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（由申请人提供）：许多癌症来自细胞中遗传变化的逐渐积累。基因治疗方法以及识别具有异常基因或某些mRNA水平升高的癌细胞的技术包括设计和递送到反义和抗基因寡核苷酸的癌细胞中或它们的合成模拟物，例如肽核酸（PNAS）。 PNA具有高度稳定，对核酸酶和蛋白酶具有抗性，并以高亲和力的序列特异性方式结合RNA和DNA靶标。基因疗法的主要障碍之一是缺乏在体内选择性递送到癌细胞中的技术。在这里，我们提出了一项新技术，以选择性地递送到靶向涉及肿瘤生长和转移的mRNA的PNA癌细胞中。众所周知，肿瘤会发展出低氧和酸性的细胞外环境，尤其是在早期阶段。我们设计了一种溶解水的短肽，能够在低pH值（<6.5）处插入膜中，作为跨膜α-螺旋（<6.5），但在正常的pH值下（7.4）。该肽充当纳米气息：它以低pH值插入膜中，在细胞质中易位和释放各种分子，包括染料，毒素和PNA（Reshetnyak等，PNAS，PNAS，2006，2006，103，6460）。将荧光PNA易位到细胞中，并染色细胞质和核。 pH低插入肽（PHLIP）的转运机制基于跨膜结构域中两个ASP残基的质子化，并且该机制与所有报道的肽递送剂根本不同。全身成像表明，荧光PHLIP在小鼠的肿瘤中积聚。肿瘤中的积累之所以发生，是因为phlips在低pH值中插入膜中，而它们仅与正常pH的组织中细胞表面相互作用。用LYS或ASN残基替换两个ASP残基可以消除PHLIP在肿瘤中积聚的能力，这证实了提出的PHLIP插入细胞中的机制。我们的目标是开发这种纳米传感器技术，用于在体外和体内选择性细胞内抗义和抗抗原PNA的抗原PNA。我们计划通过二硫键连接到插入细胞内的肽末端的各种PNA。通过荧光显微镜，流式细胞仪，光谱法，全身成像，以及测量靶蛋白的表达水平和靶蛋白的表达水平，在体外和体内介导的PNA易位效率将在小鼠的不同细胞系上进行测试。细胞增殖和肿瘤生长。 phlip纳米喷射可能是对癌细胞分子分析以及诊断和治疗癌症的非常有效的工具。