Humanized Pigs
A new generation of genetically compatible pigs has been born in China
Polina Loseva, N+1
The company eGenesis, which specializes in the production of genetically modified pigs for human organ transplantation, announced the birth of repeatedly edited animals. They lost three genes, acquired nine, and also lost 25 viruses multiplying in the genome. At the same time, pigs are perfectly healthy and fertile, and their cells do not cause aggression in the components of the human immune system. Clinical trials of transplants in humans are planned in the coming years. A preprint of the work is posted on the bioRxiv portal (Yue et al., Extensive Mammalian Germline Genome Engineering).
There are three obstacles on the way to the transplantation of animal organs to humans. The first is physiological compatibility: the organ should 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 – are not significantly different from human ones.
The second obstacle is immune rejection. The human body reacts warily 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 a 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 the pig's organ to become compatible with humans, taking into account all these requirements, it is necessary to make changes to a number of genes at once. At first, they tried to achieve this through gene knockouts, and the pig's heart with several knockouts lasted in the baboon's body for three years.
But in 2015, American geneticist George Church and Chinese biologist Luhan Yang founded the company eGenesis, whose goal was to create pigs modified by multiple genes at once. After a while, scientists led by Church reported that they had managed to completely purify pig cells from endogenous retroviruses. Now Church and Yan have taken the next step: to create both "virus-free" and immunologically compatible animals.
To do this, researchers from eGenesis had to develop a multi-stage protocol. To begin with, they took a culture of fibroblasts from the ear of an ordinary pig. Using electroporation, CRISPR/Cas9 molecules were injected into them to cut out three genes from DNA that cause the most severe rejection in the human body. At the same time, a plasmid was injected into the cells with nine new, already human genes that are responsible for suppressing the immune response and blood clotting processes. Thus, the resulting cells should not only not cause immune aggression, but also suppress it.
After making sure that the editing was successful, the scientists extracted the fibroblast nuclei and placed them in nuclear–free eggs - this is a long-established cloning method. Embryos developed from the eggs, which were planted to surrogate mothers, and they gave birth to piglets of the first generation. These piglets still carried viruses, but they should have already turned out to be immunologically compatible with humans.
The scientists isolated the fibroblast culture from their body again and carried out the next stage of editing in it: again, with the help of electroporation, the CRISPR/Cas9 system was introduced into the cells, which attacked the reverse transcriptase gene, a key enzyme by which endogenous viruses multiply in the genome. After that, the cell nuclei were also isolated, placed inside the eggs and received piglets of the second generation. Pigs, thus, lost three genes, got nine, and also lost twenty-five actively reproducing viruses.
The researchers verified that the edited pigs are genetically stable. Eight out of nine human genes were actually expressed in their cells and the genes causing 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 interference with their immunity and blood clotting system, their blood tests 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 checked whether the genetically edited pigs 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 bound to the modified cells 90 percent less than with conventional ones. Then these cells were affected 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 by recognizing rarer proteins on them, this can be dealt with with the help of immunosuppressors. Editing also allows you to avoid acute rejection and gain time for which the organ will have time to take root inside the body. In an interview with Science, Yang clarified that in 2020 the company plans to focus on preclinical trials, but expects to switch to human research in the next five years.
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