Muscle regeneration affects Adeno Associated Virus 1 mediated transgene transcription.

Duchenne muscular dystrophy is a severe neuromuscular disease causing a progressive muscle wasting due to mutations in the DMD gene that lead to the absence of dystrophin protein. Adeno-associated virus (AAV)-based therapies aiming to restore dystrophin in muscles, by either exon skipping or microdystrophin expression, are very promising. However, the absence of dystrophin induces cellular perturbations that hinder AAV therapy efficiency. We focused here on the impact of the necrosis-regeneration process leading to nuclear centralization in myofiber, a common feature of human myopathies, on AAV transduction efficiency. We generated centronucleated myofibers by cardiotoxin injection in wild-type muscles prior to AAV injection. Intramuscular injections of AAV1 vectors show that transgene expression was drastically reduced in regenerated muscles, even when the AAV injection occurred 10 months post-regeneration. We show also that AAV genomes were not lost from cardiotoxin regenerated muscle and were properly localised in the myofiber nuclei but were less transcribed leading to muscle transduction defect. A similar defect was observed in muscles of the DMD mouse model mdx. Therefore, the regeneration process per se could participate to the AAV-mediated transduction defect observed in dystrophic muscles which may limit AAV-based therapies.

Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice.

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients.

miR-708-5p and miR-34c-5p are involved in nNOS regulation in dystrophic context.

BACKGROUND: Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context. METHODS: By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3'UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS. RESULTS: TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3'UTR-luciferase assay, confirming their interaction with the NOS1-3'UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts. CONCLUSION: These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle.

RFX1 and RFX3 Transcription Factors Interact with the D Sequence of Adeno-Associated Virus Inverted Terminal Repeat and Regulate AAV Transduction.

Adeno-associated virus (AAV) transduction efficiency depends on the way in which cellular proteins process viral genomes in the nucleus. In this study, we have investigated the binding of nuclear proteins to the double stranded D (dsD) sequence of the AAV inverted terminal repeat (ITRs) by electromobility shift assay. We present here several lines of evidence that transcription factors belonging to the RFX protein family bind specifically and selectively to AAV2 and AAV1 dsD sequences. Using supershift experiments, we characterize complexes containing RFX1 homodimers and RFX1/RFX3 heterodimers. Following transduction of HEK-293 cells, the AAV genome can be pulled-down by RFX1 and RFX3 antibodies. Moreover, our data suggest that RFX proteins which interact with transcriptional enhancers of several mammalian DNA viruses, can act as regulators of AAV mediated transgene expression.

Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.

In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients.

Intrinsic transgene immunogenicity gears CD8(+) T-cell priming after rAAV-mediated muscle gene transfer.

Antitransgene CD8(+) T-cell responses are an important hurdle after recombinant adeno-associated virus (rAAV) vector-mediated gene transfer. Indeed, depending on the mutational genotype of the host, transgene amino-acid sequences of foreign origin can elicit deleterious cellular and humoral responses. We compared here two different major histocompatibility complex (MHC) class I epitopes of an engineered ovalbumin transgene delivered in muscle tissue by rAAV1 vector and found very different strength of CD8 responses, muscle destruction being correlated with the course of the immunodominant response. We further demonstrate that robust CD8(+) T-cell priming can occur through the cross-presentation pathway but requires the presence of either a strong MHC class II epitope or antibodies to the transgene product. Finally, manipulating transgene subcellular localization, we found that provided we avoid transgene expression in antigen presenting cells, the poorly accessible cytosolic form of ovalbumin transgene lacking strong MHC II epitope, evades CD8(+) T-cell priming and remains permanently expressed in muscle with no immune cell infiltration. Our results demonstrate that the intrinsic immunogenicity of transgenes delivered with rAAV vector in muscle can be manipulated in a rational manner to avoid adverse immune responses.

Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches.

RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre-trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations.

AAV genome loss from dystrophic mouse muscles during AAV-U7 snRNA-mediated exon-skipping therapy.

In the context of future adeno-associated viral (AAV)-based clinical trials for Duchenne myopathy, AAV genome fate in dystrophic muscles is of importance considering the viral capsid immunogenicity that prohibits recurring treatments. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx dystrophic muscles within 3 weeks after intramuscular injection. In contrast, AAV genomes encoding U7ex23 restoring expression of a slightly shortened dystrophin were maintained endorsing that the arrest of the dystrophic process is crucial for maintaining viral genomes in transduced fibers. Indeed, muscles treated with low doses of AAV-U7ex23, resulting in sub-optimal exon skipping, displayed much lower titers of viral genomes, showing that sub-optimal dystrophin restoration does not prevent AAV genome loss. We also followed therapeutic viral genomes in severe dystrophic dKO mice over time after systemic treatment with scAAV9-U7ex23. Dystrophin restoration decreased significantly between 3 and 12 months in various skeletal muscles, which was correlated with important viral genome loss, except in the heart. Altogether, these data show that the success of future AAV-U7 therapy for Duchenne patients would require optimal doses of AAV-U7 to induce substantial levels of dystrophin to stabilize the treated fibers and maintain the long lasting effect of the treatment.

Exon exchange approach to repair Duchenne dystrophin transcripts.

BACKGROUND: Trans-splicing strategies for mRNA repair involve engineered transcripts designed to anneal target mRNAs in order to interfere with their natural splicing, giving rise to mRNA chimeras where endogenous mutated exons have been replaced by exogenous replacement sequences. A number of trans-splicing molecules have already been proposed for replacing either the 5' or the 3' part of transcripts to be repaired. Here, we show the feasibility of RNA surgery by using a double trans-splicing approach allowing the specific substitution of a given mutated exon. METHODOLOGY/PRINCIPAL FINDINGS: As a target we used a minigene encoding a fragment of the mdx dystrophin gene enclosing the mutated exon (exon 23). This minigene was cotransfected with a variety of exon exchange constructions, differing in their annealing domains. We obtained accurate and efficient replacement of exon 23 in the mRNA target. Adding up a downstream intronic splice enhancer DISE in the exon exchange molecule enhanced drastically its efficiency up to 25-45% of repair depending on the construction in use. CONCLUSIONS/SIGNIFICANCE: These results demonstrate the possibility to fix up mutated exons, refurbish deleted exons and introduce protein motifs, while keeping natural untranslated sequences, which are essential for mRNA stability and translation regulation. Conversely to the well-known exon skipping, exon exchange has the advantage to be compatible with almost any type of mutations and more generally to a wide range of genetic conditions. In particular, it allows addressing disorders caused by dominant mutations.

Protein- and mRNA-based phenotype-genotype correlations in DMD/BMD with point mutations and molecular basis for BMD with nonsense and frameshift mutations in the DMD gene.

Straightforward detectable Duchenne muscular dystrophy (DMD) gene rearrangements, such as deletions or duplications involving an entire exon or more, are involved in about 70% of dystrophinopathies. In the remaining 30% a variety of point mutations or "small" mutations are suspected. Due to their diversity and to the large size and complexity of the DMD gene, these point mutations are difficult to detect. To overcome this diagnostic issue, we developed and optimized a routine muscle biopsy-based diagnostic strategy. The mutation detection rate is almost as high as 100% and mutations were identified in all patients for whom the diagnosis of DMD and Becker muscular dystrophy (BMD) was clinically suspected and further supported by the detection on Western blot of quantitative and/or qualitative dystrophin protein abnormalities. Here we report a total of 124 small mutations including 11 nonsense and frameshift mutations detected in BMD patients. In addition to a comprehensive assessment of muscular phenotypes that takes into account consequences of mutations on the expression of the dystrophin mRNA and protein, we provide and discuss genomic, mRNA, and protein data that pinpoint molecular mechanisms underlying BMD phenotypes associated with nonsense and frameshift mutations.