Purging Mutant mtDNA Using Mitochondrially‐Targeted 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：
9808265
负责人：
Bruce A. ARMITAGE
金额：
$ 23.02万
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 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9808265
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 Stem cells 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 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.

项目摘要许多疾病是由线粒体DNA（mtDNA）的正常和突变形式的存在引起的。在这种异质中，适度的转移有利于正常的mtDNA可以具有显着的患者益处。我们建议使用PNA低聚物选择性地结合突变mtDNA并阻止其复制，从而导致A 进行性转移，有利于正常mtDNA。 PNA是唯一能够结合到与的合成寡核苷酸任何双链DNA序列成年小鼠以及子宫内。我们将用线粒体 - 渗透肽功能化PNA，以促进细胞吸收和本地化。 PNA将通过标准固相方法合成，然后在以生物物理（凝胶迁移率转移）和生化（抑制引物扩展）实验。展示的PNA 然后，最高亲和力和最大的聚合酶活性阻塞将在细胞培养中研究吸收，定位和表型对异质和线粒体氧的影响将是决定。