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

描述（由申请人提供）：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mrna水平升高的癌细胞的技术包括设计和将反义和抗原寡核苷酸或其合成模拟物（如肽核酸（PNAs））递送到癌细胞中。PNAs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。基因治疗的主要障碍之一是缺乏在体内将基因制剂选择性地递送到癌细胞中的技术。在这里，我们提出了一种新的技术，选择性递送到肿瘤细胞的PNAs靶向mrna参与肿瘤生长和转移。众所周知，肿瘤发展为缺氧和酸性的细胞外环境，特别是在早期阶段。我们设计了一种短肽，它可溶于水，在低pH值（<6.5）下能够以跨膜α -螺旋的形式插入膜中，而在正常pH值（7.4）下则不能。肽就像一个纳米注射器：它在低pH下插入细胞膜，在细胞质中移位并释放各种分子，包括染料、毒素和PNAs （Reshetnyak et al., PNAs, 2006,103, 6460）。荧光PNAs被转移到细胞中并染色细胞质和细胞核。pH低插入肽（pHLIPs）的易位机制是基于跨膜结构域中两个Asp残基的质子化，这一机制与所有报道的肽递送剂有着根本的不同。全身成像显示荧光philips在小鼠肿瘤中积累。在肿瘤中积累是因为pHLIPs在低pH下插入细胞膜，而在正常pH下它们与组织细胞表面的相互作用很弱。用Lys或Asn残基取代两个Asp残基消除了pHLIPs在肿瘤中积累的能力，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，在体外和体内选择性地将反义和抗原PNAs输送到癌细胞中。我们计划通过二硫键将各种PNAs偶联到插入细胞内的肽的末端。通过荧光显微镜、流式细胞术、光谱学、全身成像，以及靶蛋白表达水平、细胞增殖和肿瘤生长速率的测量，我们将在小鼠体内和体外不同细胞系上测试philips介导PNAs易位的效率。philips纳米注射器可以成为一种非常有效的工具，用于癌细胞的分子分析和癌症的诊断和治疗。

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.