Genome engineering in mammalian systems: Challenges for rewriting pig genomes for xenotransplantation

Luhan Yang, eGenesis

We understand there are 100,000 people on transplantation waiting list. Only 20% of them can get transplants each year. There are millions of patients that suffer from late stage organ fialure and their life could be saved by new organs. The solution and strategy that eGenesis is using is xenotransplantation, use pig as bioreactor to mature the organ. We start with somatic cell, do the modification, then we clone a pig out of this cell, and then use the organ for transplant.

The concept for xenotransplantation has been attempted for decades. There were some problems that they couldn't address before. The first problem was endogenous pig virus in the genome could jump to human, so it's a real safety concern that we have to address because they could also jump to other genomes. There were no tools to remove PERVs in the past. With CRISPR, eGenesis is trying to elaborate this tool to address the problems.

In 2015, our team at Church lab demonstrated that we can use CRISPR to eradicate 62 copies in the pig cell line. People got very excited, we broke the record as to how many modifications you can do per mammalian genome. I think we still have the record. We also show that after modification, there's no infection. It's only the start of our journey.

At eGenesis, we are repeating the procedure in a primary cell line instead of a tumor cell line. It's an iterative process before we will be able to get this into a human. I don't have time to go into detail about progress, but we can talk about challenges.

There are some unique features about the mammalian genome that makes it difficult to engineer. We don't fully understand how the epigenetic factor and how the 3d structure interferes with gene expression, not to mention the problems of foregin genetic circuits. CRISPR is powerful to do knock-out, but we don't have a good tool for 1 kb knock-out at a specific locus. To understand how the gene circuit will function is another problem, we're thinking about using transgenic animals for this.

Here is a snapshot-- the problem for transgene expression in pig fibroblast cell. For this transcription unit, it was fine, but once we put it into the circuit, it became silent. We are also in parallel looking at silencing and microdilution for our circuit. If we're growing knowledge of epigenetic factor, ... with single cell RNA transcriptome analysis maybe we can have some design principles and structure to design mammalian circuits.

We need to modify genomes in the range of 1 to 100 kbp. The engineering challenge is how to make it programmable. If we do more than 100 kbp, too. Intra cellular chromosome translocation and lost-- coincident with multiple cas9 cutting sites, has 1-50% efficiency. Bacteria has conjugation system and yeast has mating system, and there are cell transfer system, can we learn from nature and our cardiomyocte will fuse from two component could we use this to enhance intracellular chromosome transfer?

Looking at our history, I think it's promivsing. Our ability to write the genome is really lagging our ability to read the genome.

Despite the complexity, there's some encouragement for mammalian genome engineering. In the mammoth project from George Church, we're trying to use somatic cell nuclear transfer. We use the pig cell as the donor cell due to availability of material and we got robust hybrid elephant blastocyte with pig after 7 days. It almost look like genome transfer. It functions fine. Life is quite adaptive and flexible as long as we use the same genomic language.

At eGenesis, we are trying to make a human transplatntable organ in pig. Also we need human immunological compatibility.