INTERACTIONS BETWEEN MECHANICAL LOADING, GENE TRANSFER,

AND GENE EXPRESSION IN SEVERAL CELL TYPES IN VITRO

Participants: M. Ominsky, M. Moalli, B. Roessler, A. Bielinska, R. Dennis, M. Stock, D. Kayner, M. Shaw, S. Goldstein

Keywords: gene therapy, cell culture, mechanical stimulation

Introduction

This study is designed to examine the effect of mechanical loading on the transgene uptake and expression in in vitro populations of cells. Changes in the mechanical environment have been shown to alter gene expression in many cell types, including both fibroblasts and osteoblasts. It is hypothesized that transfected cells will be likewise effected, possibly yielding higher transgene production per cell and a larger percentage of transfected cells. The elucidation of a symbiotic effect of mechanical factors on gene therapy could increase its effectiveness in many in vivo and ex vivo applications including tissue engineering approaches to fracture healing, cartilage repair, and engineered blood vessels. Other applications of this study are in the development of gene knockout strategies in hard-to-transfect cells or to more closely examine signal transduction pathways.

Materials and Methods

Several different cell types, modes of mechanical stimulation, and plasmid vectors have been chosen to characterize this relationship. Plasmid uptake will be evaluated using the techniques of Professor Kevin Rice of the school of Pharmacology. Briefly, ¹²⁵I will be incorporated into supercoiled plasmid, allowing post-transfection plasmid uptake to be measured in situ via autoradiography. Transgene protein expression will be measured using a firefly luciferase expression plasmid, which when translated reacts with an enzyme to luminesce. The luciferase expression will be quantified using a luminometer, and normalized to total protein quantity, as determined by spectrophotometry. Transgene expression at the transcriptional level will be evaluated using Northern blotting techniques with a designed luciferase mRNA oligonucleotide probe.

Plasmid vectors will be designed and constructed with different promoters and conformations to see how mechanical factors affects transcription. Use of transfection enhancers such as liposomes may be necessary for some cell types to get a baseline plasmid expression in vitro. Gene transfer into fibroblasts, myoblasts, and osteoblasts will all be examined with respect to their dynamic mechanical environments.

Progress

Preliminary work has focused on the development of a cyclic hydrostatic pressure device and protocols to transfect with plasmid DNA. The current device has applied controlled cyclic pressures to an enclosed cell chamber up to 3.5psi. Initial experiments have determined optimum quantities of DNA (CMV-luciferase plasmid) and liposomal complex (Lipofectamine) for the transfection of a fibroblastic cell line. Subsequent experiments revealed a possible downregulation of CMV promoter transcriptional activity due to 30 minutes of cyclic hydrostatic loading, an unexpected but not unreasonable finding. Other promoters which may be upregulated by cyclic pressure (a - & b -actin) will be examined, and DNA uptake will be closely monitored. A new adjunct system to the pressure system is currently being calibrated which will allow the cell seeded flexible substrates to be cyclically stretched up to 15% with and without additional hydrostatic pressure.

A collaboration with Dr. Robert Dennis in the Institute of Gerontology has been undertaken to study gene uptake and expression in his in vitro muscle fiber (or myooid) system. The engineered fibers can be stretched or can actively contract, making it an ideal system for the proposed hypotheses, and a step toward possible in vivo studies.