Endogenous MicroRNA Competition as a Mechanism of shRNA-Induced Cardiotoxicity.

Gene knockdown using short hairpin RNAs (shRNAs) is a promising strategy for targeting dominant mutations; however, delivering too much shRNA can disrupt the processing of endogenous microRNAs (miRNAs) and lead to toxicity. Here, we sought to understand the effect that excessive shRNAs have on muscle miRNAs by treating mice with recombinant adeno-associated viral vectors (rAAVs) that produce shRNAs with 19-nt or 21-nt stem sequences. Small RNA sequencing of their muscle and liver tissues revealed that shRNA expression was highest in the heart, where mice experienced substantial cardiomyopathy when shRNAs accumulated to 51.2% ± 13.7% of total small RNAs. With the same treatment, shRNAs in other muscle tissues reached only 12.1% ± 5.0% of total small RNAs. Regardless of treatment, the predominant heart miRNAs remained relatively stable across samples. Instead, the lower-expressed miR-451, one of the few miRNAs processed independently of Dicer, changed in relation to shRNA level and toxicity. Our data suggest that a protective mechanism exists in cardiac tissue for maintaining the levels of most miRNAs in response to shRNA delivery, in contrast with what has been shown in the liver. Quantifying miRNA profiles after excessive shRNA delivery illuminates the host response to rAAV-shRNA, allowing for safer and more robust therapeutic gene knockdown.

Progress toward Gene Therapy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) has been a major target for gene therapy development for nearly 30 years. DMD is among the most common genetic diseases, and isolation of the defective gene (DMD, or dystrophin) was a landmark discovery, as it was the first time a human disease gene had been cloned without knowledge of the protein product. Despite tremendous obstacles, including the enormous size of the gene and the large volume of muscle tissue in the human body, efforts to devise a treatment based on gene replacement have advanced steadily through the combined efforts of dozens of labs and patient advocacy groups. Progress in the development of DMD gene therapy has been well documented in Molecular Therapy over the past 20 years and will be reviewed here to highlight prospects for success in the imminent human clinical trials planned by several groups.

Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy.

Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx4cv mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.

Therapeutic impact of systemic AAV-mediated RNA interference in a mouse model of myotonic dystrophy.

RNA interference (RNAi) offers a promising therapeutic approach for dominant genetic disorders that involve gain-of-function mechanisms. One candidate disease for RNAi therapy application is myotonic dystrophy type 1 (DM1), which results from toxicity of a mutant mRNA. DM1 is caused by expansion of a CTG repeat in the 3' UTR of the DMPK gene. The expression of DMPK mRNA containing an expanded CUG repeat (CUG(exp)) leads to defects in RNA biogenesis and turnover. We designed miRNA-based RNAi hairpins to target the CUG(exp) mRNA in the human α-skeletal muscle actin long-repeat (HSA(LR)) mouse model of DM1. RNAi expression cassettes were delivered to HSA(LR) mice using recombinant adeno-associated viral (rAAV) vectors injected intravenously as a route to systemic gene therapy. Vector delivery significantly reduced disease pathology in muscles of the HSA(LR) mice, including a reduction in the CUG(exp) mRNA, a reduction in myotonic discharges, a shift toward adult pre-mRNA splicing patterns, reduced myofiber hypertrophy and a decrease in myonuclear foci containing the CUG(exp) mRNA. Significant reversal of hallmarks of DM1 in the rAAV RNAi-treated HSA(LR) mice indicate that defects characteristic of DM1 can be mitigated with a systemic RNAi approach targeting the nuclei of terminally differentiated myofibers. Efficient rAAV-mediated delivery of RNAi has the potential to provide a long-term therapy for DM1 and other dominant muscular dystrophies.

Systemic RNAi delivery to the muscles of ROSA26 mice reduces lacZ expression.

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 ß-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.

Adeno-associated viral (AAV) vectors do not efficiently target muscle satellite cells.

Adeno-associated viral (AAV) vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Several recombinant AAV vectors (rAAV) have the ability to transduce striated muscles in a variety of animals following intramuscular and intravascular administration, and have attracted widespread interest for therapy of muscle disorders such as the muscular dystrophies. However, most studies have focused on the ability to transduce mature muscle cells, and have not examined the ability to target myogenic stem cells such as skeletal muscle satellite cells. Here we examined the relative ability of rAAV vectors derived from AAV6 to target myoblasts, myocytes and myotubes in culture and satellite cells and myofibers in vivo. AAV vectors are able to transduce proliferating myoblasts in culture, albeit with reduced efficiency relative to post-mitotic myocytes and myotubes. In contrast, quiescent satellite cells are refractory to transduction in adult mice. These results suggest that while muscle disorders characterized by myofiber regeneration can be slowed or halted by AAV transduction, little if any vector transduction can be obtained in myogenic stems cells that might other wise support ongoing muscle regeneration.

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.

Muscling in: Gene therapies for muscular dystrophy target RNA.

Muscle diseases can take many forms, from the progressive muscle degeneration of dystrophies to the childhood cancer rhabdomyosarcoma. In 'Bench to Bedside', Joel R. Chamberlain and Jeffrey S. Chamberlain discuss studies using antisense oligonucleotides to treat Duchenne muscular dystrophy and myotonic dystrophy. In 'Bedside to Bench', Simone Hettmer and Amy J. Wagers examine the implications of clinical studies describing a type of rhabdomyosarcoma that resembles acute leukemia. The findings dovetail with other studies suggesting that some of these cancers might originate outside of muscle tissue and highlight the need for a better understanding of the cells that give rise to this condition.

Therapy for neuromuscular disorders.

Research into therapeutic approaches for both recessive and dominant neuromuscular disorders has made great progress over the past few years. In the field of gene therapy, antisense-mediated exon skipping is being applied to bypass deleterious mutations in the dystrophin gene and restore dystrophin expression in animal models of muscular dystrophy. Approaches for the dominant genetic muscle diseases have turned toward elimination of the mutant gene product with anti-sense oligonucleotide therapy and RNA interference techniques. Refinements of adeno-associated viral vectors and strategies for their delivery are also leading towards future clinical trials. The discovery of new, multipotent cell lineages, some of which possess the ability to successfully engraft muscle following vascular delivery, presents exciting prospects for the field of stem cell therapy. These discoveries represent steady progress towards the development of effective therapies for a wide range of neuromuscular disorders.

Gene targeting of mutant COL1A2 alleles in mesenchymal stem cells from individuals with osteogenesis imperfecta.

Mesenchymal stem cells (MSCs) are adult cells with the capacity to differentiate into multiple cell types, including bone, fat, cartilage, and muscle cells. In order to effectively utilize autologous MSCs in cell-based therapies, precise genetic manipulations are required to eliminate the effects of disease-causing mutations. We previously used adeno-associated virus (AAV) vectors to target and inactivate mutant COL1A1 genes in MSCs from individuals with the brittle bone disorder, osteogenesis imperfecta (OI). Here we have used AAV vectors to inactivate mutant COL1A2 genes in OI MSCs, thereby demonstrating that both type I collagen genes responsible for OI can be successfully targeted. We incorporated improved vector designs so as to minimize the consequences of random integration, facilitate the removal of potential antigens, and avoid unwanted exon skipping. MSCs targeted at mutant COL1A2 alleles produced normal type I procollagen and formed bone, thereby demonstrating their therapeutic potential.

Chimeric Ad5 vectors expressing the short fiber of Ad41 show reduced affinity for human intestinal epithelium.

Altering adenovirus tropism has attracted increased attention in recent years to improve gene delivery. We constructed a recombinant Ad5 vector carrying the non-CAR (coxsackievirus and adenovirus receptor) binding short fiber of enterotropic Ad41 (Ad5SHORT) and tested its transduction efficiency on enterocytes. Ad5SHORT was engineered, in high titers similar to the parent vector, by homologous recombination in Escherichia coli BJ5183 (recBC sbcBC) and propagated on C7 cells. Western blotting confirmed the presence of Ad41 short fiber on Ad5SHORT while lack of CAR-binding was evident by the low transduction of CHO-CAR cells. Transduction efficiency of enterocytes, the natural target tissue for the fiber-"donor" virus Ad41, was tested in human intestinal biopsy cultures and in Caco-2 cells, including ulcerative colitis tissue and mucosal wound healing models. Ad5SHORT exhibited up to 23-fold lower transduction levels compared to Ad5 in human intestinal biopsy cultures and up to 13-fold in the in vitro systems. The differences with the in vitro systems were more pronounced when less differentiated cells were used. These studies highlight the potential for using this chimeric Ad5/Ad41 vector as a scaffold for the development of retargeted adenoviral vectors. Finally, our results suggest that the short fiber does not appear to be mediating, at least by itself, the increased enterocyte affinity of Ad41.

Gene targeting in stem cells from individuals with osteogenesis imperfecta.

Adult stem cells offer the potential to treat many diseases through a combination of ex vivo genetic manipulation and autologous transplantation. Mesenchymal stem cells (MSCs, also referred to as marrow stromal cells) are adult stem cells that can be isolated as proliferating, adherent cells from bones. MSCs can differentiate into multiple cell types present in several tissues, including bone, fat, cartilage, and muscle, making them ideal candidates for a variety of cell-based therapies. Here, we have used adeno-associated virus vectors to disrupt dominant-negative mutant COL1A1 collagen genes in MSCs from individuals with the brittle bone disorder osteogenesis imperfecta, demonstrating successful gene targeting in adult human stem cells.