Transcending Genetic Demarcations

Harnessing CRISPR-Cas9 for Xenotransplantation

Traditionally held in a theoretical aspect, xenotransplantation’s clinical feasibility has gained credence with recent notable breakthroughs attributed to CRISPR-Cas9. This process of grafting animal organs, tissues and cells between different species carries a revolutionary weight of salience regarding the shortage of organs around the world. Genetic engineering tool CRISPR-Cas9 first evolved as a bacterial defense mechanism and has been technologically advanced to allow precision modifications of DNA. Relevant to the application of porcine models in transplantation, advancements driven by CRISPR-Cas9 technology have transformed the area of xenotransplantation. In particular, the creation of 10-GE swine, successful organ transplants, CRISPR-based PERV elimination and knock in/knock outs. Additional progressions in cloning and immunology have further encouraged the evolution of xenotransplantation. Namely, tolerance strategies relating to immunity, insertion of human regulatory proteins, somatic cell reprogramming, T-cell depletion, and the combination of CRISPR-Cas9 with SCNT. While these milestones have brought this evolving discipline closer to becoming an empirically supported reality, regulatory barriers, rejection, long-term viability, ethical and safety concerns still present challenges in this field.

CRISPR-Cas9 exemplifies a preeminent foundation for iterative augmentation, allowing researchers to enhance compatibility of porcine organs with the human genome. Since the early 2000s, scientists have made efforts to eradicate the a-gal sugar which catalyzes rejection of xenotransplants in humans. Preclinical studies performed in 2013, which used older models of gene editing (Zinc finger nucleases, transcription activator-like effector nucleases, homologous recombination) displayed successful mitigation of hyperacute rejection with the use of alpha-gal knockout pigs. The most prominent xenogeneic antigens, 3-galactosyltransferase (GGTA1), B4GALNT2 and CMAH (Neu5Gc) are knocked out using CRISPR-Cas9. Removal of these antigens is critical for acceptance of porcine organs when considering that humans produce antibodies which contrast certain glycans, genes, and enzymes leading to hyperacute rejection. One major setback to xenotransplantation is porcine endogenous retroviruses (PERVs). In 2016, researchers used CRISPR-Cas9 to effectively define and eliminate proviral DNA within the pig genome, inactivating PERVs. Between 2015-2017, CRISPR-Cas9 was used to prune sixty-two copies of PERV genes, serving as a massive leap in the safety of transplants and prevention of viruses.

In addition to the knock-out of incompatible genes, CRISPR-Cas9 also knocks-in those which promote organ longevity, immunity, size and compatibility. Prior to transplantation, pigs are genetically enhanced to become more “humanized” with intentions of decreasing rejection and increasing congruence. Knocking in human proteins CD46, CD55 and CD59 inhibits typical immune activation and synthesis of the complement membrane attack complex. Integration of PD-1 and PD-L1 genes help to enrich immune tolerance. Transfection of insulin-like growth factor (IGF-1), fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), PROCR as well as genes that regulate organogenesis work to regulate organ size and growth. Knocking in genes which specifically correlate to the organ being transplanted, such as cytochrome P450 with a liver transplant or MYH7, ACTC1, or SCN5A with a heart transplant, fosters an optimal environment for survival capacity.

Additional technological advances that have enabled development of xenotransplantation include SCNT, NMP, and HLA-MHC engineering. Somatic Cell Nuclear Transfer (SCNT) when used in combination with CRISPR-Cas9 implements proper genetic modifications and allows for multiplex editing. Normothermic Machine Perfusion (NMP) is a technology which assists in the preservation of organs by providing a replica of normal bodily conditions and regulation of blood flow. Researchers also use NMP to test organs prior to transplantation. T-cell mediated rejection is prominently caused by Human Leukocyte Antigens (HLA) and the Major Histocompatibility Complex (MHC). Within HLA-MHC engineering CRISPR-Cas9 is used to knock out pig MHC I genes (SLA-1, SLA-2 and SLA-3) and pig MHC II genes (SLA-DR, SLA-DQ and SLA-DP) and knock-in HLA-E and HLA-G. These edits encourage the suppression of CD8+ T-cells and reduces the activation of CD4+ T-cells.

Xenotransplantation’s most substantial hinderance is the human immune system. Hyperacute rejection and acute cellular rejection are ubiquitous in foreign transplant practices. To neutralize the impacts of this challenge, CRISPR-Cas9 prime and base editing mechanisms are continuously evolving to minimize and potentially eliminate rejections in the future. Immunosuppressive drugs and immune cell reprogramming are correspondingly being refined to combat rejection issues. The variegated association amongst bone morphogenetic proteins (BMPs) and bone marrow allow for a more natural enhancement of immune tolerance, and potentially lessen the requirement of long-term or lifelong immunosuppressive therapies. Xenosis, the transference of bacteria, viruses or microorganisms into humans from animal donors, has proven to be a challenge. Though CRISPR-Cas9 has effectively inhibited PERVs, zoonotic diseases and other viruses are still risk factors. Scientists have been able to detect viruses early on through continual monitoring and tests, however there is a dire need of additional research concerning this issue. Xenotransplants have occasionally shown to have short-term survival rates and poor functionality. This challenge is accredited to blood flow, perfusion, and immune adaptation.  Complement regulation and NMP have helped alleviate this challenge, allowing ex vivo perfusion and tests on organs.

Regulations surrounding xenotransplantation vary across the globe, causing challenges with therapeutic integration. Many agencies have yet to lay out clear guidelines for researchers to effectively pursue this field. However, collaborations between academic institutions, companies and regulatory bodies have pushed advancements along. Scientists understand the importance of orchestrating stringently modulated trials, particularly in this current juncture of research and development. Preclinical trials have been and continue to be diligently monitored with scientifically adept researchers. Exercising prophylactic measures when moving forward into clinical studies is vital in order to correctly navigate this field. Despite sustainable outcomes and constructive evolution, many individuals question xenotransplantation’s ethical vicinity and economic pragmatism. Animal rights, human-animal chimeras, recrudescence of social stratification and high development costs are the most prominent skepticisms. Encouraging intellectual openness by use of public discourse, informed consent, equitable access and provision of information for individuals to appropriately consider the long term potentials of xenotransplantation could promote credibility. When considering future potentialities of this field, the cost and use of dialysis would lower and the overarching concern of organ shortages could be mitigated, validating startup costs and demonstrating long-term worth.

The xenotransplantation industry houses a wide array of academic researchers, startups and established companies. Academia is rich in advanced scientists at New York University, Harvard, Stanford, Johns Hopkins, University of Edinburgh and other institutions. Notable startups include eGenesis, Xenocell, Xenotherapuetics, and Ouroboros Technologies. The competitive milieu of xenotransplantation is composed of sophisticated companies such as Revivicor (subsidiary of United Therapeutics), CRISPR Therapeutics, Sangamo Therapeutics, Vertex Pharmaceuticals, Novartis, and Regeneron Pharmaceuticals. Therefore, innovative  rivalry amongst these key players promotes the expansion of research initiatives.

Dr. Robert Montgomery, director of the Transplant Institute at NYU Langone, has fostered the progression of genetically modified porcine models and successfully performed a pig-to-human kidney transplant in 2021. He has also converged efforts with Revivicor on genetic modifications and analyzed ethical quandaries encapsulating xenotransplantation. At Stanford University, Dr. Mark Shlomchik and Dr. David Sachs have engaged in preclinical trials, focused on breaking down immunological barriers and are accredited with the discovery of tolerogenic dendritic cells. Through manipulation of checkpoint inhibitors they have promoted immune tolerance while steering away from the need for long-term immune suppression. At the Roslin Institute (University of Edinburgh), Prof. David Hume has been a pioneer in the production of PERV-free and humanized MHC pigs. Dr. George Church, professor of genetics at Harvard Medical School, has had a powerful influence in defining and displaying the use of CRISPR-Cas9. In 2015 he co-founded eGenesis, institutionalizing the base infrastructure and underlying framework of genetic modification. Dr. Church also assisted the creation of transgenic pigs in 2017, performed trials which transplanted pig hearts into non-human primates in 2020 and continues to focus on the preliminary parameters of specified genetic modifications in pig hearts, kidneys and lungs.  He has also collaborated with Revivicor to further viral safety and amplify humanization of pig organs. Revivicor has driven the evolution of xenotransplantation through collaborations with leading academic institutions and premier medical facilities. Within these cross-institutional collaborations and through their own research, Revivicor has contributed to the effective performance of preclinical studies. After years of research, they have achieved a seminal accomplishment with their most recent FDA approval for clinical trials in February, 2025. This is a fundamental milestone for Revivicor, revolutionizing the progression of scientific exploration and now making it the most influential global company in association with xenotransplantation.

While CRISPR-Cas9 has allowed for major transformation of the xenotransplantation field, it has also assisted other intellectual territories of genetic progression and enlarges the horizon for subsequent possibilities. One key prospect, 3D bioprinting - bolstered by CRISPR-Cas9, has elicited comparable prominence to xenotransplantation maturation. The use of intercalated cells, growth factors and biomaterials are used to print three-dimensional organs. If they can be printed in a state of complete viability, the exigency of xenotransplantation would diminish. Other novel elevations such as OoC technology, innovations in induced pluripotent stem cells (iPSCs), artificial organs and biohybrids all imply an antidote of competition towards xenotransplantation.

The harmonization of CRISPR-Cas9 into the orchestrative challenge of xenotransplantation has provided monumental advances and an upbeat future of developmental composition. This technology has pushed immunological integrations and functional potentials by means of precision modifications, knock in/knock outs, and control of viruses. A molecular nexus fused of propelling research, genetic modifications, honorable ethics, tolerance induction, technological and regulatory advances is constituted by virtue of genetic engineering technology, CRISPR-Cas9. Despite anomalies, the cellular adaptability of xenotransplantation emits optimistic prognostic trends for future research and a paradigm shift in the global organ shortage.

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