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New Technology for Selective Delivery of PNAs in Cancer Cells In Vitro and In Viv

体外和体内癌细胞中选择性递送 PNA 的新技术

基本信息

批准号：
7483276
负责人：
Oleg A Andreev
金额：
$ 12.98万
依托单位：
UNIVERSITY OF RHODE ISLAND
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-10 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7483276
关键词：
Acidity Affinity Amides Attention Base Pairing Binding Biological Biological Assay Breast Adenocarcinoma Breast Carcinoma CCNB1 gene Cancerous Cell Line Cell Nucleus Cell Proliferation Cell Survival Cell membrane Cell surface Cells Cleaved cell Codon Nucleotides Complementary DNA Cyclic Peptides Cytoplasm Cytosol DNA Development Diagnosis Diagnostic Disease Disulfide Linkage Drug Delivery Systems Dyes Endopeptidases Engineering Environment Evaluation Flow Cytometry Fluorescence Microscopy Fluorescent Dyes Gene Delivery Gene Expression Gene Expression Regulation Gene Targeting Genes Goals Human Hypoxia Imagery In Vitro Investigation Label Lead Link MCF7 cell Malignant Epithelial Cell Malignant Neoplasms Maturation-Promoting Factor Measures Mediating Membrane Messenger RNA Modeling Molecular Molecular Analysis Mus Mutation Neoplasm Metastasis Nucleic Acids Nude Mice Object Attachment Oligonucleotides Open Reading Frames Organ Peptide Hydrolases Peptide Nucleic Acids Peptides Personal Satisfaction Pharmacologic Substance Protein Kinase Proteins RNA RNA Binding Rate Reporting Resistance Spectrum Analysis Staging Staining method Stains Surface Syringes Techniques Technology Testing Therapeutic Time Tissues Toxin Translations Transmembrane Domain Tumor Tissue Vertebral column Water Western Blotting alpha helix analog antigene base cancer cell cancer therapy cyclin B1 design extracellular fluorescence imaging gene therapy imaging probe in vivo mutant neoplastic cell new technology nuclease protonation research study tool tumor tumor growth whole body imaging

项目摘要

DESCRIPTION (provided by applicant): Many cancers arise from the gradual accumulation of genetic changes in cells. Gene therapy approaches as well as techniques for recognizing cancer cells with abnormal genes or elevated levels of certain mRNAs include the design and delivery into cancerous cells of antisense and antigene oligonucleotides or their synthetic mimics such as peptide nucleic acids (PNAs). PNAs are highly stable, resistant to nucleases and proteases, and bind RNA and DNA targets in a sequence-specific manner with high affinity. One of the main obstacles for gene therapy is a lack of technology for selective delivery of gene agents into cancer cells in vivo. Here we propose a new technology for selective delivery into cancer cells of PNAs targeting mRNAs involved in tumor growth and metastasis. It is well established that tumors develop a hypoxic and acidic extracellular environment, especially in the earlier stages. We designed a short peptide that is soluble in water and able to insert into the membrane as a transmembrane alpha-helix at low pH (<6.5) but not at normal pH (7.4). The peptide acts as a nanosyringe: it inserts in the membrane at low pH, translocates and releases in the cytoplasm various molecules, including dyes, toxins, and PNAs (Reshetnyak et al., PNAS, 2006, 103, 6460). The fluorescent PNAs are translocated into cells and stain the cytoplasm and nuclei. The mechanism of translocation of pH Low Insertion Peptides (pHLIPs) is based on a protonation of two Asp residues in the transmembrane domain, and this mechanism is fundamentally different from all reported peptide delivery agents. Whole-body imaging revealed that fluorescent pHLIPs accumulate in tumors in mice. The accumulation in tumors occurs because pHLIPs insert in the membrane at low pH while they interact only weakly with the surfaces of cells in tissues at normal pH. The replacement of two Asp residues by Lys or Asn residues eliminates the ability of pHLIPs to accumulate in tumors, which confirms the proposed mechanism of insertion of pHLIPs into cells. Our goal is to develop this nanosyringe technology for selective intracellular delivery of antisense and antigene PNAs into cancer cells in vitro and in vivo. We plan to conjugate various PNAs via disulfide linkages to the end of the peptide that inserts inside a cell. The efficiency of translocation of PNAs mediated by pHLIPs will be tested on different cell lines in vitro and in vivo in mice using fluorescence microscopy, flow cytometry, spectroscopy, whole-body imaging, and by measuring of level of expression of target proteins and rates of cell proliferation and tumor growth. The pHLIP nanosyringe could be a very effective tool for molecular analysis of cancer cells and diagnosis and treatment of cancer.

描述(申请人提供)：许多癌症是由细胞中基因变化的逐渐积累引起的。基因治疗方法以及识别具有异常基因或某些mRNAs水平升高的癌细胞的技术包括设计反义和反基因寡核苷酸或其合成模拟物，如肽核酸(PNA)，并将其输送到癌细胞中。PNA高度稳定，对核酸酶和蛋白水解酶具有抗性，并以序列特异性的方式与RNA和DNA靶标结合，亲和力高。基因治疗的主要障碍之一是缺乏将基因制剂选择性地输送到体内癌细胞的技术。在这里，我们提出了一种新的技术，以靶向参与肿瘤生长和转移的mRNAs为靶点，将PNA选择性地输送到癌细胞中。众所周知，肿瘤形成一个低氧和酸性的细胞外环境，特别是在早期阶段。我们设计了一种可溶于水的短肽，能够在低pH(6.5)下以跨膜α-螺旋的形式插入膜中，但在正常pH(7.4)下不能。多肽充当纳米注射器：它在低pH值下插入细胞膜，在细胞质中转移和释放各种分子，包括染料、毒素和PNA(Reshetnyak等人，PNAS，2006,103,6460)。荧光的PNA被转移到细胞内，并对细胞质和细胞核进行染色。PH低插入多肽的转运机制是基于跨膜区两个天冬氨酸残基的质子化，这一机制与所有已报道的多肽递送剂有根本的不同。全身成像显示，荧光PHLIP在小鼠肿瘤中积聚。在肿瘤中的蓄积是因为PHLIP在低pH时插入到膜中，而在正常pH下它们只与组织中的细胞表面弱相互作用。两个天冬氨酸残基被赖氨酸或天冬氨酸残基取代后，PHLIPs在肿瘤中的蓄积能力消失，这证实了PHLIPs插入细胞的机制。我们的目标是开发这种纳米注射器技术，在体外和体内将反义和反基因PNA选择性地输送到癌细胞中。我们计划通过二硫键将不同的PNA连接到插入细胞内的多肽的末端。利用荧光显微镜、流式细胞仪、光谱分析、全身成像技术，通过检测靶蛋白的表达水平、细胞增殖率和肿瘤生长速度，在不同的细胞系上测试PHLIPs介导的PNAs的转位效率。PHLIP纳米注射器可能是一种非常有效的肿瘤细胞分子分析和癌症诊断和治疗的工具。