Endocrine Abstracts

BACKGROUND: Atherosclerosis is a major cause of mortality in diabetes mellitus. Gene therapy may be a useful approach to the treatment of this disorder. One of the main obstacles to the use of this therapeutic modality is the difficulty in obtaining safe and efficient gene transfer in vivo. Adenoassociated virus vector have been used in a number of previous studies but have never been used for vascular gene delivery. OBJECTIVE: Our goal was to characterize AAV mediated gene delivery to vascular cell lines in vitro.

METHODS: HCSMC (Human coronary smooth muscle cell), HAEC (human aortic endothelial cell), HUVEC (human umbilical vein endothelial cell) were plated in 6 well plates and transduced with MOIs of 0 to 100,000 using AAV beta Gal. Efficiencies of gene transfer were assessed by observers at 3, 9 and 14 days. The effects of Etoposide(DNA synthesis inhibitor) and Mg-132 (proteasome inhibitor) on efficiency were also studied.

RESULTS: No transgene expression was noted following AAV mediated gene transfer of beta-Galactosidase to HAEC and HUVEC at3,9 and 14 days. In contrast, inefficient gene transfer to HCSMC was demonstrated in a dose-dependant manner with optimal expression at 9 days, as compared to 3 and 14 days. Use of Etoposide and MG-132 resulted in more efficient gene transfer.

CONCLUSION: AAV-2 was unable to transduce endothelial cells and resulted in inefficient gene delivery to human smooth muscle cells. High viral titres are required and optimal time point of expression appears to be 9 days. Addition of Etoposide and Mg132 enhances expression. Further studies with AAV beta-Gal in vivo will help to further characterize this vector system.