AAV-mediated transfer of FKRP shows therapeutic efficacy in a murine model but requires control of gene expression

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AAV-8 and AAV-9 Vectors Cooperate with Serum Proteins Differently Than AAV-1 and AAV-6

International audienceUnder intravenous delivery, recombinant adeno-associated vectors (rAAVs) interact with blood-borne components in ways that can critically alter their therapeutic efficiencies. We have previously shown that interaction with human galectin 3 binding protein dramatically reduces rAAV-6 efficacy, whereas binding of mouse C-reactive protein improves rAAV-1 and rAAV-6 transduction effectiveness. Herein we have assessed, through qualitative and quantitative studies, the proteins from mouse and human sera that bind with rAAV-8 and rAAV-9, two vectors that are being considered for clinical trials for patients with neuromuscular disorders. We show that, in contrast to rAAV-1 and rAAV-6, there was a substantial similarity in protein binding patterns between mouse and human sera for these vector serotypes. To establish an in vivo role for the vector binding of these sera proteins, we chose to study platelet factor 4 (PF4), which interacts with both vectors in both mouse and human sera. Experiments using PF4-knockout mice showed that a complete lack of PF4 did not alter skeletal muscle transduction for these vectors, whereas heart transduction was moderately improved. Our results strongly support our position that the impact of serum proteins on the transduction properties of rAAV-8 and rAAV-9, already observed in mouse models, should be similar in human preclinical trials

Proteomics profiling of urine reveals specific titin fragments as biomarkers of Duchenne muscular dystrophy.

International audienceDiagnosis of muscular dystrophies is currently based on invasive methods requiring muscle biopsies or blood tests. The aim of the present study was to identify urinary biomarkers as a diagnostic tool for muscular dystrophies. Here, the urinary proteomes of Duchenne muscular dystrophy (DMD) patients and healthy donors were compared with a bottom-up proteomic approach. Label-free analysis of more than 1100 identified proteins revealed that 32 of them were differentially expressed between healthy controls and DMD patients. Among these 32 proteins, titin showed the highest fold change between healthy subjects and DMD patients. Interestingly, most of the sequenced peptides belong to the N-terminal and C-terminal parts of titin, and the presence of the corresponding fragments in the urine of DMD patients was confirmed by Western blot analysis. Analysis of a large cohort of DMD patients and age-matched controls (a total of 104 individuals aged from 3 to 20 years) confirmed presence of the N-ter fragment in all but two patients. In two DMD patients aged 16 and 20 years this fragment was undetectable and two healthy controls of 16 and 19 years with serum CK >800 IU/L demonstrated a low level of the fragment. N- and C-terminal titin fragments were also detected in urine from patients with other muscular dystrophies such as Becker muscular dystrophy and Limb-girdle muscular dystrophy (type 1D, 2D and 2J) but not in neurogenic spinal muscular atrophy. They were also present in urine of dystrophin-deficient animal models (GRMD dogs and mdx mice). Titin is the first urinary biomarker that offers the possibility to develop a simple, non-invasive and easy-to-use test for pre-screening of muscular dystrophies, and may also prove to be useful for the non-invasive follow up of DMD patients under treatment

Evaluating the Impact of Variable Phosphorothioate Content in Tricyclo-DNA Antisense Oligonucleotides in a Duchenne Muscular Dystrophy Mouse Model

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Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides

International audienceAbstract Tricyclo-DNA (tcDNA) is a conformationally constrained oligonucleotide analog that has demonstrated great therapeutic potential as antisense oligonucleotide (ASO) for several diseases. Like most ASOs in clinical development, tcDNA were modified with phosphorothioate (PS) backbone for therapeutic purposes in order to improve their biodistribution by enhancing association with plasma and cell protein. Despite the advantageous protein binding properties, systemic delivery of PS-ASO remains limited and PS modifications can result in dose limiting toxicities in the clinic. Improving extra-hepatic delivery of ASO is highly desirable for the treatment of a variety of diseases including neuromuscular disorders such as Duchenne muscular dystrophy. We hypothesized that conjugation of palmitic acid to tcDNA could facilitate the delivery of the ASO from the bloodstream to the interstitium of the muscle tissues. We demonstrate here that palmitic acid conjugation enhances the potency of tcDNA-ASO in skeletal and cardiac muscles, leading to functional improvement in dystrophic mice with significantly reduced dose of administered ASO. Interestingly, palmitic acid-conjugated tcDNA with a full phosphodiester backbone proved effective with a particularly encouraging safety profile, offering new perspectives for the clinical development of PS-free tcDNA-ASO for neuromuscular diseases

Serum proteomic profiling reveals fragments of MYOM3 as potential biomarkers for monitoring the outcome of therapeutic interventions in muscular dystrophies

Therapy-responsive biomarkers are an important and unmet need in the muscular dystrophy field where new treatments are currently in clinical trials. By using a comprehensive high-resolution mass spectrometry approach and western blot validation, we found that two fragments of the myofibrillar structural protein myomesin-3 (MYOM3) are abnormally present in sera of Duchenne muscular dystrophy (DmD) patients, limb-girdle muscular dystrophy type 2D (LGMD2D) and their respective animal models. Levels of MYOM3 fragments were assayed in therapeutic model systems: (1) restoration of dystrophin expression by antisense oligonucleotide-mediated exon-skipping in mdx mice and (2) stable restoration of a-sarcoglycan expression in KO-SGCA mice by systemic injection of a viral vector. Following administration of the therapeutic agents MYOM3 was restored toward wild-type levels. In the LGMD model, where different doses of vector were used, MYOM3 restoration was dose-dependent. MYOM3 fragments showed lower inter-individual variability compared with the commonly used creatine kinase assay, and correlated better with the restoration of the dystrophin-associated protein complex and muscle force. These data suggest that the MYOM3 fragments hold promise for minimally invasive assessment of experimental therapies for DMD and other neuromuscular disorders.Peer reviewe

γ-sarcoglycan and dystrophin mutation spectrum in an Algerian cohort

International audienceWe report the genetic analysis of a large series of 76 Algerian patients from 65 unrelated families who presented with early onset severe muscular dystrophy and a clinical phenotype resembling limb-girdle muscular dystrophy type 2C