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

Abstract

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.

Figure 1

Following intravascular injection of AAV9 at E14 with a GFP reporter gene, a mouse examined at P30 under a fluorescent stereomicroscope reveals GFP positive muscles of the (a) upper body, (b) lower body, Panniculus carnosus (PC) muscle underneath the (c) skin, (d) intercostal muscles, (e) diaphragm (Image taken from above the diaphragm) and (f) muscles of the face. Injection at (g) E12 and (h) E14 result in expression of the endocardium of the heart. Injection at (i) E16 and (j) P1 result in expression throughout the entire myocardium. Tibialis anterior muscle, following intravascular injection at (k) E12, (l) E14, (m) E16, and (n) P1. (o–q) E12 injection results in GFP expression in slow twitch fibers (Mab 484 shown in red). (r) E14 injection results in GFP positive myofibers up to 1 year of age.

Figure 2

Myofibers were isolated from the legs of Myf5-nlacZ^Het mice injected at E14 with AAV 9 carrying GFP as a reporter and stained with β-galactosidase (βgal) antibody. βgal antibody positive (red), GFP positive cells were observed on the edge of transduced myofibers. (a–c) Transduced GFP positive and untransduced GFP negative satellite cells that are (e–g) βgal positive satellite cells (red) on the edge of a transduced myofiber. Satellite cells migrate off of a transduced myofiber during satellite cell migration assay (fiber loses GFP after dying in the assay). (d) Confocal microscopy of transduced satellite cells in tibialis anterior muscle imaged at 100× of P30 mice following (h–k) E14 and (l–o) E16 injection. βgal in red. GFP in green. β-2-Laminin in blue.

Figure 3

FACS analysis. Myf5^nlacZ/+ mice underwent Intravascular injection with AAV 9 carrying GFP as a reporter gene at E12, E14, E16, and P1. Following myoblast prep of injected legs, cells were analyzed by FACS. Lineage negative cells were gated upon (a). These cells were analyzed for α 7 integrin (y axis) and CD34 (x axis) (b) and revealed two peaks: lineage negative, α 7 positive, and CD34 positive satellite cells and lineage negative, α 7 positive and CD34 negative myoblasts. Animals injected at E12, E14, E16, and P1 were analyzed for GFP positive satellite cells and myoblasts. (c,d) E12 injected myoblasts and satellite cells revealed 0.1 ± 0.1 and 0.2 ± 0.1% GFP transduction respectively. (e,f) E14 injected myoblasts and satellite cells revealed 16.2 ± 3.6 and 13.1 ± 2.6% GFP transduction respectively. (g,h) E16 injected myoblasts and satellite cells revealed 26.6 ± 2.4 and 27.7 ± 10.0% GFP transduction respectively. (i,j) P1 injected myoblasts and satellite cells revealed 2.3 ± 0.3 and 1.8 ± 0.5% GFP transduction respectively. (f) The GFP positive Satellite cells were injected into irradiated Rag mice and following engraftment, injury with notexin and healing failed to show GFP positive muscle fibers but (k) X-gal (blue) cells could be found in the satellite cell position.