10-Gene edited pig
10-Gene edited pig (10-GE pig) refers to pigs used in pig-to-human xenotransplantation experiments. The aim of this field is to supply enough organs from animals for transplants because there are not enough human donors to meet demand. Modifications of the donors are necessary to overcome the genetic barrier between species. 10-GE pigs harbor genetic modifications of 10 genes that contribute significantly to graft rejection. This makes it possible for their organs to be transplanted into humans while retaining their function, although the accompanying pharmacological immunosuppression still needs to be very strong.
Pigs with these modifications were used in the most notable xenotransplantation experiments: a kidney transplant to a brain-dead patient and a heart transplant to a living patient with heart failure. They were developed by Revivicor, a subsidiary of United Therapeutics.
The genome is edited in the following way:
- 4 genes are knocked out: the α-1,3-galactosyltransferase gene, the β1,4-N-acetylgalactosyltransferase gene, the CMP-N-acetylneuraminic acid hydroxylase gene, and the pig growth hormone receptor gene
- 6 human genes are added in 2 inserts: CD46, CD47, CD55, THBD, PROCR, HMOX1
Since the last common ancestor between humans and pigs around 80 million years ago, ancestors of humans have lost the ability to synthesize some carbohydrates on the surface of their cells. This makes the human immune system recognize these carbohydrates as foreign and mount a reaction, leading to hyperacute rejection of such tissue. Only knockout of all 3 suppresses this response.
Alpha gal – Galactose-α-1,3-galactose, also known as alpha-gal, is synthesized by α-1,3-galactosyltransferase encoded by GGTA1. It is present in most mammals but not in Catarrhini (a group that includes humans). Antibodies against alpha-gal are abundant in humans, elicited by gut flora. Alpha gal antibodies of class IgE are the cause of acquired red-meat allergy following the bite of certain tick species.
Neu5Gc – N-Glycolylneuraminic acid is synthesized by CMP-N-acetylneuraminic acid hydroxylase encoded by CMAH. With a mutation in CMAH, this antigen is missing in humans and also new world monkeys, possibly due to malaria.
Sd(a) – The Sd(a) antigen is synthesized by β1,4-N-acetylgalactosyltransferase encoded by B4GALNT2. In contrast to the previous 2 this carbohydrate is not lost in most humans and forms the basis of the Sid blood group system. The mechanism responsible for eliciting an immune reaction in xenotransplantation is being investigated, but it seems that Sd(a) is compensatorily over-expressed after alpha-gal knockout.
Growth hormone receptor
Domestic pigs have historically been bred for the biggest size and quickest growth. To make the size of donor organs similar to human ones and to reduce the negative impact on tissue quality caused by rapid growth, the growth hormone receptor gene GHR is knocked out.
Insertion of human genes
These genes have partake in fundamental systems for differentiating between self and foreign cells, inhibit excessive coagulation and inflammation.
Also known as integrin associated protein (IAP). It is expressed on all cells and can be over-expressed in tumors. It acts as a “don’t eat me” signal after binding with the receptor SIRP-α. Pig CD47 does not bind human SIRP-α.
Heme-oxygenase 1 has anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Degradation of free heme modulates the immune response in multiple ways. The complete mechanism is still a subject of research.
Coagulation cascade regulators
Blood clotting is one of the most detrimental processes in graft rejection. Without these 2 inserted genes the coagulation is to severe to be controlled pharmacologically.
THBD – Thrombomodulin (CD141) activates protein C in the presence of thrombin in a regulatory feedback loop.
PROCR – Endothelial protein C receptor (CD201) binds protein C regardless of its state to facilitate its function. It can also activate γ:δ T cells.
Complement cascade regulators
Human cells have multiple ways of defending themselves from the complement cascade, these 2 have shown the most benefit when inserted into pigs:
CD55 – Decay accelerating factor (DAF) binds C3b and C4b, preventing the formation of C3-convertases. In humans, a deficiency leads to Paroxysmal nocturnal hemoglobinuria.
CD46 – Membrane Cofactor Protein (MCP) facilitates the cleavage of C3b and C4b by complement factor I.