Mitochondrial Expression of Therapeutics Proteins

描述（由申请人提供）： 最近的一份报告显示，在法国进行的一项针对X连锁严重联合免疫缺陷（X-SCID）的基因治疗试验中，第二名儿童患上了白血病，这突出表明了将治疗基因随机整合/重组到细胞核中的问题。为了规避这种可能性，Gencia公司提出了一种新的线粒体转染方法，在线粒体内表达治疗基因，并将基因产物靶向递送到线粒体外的位置。考虑到线粒体DNA（mtDNA）不经历重组;靶向亚细胞区室的治疗基因的线粒体表达提供了在细胞核中表达基因的新的替代方案，其中DNA的随机整合/重组可以破坏重要的细胞过程。 在该项目的第一阶段，我们建议开发一种方法，用于克隆到工程全长线粒体基因组中的外源基因的线粒体表达，并将基因产物递送到特定的线粒体外位置。为了实现这一目标，Gencia提出使用一种新的线粒体转染技术Protofection（TM），在初步研究中显示能够转染全长mtDNA。Gencia提出设计一种编码荧光报告基因的mtDNA，这种荧光报告基因能够从线粒体输出，随后靶向细胞核。缺乏mtDNA重组允许含有外源基因的mtDNA作为模板，用于治疗性蛋白质的线粒体内表达，然后可以将其导向特定的亚细胞位置。本申请中概述的具体目的将提供必要的原理证明，以实现使用线粒体作为治疗性蛋白质工厂的概念。 如果成功的话，在第二阶段，我们将在体内测试我们的protofection方法-递送含有荧光报告基因的线粒体基因组用于线粒体输出和随后的线粒体外靶向。在体内研究的同时，我们将创建一个诱导型线粒体表达系统，这将进一步帮助原型转染技术的发展，并提供一个线粒体转染系统，在能力和易用性方面与核转染系统相当，同时最大限度地提高生物安全性。

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.