Construction of gene transfer systems that efficiently transduce non-dividing human hematopoietic cells

Transfer of genetic material into target cells represents a key technique in molecular biology and underlies various attempts to correct disease states using gene therapy. While numerous concepts for applications exist, highly effective procedures to deliver candidate genes into primary human target cells are still missing, thus limiting progress in various areas of experimental biology and medicine. Of particular relevance are those procedures which allow transfer of genetic material into non-dividing cells, which are often the most desired cell types These include such cells as pluripotent hematopoietic stem cells or naive T lymphocytes, which mainly reside in a non-activated state in vivo. Stimuli which allow gene transfer via conventional approaches may cause such primitive stern cells to lose their critical physiological functions upon in vitro proliferation induction. The most effective gene transfer systems to date use the molecular machinery of retroviruses for delivering and inserting genes into target cells. Despite being considerably effective, standard retroviral vector systems are limited to the infection of replicating cells. Conversely, lentiviruses such as feline immunodeficiency virus (FIV-1), or human immunodeficiency Virus (HIV) are able to infect both dividing and non-dividing cells both in vitro and in vivo. During the course of this project we will create lentiviral vectors based on FIV-1. All the necessary molecular modifications will be introduced in wild type FIV-1, and appropriate producer cell lines will be generated, in order to create high titer vector production systems. These FIV-1 based vectors will be extensively tested for their capacity to infect primary non-dividing human hematopoietic progenitor/stem cells, T cells and dendritic cells. This gene transfer system will provide a basis for further studies on the physiology of hematopoietic/immune cells, and may enable to test more clinically relevant gene therapy concepts.