Cross-Species Transplantation

One application benefiting from CRISPR/Cas9 technology is xenotransplantation, or cross-species transplantation. It offers the prospect of an unlimited supply of organs and cells, and it could resolve the critical shortage of human tissues.

For ethical and compatibility reasons, xenotransplantation shifted away from nonhuman primates as a potential source of donor tissues. Instead, the discipline began to focus on porcine organs. Nonetheless, in 1997, pig-to-human transplants were banned worldwide due to concerns about the transmission to humans of porcine endogenous retroviruses (PERVs), which are integrated into the genome of all pigs.

According to George Church, Ph.D., professor of genetics, Harvard Medical School, work was undertaken in his laboratory on PK15 porcine kidney epithelial cells to determine if PERVs could be eradicated. It was crucial to avoid disrupting the envelope gene and the terminal regulatory elements, as both of these could be important during normal pig fetal growth. In addition, a highly conserved target in the viral polymerase gene was desired for the guide RNA (gRNA) to bind.

First, the copy number of PK15 PERV was determined to be 62. Then, when CRISPR/Cas9 was used along with two gRNAs, one which did the bulk of the work, all 62 copies of the PERV pol gene were disrupted, demonstrating the possibility that PERVs could be inactivated for potential clinical pig-to-human xenotransplantation. The repeats were well separated, and not clustered, which could have meant higher toxicity.

After two weeks of cell culture, about 8% of clones were 100% altered, and no rearrangements were found. Although a few off-target effects and point mutations were expected, they were deemed unlikely to have an impact on pig fetal development. As with conventional breeding, PERV-free clones were empirically selected as they were the healthiest.

In addition to disrupting dozens of endogenous viral elements, Dr. Churchs group altered dozens of genes involved in immune and blood-clotting functions to increase human compatibility. Some of the changes were so extensive that more powerful DNA recombination tools, and not CRISPR, were utilized.

This work may benefit eGenesis, a Cambridge biotech focused on leveraging CRISPR technology to deliver safe and effective human transplantable cells, tissues, and organs. eGenesis was cofounded by Dr. Church and Luhan Yang, Ph.D., in early 2015 and is based on their research.

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More Tooth, More Tail in CRISPR Operations | GEN - Genetic Engineering & Biotechnology News (press release)

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