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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.

项目摘要许多疾病都是由正常和突变形式的线粒体DNA（mtDNA）的存在引起的。这种异质性中有利于正常mtDNA的适度变化可以对患者产生显著的益处。我们我建议使用cDNA-PNA寡聚体选择性地结合突变mtDNA并阻断其复制，导致逐渐向正常的mtDNA转变。BMPNA是唯一能够结合到双链DNA的任何序列，最近已被证实可有效靶向活体细胞核DNA 成年小鼠以及子宫内。我们将用穿膜肽功能化TNF-PNAs，细胞摄取和定位。将通过标准固相方法合成α-PNA，然后表征，生物物理（凝胶迁移率变化）和生物化学（引物延伸的抑制）实验。展示了然后将在细胞培养中研究聚合酶活性的最高亲和力和最大有效阻断，其中吸收，定位和表型对异质性和线粒体耗氧量的影响将是测定