The organs in pigs can be suitable for human transplantation. They are nearly identical to human organs in terms of activity and size. However, every animal’s genome is loaded with integrated viruses. The viruses that are present in pigs are mostly harmless to them, but could lead to tumor growth and defects in the immune system if they were to be passed on to humans. A recent research paper tried to tackle these problems with the use of molecular scissors to edit the viruses out of the pig genome.
Every day, there is a shortage of organs available for organ transplantation. Many patients are put on a waitlist and have to wait for organs to be available. The vast majority of these patients will die before ever receiving the organ transplant. In the United States, 17 patients die every day waiting for an organ to be available. But, could there be a solution for the lack of organs?
Currently, there is tremendous focus on the development of xenotransplantation as a possible solution to these organ shortages. Xenotransplantation is the use of nonhuman organs, tissues or even cells for human transplantation. One particular animal, the pig, is slowly becoming the preferred animal to be used. This is because the organs in pigs have a similar size and function to human organs. In addition, pig organs present fewer problems in terms of infections compared to non-human primates. With all that said, why don’t we use pig organs for organ transplants?
Unfortunately, the genome of pigs have porcine (pig) endogenous retroviruses. Endogenous retroviruses are viral genes accumulated in the genome from retrovirus infections over time, and are inherited every generation. They are generally harmless to pigs, but can potentially cause tumor development and immunodeficiencies in humans. Now a new problem arises in solving the shortage of organs.
Recently, Dong Niu et al. were able to solve the porcine endogenous retrovirus problem. The group of researchers used the CRISPR-Cas9 system to fully inactivate the porcine endogenous retrovirus within fetal fibroblast cells.
General layout of the CRISPR-Cas9 system, and how it can be used for DNA editing. CRISPR is originally a locus in the genome of some bacteria that is able to produce molecules called short guide RNAs (gRNAs). In biotechnologies, gRNAs are created artificially to be able to find matching DNA sequences and bring the molecular scissors Cas9 onto them. Cas9 cuts the DNA, which introduces mutations, insertions, and deletions upon DNA repair (not shown in the figure).
Briefly, the researchers first found the locations of all the endogenous retroviruses in the fetal fibroblast cells. Short guide RNAs were then made to target the reverse transcriptase genes on all the endogenous retroviruses; reverse transcriptase is an enzyme that makes DNA from the retroviral RNAs. A population of fetal fibroblast cells were then treated with the short guide RNAs and with CRISPR-Cas9. This only produced a small number of fetal fibroblast cells that were fully inactivated for endogenous retroviruses. To make matters worse, when those fetal fibroblast cells grew, they were not able to maintain high inactivation for endogenous retroviruses (cells without inactivation took over the population).
To solve this second problem, the researchers found a way to maintain the growth of those cells with inactivated endogenous retroviruses. They came up with a chemical mixture that consists of pifithrin alpha (an molecule that prevents cell death) and a basic growth factor (promoting cell growth). This chemical mixture was applied to a fetal pig fibroblast cell population that showed high inactivation of endogenous retroviruses with CRISPR-Cas9. When these fetal fibroblast cells grew, they were able to maintain fully inactivated endogenous retroviruses.
Growth of fetal pig fibroblast cells edited with CRISPR-Cas9 for inactivating porcine endogenous retroviruses (PERV). PERV NHEJ efficiency (x-axis) represents the percentage of inactivated endogenous retroviruses within fetal fibroblast cells, after a DNA repair mechanism (Non-Homologous End Joining; NHEJ) has occurred. The number of clones (y-axis) represent the number of populations that were able to be replicated and grow after a single cell cloning event. Some of the fibroblast cell populations treated with the chemical mixture have 100% inactivated endogenous retroviruses.
To conclude their experiment, the researchers used the fully inactivated porcine endogenous retrovirus fetal fibroblast cells to produce live pigs. They were able to produce the first born fully inactivated porcine endogenous retrovirus pigs. From their experiment, they were able to solve the problem of endogenous retroviruses in pigs and inactivated them.
Like in any experiment, there are many challenges that have to be overcome. One, there are many ethical concerns working with animals, and using them as potential organ donors. In addition, it still remains unknown if humans can be infected by porcine endogenous retroviruses inside the body. This is difficult to determine, as not much is known about the activities of these endogenous retroviruses. In their previous study, the authors removed all the porcine endogenous retroviruses in pig kidney epithelial cells. In their present study, they showed that human cells infected by porcine endogenous retroviruses can also infect human cells outside the body. These uninfected human cells were not subjected to any pig cells beforehand. Therefore, it is important to figure out how porcine endogenous retroviruses would work inside a human body before it can be used for organ transplant.
In summary, this study helps move closer to solving the organ shortages in the world with xenotransplantation as the solution. Specifically, using CRISPR-Cas9 and pifithrin alpha can be really important in creating a variety of non-human animal organs.
About the author
This post was written by Mendy Guan. He is a 4th year student at the University of Toronto Scarborough, and doing a double major in Human Biology and Molecular Biology. In his spare time, he enjoys playing sports and video games with friends. In the future, he wants to go into the medical field and help as many people as possible.