Genomics: Insight

Xenotransplantation, the process of transplanting tissues or organs of one species to another species, has recently received much attention on behalf of the University of Maryland Medicine’s latest surgery on porcine cardiac transplantation. On January 7th, 2022, David Bennett, a 57-year-old with terminal heart disease, underwent surgery to replace his heart with the heart of a genetically modified pig. Bennett’s heart complications and medical history deemed a porcine heart the only available choice. Although he passed away on March 9th, 2022, he survived for two additional months with the transplanted porcine heart ^[1]. During that period, he spent time with his family, watched the Super Bowl, and participated in other enjoyable activities.

Throughout history, xenotransplantation has woven a distinct although obscured path. Some of the field’s notable experiments include the human use of frog skin grafts during the 19th century and Keith Reemtsma’s transplantations of chimpanzee kidneys into patients in 1964, which led to one female patient living for an additional nine months after the surgery ^[2]. In the latest decades, xenotransplantation has been studied with the purpose of animal organs – specifically livers and hearts – being replacements for humans suffering from chronic organ diseases and organ failure. Currently, 106,275 people are on the national transplant waiting list, with 17 people dying every day due to the disparity between the organs needed and the organs supplied ^[3]. Pigs are often portrayed as the model animals for xenotransplantation due to their large litter, physiological similarities with humans, especially the heart, and the short time it takes to mature into adult animals ^[4]. However, despite their similarities, there are significant immune cells and pathways leading to HAR that can cause severe rejections that eventually lead to the recipient’s death.

Within the studies conducted on xenotransplantation, three main types of rejection have been identified: hyperacute xenograft rejection (HAR), acute humoral xenograft rejection (AHXR), and acute cellular rejection (ACR). HAR occurs between minutes to a couple of days after the surgery, when the existing non-porcine antibodies bind to porcine antigens and activate proteins that lead to the destruction and failure of the tissue and can arguably be the most violent and severe type of rejection. AHXR takes place several weeks or months after the transplantation and is caused by immune responses and medical reactions such as inflammation. Lastly, ACR happens several months up to a year after surgery and is influenced by the activities of natural killer (NK) cells, macrophages, T cells, and more ^[4].

However, in recent years, CRISPR-Cas9 has shown potential in overcoming problems leading to cardiac rejection, specifically by modifying the genes related to the immunological cells and responses causing HAR with its straightforward yet precise way of refashioning organism genomes ^[5]. For instance, the porcine heart that was transplanted into Bennett had three genes tied to porcine cardiac rejection and one gene relating to excessive tissue growth removed to lower the chance of organ failure and rejection. In addition, six additional human genes regulating immune acceptance were added ^[6]. Although the identification of the genes used in Bennett’s case were not released to the public, multiple studies on the role certain genes play in the acceptance of xenografts have been performed. One of the significant genes in interest was the porcine gene GGTA1.

GGTA1 encodes for Galactose-alpha-1,3-galactose (Gal alpha-1,3-Gal). The latter is a protein that creates the alpha-Gal sugar epitope, the part of the antigen where the antibody attaches itself. One of its most important characteristics is that this specific gene is found in pigs but not humans. Therefore, natural antibodies found in humans can be produced as a reaction to Gal alpha-1,3-Gal, triggering HAR by forming a membrane attack complex, a complex of proteins that attacks pathogens. Eventually, graft destruction and failure take place. As noted in previous research, the absence of Gal alpha-1,3-Gal may decrease the possibility of HAR and increases the chances of survival for cardiac porcine xenotransplantation ^[7][8].

Masahiro Sato led the first recorded study to prevent the synthesis of Gal alpha-1,3-Gal in 2014. They first cultivated a generation of GGTA1 biallelic knockout (KO) cells. Then, the CRISPR-Cas9 components were inserted to knockout the GGTA1 gene in porcine embryonic cells. The cells were incubated with IB4 (a protein that binds to alpha-Gal residues), conjugating with saporin, a protein that triggers cell death, to verify the absence of Gal alpha-1,3-Gal. The cells that were still alive after IB4SAP (IB4 conjugated with saporin) treatment were the cells that did not contain alpha-Gal epitopes. The study concluded with successful Gal alpha-1,3-Gal epitope negative colonies predicting the potential CRISPR-Cas9 holds within the field of xenotransplantation ^[9]. Another study conducted by Sato in 2015 concluded that Gal alpha-1,3-Gal expression could possibly be regulated through CRISPR-Cas9 when his team conducted a knock-in of the GGTA1 through targeted homologous recombination, a controlled process of homologous strands crossing over ^[10][11].

CRISPR-Cas9 is not the only gene-editing system used to produce tissues with disruptions in GGTA1 expression. Cheng et al. (2016) utilized transcription activator-like effector nucleases (TALENs) to establish GGTA1-knockout cell lines for somatic cell nuclear transfer, a process to create viable embryos from cells by transferring a somatic cell’s nucleus into an egg cell. TALENs are artificial restriction enzymes that consist of several components: transcription activator-like effectors (TALEs) that bind to specific DNA sequences attached to a modified FokI restriction enzyme ^[12]. The FokI is a part that only becomes active when it is dimerized with another part. On the corresponding strand, another FokI will become attached with its TALEs. When the two FokIs undergo dimerization, a double-stranded break in DNA will be created, and in this case, eventually leads to GGTA1 knockout. This resulted in multiple GGTA1-knockout piglets, with one piglet having no Gal alpha-1,3-Gal epitopes in some of its organs; therefore, if that piglet was used for xenotransplantation for one of its Gal alpha-1,3-Gal absent organs, the possibility of the recipient experiencing an HAR would significantly decrease ^[7].

Numerous other genes influence the possibility of an HAR occurrence, and a notable amount of these have the potential to be genetically edited to increase chances of xenotransplantation survival, such as the Human GLA+ and Human H-transferase, both of which can be tied in with Gal alpha-1,3-Gal expression regulation. Modifying the Human GLA+ gene would reduce the interaction between the antibodies and antigens involved with Gal alpha-1,3-Gal expression, thus lowering the chance of an immune rejection. Human H-transferase would generally reduce the Gal alpha-1,3-Gal expression in human cells, decreasing the possibility of an adverse reaction to transplantation ^[8]. With the use of CRISPR-Cas9 for these genes, the gene-editing aspect of the xenotransplantation field will yield more accurate results.

GGTA1 is a significant gene that bears close observation and research as removing GGTA1 expression alleviates the looming concern of porcine organ rejection. The gene itself is significant, but the success of CRISPR-Cas9 systems in knockout GGTA1 also bears importance as it signifies a new era of progress, innovation, and acceleration and sheds light on the possibility of future clinical trials official experiments. Continuation of gene-editing research in xenotransplantation may eventually lead to medical applications to combat the tragic organ shortage.

Related Links

1. University of Maryland Medical Center. (2022, March 9). In Memoriam: David Bennett [Press release]. https://www.umms.org/ummc/news/2022/in-memoriam-david-bennett
2. Cooper, D. K.C., Ekser, B., & Tector, J. (2015). A brief history of clinical xenotransplantation. International Journal of Surgery, 23(B), 205-210. https://doi.org/10.1016/j.ijsu.2015.06.060
3. Health Resources & Services Administration. (2022, March). Organ Donation Statistics. OrganDonor.gov. Retrieved March 27, 2022, from https://www.organdonor.gov/learn/organ-donation-statistics
4. Lu, T., Yang, B., Wang, R., & Qin, C. (2020). Xenotransplantation: Current Status in Preclinical Research. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.03060
5. Bhattacharjee, G., Gohil, N., Khambhati, K., Mani, I., Maurya, R., Karapurkar, J. K., Gohil, J., Chu, D.-T., Vu-Thi, H., Alzahrani, K. J., Show, P.-L., Rawal, R. M., Ramakrishna, S., & Singh, V. (2022). Current approaches in CRISPR-Cas9 mediated gene editing for biomedical and therapeutic applications. Journal of Controlled Release, 343, 703-723. https://doi.org/10.1016/j.jconrel.2022.02.005
6. Hassan, C., & Dillinger, K. (2022, March 9). Patient who received genetically modified pig heart in groundbreaking transplant surgery dies. CNN. Retrieved March 27, 2022, from https://www.cnn.com/2022/03/09/health/pig-heart-transplant-death/index.html
7. Cheng, W., Zhao, H., Yu, H., Xin, J., Wang, J., Zeng, L., Yuan, Z., Qing, Y., Li, H., Jia, B., Yang, C., Shen, Y., Zhao, L., Pan, W., Zhao, H.-Y., Wang, W., & Wei, H.-J. (2016). Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer. Reproductive Biology and Endocrinology, 14. https://doi.org/10.1186/s12958-016-0212-7
8. Kararoudi, M. N., Hejazi, S. S., Elmas, E., Hellström, M., Kararoudi, M. N., Padma, A. M., Lee, D., & Dolatshad, H. (2018). Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Gene Editing Technique in Xenotransplantation. Frontiers in Immunology, 9. https://doi.org/10.3389/fimmu.2018.01711
9. Sato, M., Miyoshi, K., Nagao, Y., Nishi, Y., Ohtsuka, M., Nakamura, S., Sakurai, T., & Watanabe, S. (2014). The combinational use of CRISPR/Cas9-based gene editing and targeted toxin technology enables efficient biallelic knockout of the α-1,3-galactosyltransferase gene in porcine embryonic fibroblasts. Xenotransplantation, 21(3), 291-300. https://doi.org/10.1111/xen.12089
10. Sato, M., Kagoshima, A., Saitoh, I., Inada, E., Miyoshi, K., Ohtsuka, M., Nakamura, S., Sakurai, T., & Watanabe, S. (2015). Generation of α-1,3-Galactosyltransferase-Deficient Porcine Embryonic Fibroblasts by CRISPR/Cas9-Mediated Knock-in of a Small Mutated Sequence and a Targeted Toxin-Based Selection System. Reproduction in Domestic Animals, 50(5), 872-880. https://doi.org/10.1111/rda.12565
11. Thomas, J. (Ed.). (2017, August 3). How Does Genome Editing Work? National Human Genome Research Institute. Retrieved March 27, 2022, from https://www.genome.gov/about-genomics/policy-issues/Genome-Editing/How-genome-editing-works
12. Becker, S., & Boch, J. (2021). TALE and TALEN genome editing technologies. Gene and Genome Editing, 2. https://doi.org/10.1016/j.ggedit.2021.100007