Lentiviral Vector for Gene Transfer A Versatile Tool for Regulated Gene Expression, Gene Silencing and Progenitor Cell Therapies

Gene therapy holds promise to improve the treatment options of both inherited and acquired diseases like cardiovascular diseases. There is still a need for optimal gene delivery vectors for enhanced efficacy and safety. The aim of this research was to apply the human immunodeficiency virus-1 (HIV-1) derived lentiviral vector (LV) for different approaches of gene therapy. LVs have the ability to integrate into the host cell genome and are thus suitable for applications requiring long-term expression of the therapeutic gene. However, in such applications, there is a need to regulate the level of therapeutic protein expression. During this research, a LV system was developed and its efficacy tested for the capacity to adjust the amount of protein expressed or to switch the expression on and off by the addition of the antibiotic, doxycycline. This study demonstrates the ability to fine-tune the expression of a LV delivered therapeutic gene by adjusting the concentration of doxycycline within a range which can be achieved by oral administration. It also shows the functionality of the system in vivo in rat brain. Another approach for therapeutic gene regulation is to utilize an endogenous, pathophysiological stimulus of the target tissue. We designed a series of vectors exploiting a novel approach, oxidative stress induced gene regulation. This is an alluring concept for cardiovascular gene therapy applications, since oxidative stress plays a role in a number of cardiovascular diseases. Our results showed that antioxidant response elements introduced into LVs can be used for oxidative stress induced gene expression. Also, we studied whether LVs can be applied in a gene knock-down approach exploiting a small hairpin RNA (shRNA) – based method. Our results demonstrate efficient, long-term gene silencing by LV-shRNA both in cell culture and mouse brain. As a potential therapeutic application for LVs, we studied their ability to transduce cord blood (CB) derived progenitor cells and found that these cells could be efficiently transduced by LVs. CB is a unique source for hematopoietic stem cells and other progenitor cells, which can be exploited for novel cell therapy approaches. We also assessed the therapeutic potential of progenitor cells in a nude mouse model of hindlimb ischemia. We did not detect engraftment of progenitor cells into the target tissue. However, our results show enhanced regeneration of the ischemic muscle by progenitor cell injections. Based on these results, we suggest that progenitor cells may be beneficial in the recovery of injured tissue by indirect mechanisms. Taken together, this study demonstrates the applicability of HIV-1 based vectors as a basic research tool and a potential gene therapy vector, particularly for ex vivo approaches such as progenitor cell therapies. National Library of Medicine Classification: QU 470, QU 475, QZ 52, QW 168.5. H6, QU 325 Medical Subject Headings: Gene Therapy; Gene Transfer Techniques; Genetic Vectors; Lentivirus; HIV-1; Gene Expression Regulation; Gene Silencing; Doxycycline; Brain; Rats; Oxidative Stress; Umbilical Cord; Blood; Stem Cells; Transduction, Genetic; Cardiovascular Diseases/therapy; Ischemia; Muscle, Skeletal; Mice