AAV6-mediated Systemic shRNA Delivery Reverses Disease in a Mouse Model of Facioscapulohumeral Muscular Dystrophy

Treatment of dominantly inherited muscle disorders remains a difficult task considering the need to eliminate the pathogenic gene product in a body-wide fashion. We show here that it is possible to reverse dominant muscle disease in a mouse model of facioscapulohumeral muscular dystrophy (FSHD). FSHD is a common form of muscular dystrophy associated with a complex cascade of epigenetic events following reduction in copy number of D4Z4 macrosatellite repeats located on chromosome 4q35. Several 4q35 genes have been examined for their role in disease, including FRG1. Overexpression of FRG1 causes features related to FSHD in transgenic mice and the FRG1 mouse is currently the only available mouse model of FSHD. Here we show that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of disease, led to a dose-dependent long-term FRG1 knockdown without signs of toxicity. Histological features including centrally nucleated fibers, fiber size reduction, fibrosis, adipocyte accumulation, and inflammation were all significantly improved. FRG1 mRNA knockdown resulted in a dramatic restoration of muscle function. Through RNA interference (RNAi) expression cassette redesign, our method is amenable to targeting any pathogenic gene offering a viable option for long-term, body-wide treatment of dominant muscle disease in humans

MyomiR-206 modulates Hmgb3 expression during myogenesis.

<p>(A) Hek cells were co-transfected with mimic-miR-206 at a concentration of 25 nM for 48 h and with pGLO-control or pGLOHmgb3 or pGLOHmgb3Mut. MiR-206 down-regulated the expression of luciferase of 32% (p = 0, 0151) in Hek cells co-transfected with pGLOHmgb3. Instead no down-regulation happened when the binding site of miR-206 was mutated. (B) 3T3 cells were transfected with mimic miR-206 at a concentration of 25 nM for 48 h, evidencing a strong down-regulation of endogenous Hmgb3 mRNAs. (C) Quantification of Hmgb3 mRNA by qRT-PCR in single fibres isolated from CTX-injured TA at day 2, 5, 7 and 10 from the injection evidenced a strong down-regulation of Hmgb3 during muscle regeneration. (D–E) Hmgb3 mRNA (D) and miR-206 (E) were respectively quantified by relative qRT-PCR and by absolute Q-PCR in proliferating C₁C₁₂ myoblasts cell line (MB) versus differentiated C₁C₁₂ myotubes (MT). The quantification analysis highlighted an opposite expression trend for Hmgb3 and miR-206, further validating inhibition of Hmgb3 by miR-206 (AU = arbitrary units). (Two-tail parametric t-test; *p value <0, 05; **p value <0,01; ***p value <0,001).</p

MiRNA profile of single fibres isolated from the mdx mouse.

<p>(A) Clustered heat map showing the expression ratios of miRNAs in TA, DIA and VA of age- and sex-matched c57bl (n = 3) and mdx (n = 3) mice. Expression data were normalized on Universal Reference. A total of 14 miRNAs were found over-expressed in dystrophic samples with distinction among myofibres isolated from different muscle type. (B) Fold change values of the 14 up-regulated miRNAs were reported in the table (ns = no significant).</p

Acute muscle damage activates miRNA machinery in isolated myofibers.

<p>Dystrophic-fibres associated miRNAs were quantified by Q-PCR in single fibres isolated from TA of c57bl mice (n = 10) after CTX-injection. Mice were sacrified at the day 2, 5, 7, 10 after CTX-injury. In the table are represented the fold change values of tested miRNAs in CTX-injected muscle compare to controlateral not injected TA. MiR-206, miR-31, miR-21, miR-335-5p, miR-27a, miR-142-5p and miR-223 were significantly up-regulated afterwards muscle damage respect damaged muscle. Otherwise the other 7 miRNAs were not triggered upon CTX-injection.</p><p>(Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

Identification of new murine dystrophic-fibres associated-miRNAs in muscle biopsies of DMD subjects.

<p>(A) The age and type of muscle of healthy subjects (12) and DMD patients (18) were listed. Moreover the type of muscle from which the biopsies were isolated were also reported. (B–C) MiR-15b, miR-128a, miR-206, miR-17 and miR-27a were quantified by absolute Q-PCR in muscle biopsies of control and dystrophic subjects listed in A. The absolute values (pg) of tested miRNAs were represented in the histogram for healthy (white bars) and dystrophic single fibres (black bars). (B) Quantitative analysis showed that only miR-128a, miR-206 and miR-17 were up-regulated in human dystrophic single fibres independently of the muscle type. (Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

Hmgb3 expression-pattern in single fibres.

<p>(A) Immunoblotting experiments confirmed the presence of Hmgb3 protein in single muscle fibers isolated from the TA and VA of c57bl (n = 3) and mdx (n = 3) mice. (B) Densitometric analysis on WB bands evidenced a decreased expression of Hmgb3 in the TA and VA (p = 0.0133) of mdx mice. (C) Quantification of Hmgb3 mRNA by qRT-PCR in single fibres isolated from the TA and VA of mdx mice (n = 10) and of c57bl (n = 10) mice confirmed a down-regulation of Hmgb3 in dystrophic muscle. (Two-tail parametric t-test; *p value <0, 05; **p value <0, 01; ***p value <0,001).</p

Dystrophin absence is not responsible for the over-expression of several muscle-enriched miRNAs in the mdx mouse.

<p>(A) MiRNAs that were observed over-expressed in the adult mdx mouse (3½ months-old) by the array analysis were quantified by Q-PCR in single fibres isolated from the hind limb of newborn mdx (n = 10) and c5bl (n = 10) mice. The absolute quantity (pg) of each miRNA was represented in the histogram as white columns for single fibres of c57bl mice and black bars for dystrophic muscle fibres. Single fibres of newborn mdx mice were characterized by control levels of muscle-enriched miRNAs, demonstrating no correlation with the genetic defect of DMD. (B) MiRNAs that were observed over-expressed in the adult mdx mouse (3½ months-old) by the array analysis were quantified by Q-PCR in single fibres isolated from the TA, DIA and VA of 6 months-old mdx mice and normalized on control samples. In the table were represented the fold change values of tested miRNAs in myofibres of 3½ and 6 months-old mdx mice in comparison to c57bl myofibers. Tested miRNAs showed a heterogeneous behaves depending on the muscle type and/or disease progression. (Two-wail parametric t-test; p value <0, 05 **p value <0, 01 ***p value <0,001).</p

Internalization of nanopolymeric tracers does not alter characteristics of placental cells

In the cell therapy scenario, efficient tracing of transplanted cells is essential for investigating cell migration and interactions with host tissues. This is fundamental to provide mechanistic insights which altogether allow for the understanding of the translational potential of placental cell therapy in the clinical setting. Mesenchymal stem/stromal cells (MSC) from human placenta are increasingly being investigated for their potential in treating patients with a variety of diseases. In this study, we investigated the feasibility of using poly (methyl methacrylate) nanoparticles (PMMA‐NPs) to trace placental MSC, namely those from the amniotic membrane (hAMSC) and early chorionic villi (hCV‐MSC). We report that PMMP‐NPs are efficiently internalized and retained in both populations, and do not alter cell morphofunctional parameters. We observed that PMMP‐NP incorporation does not alter in vitro immune modulatory capability of placental MSC, a characteristic central to their reparative/therapeutic effects in vitro. We also show that in vitro, PMMP‐NP uptake is not affected by hypoxia. Interestingly, after in vivo brain ischaemia and reperfusion injury achieved by transient middle cerebral artery occlusion (tMCAo) in mice, iv hAMSC treatment resulted in significant improvement in cognitive function compared to PBS‐treated tMCAo mice. Our study provides evidence that tracing placental MSC with PMMP‐NPs does not alter their in vitro and in vivo functions. These observations are grounds for the use of PMMP‐NPs as tools to investigate the therapeutic mechanisms of hAMSC and hCV‐MSC in preclinical models of inflammatory‐driven diseases.ISSN:1582-1838ISSN:1582-493