Towards Xenografts in Clinical Transplantation: Multiplexed Negative Selection of Porcine Endogenous Retroviruses with CRISPR-Cas9

Clinical organ transplantation is now a large medical enterprise, with more than 29,000 organ transplants performed in 2014 in the United States alone (https://www.unos.org/data/transplant-trends/#transplants_by_organ_type+year+2014). Nevertheless, the number of organ donors is insufficient to meet the demand for new organs. For example, in the U.S. during 2014, there were 17,104 kidney transplants but 101,035 individuals on the waiting list for such transplants. Therefore, a recent study in Science (Yang et al., 2015) offers an important proof of principle for a necessary but not necessarily sufficient step on the path to safely using pig organs to substitute for failing human organs.

George Church and colleagues recognized that a major (though not the only) impediment to tranplanting organs from pigs to humans, which is a conceivable means of substantially improving the currently inadequate supply of organs for clinical transplantation, is the fear of pig-to-human transmission of retroviruses encoded in multiple sites within the pig genome. They therefore designed a scheme to perform gene editing using a bacterial system to inactivate the polymerase (i.e., reverse transcriptase) gene for all 62 known loci encoding porcine endogenous retroviruses (PERVs) in the genome of a porcine kidney epithelial cell line (PK15). The PERV polymerase gene is critical for endogenous retrovirus proliferation and infectious transmission. buy premarin 0.625mg

The genome editing system employed by Yang et al. is called: clustered regularly interspaced short palindromic repeats (CRISPR)-(CRISPR-associated 9) Cas9 (reviewed in Doudna and Charpentier, Science 2014). In bacterial cells, CRISPR-Cas9 mediates a form of adative immunity against foreign DNA that integrates into the genome. Cas9 is the RNA-guided DNA endonuclease that cuts the DNA targeted by a so-called single guide RNA (sgRNA). When engineering loss-of-function mutations, as in this instance, the Cas9-mediated cleavages are followed by the process known as non-homologous end-joining. The molecular details of these processes are addressed in the review cited above. buy zovirax 400mg

Initial results with genes encoding the CRISPR-Cas9 and guide RNAs transiently transfected into PK15 cells were disappointing. The investigators then designed a method to obtain expression of the genes able to carry out gene editing in PK15 cells by exposure to a small molecule, in this case doxycycline, i.e. inducible gene expression. This method was substantially more effective.

Yang et al. assessed numerous PK15 clones carrying the inserted genes for CRISPR-Cas9 and two guide RNAs for targeting the catalytic region of the polymerase gene. The authors used flow cytometry to select single transduced PK15 clones and determined the extent of polymerase inactivation by deep sequencing. The extent of PERV inactivation followed a bimodal distribution with 100% or nearly 100% of PERVs inactivated in some clones (about 10% of the total number analyzed) and less than 20% in the remaining 90% of the clones. buy strattera 40mg

After verifying, by multiple methods, that the clones with high levels of PERV inactivation did not exhibit genomic instability, the authors compared the highly edited clones and the lightly edited clones for ability to transmit PERVs to human cells. The PK15 clones with 100% PERV polymerase gene inactivation were at least 1,000-fold less efficient than the wild-type PK15 cells at transmitting the retoviruses to the human embryonic kidney cells.

So, where does this demonstration of the possibility of eliminating many endogenous retroviruses at once leave the prospects for clinical transplantation using pig kidneys, livers, hearts, and lungs? First, in order for this approach to successfully be employed for clinical transplants, pig embryonic stem cells will have to be created. Then, investigators will need to demonstrate that the same methods as described above applied to such primary cells can be comparably effective in eliminating PERVs and diminishing transmission to human cells. The next step will be creating cloned pigs with genomes devoid of endogenous retroviral sequences encoding viruses capable of infecting the cells of transplant recipients.

Additional questions remain to be addressed. Will currently effective methods of immunosuppression for allografts (organs from genetically-different humans) be sufficiently effective for xenografts (organs from animals of other species)? If they are less effective, can they be modified to reach comparable degrees of effectiveness? Should these modified regimens prove incapable of reaching levels of effectiveness seen for allografts, in what clinical situations will this increased risk of graft loss associated with pig organs be accepted as tolerable? Would recipients of porcine xenografts be susceptible to infections from standard pig pathogens that do not normally pose a threat to humans? How effective will human immune systems be at monitoring pig tissues for human pathogens that can infect the xenograft? Will the genetically engineered pigs have a significant risk of acquiring new endogenous retroviruses that could subsequently pose a threat to human xenograft recipients? How will xenografts from genetically modified pigs affect the costs of organ transplants?

In summary, a new technology, genome editing using CRISPR-Cas9, has facilitated multiplexed negative selection against PERVs in porcine cell line. If the same methods can be extended to porcine embryonic stem cells and these cells can be used to create genetically altered pigs, this approach could potentially massively diminish or eliminate the risks for clinical transplantation associated with these genomic legacies (i.e., PERVs) of porcine evolution. As suggested by the questions directly above, it is as yet unclear if the eventual ability to transplant pig organs into people would have the potential to alter the evolution of human-pathogen interactions.

References

Transplants By Organ Type – 2014 Based on OPTN data as of December 11, 2015; https://www.unos.org/data/transplant-trends/#transplants_by_organ_type+year+2014

Waiting List Candidates by Organ Type – All States Based on OPTN data as of December 11, 2015; https://www.unos.org/data/transplant-trends/#waitlists_by_organ

Yang L, Güell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson RA, Fishman JA, Church G. Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science. 2015 Nov 27;350(6264):1101-4. doi: 10.1126/science.aad1191. Epub 2015 Oct 11. PubMed PMID: 26456528.

Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014 Nov 28;346(6213):1258096. doi: 10.1126/science.1258096. Review. PubMed PMID: 25430774.