This proposal is designed to create lentiviral based packaging systems for delivery of gene therapy constructs to HIV-1 infected individuals using Feline Immune Deficiency Virus (FIV) and Simian Immunodeficiency Virus (SIV) based vectors systems. Many current human gene therapy protocols utilize the retrovirus. Moloney murine leukemia virus (MMLV) because of its properties of long-term integration, low copy number, adequate expression and lack of an immune response against the vector. While these systems have been show to work well in mice and other small animals, the transduction efficiency is reduced in primates and humans and long-term expression is variable. The reasons for the decreased transduction efficiencies in humans are not clear, but are thought to be partly due to the inability of these retroviral systems to infect non-dividing or quiescent cells, such as hematopoietic stem cells and hepatocytes. To achieve, others have developed HIV-1 based lentiviral packaging systems (ref) to infect non-dividing cells. Lentiviruses, as a subclass of retroviruses, have the ability to infect non-dividing cells that are metabolically active. Using molecular clones of FIV and SIV, we will dissect their genomes into multiple non-overlapping elements that provide the necessary trans-proteins for synthesis of viral cores and envelope proteins. Other cis elements required for vector delivery will be engineered into safe, high efficiency vector systems for infection of non- dividing human cells of the immune system. Multiple individual vector constructs will be created for testing. We will work closely with Dr. Nabel's group in Project 1 to accommodate vector designs that express RevM10, ribozymes, and single chain variable regions with appropriate marker genes. Cell tested will be model systems, PBL-derived macrophages and hematopoietic stem cells, and T cells. As vector designs continue and progress is made in the first and second specific aims, we will work with Dr. Kohn's group for delivery go genes to be isolated stem cells. We will work closely with Dr. Daniel Littman in Project 4, who has experience with infection of T cells using MMULV, as well as extensive experience in T cell gene expression, to compare directly the abilities of our vector systems to expression at various stages of in vitro and in vivo development after transfer to SCID/Hu models. Several internal promoter elements will be tested, and these will also be tested in the SCID hum model for efficacy and cell subtype expression of inserted genes.
| Curran, Michael A; Ochoa, M Sofia; Molano, R Damaris et al. (2002) Efficient transduction of pancreatic islets by feline immunodeficiency virus vectors1. Transplantation 74:299-306 |
