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HIV Treatment: Zinc Finger Show

The zinc finger nucleases are proteins produced artificially, capable of detecting specific DNA segments and cut. The transport across the cell membrane of these molecular tool's precision cutting succeeded for the first time without using potentially dangerous viral vector.

Previously, it was assumed that the zinc finger nuclease (ZFN) could not pass cell membranes. Thus, these molecular tools for precision cutting used for gene therapy were hitherto only introduced using viral vectors into cells. Once on site, the ZFN genes included in the viral vectors produce many proteins ZFN becomes active in the cellular DNA. The principle is simple: the zinc finger domain binds to DNA, the nuclease domain breaker - but not anywhere. Like heat-seeking missiles, the zinc finger nucleases are constructed so that they can identify particular DNA segments and predetermined cut. This takes place through a kind of navigation sequence, a small piece of DNA, which adheres to specific sites in the genome - and only these. The binding specificity of this zone can be changed and adapted to the laboratory selected destinations. This allows scientists to manipulate targeted almost any site in the genome.

Gene therapy can be dangerous.

It seems easy at first, but this method is associated with certain risks. One of the dangers of this approach is that gene therapy viral DNA - even if it is not a retrovirus - integrates randomly into the cellular DNA and may cause, depending on the site of integration, of more or less severe damage in the body. Another risk of such a method of transport is the overproduction of ZFN molecules ZFN virus vector, which leads to a large number of cut DNA segments not targeted. For example, if a tumor suppressor gene is reached, a cancer may develop.

ZFN: better naked

Dr. Carlos Barbas then sought a vector with his colleague safer for these artificial restriction enzymes - if possible without the involvement of viruses and other genetic material. The chemistry of the Scripps Research Institute U.S. invented in the early 1990s, the ZFN technology-specific sequence. The working group around Barbas first tried to use ZNF proteins, which possess additional protein segments. This would facilitate passage through cell membranes. However, it was difficult to obtain sufficient quantities of these proteins. The scientists then returned to ZFN "naked." "We tried to work with unmodified ZFNs, and we now have the expected result: they are easy to produce and very effectively penetrate into the cells," says Dr. Barbas, commenting on his approach on the website of the Institute. Scientists began simply ZFN proteins directly on human cells in a petri dish and a little later they showed how artificial restriction enzymes could work efficiently in the cells, accurately and with a minimum of collateral damage. "This work removes the main obstacle to the effective use of ZFN proteins as a tool for gene therapy in humans," said Michael R. Reddy U.S. Food and Drug Administration, which co-financed the project, the importance of the discovery of Dr. Barbas.

HIV treatment with ZFN

Scientists with Dr. Barbas showed how this could have practical application discovered during an experiment with T cells from patients infected with HIV. The virus that causes AIDS infects T lymphocytes, usually through a surface receptor called CCR5 cells. T cells without this receptor are highly resistant to infection by HIV. In 2006, we observed exactly this effect on an HIV patient who had been treated with stem cells against leukemia Berlin. The graft of bone marrow donor cells contained with a variant of the CCR5 gene, wherein the CCR5 receptor was expressed in a form that substantially reduced on the T cells. Shortly after the bone marrow transplant, the patient lost all signs of HIV infection. This effect may also occur, if possible, destroying the CCR5 gene in T cells through a targeted therapy by ZFN, suggested Dr. Barbas and colleagues. "Our idea was to protect certain HIV T cells of a patient so that the immune system is strong enough to overcome the infection," said Dr. Barbas.

Currently, clinical trials of gene therapy in which the ZFN destroyed the CCR5 gene in T cells are already underway. Barbas and colleagues may be able to get the same effect in T cells with significantly fewer side effects than their colleagues who perform gene therapy approaches using viral vectors. In their experiments, they applied just ZFN proteins directly on human T cells in culture and measured each few hour a sharp reduction in the activity of the CCR5 gene. However, this is not enough. The research group led by Dr. Barbas Bricola and put through a special cooling method that facilitates the passage of ZFN proteins through cell membranes, inactivate the CCR5 gene as effectively with gene therapy. The new approach also seems to be safer than gene therapy: in comparative tests with methods based on DNA or viruses, the cells produced during the same period as the ZFN excess cells that did respond to damage created by non-specific cuts in DNA. In contrast, the bare ZFN directly infiltrated were active only for a few hours in the cells, leaving very little cut DNA in a non-specific. "For many non-target sequences on which the gene therapy approach often leaves damage, we have seen with our technique absolutely no side effects," said Dr. Barbas.

Therapeutic cell factories

Explore team examined the number of cells ZFN introduced a new method, that is found inhuman skin fibro blasts that operate more efficiently. Scientists are currently working on complementary therapies, in which they collect in such a patient's fibro blasts, and reprogram gene expression of these cells to become stem cells. Stem cells may then be modified by ZFN based on patient needs. Back into the patient's body, these cells can produce over a long period of millions of therapeutic cells."With this technology, many diseases one day be treated," the authors speculate in the paper published in July in Nature Methods. Dr. Barbas now wants to tackle the creation of small production sites of T-cells immune to HIV through the development of a therapy based on ZFN in hematopoietic stem cells. Barbas explains the great potential of its discovery as follows: "Even a small number of stem cells resistant to HIV can replace the total population of T cells of a patient susceptible to HIV. '

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