Impairment of Force Generation after Adenovirus-Mediated Gene Transfer to Muscle Is Alleviated by Adenoviral Gene Inactivation and Host CD8 + T Cell Deficiency

Abstract

Recombinant adenovirus vectors (AdV) hold promise as a means of delivering therapeutic genes to muscle in diseases such as Duchenne muscular dystrophy (DMD). However, we have previously shown that the use of AdV is hampered by the development of reduced force-generating capacity, which occurs within 1 week and is progressive up to at least 1 month after AdV delivery in immune-competent animals. Determinations of muscle force production provide a sensitive and clinically important measure of potential adverse effects of AdV-mediated gene transfer on muscle cell function. In the present study, we investigated the role of AdV-related gene expression and host T lymphocyte responses in the genesis of muscle dysfunction following AdV injection of muscle. We report that UV-irradiation of AdV particles, which reduced AdV transcriptional activity without impairing infectivity (as confirmed by in situ polymerase chain reaction), significantly reversed early (4 days post-injection) AdV-induced contractile impairment in immune-competent mice as well as in mice lacking effective CD8⁺ T cell activity. The superimposed additional reduction in force-generating capacity normally found between 4 and 30 days post-AdV delivery in immune-competent mice, along with the associated loss of transgene (β-galactosidase) expression, was largely abrogated by the absence of an intact CD8⁺ T lymphocyte response. Furthermore, short-term administration of a neutralizing antibody against CD4⁺ T cells significantly prolonged transgene expression and showed a trend toward mitigation of AdV-induced reductions in force-generating capacity. Cellular infiltration and humoral immune responses against the vector and transgene product were also blunted to varying degrees in the setting of CD8⁺ or CD4⁺ T cell deficiency. We conclude that AdV-related gene expression has an early negative (probably toxic) effect on muscle cell function that is independent of CD8⁺ T cell-mediated immunity. In contrast, further progression of contractile impairment and the accompanying loss of transgene expression from AdV-injected muscle are largely dependent upon the activity of CD8⁺ T cells. These results have implications for the design of future generation vectors and the potential need for immunosuppressive therapy after AdV-mediated gene transfer to muscle.

Overview summary

The effects of recombinant adenovirus vector (AdV) particle inactivation and interference with host CD4⁺ or CD8⁺ T cell function were examined with respect to muscle contractile function, transgene expression, and host immune responses (cellular and humoral). Both UV inactivation of AdV and CD8⁺ T cell deficiency were found to alleviate significantly AdV-induced reductions in muscle force-generating capacity. Brief (2-day) administration of a neutralizing antibody against host CD4⁺ T cells also produced a trend toward mitigation of AdV-induced contractile dysfunction. In addition, transgene expression 1 month after AdV delivery was significantly enhanced with inhibition of either CD4⁺ or CD8⁺ T cell function. The data indicate at least two sources of reduced force generation after AdV-mediated gene transfer to muscle: (i) a nonimmune toxic component of early onset associated with AdV transcriptional activity, and (ii) an immune-based component of more delayed onset that is dependent upon CD8⁺ T cell activity.