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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. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSμ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSμ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

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., Graphical Abstract, The AAV-based gene therapies used in DMD have the major limitation of maintaining the therapeutic effect over time. Forand et al. demonstrated an undeniable advantage of the PPMO and AAV combination on the striated muscles, notably on heart and diaphragm structure and function with an unrivalled extent of survival.

International audience; 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 0-RNA pretrans-splicing molecules containing the first five exons of Lmna and targeting intron 5 of Lmna pre-mRNA. Among nine pre-trans-splicing molecules, differing in the targeted sequence in intron 5 and tested in C2C12 myoblasts, three induced trans-splicing events on endogenous Lmna mRNA and confirmed at protein level. Further analyses performed in primary myotubes derived from an LMNA-related congenital muscular dystrophy (L-CMD) mouse model led to a partial rescue of the mutant phenotype. Finally, we tested this approach in vivo using adeno-associated virus (AAV) delivery in newborn mice and showed that trans-splicing events occurred in WT mice 50 days after AAV delivery, although at a low rate. Altogether, while these results provide the first evidence for reprogramming LMNA mRNA in vitro, strategies to improve the rate of trans-splicing events still need to be developed for efficient application of this therapeutic approach in vivo.

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 (TRM), in parallel to the generation of systemic memory CD8+ T cell responses. The potential to induce TRM 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. Using a model rAAV2/1-based vaccine, we determined that a single intradermal immunization with rAAV2/1 vectors in mice induces fully functional TRM at the local site of immunization. The optimal differentiation of rAAV-induced transgene-specific skin TRM was dependent on local transgene expression and additional CD4+ T cell help. Transgene expression in dendritic cells, however, appeared to be dispensable for the priming of transgene-specific skin TRM, suggesting that this process solely depends on the cross-presentation of transgene products. Overall, this study provides needed information to properly assess rAAV vectors as T cell-inducing vaccine carriers.IMPORTANCE rAAVs display numerous characteristics that could make them extremely attractive as vaccine carriers, including an excellent safety profile in humans and great flexibility regarding serotypes and choice of target tissue. Studies addressing the ability of rAAV to induce protective T cell responses, however, are scarce. Notably, the potential to induce a tissue-resident memory T cell response has never been described for rAAV vectors, strongly limiting further interest for their use as vaccine carriers. Using a model rAAV2/1 vaccine delivered to the skin, our study demonstrated that rAAV vectors can induce bona fide skin resident TRM and provides additional clues regarding the cellular mechanisms underlying this process. These results will help widen the field of rAAV applications.

International audience; 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. We report here that intradermal immunization with a model rAAV2/1-based vaccine drives the development of bona fide long-term memory CD8+ T cell responses. The intradermal route of immunization and the presence of potent major histocompatibility complex (MHC) class II responses showed synergistic effects on the overall quantity and quality of systemic long-term effector memory transgene-specific CD8+ T cells being generated against the transgene. Of key interest, we found that the induction of memory cytotoxic T lymphocytes (CTLs) following intradermal immunization was solely dependent on the cross-presentation of skin-expressed transgene products, which appeared highly enhanced as compared to muscle-expressed transgene products. Overall our results highlight key tissue-specific differences in transgene presentation pathway requirements of importance for the design of rAAV-based T cell-inducing vaccines.

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. © The American Society of Gene and Cell Therapy.

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.

International audience; 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.

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). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.

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. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (microKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in microKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge.

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. We tested 17 different molecules targeting the pre-mRNA intron 1, by transient transfection of HEK-293T cells, with subsequent trans-splicing quantification at the transcript level. We found that the targeting of some parts of the intron promoted trans-splicing more efficiently than the targeting of other areas, and that trans-splicing rate could be increased by modifying the replacement sequence. We then developed cell lines stably expressing the rhodopsin gene, for the assessment of phenotypic criteria relevant to the pathogenesis of retinitis pigmentosa. Using this model, we showed that trans-splicing restored the correct localization of the protein to the plasma membrane. Finally, we tested our best candidate by AAV gene transfer in a mouse model of retinitis pigmentosa that expresses a mutant allele of the human rhodopsin gene, and demonstrated the feasibility of trans-splicing in vivo. This work paves the way for trans-splicing gene therapy to treat retinitis pigmentosa due to rhodopsin gene mutation and, more generally, for the treatment of genetic diseases with dominant transmission.

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.

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. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1., The splicing of the penultimate exon of the dystrophin gene is developmentally regulated. Here the authors show that the dysregulation of this exon's splicing leads to the expression of an embryonic dystrophin form with a C-terminus distinct from the adult isoform, which leads to muscle wasting in zebrafish and mice.

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. We have designed relevant plasmids directed either to mouse or to human GNE. Following transfection of C2C12 murine muscle cells with the mouse TS vectors, we have been able to detect by nested RT-PCR trans-spliced molecules carrying the wild-type exon 12 of GNE. Similarly, transfection of HEK293 human cells with the human-directed TS vectors resulted in the generation of trans-spliced human GNE RNA molecules. Furthermore, infection of primary muscle cells from a GNE myopathy patient carrying the homozygous M712T mutation, with an AAV8-based viral vector carrying a human-directed TS construct, resulted in the generation of wild-type GNE transcripts in addition to the mutated ones. These studies provide a proof of concept that the TS approach could be used to partially correct the Middle Eastern mutation in GNE myopathy patients. These results provide the basis for in vivo research in animal models using the AAV platform with TS plasmids as a potential genetic therapy for GNE myopathy.

International audience; 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. We identified an alternative variant (Var-4) deleted of exons 5–6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5þ6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5þ6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.

International audience; 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. In order to develop therapeutic strategy, we evaluated here the potential of Spliceosome-Mediated RNA Trans-splicing technology to reprogram the Dnm2-mRNA in vitro and in vivo in mice. We show that classical 3′-trans-splicing strategy cannot be considered as accurate therapeutic strategy regarding toxicity of the pre-trans-splicing molecules leading to low rate of trans-splicing in vivo. Thus, we tested alternative strategies devoted to prevent this toxicity and enhance frequency of trans-splicing events. We succeeded to overcome the toxicity through a 5′-trans-splicing strategy which also allows detection of trans-splicing events at mRNA and protein levels in vitro and in vivo. These results suggest that the Spliceosome-Mediated RNA Trans-splicing strategy may be used to reprogram mutated Dnm2-mRNA but highlight the potential toxicity linked to the molecular tools which have to be carefully investigated during preclinical development.

Adeno Associated virus serotype 8 (AAV8) is of particular interest as a vector for pre-clinical and clinical trial for Duchenne Muscular Dystrophy (DMD). In several cell lines, this vector has been shown to enter cells through clathrin-mediated endocytosis followed by a trafficking through the microtubule network in various endosomal compartments toward the nucleus. To efficiently transduce cells, AAV must undergo multiple levels of regulation in these cellular compartments. In DMD, dystrophin deficiency results in disturbed balance of cellular events i.e., fiber centronucleation, disorganized cytoskeleton, presence of fibrosis. We have recently described a loss of virion genomes from both dogs and mice models of DMD treated with therapeutic molecules vectorized in AAV. Indeed, the pathophysiological state of DMD muscle should impact on virions fate and subsequently affect crucial steps for AAV effectiveness as viral uncoating, viral genome maintenance and consequently, the transduction efficiency of AAV. Our project aims to characterize cellular uptake and intracellular transport of AAV8 in DMD muscular cells, with the goal of optimizing AAV vector use to get the best transduction efficiency with the lowest AAV dose. Our first data showed that AAV8-GFP was less efficient to transduce DMD and control primary muscular cells compared to HeLa cells. Moreover, AAV8 traffics through same endosomal compartment in DMD and control myoblasts, but at different rates during early time points of the transduction. These results suggest that in muscle cells, AAV8 uses different entry and trafficking pathways from those previously described in HeLa cells and that dystrophic cellular status could affect subcellular processing of the vector particles. We will specify the relationship between AAV8 vector entry, trafficking, uncoating, and transduction efficiency in vitro in primary myoblasts/myotubes of DMD patients and controls.

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 59 or the 39 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.

The most frequent cause of retinitis pigmentosa (RP), a group of hereditary retinal dystrophies leading to blindness, is the occurrence of point mutations in the Rhodopsin (RHO) gene. Most of these mutations lead to gain of functions or dominant negative effects deleterious for photoreceptors, thus resulting in a dominant mode of transmission. Moreover, it has been shown that variations in RHO expression level are also deleterious for the retina. Gene transfer strategies modifying RHO mutations should therefore lead both to suppression of mutant protein expression and restoration of the normal one at a physiological level. Spliceosome-mediated RNA trans-splicing should in theory respect these constraints by repairing mutations at the pre-mRNA level. Using this technology, the expression level of the repaired mRNA will indeed depend solely on that of the gene endogenous promoter. To achieve this goal, this approach consists of introducing, by gene transfer, an exogenous RNA – called PTM, for Pre-Trans-splicing Molecule – able to bind the pre-mRNA and promote splicing in trans, leading to replacement of the mutant part of the RHO pre-mRNA.We engineered fourteen different RHO-PTMs able to repair any mutation in RHO exons 2, 3, 4 and 5, which differ only on their binding sequence to RHO pre-mRNA. To determine the efficiency of each PTM, we transiently co-transfected HEK293T cells with a plasmid encoding a PTM and another encoding the wild-type or mutant RHO. The maximum trans-splicing efficiency observed at the mRNA level was about 25%. We improved this efficiency to 40% by refinement of the PTM sequence, through reintroduction of an endogenous RHO intron in the cDNA part of the PTM. We then tested PTM at the protein level in a cellular model expressing RHO stably. While wild-type RHO was localized to the plasma membrane, mutant RHO was sequestrated in the cytoplasmic compartment. Using a flow-imaging methodology (ImageStreamX), we were able to precisely quantify this phenotype and showed that trans-splicing partially restored a correct localization of RHO in this cellular model. Thus, we constructed an AAV vector containing our best PTM to assess its activity in vivo in a humanized mouse model of rhodopsin mutation following subretinal injection. Using co-injection with an AAV-GFP and micro-dissection of the transduced part of the retina, we demonstrated that trans-splicing reached 30% at the RNA level, while it was not detectable in the untransduced part, or in animals that received a control PTM. This study demonstrates that trans-splicing may provide a therapeutic benefit in case of dominant mutations of the rhodopsin gene.

Top of pageAbstract Most cases of Duchenne muscular dystrophy (DMD) are caused by dystrophin gene mutations that disrupt the mRNA reading frame. In some cases, forced exclusion of a single exon can restore the reading frame, given rise to a shorter, but still functional dystrophin protein (so called quasi-dystrophin). One potential treatment of the disorder has utilized antisense oligoribonucleotide (AO) to induce removal of disease-associated exons during pre-mRNA processing. Indeed, this approach has been successfully used in DMD cells in vitro with antisense sequences against splice junctions of exon 51. Skipping of this exon would theoretically restore a functional quasi- dystrophin in a significant subset of DMD patients with |[Delta]|45-50,|[Delta]|47-50,|[Delta]|48-50, |[Delta]|49-50, |[Delta]|50 and |[Delta]|52 genotypes. However, since the AO are not self-renewed, they can not achieve long term correction. To overcome this limitation, we have introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized them in AAV and lentiviral vectors. We have designed AAV and lentiviral vectors harboring chimeric U7 snRNA carrying antisense sequences against exon 51 of the human dystrophin gene (U7-ex51). Lentiviral vectors expressing this U7-ex51 were tested on human myoblasts, whereas AAV vectors were injected in the transgenic hDMD mice (carrying the human dystrophin gene). We confirmed the skipping of the exon 51 in vitro in human myoblasts after transduction with the lentiviral vector encoding U7-ex51 by RT-PCR. We also detected the skipping of the exon 51 after intramuscular injection of an AAV-U7ex51 vector in the transgenic hDMD mouse. We have also tested the efficacy of these vectors to restore dystrophin expression in myoblasts from patients with |[Delta]|49-50 and |[Delta]|52 deletions. In this study, we provide evidence that efficient skipping of exon 51 can be achieved in human cells and also in vivo after intramuscular injection in a transgenic hDMD mice through U7snRNA shuttle. These results offer very promising tools for clinical treatment of DMD.

GNE Myopathy is a rare neuromuscular worldwide autosomal recessive disease, which is very common among Persian Jews. The disorder results from mutations in the gene UDP N -Acetylglucosamine 2-epimerase/ N -Acetylmannosamine kinase (GNE). To date, over 60 different mutations in GNE have been reported to cause the disease worldwide, but a single homozygous mutation, M712T, has been identified in all patients of Persian descent, 10 amino acids before the end of the protein. The same mutation was found in other Jews and non-Jews families in the Middle East therefore we term it the Middle East mutation. The mechanism leading from GNE mutations to the myopathy phenotype is not yet understood. Since the disease is recessive, it most likely results from the lack of wild type GNE protein; therefore the production of wt protein in patients might alleviate the disease phenotype. Trans-splicing is a phenomenon where splicing between two different pre-mRNA molecules occurs. This mechanism can be used to address mutated gene products and correct transcripts defects on the transcript itself, by altering the cis-splicing process but conserving the original endogenous regulation of the gene. Based on an Adeno Associated Virus platform we have generated a series of transplicing vectors designed to transplice the last exon of GNE which carries the most frequent mutation occurring in GNE myopathy. Transfection and infection of these vectors result in transpliced transcripts both in mouse and human cells. Furthermore, human transplicing vectors are able to partially correct the M712T mutation in patients derived muscle cells. The goal of this project is to use an AAV based trans-splicing vector as a gene therapy tool to overcome the GNE myopathy mutation M712T.

The human HIRA protein is encoded from a region of chromosome 22q that is critical for the DiGeorge syndrome and the velocardiofacial syndrome. We have previously reported that it directly interacts with core histones, with a novel histone-binding protein, HIRIP3, and during mouse embryogenesis, with the developmentally regulated homeodomain protein Pax3, suggesting a promoter-targeted function at the chromatin level. We here report on HIRA-interacting protein 5 (HIRIP5), a small acidic protein that interacted with HIRA in a double-hybrid screen performed in yeast and in in vitro protein interaction experiments. HIRIP5 has highly conserved homologs in both prokaryotes and eukaryotes, including the NFU1 gene product which has been implicated in iron metabolism in mitochondria of the yeast Saccharomyces cerevisiae. By radioactive in situ hybridization, the HIRIP5 gene was mapped to the 2p13-p15 chromosomal region, separate from any region previously associated with DiGeorge syndrome.

HIRA maps to the DiGeorge/velocardiofacial syndrome critical region (DGCR) at 22q11 (refs 1,2) and encodes a WD40 repeat protein similar to yeast Hir1p and Hir2p. These transcriptional co-repressors regulate cell cycle-dependent histone gene transcription, possibly by remodelling local chromatin structure. We report an interaction between HIRA and the transcription factor Pax3. Pax3 haploinsufficiency results in the mouse splotch and human Waardenburg syndrome (WSI and WSIII) phenotypes. Mice homozygous for Pax3 mutations die in utero with a phenocopy of DGS, or neonatally with neural tube defects. HIRA was also found to interact with core histones. Thus, altered stoichiometry of complexes containing HIRA may be important for the development of structures affected in WS and DGS.

Developmental anomalies are frequently observed in humans in association with deletions affecting the proximal region of the long arm of chromosome 22. These 22q deletion disorders (22DD) include the DiGeorge syndrome (Mendelion inheritance in man [MIM] 188400) and the velocardiofacial syndrome (MIM 192430), whose phenotypes overlap partially. Main clinical features associated with 22DD comprise abnormalities of the face and palate, hypoplastic parathyroid glands, and conotruncal malformations (38), all probably resulting from anomalies of neural crest cells in the embryological region of the pharyngeal arches and pouches (26). Genetically, 90% of all patients have a large (approximately 3-Mb-long) 22q deletion. Although most deletions occur de novo, up to 28% could be inherited (38). In these familial cases whose transmission is autosomal dominant, the phenotypic expression of the same chromosomal defect is largely variable. The additional lack of correlation between the extent of the deletion and the intensity of the phenotype seems to argue against different contiguous genes being each responsible for distinct clinical features. Recently, however, the hypothesis that two causative genes each mapping to the same 22q region may together be responsible for the disorders has been reconsidered (5, 9). In the absence of any mutation identified in the minority of patients without a confirmed 22q deletion, none of the genes cloned from the large commonly deleted region has been definitely linked to 22DD, making it necessary to study each plausible candidate in detail in order to evaluate its possible implication. This task will be facilitated by recent reports of a few patients whose unusually small 22q deletions with variant proximal and distal chromosomal breakpoints have reduced the critical region to less than 500 kb (6, 15, 28). Of the five genes characterized within the region, neither CTP, which codes for a mitochondrial citrate transport protein (20), CLTD (21), a clathrin heavy chain-like gene, nor the ubiquitously expressed TMVCF gene, which encodes a putative transmembrane protein (41), appears to be a plausible candidate. GSCL, a small (less than 4-kb) gene identified by systematic genomic sequencing, is more intriguing since it contains regions of homology to Goosecoid, a homeodomain gene whose specific expression pattern in the mouse suggests a role in the development of neural tissues (15). The HIRA gene (27), first reported as TUPLE1 (18) for its partial similarity to the yeast general transcriptional repressor TUP1 (51), also appears to be an interesting candidate. It consists of 25 exons scattered over 100 kb of genomic DNA which is entirely reduced to single copy in 22DD patients (30). In situ hybridization experiments have demonstrated high levels of transcripts in the heart, cranial neural folds, pharyngeal arches, and circumpharyngeal neural crest of murine embryos (53) and in the neuroepithelium, neural crest-derived regions of the head, branchial arches, and pharyngeal pouches of chicken embryos (37). These evolutionarily conserved spatiotemporal expression patterns suggest that haploinsufficiency of HIRA could play an important part in the genesis of 22DD. HIRA was named for its sequence similarity to two yeast proteins, Hir1p and Hir2p (40). In the HIR family, which also comprises HIRA homologs subsequently identified in mice, chickens, and the fish Fugu rubripes, all protein sequences can be aligned over their entire lengths (29, 37, 39, 53). Shared features include basic nuclear localization signals, absence of an identifiable DNA- or RNA-binding motif, and seven characteristic WD repeats conserved at the amino terminus of all family members but Hir2p. WD repeats are ancient motifs that have been detected throughout the eukaryotic kingdom (33). They are present in a set of functionally diverse proteins that are part of macromolecular complexes (33) and in several instances have been shown to provide interfaces for protein interactions (14, 24, 25, 42, 50). Contrasting with the single HIRA gene present in higher eukaryotes, the two budding yeast genes HIR1 and HIR2 likely have diverged and specialized functionally following an ancestral duplication event. HIR1 and HIR2 had been identified in the course of a genetic screen for Saccharomyces cerevisiae mutants with deregulated core histone gene transcription (34). In a wild-type strain, transcription of core histone genes is repressed outside late G1 and S phases, while in either a hir1 or hir2 mutant, transcription becomes constitutive throughout the cell cycle. To perform their cyclic repressive function, Hir1p and Hir2p, which can be coimmunoprecipitated (43), require the presence of other proteins, including the products of the SPT4, SPT5, and SPT6 genes, which are known for their impact on chromatin (3, 44) and transcription elongation by RNA polymerase II (19, 49). A regulatory protein complex targeted to the negative element identified in the promoter region of regulated histone gene loci by a DNA-binding factor that has been detected but not characterized would provoke transcriptional repression, probably in association with a local remodeling of chromatin structure (43). In humans, where the number and complexity of core histone genes (1) far exceed those in yeast, it appeared that the main function of HIRA proteins would not necessarily involve the regulation of histone gene transcription. Rather, we hypothesized that during evolution, HIRA proteins could have built upon ancient biochemical properties to acquire the developmental role that is suggested by their regulated expression pattern during vertebrate embryogenesis. To explore the function of HIRA in multicellular species, we have looked for HIRA-interacting proteins (HIRIPs). We here report that human HIRA is a primarily nuclear protein that directly binds core histones H2B and H4, using overlapping but distinguishable domains outside its WD repeat region. HIRIP3, one of the other HIRA interactors identified, is a novel protein which also displays binding to two core histones, H2B and H3, suggesting that HIRA and HIRIP3 can assemble into a protein complex with a role at the level of chromatin components or structure.

In the context of future 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 followed AAV genome copy numbers after AAV-U7 delivery in the mdx dystrophic mouse. We showed that AAV genomes encoding non-therapeutic U7 were lost from mdx muscles within three weeks after intra-muscular 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.

A full-length cDNA has been isolated for the murine homolog of the human HIRA protein, a member of the HIR family of nuclear proteins that is encoded from the chromosome 22 region critical for the DiGeorge syndrome. This family also contains Hir1p and Hir2p, two proteins identified as regulators of histone gene transcription in yeast. The murine and human amino acid sequences are 95.3% identical, with a striking 99.2% identity in the N-terminal WD repeat domain that is characteristic of the family. The two cDNAs are highly conserved within the coding regions, but also in the entire 5′ untranslated region and in a strikingly long stretch of nucleotides in the 3′ untranslated region.

The human gene HIRA lies within the smallest critical region for the DiGeorge syndrome (DGS), a haploinsufficiency developmental disorder associated with instertitial deletions in most patients in a juxtacentromeric region of chromosome 22. The HIRA protein sequence can be aligned over its entire length with Hir1 and Hir2, two yeast proteins with a regulatory function in chromatin assembly. The HIRA transcription unit was found to spread over approximately 100 kb of the DGS critical region. The human transcript is encoded from 25 exons between 59 and 861 bp in size. Domains of highest conservation with Hir1 and Hir2 are encoded from exons 1-11 and 13-25, respectively. The amino- and carboxy-terminal regions of homology are separated from each other by a domain unique to HIRA that is encoded from a single exon. Seven WD repeats are conserved between yeast and man in the amino-terminal region of the HIR proteins. Individual repeats were found to be encoded from one, two, or three exons of the HIRA gene. End sequences have been obtained for all 24 introns, opening the way to PCR amplification of the entire coding sequence starting from genomic DNA. Point mutations can also be sought in 16 of the 24 introns that are readily PCR-amplifiable.

Dans la cellule eucaryote, l'ADN genomique s'associe a des proteines nucleaires pour former la chromatine, dans laquelle il est plus ou moins accessible selon les circonstances. La transcription genique, la replication de l'ADN comme sa reparation impliquent une regulation extremement precise de cette accessibilite. Pour une large part, celle-ci depend de diverses activites enzymatiques qui jouent un role essentiel dans la dynamique chromatinienne en modifiant ses composautes nucleique et proteique. Or, des anomalies dans ces mecanismes de controle semblent susciter nombre de pathologies humaines comme le suggere une serie d'observations publiees ces deux dernieres annees. C'est un assemblage nucleoproteique particulier, le nucleosome - huit molecules d'histones autour desquelles l'ADN s'enroule - qui a ete reconnu il y a un quart de siecle comme unite de base de la chromatine. Nous passons ici en revue quelques avancees majeures dans le domaine de la chromatine, avec un regard specifique porte sur certaines machineries moleculaires tres complexes qui utilisent l'energie de l'ATP pour mobiliser le nucleosome et remodeler la structure locale de la chromatine.