Previously, it was assumed that zinc finger nucleases (ZFN) could not penetrate the cell membranes. Thus, these molecularly precise cutting tools used for gene therapy purposes had until now been introduced into cells with the aid of viral carryovers cells. Once there, the ZFN genes within the viral carriers produce a multitude of ZFN proteins which become active in the cellular DNA. The principle is simple: the zinc finger domain binds to the DNA, the nuclease domain slices it – but not just anywhere. Like a sort of heat-seeking missile, zinc finger nucleases are constructed in such a way that they can identify and cut precise predetermined DNA segments. They achieve this with a kind of navigation sequence: a short piece of DNA with which they attach at very specific points in the genetic material, and only at these points. The binding specificity of this region can be altered in the laboratory and adjusted to selected targets. This allows scientists to manipulate almost any site in the genome in a targeted way.
Gene therapy conceals dangers
It sounds simple at first, but is associated with some risks. One hazard of this gene therapy approach is that the viral DNA – even if it is not a retrovirus – randomly integrates into the cellular DNA and can cause, more or less depending on the integration site, major damage in the organism. Another risk of such a ZFN transportation method exists with the over-production of ZFN molecules via the carrier virus, which leads to a high number of non-targeted DNA cuts. If, for example, a tumour suppressor gene is sliced, cancer can arise.
ZFN: best naked
Dr. Carlos Barbas together with his colleagues has now sought a safer method of transportation for the man-made restriction enzymes – if possible without the involvement of viruses and other genetic material. In the early 1990s the chemist working at the Scripps Research Institute in the U.S. invented the technology of sequence-specific ZFN. At first the working group under Barbas experimented with ZNF proteins that possessed additional protein segments. These were supposed to facilitate movement through cell membranes. Such proteins can however only be produced in useful quantities with difficulty. Then the scientists reflected on using “naked” ZFN. “We tried to work with unmodified ZFN and, lo and behold, they were easy to make and penetrated the cells very efficiently”, says Dr. Barbas to the Institute’s website about his approach. The scientists simply added the ZFN proteins directly to human cells in a petri dish and a short time later showed how the man-made restriction enzymes executed their work efficiently and with precision in the cells, with minimal collateral damage. “This work removes the main conceptual bottleneck in the efficient use of ZFN proteins as gene therapy tools for humans”, Michael R. Reddy of the U.S. Food and Drug Administration, which has co-financed the project, says, explaining the importance of Dr. Barbas’ discovery.
HIV therapy with ZFN
What practical applications their discovery might have were shown by the scientists led by Dr. Barbas in an experiment using T cells from HIV patients. The AIDS-causing virus usually infects T cells via a T cell surface receptor called CCR5. T cells without the receptor are largely resistant to HIV infection. In 2006 an HIV patient who was treated in Berlin for leukemia with stem cells experienced exactly this effect. The Bone marrow transplant donor cells contain a CCR5 gene variant, in which the CCR5 receptor was expressed on the T-cells only in strongly diminished form. Shortly after the bone marrow transplant the patient lost all signs of HIV infection. This effect might also occur if the CCR5 gene in a T cell were successfully destroyed via ZFN targeted therapy, Dr. Barbas and his colleagues speculate. “Our idea was to protect some T cells of a patient from the HIV virus so that the immune system would remain strong enough to defeat infection”, says Dr. Barbas.
There are already clinical trials for gene therapy running in which ZFN has been studied wherein the CCR5 gene in T-cells is supposed to have been destroyed. Barbas and his colleagues could possibly achieve the same effect in the T cells with far fewer side effects than their colleagues who employ gene therapy approaches using viral carriers. In their tests, they simply administered ZFN proteins directly to human T cell cultures and already in a few hours measured greatly reduced CCR5 gene activity. But it doesn’t stop there. The research group led by Dr. Barbas tinkered further and was able, using a special cooling method which facilitates the passage of the ZFN proteins across cell membranes, to inactivate CCR5 genes at a similar efficiency to gene therapy. The new approach also seems to be significantly more safe than gene therapy: in comparison studies using DNA or virus-based methods, the cells over days produced so many excess ZFNs that the cells to some extent caused substantial damage via non-specific DNA slicing. Directly infiltrated, naked ZFNs by contrast were active for only a few hours in the cells, leaving very little improperly cut DNA. “With respect to non-target sequences, where the gene therapy approach often leaves damage behind, we saw with our technique absolutely no adverse effects”, according to Dr. Barbas.
Therapeutic cell factories
The research team examined his novel ZFN penetration method in numerous other cell types, and found that they act most effectively in fibroblasts from human skin. Scientists are now working on further therapies in which such fibroblasts are removed from patients and the gene expression of these cells is reprogrammed so that they revert to being stem cells. Such stem cells could then be modified, according to the needs of the patient, using ZFN. Once back within the body of the patient, these cells could produce millions of therapeutic cells over a long period of time.
“With this technology one day it could be possible to treat many diseases”, the authors surmise in their article in Nature Methods published in July. Dr. Barbas however first wants to occupy himself with small production facilities for HIV-resistant T cells by developing a ZFN-based therapy in hematopoietic stem cells.
Barbas announces the great potential of his discovery as follows: “Even a small number of stem cells which are resistant to HIV could replace the original HIV-sensitive T cell population of a patient completely”.