Genomics: Insight

End Stage Renal Disease (ESRD):

From 2000 to 2019, the incident count of newly diagnosed patients with ESRD (the final stage of Chronic Kidney Disease) has drastically increased from 94,466 patients in 2000 to 134,862 patients in 2019.1 African Americans are particularly vulnerable to ESRD. In 2019, African Americans had the highest prevalence for ESRD among all race/ethnicity groups at 6,437 ESRD patients per million population.¹

Importance of Transplantation and Transplantation Shortage:

Treatments of ESRD include kidney transplantation and dialysis. However, with an 18.6% mortality rate in 2020, dialysis has been proven to be less effective than transplantation, which had a 6.4% mortality rate in the same year.¹ Despite transplantation being the best option to treat ESRD, many patients are often unable to undergo a transplant due to a lack of kidney donations from either living or deceased donors. Between 2009 and 2020, the number of candidates waiting for a kidney transplant has never dropped below 100,000, and in 2020 alone, 4,879 candidates died while waiting for a kidney transplant.²

Between 2009 and 2020, the number of candidates waiting for a kidney transplant has never dropped below 100,000 and in 2020 alone, 4,879 candidates died while waiting for a kidney transplant.

Porcine Kidney Xenotransplantation:

Because the number of kidneys is the limiting factor in transplantation treatment, porcine to human xenotransplantation has been considered due to porcine organs’ similar anatomic size and pigs' reproductive efficiency. In 2021, researchers at New York University performed a porcine kidney xenotransplant on two brain-dead patients using a porcine kidney that was genetically modified to increase transplantation efficiency.³ In both patients, the estimated glomerular filtration rate (eGFR, a measurement of kidney filtration) increased after the transplant, and there were no signs of organ rejection.³ Porcine organs pose two significant risks to humans: hyperacute rejection, which is a severe immune response to grafted cells, and major viral infection from retroviruses endogenous to porcine genome.

Porcine Genes Targeted for Editing to Improve Transplantation Efficiency:

The GGTA1, CMAH, B4GALNT2, and SLA genes have all been identified as potential causes for hyper-acute rejection (HAR) due to the high immunoglobulin G (IgG) and immunoglobulin M (IgM) response. IgG and IgM are antibodies that make up 80% of all immunoglobulins in humans, making them particularly likely to generate a dangerous autoimmune response. Due to the risks that HAR poses, scientists have genetically engineered pigs to remove all 4 of these genes, creating 4xKO pigs.⁴

In addition to the risk of HAR, another major concern about porcine to human xenotransplantation comes in the form of Porcine Endogenous Retroviruses (PERVs). Retroviruses are a serious concern due to their association with diseases such as AIDS and various forms of cancer.5 Unlike many other zoonotic pathogens (diseases that can be spread from non-human animals to humans), PERVs genes could not be knocked out through early gene-editing techniques.⁶

However, the novel and efficient gene-editing technology, CRISPR/Cas-9, has been shown to effectively eliminate PERV genes.^{6, 7}

CRISPR/ Cas-9 PERV Gene Elimination:

In-vitro co-culturing of HEK-293 (human cell line) and PK-15 (porcine cell line) cells have been used as a model system to study xenotransplantation and specifically the susceptibility of human cells to infection during transplantation. Two independent studies have shown that HEK-293 cells that are co-cultured with PERV-inactivated PK-15 cells are significantly less likely to be infected with PERV than HEK-293 cells that are co-cultured with wild type PK-15 cells. The research report by Luhan Yang et al. indicates that co-cultures of human (HEK293) cells with porcine cells that have undergone CRISPR/Cas-9 inactivation of PERV pol (a gene involved in viral replication of PERV) decreases infection of human cells in comparison to human cells co-cultured with porcine cells that have fully functional pol genes.⁶ In two gene-expression-tracking qPCR, minimal to no PERV DNA was detected in the human cells for the CRISPR/Cas-9 edited PERV pol genes even with the DNA equivalent of 100 porcine cells. With the porcine cells containing intact PERV pol genes, PERV DNA was detected in human cells with the DNA equivalent of 1 porcine cell. Also, research by Marc Güell et al. showed that all of the major components of the PERV virus were present in HEK-293 cells that had been co-cultured with wild type PK-15 cells; by contrast, none of the major components of PERV were present in HEK-293 cells that had been co-cultured with CRISPR-Cas 9 PERV inactivated PK-15 cells.⁷ These findings indicate that, with the use of CRISPR/Cas-9 gene editing, scientists can successfully eliminate PERV infection of human cells to increase the efficacy of porcine to human xenotransplantation.

CRISPR/Cas-9 GGTA1, CMAH, B4GALNT2, and SLA Class I Gene Elimination:

IgG and IgM hyperacute rejection has been modeled through an in-vitro co-culture with porcine peripheral mononuclear cells (PBMC) and human serum. Multiple studies have shown that the CRISPR/Cas-9 inactivation of GGTA1, CMAH, B4GALNT2, and SLA Class I genes in porcine cells (4xKO pig) significantly reduces the IgG and IgM response in co-culture with the human serum relative to wild type porcine cells. In the paper by Konrad Fischer et al., there was an 18 fold reduction in IgG response and a 9 fold reduction in IgM response when the CRISPR/Cas-9 mediated 4xKO porcine cells are used relative to the wild type porcine cells.⁴ Independently, Gregory R. Martens et al. tested the 4xKO pig IgG and IgM responses using the same methods except they separated the GGTA1, CMAH, and B4GALNT2 (3xKO pig) from the SLA Class I Gene in testing.⁸ The 3xKO pig had a 100 fold reduction in IgG response and a 10 fold reduction in IgM response relative to the wild type pigs.⁸ The study also shows that 14 out 22 patients tested with the SLA Class I gene inactivated had a reduced IgG response and 11 out of 17 patients tested with the same inactivation had a reduced IgM response relative to the wild type pigs.⁸ These findings indicate that CRISPR/Cas-9 editing was effective in limiting the autoimmune response to the GGTA1, CMAH, B4GALNT2, and SLA Class I genes, eliminating one of the major threats caused by xenotransplantation.

CRISPR/Cas-9 editing was effective in limiting the autoimmune response to the GGTA1, CMAH, B4GALNT2, and SLA Class I genes, eliminating one of the major threats caused by xenotransplantation.

Conclusion:

CRISPR/Cas-9 gene-edited porcine kidneys could be the most effective treatment option for ESRD, preventing thousands of people from dying every year while on the waiting list. As a result of CRISPR/Cas-9 editing of the PERV genes and GGTA1, CMAH, B4GALNT2, and SLA Class I genes, both human cell PERV infection and IgG and IgM response to GGTA1, CMAH, B4GALNT2, and SLA Class I genes (two of the major barriers to porcine xenotransplantation) were severely limited. Promisingly, CRISPR/Cas9 editing has been used to great effect in porcine to human xenotransplantation trials, allowing porcine kidneys to function in human patients for 54 hours, although the kidneys likely would have functioned for longer if the trial was extended.³ The use of living beings purely for human gain warrants ethical consideration. The organ-donating pigs will likely be raised in lab-like conditions that are unable to provide for the animals’ biological and psychological needs.⁹ Despite this concern, with genetically modified porcine kidneys, the lifespan and quality of life for patients with ESRD can be significantly improved. Although the PERV, GGTA1, CMAH, B4GALNT2, and SLA Class I genes are the major pathogen risks associated to porcine to human xenotransplantation, gene-editing must be implemented to eliminate other pathogens in order to ensure the safety of xenotransplantation on a widespread level. Given the highly advanced technique of kidney transplantation and its effectiveness in improving and extending the lives of ESRD patients, the limiting factor should not be the number of available organs. To this end, porcine to human xenotransplantation is a promising candidate that ensures that this is not the case. While CRISPR/Cas-9 mediated porcine xenotransplantation might allow medical professionals to save thousands of lives every year, the unprecedented technology could be most impactful only as long as the scientific community is provided with enough resources to further research and eventually implement the revolutionary treatment.

References

1. United States Renal Data System. 2022 USRDS Annual Data Report: Epidemiology of kidney disease in the United States. National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2022. https://usrds-adr.niddk.nih.gov/2022/end-stage-renal-disease/1-incidence-prevalence-patient-characteristics-and-treatment-modalities
2. Lentine, K. L., et al. (2022). OPTN/SRTR 2020 Annual Data Report: Kidney. American Journal of Transplantation, 22(S2), 21-136.https://onlinelibrary.wiley.com/doi/10.1111/ajt.16982
3. Montgomery, R. A., et al. (2022). Results of Two Cases of Pig-to-Human Kidney Xenotransplantation. The New England Journal of Medicine, 1889-1898. https://www.nejm.org/doi/full/10.1056/NEJMoa2120238
4. Fischer, K., et al. (2019). Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2. Xenotransplantation, 27(1). https://doi.org/10.1111/xen.12560
5. Ganguly, P., Ph.D. (2023, February 1). Retrovirus. National Human Genome Research Institute. https://www.genome.gov/genetics-glossary/Retrovirus#:~:text=Upon%20infection%20with%20a%20retrovirus,and%20some%20forms%20of%20cancer
6. Yang, L., et al. (2015). Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science, 350(6246), 1101-1104. https://www.science.org/doi/10.1126/science.aad1191?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub%20%200pubmed
7. Güell M, et al. PERV inactivation is necessary to guarantee absence of pig-to-patient PERVs transmission in xenotransplantation. Xenotransplantation. 2017;24:e12366. https://doi.org/10.1111/xen.12366
8. Martens, Gregory R. MD; et al. Humoral Reactivity of Renal Transplant-Waitlisted Patients to Cells From GGTA1/CMAH/B4GalNT2, and SLA Class I Knockout Pigs. Transplantation 101(4):p e86-e92, April 2017. | DOI: 10.1097/TP.0000000000001646. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7228580/
9. Rollin, B. E. (2020). Ethical and Societal Issues Occasioned by Xenotransplantation. Animals. https://doi.org/10.3390/ani10091695