RETROVIRAL MEDIATED GENE TRANSFER INTO PRIMATE HEMATOPOIETIC CELLS

Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Over the past year, studies have continued using murine and human retroviral vectors to optimize gene transfer to non-human primate hematopoietic cells. The vectors predominantly used were constructed to express the enhanced green flourescent protein (EGFP) reporter gene. After having demonstrated that a commercially available RGD-containing fibronectin (FN) fragment, RetroNectin? (CH-296), can improve gene transfer to rhesus CD34+ cells, a number of autologous transplant studies were performed using the FN fragment CH-296. The retroviral vectors used include a murine stem cell virus(MSCV)-based vector and a VSV-G pseudotyped human immunodeficiency virus type-1 (HIV-1) lentiviral vector. For the amphotropic MSCV-based vector, it was observed that up to 55% of circulating granulocytes expressed the EGFP gene during the early post-transplant period. This level, however, decreased to 0.1% or lower within two weeks following transplant. In contrast to granulocytes, EGFP gene expression rose in peripheral blood (PB) lymphocytes from 2-5% shortly following transplant to 10% or greater by week 5. After 30 weeks, the level of expression in PB lymphocytes remained at 1-3% by both flow cytometry and Southern blot analysis, with EGFP expression being observed in CD4+, CD8+, CD20+, and CD16/56+ lymphocyte subsets. EGFP expression was only transiently detected in red blood cells and platelets early following transplantation. Such sustained levels of lymphocyte marking is encouraging and may be therapeutic in a number of human gene therapy applications targeting the lymphoid compartment. The transient appearance of EGFP+ myeloid cells, however, suggests that transduction of a lineage restricted myeloid progenitor, capable of short term engraftment, was only achieved with this protocol. In order to improve upon this level of marking, a lentiviral first generation HIV-1-based vector was evaluated. Lentiviral vectors have gained increasing use as a gene delivery vehicle, particularly because of their ability to infect non-dividing cells at most points of the cell cycle. The use of lentiviral vectors, however, in a clinical setting has raised concerns that these vectors, most of which are based upon HIV-1, may be unsafe. Following transplantation of cytokine mobilized autologous PB CD34+ cells transduced with a VSV-G pseudotyped lentiviral vector using RetroNectin?, reconstitution resulted in the genetic marking of approximately 1% of circulating leukocytes in rhesus macaques. Unlike the amphotropic MSCV-based vector, however, prolonged detectable levels of EGFP gene expression were observed in granulocytes, monocytes, and lymphocytes, with the proportion of marked cells increasing somewhat over time. In one animal, red blood cells and platelets also had persistent EGFP gene expression. There was no evidence for replication competent HIV-1 or any of the pathogenic effects associated with HIV-1 infection. These results therefore provide a proof of concept for the use of lentiviral vectors in human clinical applications. Future studies for retroviral vectors will continue to focus on improving the frequency of genetically marked cells within the circulation. Other studies in the gene therapy program evaluate vectors that express human Factor IX in vivo, and studies which help delineate the potential immunogenicity of protein products transduced into either CD4+ lymphocytes and CD34+ cells. In addition, outside the gene therapy program, studies continue to determine the best adjuvant for a B19 capsid vaccine, attempts at isolating potential human viral agents that may be associated with hepatitis in aplastic anemia patients in tamarins, and the demonstration that non-anemic rhesus macaques are an animal model for testing biological and chemical agents that stimulate F-cell expansion and HbF levels. - gene therapy, non-human primate, retroviral vectors, lentiviral vectors