GENE TRANSFER INTO STEM CELLS BY FOAMY VIRUS VECTORS

通过泡沫病毒载体将基因转移到干细胞中

• 批准号: 6233010
• 负责人: Neil Josephson
• 金额: $ 12.03万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6233010
• 关键词: NOD mouse; Retroviridae; SCID mouse; baboons; cell cycle; cord blood; gene delivery system; hematopoietic stem cells; human tissue; stem cell transplantation; technology /technique development; transfection /expression vector; xenotransplantation

DESCRIPTION (adapted from the application) This research project is designed to provide the applicant, Neil Josephson, with training in the areas of retroviral vector development and gene transfer into hematopoietic stem cells. Dr. Josephson is a board certified hematologist with an interest in stem cell disorders and gene therapy. Stem cell gene therapy offers the promise of treating hereditary disorders such as sickle cell anemia and thalassemia. It also may play a role in the therapy of acquired diseases such as cancer and HIV. This promising technology has not yet yielded clinical results because current retroviral vectors do not efficiently transfer genes into hematopoietic stem cells. The work proposed in this application will develop and test a new retroviral gene transfer system based on the human foamy virus (HFV). HFV vectors have many qualities that make them good candidates for use in stem cell gene transfer. They are non-pathogenic, have a wide host range, and can transduce quiescent cells. Aim 1 focuses on vector development. Using currently available HFV vector backbones, new constructs will be generated with a variety of different internal promoters and reporter genes. Current HFV vector production methods rely on transient transfection of vector constructs and yield a crude stock contaminated with toxins. Density centrifugation techniques for purifying HFV stocks will be investigated. To allow for easier and more pure vector production an HFV packaging line will be developed. Aim 2 looks at the ability of HFV vectors to transduce human hematopoietic cells. The impact of multiplicity of infection and length of exposure to vector stock on hematopoietic cell transduction will be explored. The role of cell cycle in transduction efficiency will also be explored. Conditions that are found to most efficiently transduce progenitor cells will be applied to marking studies of human pluripotent repopulating cells in the NOD/SCID xenotransplantation model. Aim 3 outlines work that will take the best HFV vectors produced in aim 1 and the optimal transduction protocols from work in aim 2 and apply them to a pre-clinical marking study of nonhuman primates. Non-human primates are the most biologically similar animal model to humans. Therefore, it is essential to use this model for testing the efficacy and safety of HFV vectors before applying them to clinical studies. Most primates kept in captivity are infected with the simian foamy virus (SFV) which is very similar to HFV. The presence of SFV in HFV vector transduced animals could complicate the interpretation of marking and toxicity results. Therefore, in vitro analysis of HFV effects on SFV will be explored. Transduction protocols from studies in aim 2 will be applied to marking studies of non-human primate hematopoietic progenitor cells. Once optimal transduction protocols have been determined, in vivo transplantation and marking studies will be performed. Marked animals will be followed for the presence of transduced cells by evaluation of reporter gene expression and proviral copy numbers. Animals will be evaluated for any potential toxic effects of the transduction and transplantation.

描述（改编自应用程序）