Study of Adenovirus-Mediated Dystrophin Minigene Transfer to Skeletal Muscle by Combined Microscopic Display of Adenoviral DNA and Dystrophin

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Abstract
In situ DNA hybridization of an E4 adenoviral sequence amplified by in situ polymerase chain reaction (PCR) was used to mark adenovirus-containing myonuclei in muscles of immunocompetent and immunosuppressed mdx mice following intramuscular injection of adenoviral recombinants. The adenoviral recombinants contained a 6.3-kb dystrophin cDNA (minigene) driven by a cytomegalovirus (CMV) promoter/enhancer and thus, immunostaining for dystrophin of the same sections permitted correlation of adenoviral recombinant-containing myonuclei with dystrophin positivity of the same muscle fiber segments. As early as 2 hr post-injection of adenoviral recombinant, an appreciable number of adenoviral recombinant-positive (AVR+) myonuclei, and some partial dystrophin positive (pdys+) fibers were observed. Some fully dystrophin-positive (dys+) muscle fibers were present as early as 6 hr. The maximum number of fibers containing AVR+ myonuclei (observed by 72 hr) was maintained until 60 days in immunosuppressed, but not in immunocompetent, animals. In immunocompetent animals, the maximum number of dys+ fibers was observed at 10 days. The vast majority of these fibers contained AVR+ myonuclei; however, by 60 days, dys+ fibers disappeared with some AVR+ myonuclei persisting. Our studies suggest that widespread delayed inactivation of the dystrophin expression cassette is probably unlikely. Thus, optimization of immunosuppression could assure successful long-term dystrophin gene transfer for gene therapy. Demonstration of adenoviral vector DNA (by in situ polymerase chain reaction) and dystrophin (by immunocytochemistry) in the same sections of mdx skeletal muscle was used to investigate important aspects of adenovirus-mediated dystrophin minigene transfer. We determined the approximate time course of journey of the injected adenoviral recombinants into myonuclei and correlated the prevalence of adenoviral recombinant-positive myonuclei with dystrophin expression in the same fiber segments at early and late postinjection time points in tibialis anterior muscles of immunocompetent and immunosuppressed (FK506) mdx mice. We demonstrated the presence of adenoviral recombinant-positive (AVR+) myonuclei and the beginning of probable dystrophin gene expression by 2 hr post-injection. The peak level of AVR+ myonuclei was already attained at 72 hr and was maintained in immunosuppressed animals at 60 days. Maximum dystrophin expression was observed at 10 days and was maintained at 60 days only in immunosuppressed animals. In the majority of fibers at 10 and 60 days, there was a good correlation between the prevalence of AVR+ myonuclei and dystrophin expression in the same fiber segments. The 60-day data also suggest that substantial delayed inactivation of the dystrophin expression cassette is unlikely in either immunocompetent or immunosuppressed animals.