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.

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.

Cross-Presentation of Skin-Targeted Recombinant Adeno-associated Virus 2/1 Transgene Induces Potent Resident Memory CD8 T Cell Responses.

A key aspect to consider for vaccinal protection is the induction of a local line of defense consisting of nonrecirculating tissue-resident memory T cells (T), in parallel to the generation of systemic memory CD8 T cell responses. The potential to induce T has now been demonstrated for a number of pathogens and viral vectors. This potential, however, has never been tested for recombinant adeno-associated virus (rAAV) vectors, which are weakly inflammatory and poor transducer of dendritic cells.

Gene Therapy via Trans-Splicing for LMNA-Related Congenital Muscular Dystrophy.

We assessed the potential of Lmna-mRNA repair by spliceosome-mediated RNA trans-splicing as a therapeutic approach for LMNA-related congenital muscular dystrophy. This gene therapy strategy leads to reduction of mutated transcript expression for the benefit of corresponding wild-type (WT) transcripts. We developed 5'-RNA pre-trans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA.

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.

Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy.

Rapid advances in allele-specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal-dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the gene encoding Dynamin 2. Allele-specific siRNA sequences were developed in order to specifically knock down the human and murine -mRNA harbouring the p.

Intradermal Immunization with rAAV1 Vector Induces Robust Memory CD8 T Cell Responses Independently of Transgene Expression in DCs.

Recombinant adeno-associated viral (rAAV) vectors exhibit interesting properties as vaccine carriers for their ability to induce long-lasting antibody responses. However, rAAV-based vaccines have been suggested to trigger functionally impaired long-term memory CD8 T cell responses, in part due to poor dendritic cell (DC) transduction. Such results, albeit limited to intramuscular immunization, undermined the use of rAAV as vaccine vehicles against intracellular pathogens.

Delivery is key: lessons learnt from developing splice-switching antisense therapies.

The use of splice-switching antisense therapy is highly promising, with a wealth of pre-clinical data and numerous clinical trials ongoing. Nevertheless, its potential to treat a variety of disorders has yet to be realized. The main obstacle impeding the clinical translation of this approach is the relatively poor delivery of antisense oligonucleotides to target tissues after systemic delivery.

Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing.

Dynamin 2 (DNM2) is a large GTPase, ubiquitously expressed, involved in membrane trafficking and regulation of actin and microtubule cytoskeletons. DNM2 mutations cause autosomal dominant centronuclear myopathy which is a rare congenital myopathy characterized by skeletal muscle weakness and histopathological features including nuclear centralization in absence of regeneration. No curative treatment is currently available for the DNM2-related autosomal dominant centronuclear myopathy.

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.

Dystrophin Threshold Level Necessary for Normalization of Neuronal Nitric Oxide Synthase, Inducible Nitric Oxide Synthase, and Ryanodine Receptor-Calcium Release Channel Type 1 Nitrosylation in Golden Retriever Muscular Dystrophy Dystrophinopathy.

At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger.

Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform.

Dysferlin rescue by spliceosome-mediated pre-mRNA trans-splicing targeting introns harbouring weakly defined 3' splice sites.

The modification of the pre-mRNA cis-splicing process employing a pre-mRNA trans-splicing molecule (PTM) is an attractive strategy for the in situ correction of genes whose careful transcription regulation and full-length expression is determinative for protein function, as it is the case for the dysferlin (DYSF, Dysf) gene. Loss-of-function mutations of DYSF result in different types of muscular dystrophy mainly manifesting as limb girdle muscular dystrophy 2B (LGMD2B) and Miyoshi muscular dystrophy 1 (MMD1). We established a 3' replacement strategy for mutated DYSF pre-mRNAs induced by spliceosome-mediated pre-mRNA trans-splicing (SmaRT) by the use of a PTM.

Repair of rhodopsin mRNA by spliceosome-mediated RNA trans-splicing: a new approach for autosomal dominant retinitis pigmentosa.

The promising clinical results obtained for ocular gene therapy in recent years have paved the way for gene supplementation to treat recessively inherited forms of retinal degeneration. The situation is more complex for dominant mutations, as the toxic mutant gene product must be removed. We used spliceosome-mediated RNA trans-splicing as a strategy for repairing the transcript of the rhodopsin gene, the gene most frequently mutated in autosomal dominant retinitis pigmentosa.

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.

Correction of the Middle Eastern M712T mutation causing GNE myopathy by trans-splicing.

GNE myopathy is a rare neuromuscular autosomal recessive disease, resulting from mutations in the gene UDP N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). The most frequent mutation is the single homozygous missense mutation, M712T-the Middle Eastern mutation-located ten amino acids before the end of the protein. We have used an adeno-associated virus (AAV)-based trans-splicing (TS) vector as a gene therapy tool to overcome this mutation by replacing the mutated last exon of GNE by the wild-type exon while preserving the natural endogenous regulatory machinery.

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.

Repair of Mybpc3 mRNA by 5'-trans-splicing in a Mouse Model of Hypertrophic Cardiomyopathy.

RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated.

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.

Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice.

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs.

Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD).

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.

Transient immunomodulation allows repeated injections of AAV1 and correction of muscular dystrophy in multiple muscles.

Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles.

Transcriptional coactivator PC4, a chromatin-associated protein, induces chromatin condensation.

Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability.

Subnuclear localization and mitotic phosphorylation of HIRA, the human homologue of Saccharomyces cerevisiae transcriptional regulators Hir1p/Hir2p.

The HIRA gene encodes a nuclear protein with histone-binding properties that have been conserved from yeast to humans. Hir1p and Hir2p, the two HIRA homologues in Saccharomyces cerevisiae, are transcriptional co-repressors whose action resides at the chromatin level and occurs in a cell-cycle-regulated fashion. In mammals, HIRA is an essential gene early during development, possibly through the control of specific gene-transcription programmes, but its exact function remains to be deciphered.