Persistent postnatal transgene expression in both muscle and liver after fetal injection of recombinant adenovirus.
OBJECTIVE
Immune responses to both vector and transgene antigens have limited the efficacy of postnatal gene therapy. We hypothesize that the fetal period may offer immunologic and developmental advantages for successful gene therapy. In this study we examined the efficacy, persistence, and immunologic effects of recombinant adenovirus after intramuscular delivery into fetal mice.


METHODS
E1-deleted adenovirus (AdCMVlacZ) containing the beta-galactosidase marker gene was used for injection. Fetal Balb/c mice (14 to 15 days' gestation) were injected with AdCMVlacZ in 10-microL volume in either the shoulder or hindlimb musculature. Animals were killed at 18 to 20 days' gestation and up to 4 months postnatally for analysis of transgene expression and adenoviral genome persistence.


RESULTS
Fetuses were injected with doses of AdCMVlacZ from 1 x 10(8) to 2 x 10(10) viral particles (n = 80). Optimal survival rate was 83% at 18 to 20 days' gestation and 55% at 4 weeks of age using a dose of 1 x 10(9) particles. Expression of beta-galactosidasae at 18 to 20 days localized to multiple muscle groups surrounding the site of injection, as well as bone marrow stroma, liver, lung, and dorsal root ganglia. Persistent muscle and liver transgene expression was observed for as long as 16 and 8 weeks, respectively, after injection. The pattern of liver expression was confined to discrete foci of hepatocytes, which appeared to increase in size in older animals. No histological evidence of muscle or liver inflammation was observed at any time after injection. No neutralizing antibodies were observed postnatally.


CONCLUSIONS
Our results confirm that gene therapy in the fetus may be advantageous. Distribution of vector in the fetus at the site of injection is clearly broader than in the adult setting. Furthermore, the absence of immune response and persistence of transgene expression suggests that fetal exposure to foreign transgene and vector antigens may induce tolerance. Although we have not proven genomic integration, the histological appearance of transgene expression in the liver supports this conclusion. By understanding the mechanisms that underlie persistent transgene expression, fetal gene therapy may become a feasible strategy for the treatment of fatal genetic diseases.
Yang
E Y
EY
The Children's Institute for Surgical Science, Department of Surgery, The Children's Hospital of Philadelphia, PA 19104, USA.
Kim
H B
HB
Shaaban
A F
AF
Milner
R
R
Adzick
N S
NS
Flake
A W
AW
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D005240:Feasibility Studies; D005315:Fetal Diseases / Q000628:therapy; D005333:Fetus / Q000502:physiology*; D015870:Gene Expression; D015316:Genetic Therapy; D016679:Genome, Viral; D007150:Immunohistochemistry; D008099:Liver / Q000196:embryology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D018482:Muscle, Skeletal / Q000196:embryology / Q000378:metabolism*; D019076:Transgenes; D001616:beta-Galactosidase / Q000378:metabolism
D001616:beta-Galactosidase