Mitochondrial Expression of Therapeutic Proteins

描述（由申请人提供）：一般来说，基因治疗和转染技术的一个主要问题是DNA随机整合/重组进入核基因组，这可能会破坏关键的生物过程。参与x连锁严重联合免疫缺陷（SCID）基因治疗试验的两名儿童的白血病诊断突出了这种风险。核基因组包含了绝大多数人类基因，但并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，线粒体内外膜将其与细胞核分开。单个细胞可以包含数千个线粒体，每个线粒体都具有复制、转录和翻译能力的16569个碱基对mtDNA的多个拷贝。如果有可能利用线粒体机制生产治疗性蛋白质，科学家和医生将拥有一种方法来规避对核基因组的操纵，并降低固有的相关风险。在一期SBIR中，Gencia Corporation成功地利用了一种将基因克隆成全长mtDNA的线粒体表达方法。这些基因产物可以专门针对线粒体外的位置，如细胞核。使用这种新的线粒体转染技术，Protofection(tm)（蛋白质介导转染），超出I期研究目标的额外数据表明，Protofection可以在体内传递和表达全长mtDNA，用于表达核靶向报告蛋白（GFP，绿色荧光蛋白）。此外，线粒体转染技术将mtDNA传递到线粒体的机制涉及线粒体脂筏。为了在II期SBIR提案中发挥该技术的治疗潜力，Gencia公司将针对弗里德赖希共济失调（FRDA）， FRDA是一种常染色体隐性遗传病，由frataxin的三重扩增引起，frataxin是一种编码线粒体蛋白的基因。Gencia公司认为，通过在FRDA动物模型的线粒体中表达frataxin，可以克服针对细胞核的治疗所固有的问题。该提案中寻求的具体目标将使常染色体疾病的第一个非核基因治疗成为可能，并为FDA/生物制品评估和研究中心的研究性新药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.