ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION IN COMBINATION WITH THE PIGGYBAC

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 乳腺癌是世界范围内诊断出的最常见的女性癌症，估计影响10%的女性。一半以上的病例发生在工业化国家，虽然由于早期发现，乳腺癌的死亡率有所下降，但在世纪的最后25年里，这些国家的乳腺癌发病率上升了30%。因此，开发治疗乳腺癌的新技术应该是主要的兴趣，基因治疗在这方面提供了一种有前途的方法。自杀基因或基因导向的酶前体药物治疗（GDEPT）是一种选择性靶向癌细胞的策略。在这里，癌症的治疗是基于将非哺乳动物基因转移到肿瘤细胞中。这些转基因编码的酶选择性地将无毒前药转化为高毒性代谢物。例如，单纯疱疹病毒胸苷激酶（HSVtk）基因在哺乳动物细胞中的表达使得这些细胞在被病毒酶磷酸化后对无害核苷酸类似物如更昔洛韦（GCV）的细胞毒性作用敏感。用HSVtk/GCV处理不仅杀死表达HSVtk的细胞，而且杀死不表达HSVtk基因的邻近细胞，大大增强了HSVtk介导的细胞毒性的功效。病毒系统是最有效的递送系统;然而，免疫原性、致突变性的可能性以及产生大量纯病毒的困难和费用的问题阻碍了它们在临床环境中的成功应用。相比之下，非病毒系统有几个优点：它们容易生产，成本低廉，能够整合大的转基因，并且不会引起免疫反应。然而，迄今为止，许多非病毒基因治疗策略受到这种基因递送系统的效率和缺乏稳定的染色体整合的限制。转座因子（转座子）是移动的遗传因子，其代表新型非病毒DNA递送系统，其提供转基因的长期表达，因为它们能够有效且永久地整合到宿主基因组中。PiggyBac（pB）是一种最初从甘蓝夜蛾Trichoplusia ni中分离的转座因子，在体外多种细胞和小鼠体内实验中显示出有效的转座和长期的转基因表达。最近，超声靶向微泡破坏（UTMD）已被提出作为基因递送的安全和有效的手段。微泡通常用作超声成像中的造影剂，并且也已经显示微泡增强超声可以短时间改变细胞膜渗透性，允许细胞外大分子如质粒DNA瞬时进入细胞而没有细胞毒性。超声的时间和空间施用允许在精确的时间点将基因受控地递送到特定组织或器官。在本申请中，我们建议评估使用UTMD与piggyBac转座子系统组合作为增强人乳腺癌细胞异种移植物中的基因导向酶前药治疗的工具的可行性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Breast cancer is the most common form of cancer diagnosed in women worldwide, affecting an estimated 10% of women. More than half of the cases are in industrialized countries and while the rate of mortality as a result of breast cancer has decreased due to early detection, the incidence of breast cancer has risen by 30% in these countries in the last quarter of a century. Developing novel techniques for treating breast cancer should therefore be of primary interest and gene therapy offers a promising approach in that regard. Suicide-gene or gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. Here, treatment of cancer is based on the transfer of non-mammalian genes into tumor cells. These transgenes encode enzymes that selectively convert non-toxic prodrugs to highly toxic metabolites. For example, expression of the herpes simplex virus thymidine kinase (HSVtk) gene in mammalian cells renders these cells susceptible to the cytotoxic effects of innocuous nucleotide analogues such as ganciclovir (GCV) after phosphorylation by the viral enzyme. Treatment with HSVtk/GCV not only kills HSVtk expressing cells but also neighboring cells that do not express the HSVtk gene, greatly enhancing the efficacy of HSVtk-mediated cytotoxicity. Viral systems are the most efficient delivery systems; however, problems with immunogenicity, the potential for mutagenicity and the difficulty and expense of producing large amounts of pure virus hamper their successful application in a clinical setting. In contrast, non-viral systems have several advantages: they are easy and inexpensive to produce, are able to incorporate large transgenes and do no illicit an immune response. However, to date many non-viral gene therapy strategies have been limited by the efficiency of this gene delivery systems and the lack of stable chromosomal integration. Transposable elements (transposons) are mobile genetic elements that represent a novel non-viral DNA delivery system that provides long-term expression of transgenes as they are able to efficiently and permanently integrate into the host genome. PiggyBac (pB), a transposable element originally isolated from the cabbage looper moth Trichoplusia ni showed efficient transposition and long-term transgene expression in vitro in a variety of cells and in vivo in mouse experiments. Recently, ultrasound-targeted microbubble destruction (UTMD) has been proposed as safe and efficient means for gene delivery. Microbubbles are commonly used as contrast agents in ultrasound imaging and it has also been shown that microbubble enhanced ultrasound can alter cell membrane permeability for a short time, allowing extracellular macromolecules such as plasmid DNA to instantaneously enter cells without cytotoxicity. Temporal and spatial administration of the ultrasound allows for controlled delivery of genes to specific tissues or organs at precise time points. In this application we propose to evaluate the feasibility of using UTMD in combination with the piggyBac transposon system as a tool to enhance gene directed enzyme prodrug therapy in human breast adenocarcinoma cell xenografts.