Mitochondrial Expression of Therapeutic Proteins

描述（由申请人提供）：一般而言，基因治疗和转染技术中的主要问题是DNA随机整合/重组到核基因组中的发生，这可能破坏关键的生物过程。两名参与X连锁严重联合免疫缺陷（SCID）基因治疗试验的儿童白血病的诊断突出了这种风险。核基因组包含绝大多数人类基因，但不是遗传信息的唯一储存库。线粒体基因组（mtDNA）编码13个参与电子传递的基因，位于线粒体基质中，通过线粒体内膜和外膜与细胞核分离。单个细胞可以包含数千个线粒体，每个线粒体都有多个复制，转录和复制能力的16，569碱基对mtDNA。如果有可能利用线粒体机制来生产治疗性蛋白质，科学家和医生将拥有一种方法来规避核基因组的操纵并降低固有的相关风险。在I期SBIR中，Gencia Corporation成功地利用了克隆到全长mtDNA中的基因的线粒体表达方法。这些基因产物可以特异性地靶向线粒体外的位置，例如细胞核。使用这种新的线粒体转染技术Protofection（tm）（蛋白质介导的转染），超出I期研究目的的额外数据表明，protofection可以在体内递送和表达经工程改造以表达核靶向报告蛋白（GFP，绿色荧光蛋白）的全长mtDNA。此外，线粒体转染技术将mtDNA递送至线粒体的机制涉及线粒体脂筏。为了解决这项技术在第二阶段SBIR提案中的治疗潜力，Gencia公司将针对弗里德赖希共济失调（FRDA），这是一种常染色体隐性遗传疾病，由共济失调蛋白（frataxin）的三联体扩增引起，共济失调蛋白是一种编码定位于线粒体的蛋白质的基因。通过在FRDA动物模型的线粒体中表达frataxin，Gencia公司认为可以克服靶向细胞核的治疗中固有的问题。本提案中寻求的特定目的将使常染色体疾病的第一个非核基因疗法成为可能，并为向FDA/生物制品评价和研究中心提交的研究性新药（IND）申请提供必要的数据。

DESCRIPTION (provided by applicant): A major problem in gene therapy and transfection technologies in general, is the occurrence of random integration/recombination of DNA into the nuclear genome which may disrupt key biological processes. The diagnosis of leukemia in two children involved in a gene therapy trial for X-linked severe combined immunodeficiency (SCID) highlights this risk. The nuclear genome contains the vast majority of human genes but is not the only repository of genetic information. The mitochondrial genome (mtDNA) encodes 13 genes involved in electron transport and is located in the mitochondrial matrix, separated from the nucleus by both mitochondrial inner and outer membranes. A single cell can contain thousands of mitochondria, each with multiple copies of the replication, transcription, and translation-competent 16,569 base pair mtDNA. If it were possible to utilize the mitochondrial machinery to produce therapeutic proteins, scientists and physicians would possess a method to circumvent manipulation of the nuclear genome and reduce the inherent associated risk. In a Phase I SBIR, Gencia Corporation successfully utilized a method for mitochondrial expression of genes cloned into full length mtDNA. These gene products could be specifically targeted to extra-mitochondrial locations, such as the nucleus. Using this novel mitochondrial transfection technology, Protofection(tm) (Protein Mediated Transfection), additional data beyond the aims of the Phase I study showed that protofection could deliver and express a full-length mtDNA engineered to express a nuclear targeted reporter protein (GFP, Green Fluorescent Protein), in vivo. Furthermore, the mechanism by which the mitochondrial transfection technology delivers mtDNA to mitochondria implicates mitochondrial lipid rafts. To address the therapeutic potential of this technology in this Phase II SBIR proposal, Gencia Corporation will target Friedreich's ataxia (FRDA), an autosomal recessive disease caused by a triplet expansion in frataxin, a gene which codes for a protein localized to mitochondria. By expressing frataxin in the mitochondria of an animal model of FRDA, Gencia Corporation believes the problems inherent in therapies targeting the nucleus can be overcome. The Specific Aims sought in this proposal would enable the first non-nuclear gene therapy for an autosomal disease and provide necessary data for an Investigational New Drug (IND) application to the FDA/Center for Biologics Evaluation and Research.