DEVELOPMENT OF RECOMBINANT ADENOVIRUSES THAT DRIVE HIGH-LEVEL EXPRESSION OF THE HUMAN METALLOPROTEINASE-9 AND TISSUE INHIBITOR OF METALLOPROTEINASE-1 AND METALLOPROTEINASE-2 GENES - CHARACTERIZATION OF THEIR INFECTION INTO RABBIT SMOOTH-MUSCLE CELLS AND HUMAN MCF-7 ADENOCARCINOMA CELLS

Remodelling of the extracellular matrix resulting from increased secre tion of metalloproteinase enzymes (MMPs) is implicated in many patholo gical conditions, including rheumatoid arthritis, restenosis following balloon angioplasty, atherosclerosis and cancer cell invasion and met astasis. Clear definition of the normal and pathological function of i ndividual MMPs will benefit from approaches that use gene transfer to produce increases in MMP levels that mimic those observed in pathologi cal conditions. Similarly, gene transfer methods leading to controlled increases in lev els of the tissue inhibitor of metalloproteinases (T IMPs) will help to define the function of MMPs both in vitro and in vi vo. Gene transfer of TIMPs may also have therapeutic potential in path ological conditions where inhibition of MMP activity may be beneficial . We have used the adenovirus serotype 5 vector system to generate rep lication-deficient recombinant adenoviruses capable of expressing the MMP-9, TIMP-1 or -2 genes. High level expression is driven by the cyto megalovirus major immediate early promoter (CMV IEP). Efficient and se lective over-production of each recombinant protein was shown by immun ofluorescence in either rabbit smooth muscle cells (SMC) or human MCF- 7 adenocarcinoma cells. High level secretion directly dependent on the multiplicity of infection (MOI) was observed for each functional tran sgene by gelatin zymography. Using a quantitative ELISA assay, levels of recombinant TIMP-1 were detected when SMC were infected with as low as three plaque forming units (pfu) of virus per cell in vitro. A lin ear increase in TIMP-1 secretion was observed up to 1000 pfu/cell of v irus (0.75 ng/10(4) cells/24 h at 3 pfu/cell to 1243 ng/10(4) cells/24 h at 1000 pfu/cell). Similar levels of secretion of MMP-9 and TIMP-2 were observed by Western blot analysis using the same MOI of adenoviru s. Thus, recombinant adenoviruses are an efficient and flexible system for high level expression of MMPs and TIMPs and will be useful tools in the study of matrix remodelling in vivo and in vitro.