In vitro differentiation of genome engineered stem cells for a novel HIV therapy

Today’s therapy of HIV offers patients the opportunity to live almost normally. The life expectancy of HIV patients increased dramatically over the last decades. Nowadays patients are hardly restricted in their way of life. Although side effects under ART are still present, a new therapy in order to be revolutionary needs to finally cure HIV. This remaining task is targeted by many new therapy concepts including genome engineering. A broader distribution of drugs as well as a better education on HIV and safe sex are both able to decrease HIV incidence and mortality worldwide, but after all the curative therapy will remain the last resort. Designer nucleases such as TALEN and CRISPR/Cas9 are highly promising tools to solve the riddle of curing HIV. The efficiency of CCR5 knock-out introduced by TALEN, is already very high. The off-target activity is rather low and the risk of severe or even lethal side effects seem to be minimal. In my studies I set up protocols for the in-vitro differentiation for both T cells and monocytes/makrophages. Additionally I produced and then delivered TALEN mRNA to CD34+ stem cells derived from cord blood and proved that if designer nucleases are applied to these stem cells, they are able to differentiate physiologically. I obtained CD33+/CD14+/CD4+ monocyte/ macrophage precursors as well as direct T cell progenitors. Especially the monocyte/ makrophage precursors represent actual target cells for HIV and can be used for further functional analysis. Moreover, I have been able to demonstrate that once established in stem cells, the knock-out remains throughout the whole process of differentiation and persists very likely in the finally differentiated mature cells, too. The cleavage efficiencies I observed in the CCR5 locus, ranged between 70% and 97%. This indicates that almost all cells were efficiently genetically modified. After the protocol for a TALEN based genomic treatment of CD34+ stem cells was introduced and optimized by Emily Meyer in the Cornu lab, I further proved in my studies that the concept of genome engineering HIV target cells by editing their stem cells is at least functional in-vitro. This data strongly suggests that the transplanted cells in human are able to support the patient’s organism in develooping immune cells. The next experiments will have to deal with the issue of in vivo cell differentiation. Furthermore functionality assays have to be performed which show that the concept of CCR5 knock-out cells is valid and really sufficient in inhibiting HIV to infiltrate the organism. Based on these results the new treatment of HIV will be established