Life-Long Transgene Expression in Skeletal Muscle Without Transduction of Satellite Cells Following Embryonic Myogenic Progenitor Transduction by Lentivirus Administered in Utero.

Embryologic events in mammalian myogenesis remain to be fully defined. Recent evidence supports the presence of a common progenitor arising in the dermomyotome that gives rise to both embryologic and adult muscle and postnatal myogenic stem cells (satellite cells). In this study, we utilize the technique of early intra-amniotic gene transfer to target nascent muscle progenitors as they traverse the primitive streak before formation of the dermomyotome. This technique robustly transduced both epaxial and hypaxial muscle groups. Marker gene expression is observed in up to 100% muscle fibers in the lower extremities and is sustained for the lifetime of the mouse. We next analyzed transduced muscle for satellite cell transduction using highly sensitive methodology. Surprisingly, despite high levels of sustained transgene expression in muscle fibers, satellite cells lacked the marker transgene. Our data suggest that dermatomyotome is a heterogeneous structure and that not all myogenic progenitors of dermatomyotome give rise to satellite cells.

Developmental stage determines efficiency of gene transfer to muscle satellite cells by in utero delivery of adeno-associated virus vector serotype 2/9.

Efficient gene transfer to muscle stem cells (satellite cells) has not been achieved despite broad transduction of skeletal muscle by systemically administered adeno-associated virus serotype 2/9 (AAV-9) in mice. We hypothesized that cellular migration during fetal development would make satellite cells accessible for gene transfer following in utero intravascular injection. We injected AAV-9 encoding green fluorescent protein (GFP) marker gene into the vascular space of mice ranging in ages from post-coital day 12 (E12) to postnatal day 1 (P1). Satellite cell transduction was examined using: immunohistochemistry and confocal microscopy, satellite cell migration assay, myofiber isolation and FACS analysis. GFP positive myofibers were detected in all mature skeletal muscle groups and up to 100% of the myofibers were transduced. We saw gestational variation in cardiac and skeletal muscle expression. E16 injection resulted in 27.7 ± 10.0% expression in satellite cells, which coincides with the timing of satellite cell migration, and poor satellite cell expression before and after satellite cell migration (E12 and P1). Our results demonstrate that efficient gene expression is achieved in differentiated myofibers and satellite cells after injection of AAV-9 in utero. These findings support the potential of prenatal gene transfer for muscle based treatment strategies.