Development of a transient gene delivery vector to rejuvenate myofibroblasts for tissue engineering

Tissue engineering involves the use of synthetic or natural materials for the replacement of non-functional tissue or organs. One approach to tissue engineering involves harvesting the patients own cells, culturing them in vitro and then seeding the cells onto scaffolds. The use of autologous cells avoids problems associated with rejection, however a major limitation of this approach is the finite lifespan of primary cells in culture. This finite replicative lifespan is due to the shortening of telomeres, short repetitive sequences of DNA located at the ends of eukaryotic chromosomes. Ectopic expression of telomerase reverse transcriptase (hTERT) is able to reconstitute telomerase activity and maintain the length of telomeres. This study investigated an alternative gene delivery vector, the baculovirus, for the expression of hTERT in primary human cells. The aim was to rejuvenate cells for use in the tissue engineering of heart valves. The optimal conditions for the baculoviral transduction of primary fibroblasts were first determined. Transduction efficiencies of greater than 50% were routinely obtained, without the need for ultracentrifugation of the viral supernatant. A recombinant baculovirus was then used to efficiently deliver the hTERT gene to primary fibroblasts. The complete telomerase enzyme was found to be active, as evidenced by the Telomere Repeat Amplification Protocol. Although no increase in telomere length was detected, expression of hTERT in primary fibroblasts resulted in a significant extension of replicative lifespan. The transience of the gene delivery was confirmed with a baculoviral DNA half-life of approximately 10 days. To our knowledge this is a novel attempt to use a recombinant baculovirus for the extension of cellular lifespan by exogenous expression of telomerase.