title: Study of adenovirus-mediated dystrophin minigene transfer to skeletal muscle by combined microscopic display of adenoviral DNA and dystrophin.
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.
authors.affiliation: Montreal Neurological Institute, Quebec, Canada.
authors.forename: J E
authors.initials: JE
authors.lastname: Zhao
authors.forename: H
authors.initials: H
authors.lastname: Lochumüller
authors.forename: J
authors.initials: J
authors.lastname: Nalbantoglu
authors.forename: C
authors.initials: C
authors.lastname: Allen
authors.forename: S
authors.initials: S
authors.lastname: Prescott
authors.forename: B
authors.initials: B
authors.lastname: Massie
authors.forename: G
authors.initials: G
authors.lastname: Karpati
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003587:Cytomegalovirus / Q000235:genetics; D004247:DNA; D004279:DNA, Viral; D016189:Dystrophin / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D018014:Gene Transfer Techniques; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D019076:Transgenes
chemical_list: D004279:DNA, Viral; D016189:Dystrophin; D004247:DNA