Gene therapy of mitochondrial hereditary diseases: it has already worked on macaques!
The first monkeys with someone else's mitochondrial DNA were bornNadezhda Markina, Infox.ru
Biologists managed to breed macaques with someone else's mtDNA.
Thus, scientists have found a way to rid humanity of many genetic diseases.
The most autonomous parts of the cellIn addition to the storage of hereditary information in the cell nucleus, there are structures that have their own DNA – these are mitochondria, cellular energy substations.
Each mitochondria contains from two to ten copies of DNA. There are many mitochondria in the cell, so there are several thousand copies of mitochondrial DNA (mtDNA) in it. It is inherited, but unlike nuclear DNA, only from the mother. This selectivity is explained by the fact that mitochondria are contained in the cytoplasm, and practically all the cytoplasm during fertilization is obtained by the zygote from the egg, because there is practically none in the sperm. In mammals, each mtDNA molecule contains 15-17 thousand base pairs and encodes 37 genes.
The peculiarity of mtDNA is that it mutates at a higher rate than nuclear DNA. The reason is that an indispensable product of biochemical reactions in mitochondria are reactive oxygen species. They are known to be able to damage DNA and other biomolecules. And with the DNA repair system in the mitochondria, the situation is worse than in the nucleus. Therefore, today there are more than 150 mutations of human mtDNA and many diseases associated with it. Among them are myopathies, neurodegenerative diseases, diabetes, and some forms of cancer. Scientists have estimated that one in 3.5-6 thousand people is burdened with the risk of disease associated with defective mtDNA.
If a mutation in mtDNA is detected, there is a desire to replace it with a healthy one so that healthy offspring can be obtained by artificial insemination. But here a difficulty arises: it is impossible to pump out all the mitochondria from the cell. Shoukhrat Mitalipov and his colleagues from the Oregon National Primate Research Center (Oregon National Primate Research Center) and the Oregon Health & Science University (Oregon Health & Science University) have proposed another way to replace defective mtDNA with normal.
Leave the nucleus and change the cytoplasmThe alternative is to take a normal nucleus from an egg with mutant mtDNA and a normal nucleus and move it to a pre-nuclear cell with normal mtDNA.
Such an operation should be performed on a mature egg cell at stage II of meiosis. At this stage, the nuclear envelope no longer exists and DNA in the form of chromosomes is connected to the spindle of division. Scientists studied the distribution of mitochondria in the cell by staining them with a special dye and made sure that the zone of the division spindle is free of mitochondria. This means that this structure (scientists called it a karyoplast, since it contains nuclear DNA) can be transplanted into a cell with healthy mtDNA.
Meiosis
Reduction cell division, during which there is a decrease in the number of chromosomes in daughter cells. Meiosis occurs when the germ cells mature. It includes two consecutive divisions of the cell nucleus: reduction and equation. As a result, four haploid cells are formed from one diploid cell.
The spindle of divisionThe structure that occurs in the cell during division.
It looks like a spindle in shape. It consists of microtubules, part of which is attached to the center — kinetochore, chromosomes. The spindle ensures uniform divergence of chromosomes to the poles of the cell.
TransferAfter staining with a fluorescent dye, the DNA in the karyoplast could be seen in ultraviolet light.
Using a micropipette, scientists removed the karyoplast from the egg of a female rhesus macaque. The same was done with another egg that had a healthy cytoplasm – with normal mtDNA. To introduce donor nuclear material into a nuclear—free cell - cytoplast, scientists used two methods. Firstly, an electric discharge – electroporation, and secondly, a viral vector.
After the fusion of the cytoplast with the karyoplast, that is, a nuclear-free egg with donor nuclear material, an egg with the necessary genome and with normal mtDNA was obtained. In fact, the cytoplasm of the cell was replaced, which was required.
Live results of the experimentScientists performed such manipulations with a lot of eggs, then checked them and made sure that all cells have a donor complex of chromosomes with a spindle.
The chromosomes moved to another cell without damage. The eggs have completed the meiosis cycle normally. And finally ripe, ready for fertilization with spermatozoa.
At the next stage, it was necessary to try to get a live embryo from an egg with a replaced cytoplasm and grow it.
The eggs were artificially fertilized and 15 embryos at different stages were planted in nine female rhesus monkeys. Three females successfully became pregnant: one developed twins, and the other two had one fetus each.
On April 24, the first pregnant female gave birth to two babies, they were given the names Mito and Tracker (they got such strange names from the name of a specific mitochondrial dye). Their birth finally proved that the operation was successful. After that, the second pregnant macaque gave birth to one cub, and in early July, the third was also delivered from the burden. All the little monkeys are healthy and develop normally.
Video: Igor Katapetyan, Nadezhda Markina (Infox.ru ),
Oregon National Primate Research Center (much more)
Scientists, of course, have isolated and studied their nuclear and mitochondrial DNA. Nuclear DNA analysis showed that they all inherited their genome from chromosome donor eggs. And the mtDNA study found practically no mutations that were in the mtDNA of these eggs. To be more precise, mutations were found in negligible amounts, apparently transferred with a small amount of cytoplasm in the karyoplast. But this, scientists believe, can be neglected.
So, scientists have shown that mutant mtDNA can be effectively replaced. This gives hope that in this way it will be possible for a person to interrupt the inheritance of defective mtDNA and concomitant diseases transmitted through the maternal line. To do this, you just need to replace it with a healthy one before artificial insemination.
You can read about the replacement of mitochondrial DNA in the next issue of Nature.
Portal "Eternal youth" http://vechnayamolodost.ru27.08.2009