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.
