Purging Mutant mtDNA Using MitochondriallyâTargeted Gamma Peptide Nucleic Acids

使用线粒体靶向 γ 肽核酸清除突变体 mtDNA

基本信息

批准号：
10000215
负责人：
Bruce A. ARMITAGE
金额：
$ 19.26万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-22 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10000215
关键词：
Adult Affect Affinity Amino Acids Animals Binding Biochemical Biological Assay Biology Biophysics Biotin Cell Culture Techniques Cells Characteristics Chemistry Child Clinical Coupling DNA DNA Restriction Enzymes DNA Sequence DNA biosynthesis Disease Dose Excision Exhibits Fertilization in Vitro Foundations Funding Mechanisms Future Gel Genes Genetic Genetic Transcription Genome Genomic Instability Guidelines Hybrids Individual Invaded Length Life Measures Methods Mitochondria Mitochondrial DNA Mitochondrial Matrix Mothers Mus Mutation Nuclear Nucleic Acids Nylons Oligonucleotides Oxygen Consumption Pathogenicity Patients Peptide Nucleic Acids Peptides Phase Phenotype Polymerase Primer Extension RNA Reagent Replacement Therapy Resolution Site Solid Solubility Techniques Testing Time Translations Variant Vertebral column Work antigene base care burden clinically relevant design ds-DNA early childhood effective therapy experimental study functional improvement gene therapy high reward high risk in utero in vivo induced pluripotent stem cell mitochondrial DNA mutation mitochondrial genome mortality mutant new technology novel nucleobase prevent protein aminoacid sequence purge reconstitution restriction enzyme stem cells synthetic nucleic acid targeted treatment uptake

项目摘要

PROJECT SUMMARY Numerous diseases arise from the presence of both normal and mutant forms of mitochondrial DNA (mtDNA). Modest shifts in this heteroplasmy in favor of the normal mtDNA can have significant patient benefits. We propose to use PNA oligomers to bind selectively to mutant mtDNA and block its replication, resulting in a progressive shift in favor of normal mtDNA. PNA is the only synthetic oligonucleotide capable of binding to any sequence of double-stranded DNA and has recently been validated to effectively target nuclear DNA in live adult mice as well as in utero. We will functionalize PNAs with mitochondrial-penetrating peptides to promote cell uptake and localization. PNA will be synthesized by standard solid phase methods, then characterized in biophysical (gel mobility shift) and biochemical (inhibition of primer extension) experiments. PNA that exhibit highest affinity and greatest potent blockage of polymerase activity will then be studied in cell culture, where uptake, localization and phenotypic effects on heteroplasmy and mitochondrial oxygen consumption will be determined.

项目概要许多疾病都是由正常和突变形式的线粒体 DNA (mtDNA) 的存在引起的。这种有利于正常 mtDNA 的异质性的适度转变可以给患者带来显着的益处。建议使用 PNA 寡聚物选择性地结合突变体 mtDNA 并阻止其复制，从而产生有利于正常 mtDNA 的逐渐转变 - PNA 是唯一能够结合的合成寡核苷酸。任何双链 DNA 序列，最近已被验证可有效靶向活体核 DNA 我们将用线粒体穿透肽对 PNA 进行功能化，以促进成年小鼠和子宫内小鼠的生长。细胞摄取和定位将通过标准固相方法合成，然后进行表征。生物物理（凝胶迁移率变化）和生物化学（引物延伸抑制）实验显示 PNA。然后将在细胞培养中研究聚合酶活性的最高亲和力和最有效的阻断，其中对异质性和线粒体耗氧量的摄取、定位和表型影响将决定。