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

Dulbecco Telethon Institute and Division of Regenerative Medicine, San Raffaele Scientific Institute, Milano, Italy.
Molecular Therapy (Impact Factor: 6.23). 08/2011; 19(11):2055-64. DOI: 10.1038/mt.2011.153
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Giovanni Sitia, Sep 28, 2015
1 Follower
55 Reads
  • Source
    • "For example, the choice of the specific sequence encoded by the shRNA can affect not only siRNA efficiency but can also modulate overall toxicity [10], [17], [22]. In this regard we have recently shown that two separate shRNA sequences are effective at targeting the FRG1 transgene in a mouse model for FSHD [25]. Recent results in several systems suggest that miR30-based hairpins may display less toxicity than vectors based on simpler shRNA hairpins [41], [42]; JRC et al, submitted. "
    [Show abstract] [Hide abstract]
    ABSTRACT: RNAi has potential for therapeutically downregulating the expression of dominantly inherited genes in a variety of human genetic disorders. Here we used the ROSA26 mouse, which constitutively expresses the bacterial lacZ gene in tissues body wide, as a model to test the ability to downregulate gene expression in striated muscles. Recombinant adeno-associated viral vectors (rAAVs) were generated that express short hairpin RNAs (shRNAs) able to target the lacZ mRNA. Systemic delivery of these rAAV6 vectors led to a decrease of β-galactosidase expression of 30-50-fold in the striated muscles of ROSA26 mice. However, high doses of vectors expressing 21 nucleotide shRNA sequences were associated with significant toxicity in both liver and cardiac muscle. This toxicity was reduced in cardiac muscle using lower vector doses. Furthermore, improved knockdown in the absence of toxicity was obtained by using a shorter (19 nucleotide) shRNA guide sequence. These results support the possibility of using rAAV vectors to deliver RNAi sequences systemically to treat dominantly inherited disorders of striated muscle.
    PLoS ONE 08/2014; 9(8):e102053. DOI:10.1371/journal.pone.0102053 · 3.23 Impact Factor
  • Source
    • "FSHD region gene 1 (FRG1) is one of the proteins proposed to be involved in FSHD (Van Deutekom et al. 1996). FRG1-overexpressing mice are an animal model for FSHD, displaying a phenotype that strikingly resembles the functional and structural characteristics of FSHD (Gabellini et al. 2006; Bortolanza et al. 2011). Thick Tokuyasu sections of vastus lateralis muscle of FRG1-overexpressing mice were imaged with FM. "
    [Show abstract] [Hide abstract]
    ABSTRACT: One of the major challenges for correlative microscopy is the preparation of the sample; the protocols for transmission electron microscopy (TEM) and fluorescence microscopy (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure for Tokuyasu sections that yields both excellent positive membrane contrast in the TEM and bright fluorescence of the probe labeled on the section. 2+Staining involves the contrasting of the immunolabeled sections with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in sequential steps, followed by embedding in 1.8% methyl cellulose. In addition, we demonstrate an amplification of the fluorescent signal by introducing additional antibody incubation steps to the immunolabeling procedure. The methods were validated using the integrated laser and electron microscope (iLEM), a novel tool for correlative microscopy combining FM and TEM in a single setup. The approaches were tested on HL-60 cells labeled for lysosomal-associated membrane protein 2 (LAMP-2) and on sections of muscle from a facioscapulohumeral dystrophy mouse model. Yielding excellent results and greatly expediting the workflow, the methods are of great value for those working in the field of correlative microscopy and indispensible for future users of integrated correlative microscopy.
    Journal of Histochemistry and Cytochemistry 12/2012; 61(3). DOI:10.1369/0022155412473756 · 1.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The muscular dystrophies (MDs) represent a diverse collection of inherited human disorders, which affect to varying degrees skeletal, cardiac, and sometimes smooth muscle (Emery, 2002). To date, more than 50 different genes have been implicated as causing one or more types of MD (Bansal et al., 2003). In many cases, invaluable insights into disease mechanisms, structure and function of gene products, and approaches for therapeutic interventions have benefited from the study of animal models of the different MDs (Arnett et al., 2009). The large number of genes that are associated with MD and the tremendous number of animal models that have been developed preclude a complete discussion of each in the context of this review. However, we summarize here a number of the more commonly used models together with a mixture of different types of gene and MD, which serves to give a general overview of the value of animal models of MD for research and therapeutic development.
    Progress in molecular biology and translational science 01/2012; 105:83-111. DOI:10.1016/B978-0-12-394596-9.00004-4 · 3.49 Impact Factor
Show more