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利用低频超声进行皮肤基因治疗的新方法，并将其具体应用于DNA疫苗接种。 与减毒活疫苗相比，基因疫苗有几个优势。此外，由于朗格汉斯细胞的存在，皮肤为基因疫苗的传递提供了一个自然的靶点，朗格汉斯细胞在诱导免疫反应中发挥着关键作用。然而，将DNA输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来介导基因疫苗。这项拟议研究的总体目标是确定超声波进行基因疫苗接种的能力。拟议研究的具体目标如下： 1.通过体外和体内实验，对超声介导的DNA导入表皮角质形成细胞和朗格汉斯细胞的特性进行研究。这将通过将绿色荧光蛋白报告基因(pEGFP-C3)和乙肝疫苗PRC/CMV-HBs(S)输送到皮肤中实现。PEGFP-C3的运送将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绿色荧光蛋白的表达来定量。乙肝疫苗的投放将通过评估HBs(S)Ig G和肠道上皮中是否存在HBs(S)特异性抗原分泌细胞来确定。 2.通过体内实验，研究低频超声作用下朗格汉斯细胞向表皮的激活和迁移。 3.使用体外模型Epiderm和体内小鼠模型，评估皮肤对低频超声波的安全性。

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.