GFP expression in RGC and bipolar cells.

<p>Representative retina sections from adult mice treated for double-immunofluorescence analysis four weeks after the injection of 2×10¹² vg of scAAV9-GFP vectors into the tail vein. (A–C) Double-labeling of transduced RGCs for GFP (green) and Brn-3a (red) (arrowheads indicate double-labeled RGCs and arrows indicate cells expressing GFP only). A transduced photoreceptor is highlighted with an asterisk. (D–F) Double-labeling of bipolar cells for GFP (green) and Chx10 (red). The arrow indicates a Chx10-positive bipolar cell with high levels of GFP. The retinal nerve fiber layer (RNFL), labeled for GFP due to the transduction of upstream RGC, is indicated by an asterisk. No RGC can be seen on this panel which illustrates the central part of the retina (as demonstrated by the thickness of the RNFL). ONL: outer nuclear layer; INL: inner nuclear layer; RGC: retinal ganglion cell layer. Scale bar: 50 µm.</p

GFP expression in the retina after the intravenous delivery of scAAV9-GFP in adult mice.

<p>Retinal cross sections were treated for GFP immunofluorescence (green) and counterstained with DAPI (blue), four weeks after the injection of 2×10¹² vg of scAAV9-GFP into the tail veins of eight-week-old mice. GFP was detected in all retina layers (A–D) and in the ciliary bodies (CB in A). Transduction efficiency was particularly high in the RGC layer (B–C) but GFP was also expressed in the various cell types of the inner nuclear layer (INL), including cells with the morphology of bipolar cells (arrowheads in C) and of Müller cells (arrows in D). Rare GFP-positive photoreceptors (asterisks in B and D) and RPE cells (arrowheads in D and F) were also detected. (E–G) High magnification of GFP-positive (E) Müller cells, (F) RPE cells and (G) photoreceptors. RPE: retinal pigment epithelium; ONL: outer nuclear layer; INL: inner nuclear layer; RGC: retinal ganglion cell layer. Scale bar: 200 µm in A and B; 70 µm in C; 50 µm in D; 20 µm in E–G. A, B and E–G are epifluorescence images; C and D are confocal images.</p

Systemic injection of AAV serotype 2 does not lead to transduction of the neural retina.

<p>GFP expression in representative cross-sections of the retina of adult mice one month after systemic administration of 2.10¹² vg scAAV-GFP of serotype 9 (A–F) or serotype 2 (G–L) in adult mice (n = 3 per condition). GFP expression was detected in the neural retina in all mice from the serotype 9 treated-group (panel A to F are from three different animals). As expected, the highest transduction efficiency was observed at the level of the RGC layer. In contrast, no GFP expression was detected in the retina after AAV serotype 2 injection (panels G to L are from three different animals). Left panels: GFP immunofluorescence; right panels: merged view of GFP (green) and dapi (blue). ONL: outer nuclear layer; INL: inner nuclear layer; RGC: retinal ganglion cell layer. Scale bar: 100 µm.</p

GFP expression in the optic nerve and the ciliary body of intravenous scAAV9-GFP injected adult mice.

<p>Representative cross sections of the optic nerve (A) and the ciliary body (B) treated for GFP immunofluorescence (green) and stained with DAPI (blue), four weeks after the injection of 2×10¹² vg of scAAV9-GFP into the tail veins of eight-week-old mice. In (A), the boundaries of the retinal nerve fiber layer (originating from the RGC) are clearly demarcated by their pattern of GFP expression (arrowheads) (arrows: GFP-positive axons in the optic nerve). (B) High magnification of the ciliary body, showing strong GFP expression in the epithelial cells. ON: optic nerve; RET: retina; TM: trabecular meshwork. Scale bar: 100 µm.</p

Additional file 1: of Skeletal muscle characteristics are preserved in hTERT/cdk4 human myogenic cell lines

Supplemental data. (DOCX 1029 kb

Clinical features of the DMD patients ordered by increasing age.

<p>Clinical features of the DMD patients ordered by increasing age.</p

Patient flow-chart.

<p>Patient flow-chart.</p

Reliability between test and retest for all the tasks evaluated for each ActiMyo^® variable.

<p><b>◇</b> MoviPlate; <b>⬜</b> BBT; <b>△</b> Minnesota test; <b>✱</b> Writing;<b>×</b> PC typing.</p

The tools.

<p><b>(A)</b> First version of ActiMyo^®, used in the current study. <b>(B)</b> Box and Block test. <b>(C)</b> Minnesota test with five discs.</p

The maximal scores on tasks completed by each patient at the two visits sorted by the decreasing age.

<p>The maximal scores on tasks completed by each patient at the two visits sorted by the decreasing age.</p