EGenesis, which specializes in the production of genetically modified pigs for human organ transplantation, has announced the birth of multiple edited animals. They lost three genes, acquired nine, and also lost 25 viruses multiplying in the genome. At the same time, pigs are completely healthy and fertile, and their cells do not cause aggression in the components of the human immune system. Human clinical trials of transplants are planned in the coming years. A preprint of the work is posted on the bioRxiv portal.
There are three obstacles to human organ transplantation. The first is physiological compatibility: the organ must be suitable in size, structure and function. In this sense, a convenient object is a pig, many of whose organs - for example, the heart or kidneys - do not differ significantly from human ones.
The second obstacle is immune rejection. The human body reacts cautiously to any unfamiliar molecules, but some - for example, sugars on the surface of foreign cells - cause especially strong aggression.
Finally, the third is the viruses that each organism carries in its genome. We are not talking about active viruses that cause epidemics, but about endogenous ones, that is, those that do not kill the cell, but multiply only within its nucleus. But if the cell still somehow keeps its own viruses under control, then if foreign cells infect it with new viruses, then it may not be able to cope with the consequences of their reproduction.
In order for a pig's organ to become compatible with humans, taking into account all these requirements, it is necessary to make changes at once in a number of genes. At first, they tried to achieve this through gene knockouts, and the heart of a pig with several knockouts lasted in the baboon's body for three whole years.
But in 2015, American geneticist George Church and Chinese biologist Luhan Yang founded eGenesis, a company whose goal was to create pigs modified by multiple genes at once. After a while, scientists led by Church reported that they were able to completely cleanse pig cells from endogenous retroviruses. Now Church and Jan have taken the next step: to create both "virus-free" and immunologically compatible animals.
To do this, the researchers at eGenesis had to develop a multi-step protocol. To begin with, they took a culture of fibroblasts from the ear of an ordinary pig. With the help of electroporation, molecules of the CRISPR / Cas9 system were introduced into them to cut out three genes from DNA that cause the strongest rejection in the human body. At the same time, a plasmid with nine new, already human genes was introduced into the cells, which are responsible for suppressing the immune response and blood coagulation processes. Thus, the resulting cells should not only not cause immune aggression, but also suppress it.
After confirming that the editing was successful, the scientists removed the fibroblast nuclei and placed them in nuclear-free eggs - a long-established method of cloning. The embryos developed from the eggs, which were planted in surrogate mothers, who gave birth to piglets of the first generation. These pigs still carried viruses, but they should have already turned out to be immunologically compatible with humans.
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Scientists again isolated a culture of fibroblasts from their body and carried out the next stage of editing in it: again, using electroporation, the CRISPR / Cas9 system was introduced into the cells, which attacked the reverse transcriptase gene, a key enzyme with which endogenous viruses reproduce in the genome. After that, the cell nuclei were also isolated, placed inside the oocytes, and the second generation piglets were obtained. The pigs thus lost three genes, gained nine, and also lost twenty-five actively multiplying viruses.
The researchers verified that the edited pigs were genetically stable. In their cells, eight out of nine human genes were actually expressed and genes that cause immune rejection were "silent". Scientists checked the animal genome for traces of off-target editing and found several CRISPR / Cas9 "misses", but they did not affect the protein-coding regions of DNA.
The animals themselves were physiologically healthy and fertile. Despite interfering with their immunity and blood clotting system, their blood counts were within normal limits. Scientists also did not find any effect of editing on the work of the heart, liver and kidneys of pigs.
Finally, the researchers tested whether the genetically edited pigs had acquired the properties required for transplantation. First, they isolated a cell culture from the vessel walls of pigs and treated it with human immunoglobulins: they bind to modified cells 90 percent less than normal ones. Then these cells were acted upon by human complement proteins - they react to the presence of foreign cells even earlier than immunoglobulins - but the complement system was activated no more often than in the case of their own, human cells.
Thus, scientists have managed to create animals whose cells do not cause immediate aggression of human immunity. Despite the fact that the immune system can react to foreign cells later, recognizing on them rarer proteins, this can be dealt with with the help of immunosuppressants. Editing, on the other hand, allows you to avoid acute rejection and gain time for the organ to take root inside the body. In an interview with Science, Yang clarified that the company plans to focus on preclinical testing in 2020, but expects to move to human research in the next five years.
Author: Polina Loseva