Mitochondrial Expression of Therapeutics Proteins

治疗蛋白的线粒体表达

• 批准号: 6882506
• 负责人: RAFAL M SMIGRODZKI
• 金额: $ 11.57万
• 依托单位: GENCIA CORPORATION
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2005-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6882506
• 关键词: apoptosis; biosensor device; biotechnology; cell morphology; cytotoxicity; fluorescence microscopy; fluorescent dye /probe; gene expression; gene therapy; genetic recombination; green fluorescent proteins; in situ hybridization; membrane potentials; mitochondrial DNA; mitochondrial membrane; oxidative phosphorylation; polymerase chain reaction; protein localization; reporter genes; technology /technique development; transfection /expression vector; western blottings

DESCRIPTION (provided by applicant): The recent report of a second child who developed leukemia in a French gene therapy trial for X-SCID (X-linked severe combined immunodeficiency) highlights the problematic nature of random integration/recombination of therapeutic genes delivered to the nucleus. To circumvent this possibility, Gencia Corporation proposes a novel mitochondrial transfection method to express therapeutic genes inside mitochondria with targeted delivery of gene products to locations outside mitochondria. Given that mitochondrial DNA (mtDNA) does not undergo recombination; mitochondrial expression of therapeutic genes targeted to subcellular compartments provides a novel alternative to expressing genes in the nucleus where random integration/recombination of DNA can disrupt important cellular processes. In Phase I of this project, we propose to develop a method for mitochondrial expression of exogenous genes cloned into an engineered full length mitochondrial genome and to deliver the gene products to specific extramitochondrial locations. To achieve this, Gencia proposes to use a novel mitochondrial transfection technology Protofection(tm), shown in preliminary research to be capable of transfecting full length mtDNA. Gencia proposes to engineer a mtDNA encoding a fluorescent reporter capable of export from the mitochondria and subsequent targeting to the nucleus. The lack of mtDNA recombination allows mtDNAs containing exogenous genes to serve as templates for the intramitochondrial expression of therapeutic proteins that may then be directed to specific subcellular locations. The specific aims outlined in this application will provide the necessary proof of principle to enable the concept of using the mitochondria as a therapeutic protein factory. If successful, in Phase II, we will test our protofection method in vivo-delivering a mitochondrial genome containing a fluorescent reporter gene for mitochondrial export and subsequent extramitochondrial targeting. In parallel with in vivo studies, we will create an inducible mitochondrial expression system, which will further aid in the development of the protofection technology and provide a mitochondrial transfection system equaling that of nuclear transfection systems in capacity and ease of use while maximizing biosafety.

描述（由申请人提供）：