Cutaneous Gene Therapy with Ultrasound: DNA Vaccination

描述（由申请人提供）： 该研究的目的是开发一种新的皮肤基因治疗方法，使用低频超声波与特定应用的DNA疫苗接种。 基因疫苗比减毒活疫苗有几个优点。此外，皮肤提供了用于递送基因疫苗的天然靶位点，这是由于朗格汉斯细胞的存在，朗格汉斯细胞在诱导免疫应答中起关键作用。然而，将DNA递送到皮肤是一个挑战。我们假设低频超声通过表皮细胞的转染和朗格汉斯细胞向淋巴结的迁移介导基因疫苗接种。拟议研究的总体目标是建立超声波进行遗传疫苗接种的能力。拟议研究的具体目标如下： 1.通过体外和体内实验，表征超声介导的DNA递送到表皮角质形成细胞和朗格汉斯细胞中。这将通过将绿色荧光蛋白（pEGFP-C3）和B型肝炎疫苗pRc/CMV-HBs（S）的报告基因递送到皮肤中来实现。通过荧光显微镜和流式细胞术测量角质形成细胞和朗格汉斯细胞中的绿色荧光蛋白表达来定量pEGFP-C3的递送。通过评估肠上皮中的HBs（S）IgG和Hbs（S）特异性抗原分泌细胞的存在来确定B型肝炎疫苗的接种。 2.通过体内实验，表征低频超声引起的朗格汉斯细胞活化和迁移到表皮中。 3.使用体外模型、Epidermal和体内小鼠模型，评估皮肤暴露于低频超声的安全性。

DESCRIPTION (provided by applicant): The objective of the proposed study is to develop a novel method of cutaneous gene therapy using low frequency ultrasound with specific application to DNA vaccination. Genetic vaccines offer several advantages over live-attenuated vaccines. Furthermore, skin offers a natural target site for the delivery of genetic vaccines due to the presence of Langerhans cells that play a critical role in inducing an immune response. However, delivery of DNA to skin is a challenge. We hypothesize that low-frequency ultrasound mediates genetic vaccination through transfection of epidermal cells and migration of Langerhans cells to the lymph node. The general goal of the proposed study is to establish the ability of ultrasound to perform genetic vaccination. The specific aims of the proposed study are as follows: 1. Through in vitro and in vivo experiments, characterize ultrasound-mediated DNA delivery into epidermal keratinocytes and Langerhans cells. This will be achieved by delivering a reporter gene for green fluorescent protein (pEGFP-C3) and hepatitis B vaccine pRc/CMV-HBs(S) into skin. Delivery of pEGFP-C3 will be quantified by measuring green fluorescent protein expression in keratinocytes and Langerhans cells by fluorescent microscopy and flow cytometry. Delivery of hepatitis B vaccine will be determined by assessing HBs(S) IgG and presence of Hbs(S) specific antigen secreting cells in the intestinal epithelium. 2. Through in vivo experiments, characterize activation and migration of Langerhans cells into epidermis due to low-frequency ultrasound. 3. Using an in vitro model, Epiderm, and in vivo mouse model, assess safety of skin exposure to low-frequency ultrasound.