From genome editing to blastocyst complementation: Anew horizon in heart transplantation?

Abstract

Central MessageRapid advances in stem cell medicine and genome editing technology may result in a paradigm shift in heart transplantation.See Commentary on page XXX. Rapid advances in stem cell medicine and genome editing technology may result in a paradigm shift in heart transplantation. See Commentary on page XXX. Heart transplantation remains the definitive therapy for end-stage heart failure; however, widespread access to transplantation is limited by donor shortages.1Dharmavaram N. Hess T. Jaeger H. Smith J. Hermsen J. Murray D. National trends in heart donor usage rates: are we efficiently transplanting more hearts?.J Am Heart Assoc. 2021; 10: e019655Crossref PubMed Scopus (1) Google Scholar In addition to the increasing burden of heart failure secondary to dietary and lifestyle factors,2Virani S.S. Alonso A. Benjamin E.J. Bittencourt M.S. Callaway C.W. Carson A.P. et al.Heart disease and stroke statistics 2020 update: a report from the American Heart Association.Circulation. 2020; 141: e139-e596Crossref PubMed Scopus (2393) Google Scholar there is a growing population of patients with congenital heart disease, including those with prior univentricular repair.3Schilling C. Dalziel K. Nunn R. Du Plessis K Shi W.Y. Celermajer D et al.The Fontan epidemic: population projections from the Australia and New Zealand Fontan Registry.Int J Cardiol. 2016; 219: 14-19Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar As such, alternative strategies for treatment of end-stage heart failure are required. Recent breakthroughs in stem cell medicine and genome editing have led to unprecedented research activity in the field of myocardial regeneration.4Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (17471) Google Scholar,5Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7791) Google Scholar Generating specialized therapeutic cells, tissue grafts, and even entire organs from stem cells appears to be within our grasp. As such, we are at the cutting edge of what previously seemed impossible, and we will be pushing into unchartered territory as we seek pioneering innovations. The field of stem cell medicine has been revolutionized by the discovery of induced pluripotent stem cells (iPSCs). In 1962, John B. Gurdon6Gurdon J.B. The developmental capacity of nuclei taken from intestinal epithelium cells of feeding tadpoles.J Embryol Exp Morphol. 1962; 10: 622-640PubMed Google Scholar cloned a functional tadpole from the nucleus of a specialized intestinal cell of a frog, demonstrating that the nucleus of a mature cell could be returned to a pluripotent state. In 2006, Yamanaka and Takahashi4Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (17471) Google Scholar discovered 4 transcription factors that can reprogram mature cells into pluripotent stem cells. The resulting iPSCs can be generated from human cells and are capable of differentiating into cell types of all 3 germ layers.7Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K et al.Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (13869) Google Scholar,8Yu J. Vodyanik M.A. Smuga-Otto K. Antosiewicz-Bourget J. Frane J.L. Tian S. et al.Induced pluripotent stem cell lines derived from human somatic cells.Science. 2007; 318: 1917-1920Crossref PubMed Scopus (7760) Google Scholar In 2012, Gurdon and Yamanaka were jointly awarded The Nobel Prize in Physiology or Medicine. Despite their enormous potential in the field of regenerative medicine, numerous obstacles need to be addressed before iPSCs can be widely utilized in clinical practice. In particular, the potential for tumorgenicity, immunogenicity, and the integration of transplanted cells are of concern.9Yamanaka S. Pluripotent stem cell-based cell therapy-promise and challenges.Cell Stem Cell. 2020; 27: 523-531Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar Nonetheless, Yamanaka's discovery laid the foundation from which a new frontier in transplantation can be explored. In combination with the progress in stem cell research, recent advances in the field of genome editing may have significant implications for transplantation. Namely, the discovery of clustered regularly interspaced short palindromic repeats (CRISPR), and CRISPR-associated 9 (Cas9) protein, has transformed the study of molecular life sciences. Derived from ancient bacterial immune pathways, CRISPR-Cas9 technology functions as a form of a molecular machinery that allows editing of virtually any genome sequence.5Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7791) Google Scholar For their work in discovering CRISPR-Cas9 as a tool for genome editing, Jennifer A. Doudna and Emmanuelle Charpentier were jointly awarded the 2020 Nobel Prize in Chemistry. Although CRISPR-Cas9 is a hugely promising technology, several challenges still face its widespread clinical applications. Most importantly, unintended on-target genome damage, as well as off-target effects, risk introducing pathogenic mutations into edited cells.10Kosicki M. Tomberg K. Bradley A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.Nat Biotechnol. 2018; 36: 765-771Crossref PubMed Scopus (45) Google Scholar,11Saha K. Sontheimer E.J. Brooks P.J. Dwinell M.R. Gersbach C.A. Liu D.R. et al.The NIH somatic cell genome editing program.Nature. 2021; 592: 195-204Crossref PubMed Scopus (14) Google Scholar Such mutations could become neoplastic with time, particularly in iPSCs, given their long replicative lifespan. As such, comprehensive methods for screening and detecting genome damage are mandatory before patient transplantation of edited cells and their derived products.10Kosicki M. Tomberg K. Bradley A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.Nat Biotechnol. 2018; 36: 765-771Crossref PubMed Scopus (45) Google Scholar,11Saha K. Sontheimer E.J. Brooks P.J. Dwinell M.R. Gersbach C.A. Liu D.R. et al.The NIH somatic cell genome editing program.Nature. 2021; 592: 195-204Crossref PubMed Scopus (14) Google Scholar Immune-mediated rejection is a critical obstacle in the widespread use of iPSCs and the potential grafts that may be derived from them.9Yamanaka S. Pluripotent stem cell-based cell therapy-promise and challenges.Cell Stem Cell. 2020; 27: 523-531Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar A source of donor iPSCs with no (autologous) or reduced (universal donor-derived or genome-edited) immunogenicity is the crucial first step for bioengineered organs in vitro and in vivo. iPSCs created from a patient's own cells provide the incredible possibility of autologous graft and organ transplantation. Despite initial controversies, it is now established that autologous iPSCs do provide the opportunity to develop rejection-free cell therapies.12Deuse T. Hu X. Agbor-Enoh S. Koch M. Spitzer M. Gravina A. et al.De novo mutations in mitochondrial DNA of iPSCs produce immunogenic neoepitopes in mice and humans.Nat Biotechnol. 2019; 37: 1137-1144Crossref PubMed Scopus (35) Google Scholar, 13Zhao T. Zhang Z.-N. Rong Z. Xu Y. Immunogenicity of induced pluripotent stem cells.Nature. 2011; 474: 212-215Crossref PubMed Scopus (1066) Google Scholar, 14Araki R. Uda M. Hoki Y. Sunayama M. Nakamura M. Ando S. et al.Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.Nature. 2013; 494: 100-104Crossref PubMed Scopus (360) Google Scholar, 15Mandai M. Watanabe A. Kurimoto Y. Hirami Y. Morinaga C. Daimon T. et al.Autologous induced stem-cell–derived retinal cells for macular degeneration.N Engl J Med. 2017; 376: 1038-1046Crossref PubMed Scopus (665) Google Scholar However, the cost per individual patient and the time required to generate sufficient cell quantities or develop grafts for transplantation is currently seen as a prohibitive factor in the utilization of autologous iPSCs.16Chakradhar S. An eye to the future: researchers debate best path for stem cell–derived therapies.Nat Med. 2016; 22: 116-119Crossref PubMed Scopus (30) Google Scholar,17Madrid M. Sumen C. Aivio S. Saklayen N. Autologous induced pluripotent stem cell–based cell therapies: promise, progress, and challenges.Curr Protoc. 2021; 1: e88Crossref PubMed Scopus (3) Google Scholar This is particularly problematic for time-sensitive clinical indications, such as ischemic cardiomyopathy. However, in conditions where the need for transplantation may be foreshadowed several years in advance, such as hypoplastic left heart syndrome, autologous iPSCs offer the prospect of tissue grafts or entire heart transplantation, without the need for lifelong immunosuppression. In hematopoietic stem cell and solid organ transplantation, immune-mediated rejection is mitigated by matching of donor and recipient human leukocyte antigen (HLA) haplotypes. However, given the variety of HLA haplotypes, the logistics required to prepare the necessary quantity of iPSC lines is currently not practical. Alternatively, it has been proposed that iPSC lines derived from donors who are homozygote for common HLA types could cover a substantial proportion of the population. Such an approach would still require significant logistical hurdles to be overcome and would likely be dependent on large-scale collaboration between existing biobanks. For instance, it has been estimated that to establish a biobank of iPSCs that would cover 90% of the Japanese population, 140 unique HLA-homozygous donors would be needed, which would require typing of ∼160,000 individuals.18Okita K. Matsumura Y. Sato Y. Okada A. Morizane A. Okamoto S. et al.A more efficient method to generate integration-free human iPS cells.Nat Methods. 2011; 8: 409-412Crossref PubMed Scopus (1227) Google Scholar For societies with a broader range of ethnicities, this number would increase.19Gourraud P.A. Gilson L. Girard M. Peschanski M. The role of human leukocyte antigen matching in the development of multiethnic "haplobank" of induced pluripotent stem cell lines.Stem Cells. 2012; 30: 180-186Crossref PubMed Scopus (100) Google Scholar,20Taylor Craig J. Peacock S. Chaudhry Afzal N. Bradley J.A. Bolton Eleanor M. Generating an iPSC Bank for HLA-matched tissue transplantation based on known donor and recipient HLA types.Cell Stem Cell. 2012; 11: 147-152Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar To supplement biobanking of HLA-homozygous donors, a recent study utilized genome-editing technology to develop pseudohomozygous iPSCs by HLA allele-specific editing of HLA heterozygote donors.21Xu H. Wang B. Ono M. Kagita A. Fujii K. Sasakawa N. et al.Targeted Disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility.Cell Stem Cell. 2019; 24: 566-578.e7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Although a previous primate study demonstrated a reduced immune response to HLA-matched allogenic iPSC-cardiomyocytes, immunosuppression is still likely to be required, albeit for a shorter duration and at a lower dose.22Kawamura T. Miyagawa S. Fukushima S. Maeda A. Kashiyama N Kawamura A. et al.Cardiomyocytes derived from MHC-homozygous induced pluripotent stem cells exhibit reduced allogeneic immunogenicity in MHC-matched non-human primates.Stem Cell Rep. 2016; 6: 312-320Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar This is in part due to the wide variety of minor histocompatibility antigens that are expressed in the cleft of major histocompatibility complex (MHC) antigens, and differ between donor and recipient, even in the case of HLA matching.23Trounson A. Boyd N.R. Boyd R.L. Toward a universal solution: editing compatibility into pluripotent stem cells.Cell Stem Cell. 2019; 24: 508-510Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar Furthermore, genome-editing technology can be used to delete the classical class Ⅰ HLA (HLA-A, HLA-B, and HLA-C), either by direct exon targeting or by interfering with the β2-microglobulin gene (β2-microglobulin forms a heterodimer with class Ⅰ HLA and is necessary for class-Ⅰ HLA presentation on the cell surface) (Figure 1, A). With deletion of classical class Ⅰ HLA, the iPSC may avoid cytotoxic T-cells. However, with complete absence of class Ⅰ HLA the iPSC will be targeted by natural killer (NK) cells, through the missing self-response. Moreover, deletion of all classical class-Ⅰ HLA renders a cell incapable of antigen presentation and risks uncontrolled proliferation of cells that are tumorogenic or infected by pathogens. Rather, targeted deletion of HLA-A and HLA-B (Figure 1, B), with retention of a single HLA-C allele, can create iPSCs that reduce both NK cell and cytotoxic T-cell destruction, because HLA-C presentation can also inhibit NK cell activity.21Xu H. Wang B. Ono M. Kagita A. Fujii K. Sasakawa N. et al.Targeted Disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility.Cell Stem Cell. 2019; 24: 566-578.e7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Furthermore, HLA-C retention provides some capacity for antigen presentation, reducing the risks related to tumorigenesis and infection.21Xu H. Wang B. Ono M. Kagita A. Fujii K. Sasakawa N. et al.Targeted Disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility.Cell Stem Cell. 2019; 24: 566-578.e7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Of note, given the relative lack of diversity in the HLA-C haplotype, it is estimated that only 12 donors with various HLA-C haplotypes would be required to provide cells that could create iPSCs for most of the world's population.21Xu H. Wang B. Ono M. Kagita A. Fujii K. Sasakawa N. et al.Targeted Disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility.Cell Stem Cell. 2019; 24: 566-578.e7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Moreover, to overcome NK cell destruction, genome editing can overexpress a single-chain HLA-E/β2-microglobulin fusion, in which the nonclassical HLA-E can inhibit NK cell activity to a degree.24Gornalusse G.G. Hirata R.K. Funk S.E. Riolobos L. Lopes V.S. Manske G. et al.HLA-E-expressing pluripotent stem cells escape allogeneic responses and lysis by NK cells.Nat Biotechnol. 2017; 35: 765-772Crossref PubMed Scopus (218) Google Scholar In addition, the class-II MHC transactivator gene can be disrupted, depleting class-II MHC and, thus, avoiding HLA-DR-activated helper T-cell toxicity.21Xu H. Wang B. Ono M. Kagita A. Fujii K. Sasakawa N. et al.Targeted Disruption of HLA genes via CRISPR-Cas9 generates iPSCs with enhanced immune compatibility.Cell Stem Cell. 2019; 24: 566-578.e7Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar Immune cloaking via genome editing, to induce expression of immune checkpoints molecules, is another strategy to inhibit immune-mediated rejection of allogenic iPSCs.25Rong Z. Wang M. Hu Z. 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