared to wild-type CD47,CD47-Ig V does not transmit the death signal induced by CD47 agonist antibodies,thereby not inhibiting cell survival and function.Unlike the expression of endogenous CD47 and transgenic wild-type CD47 isoform II,the expression of CD47-Ig V in endothelial cells does not inhibit angiogenesis,allowing its expressing cells to exhibit angiogenesis similar to CD47 KO endothelial cells.The expression of CD47-Ig V across different species can protect the target cell from being engulfed by macrophages of the corresponding species.Since it does not transmit death signals to the cell,it offers better in vivo rejection protection than type II CD47.Consistent with the expression of wild-type CD47,CD47-Ig V expression does not affect the function of hematopoietic stem cells.The long-term survival of CD47 KO stem cells with CD47-Ig V transgenic expression provides additional evidence for CD47-Ig V inhibition of phagocytosis.Therefore,CD47-Ig V can effectively cultivate donor animals that can maximize xenograft rejection and produce low-immunogenic cell therapy products.