Mitochondrial Expression of Therapeutic Proteins

治疗性蛋白质的线粒体表达

• 批准号: 7154884
• 负责人: RAFAL M SMIGRODZKI
• 金额: $ 45.62万
• 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 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7154884
• 关键词: Friedreich' s ataxia; bioengineering /biomedical engineering; biological models; gene therapy; genetically modified animals; green fluorescent proteins; laboratory mouse; lipid raft; mitochondrial DNA; nonhuman therapy evaluation; phenotype; phosphotransferases; protein engineering; technology /technique development; therapy design /development; tissue /cell culture; transfection; transfection /expression vector

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.

描述(申请人提供)：基因治疗和转基因技术的一个主要问题是DNA随机整合/重组到核基因组中，这可能会扰乱关键的生物学过程。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一风险。核基因组包含绝大多数人类基因，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，由线粒体内外膜与细胞核隔开。单个细胞可以包含数千个线粒体，每个线粒体都有复制、转录和翻译能力强的16,569个碱基对的线粒体DNA的多个副本。如果有可能利用线粒体机制来产生治疗性蛋白质，科学家和医生就会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。在第一阶段的SBIR中，Gencia公司成功地利用了一种方法，将克隆到全长mtDNA中的基因进行线粒体表达。这些基因产物可以专门针对线粒体外的位置，如细胞核。利用这一新的线粒体转染技术Protofect(Tm)(蛋白质介导的转染)，超出第一阶段研究目标的额外数据表明，Protofect可以在体内传递和表达经工程处理的全长mtDNA，以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运送到线粒体的机制涉及线粒体脂筏。为了在这项第二阶段的SBIR提案中解决这项技术的治疗潜力，Gencia公司将瞄准Friedreich共济失调(FRDA)，这是一种常染色体隐性遗传病，由Frataxin的三重扩增引起，Frataxin是一种编码定位于线粒体的蛋白质的基因。Gencia公司相信，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中固有的问题。该提案寻求的具体目标将使第一次针对常染色体疾病的非核基因治疗成为可能，并为向FDA/生物制品评估和研究中心申请研究性新药(IND)提供必要的数据。