STUDY OF ADENOVIRUS-MEDIATED DYSTROPHIN MINIGENE TRANSFER TO SKELETAL-MUSCLE BY COMBINED MICROSCOPIC DISPLAY OF ADENOVIRAL DNA AND DYSTROPHIN

In situ DNA hybridization of an E-4 adenoviral sequence amplified by i n situ polymerase chain reaction (PCR) was used to mark adenovirus-con taining myonuclei in muscles of immunocompetent and immunosuppressed m dx mice following intramuscular injection of adenoviral recombinants, The adenoviral recombinants contained a 6.3-kb dystrophin cDNA (minige ne) driven by a cytomegalovirus (CMV) promoter/enhancer and thus, immu nostaining for dystrophin of the same sections permitted correlation o f adenoviral recombinant-containing myonuclei with dystrophin positivi ty of the same muscle fiber segments, As early as 2 hr post-injection of adenoviral recombinant, an appreciable number of adenoviral recombi nant-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 delay ed inactivation of the dystrophin expression cassette is probably unli kely, Thus, optimization of immunosuppression could assure successful long-term dystrophin gene transfer for gene therapy.