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 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.
Zhao
J E
JE
Montreal Neurological Institute, Quebec, Canada.
Lochumüller
H
H
Nalbantoglu
J
J
Allen
C
C
Prescott
S
S
Massie
B
B
Karpati
G
G
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
D004279:DNA, Viral; D016189:Dystrophin; D004247:DNA