Gene therapy has long been touted to hold the promise of cure for a variety of congenital diseases. However, gene therapy trials are still small in number and scale due to several problems that have prevented wider application of current technology. Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy. Therefore, the ability to select for or amplify a genetically corrected population of cells from patients with single gene abnormalities might enhance the applicability of gene therapy. Previously investigated strategies for in vivo cell selection have had limited effectiveness and/or require the use of highly toxic medications. We have found that the provision of interfering RNA mediated purine analog resistance (iPAR) allows for efficient in vitro and in vivo selection of lentivirus transduced murine hematopoietic progenitor cells using the purine analog, 6-thioguanine (6TG). We hypothesize that iPAR and purine analogs (iPAR/PA) can be used as a safe and effective means for in vivo selection of autologous, genetically corrected hematopoietic cells to cure human diseases. While we believe that our preliminary data are compelling, substantial pre-clinical studies are essential to determine the applicability of iPAR for use in human patients. With the studies proposed herein, we plan to provide further experimental evidence of the effectiveness of iPAR/PA in hematopoietic cell selection. The specific aims of this project are to 1) determine the effectiveness of iPAR/PA in human hematopoietic progenitor cells and 2) determine the toxicity of lentivirus delivered iPAR and treatment with 6TG as it relates to virus integration into the genome. These aims will be accomplished primarily by using 1) xenografts of human CD34+ umbilical cord blood cells (UCBC) in immunocompromised NOD/SCID mice and 2) identification of lentiviral integration sites by LAM-PCR and sequencing, as well as functional transformation assays including a modified myeloid immortalization assay and a tumor prone mouse model. Positive findings from these studies will justify further investigation of iPAR/PA in larger animals or non-human primates and eventually human patients. A variety of human hematologic diseases would benefit from this strategy of in vivo cell selection, including, but not limited to congenital bone marrow failure syndromes (Fanconi and Diamond-Blackfan anemias), chronic granulomatous disease, Wiscott-Aldrich syndrome, hemoglobinopathies (sickle cell disease, thalassemias), and even hemophilias. PUBLIC HEALTH RELEVANCE: The goal of these experiments is to improve current efforts to cure genetic diseases of the blood system. The strategy involves providing drug resistance to the cells that have been genetically corrected, followed by selection of these cells within the body using a medication that may be safer than others that have been previously studied. This strategy could be applied to a variety of human diseases such as bone marrow failure syndromes, hemoglobinopathies, and hemophilias.

R21HL094921

2009-04-15

2012-03-31