J.L. Thibaud

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). 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.

Forelimb Treatment in a Large Cohort of Dystrophic Dogs Supports Delivery of a Recombinant AAV for Exon Skipping in Duchenne Patients

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

Gait analysis using accelerometry in dystrophin-deficient dogs

Dogs affected with Golden Retriever Muscular Dystrophy (GRMD) exhibit striking clinical similarities with patients suffering from Duchenne muscular dystrophy (DMD), particularly gait impairments. The purpose of this study was to describe the use and reliability of accelerometry in gait assessment of dogs with muscular dystrophy. Eight healthy and 11 GRMD adult dogs underwent three gait assessment sessions, using accelerometry. Three-axial recordings of accelerations were performed, and gait variables calculated. Total power, force and regularity of accelerations, stride length and speed, normalized by height at withers, stride frequency, and cranio-caudal power were significantly decreased, whereas medio-lateral power was significantly increased in GRMD dogs. Moreover, these variables were repeatable within and between sessions. Accelerometry provides reliable variables which highlight specific gait patterns of GRMD dogs, describing objectively and quantitatively their slow, short-stepped, and swaying gait. As it is easy to set-up, quick to perform and inexpensive, accelerometry represents a useful tool, to assess locomotion during pre-clinical trials.

Longitudinal ambulatory measurements of gait abnormality in dystrophin-deficient dogs

BackgroundThis study aimed to measure the gait abnormalities in GRMD (Golden retriever muscular dystrophy) dogs during growth and disease progression using an ambulatory gait analyzer (3D-accelerometers) as a possible tool to assess the effects of a therapeutic intervention.MethodsSix healthy and twelve GRMD dogs were evaluated twice monthly, from the age of two to nine months. The evolution of each gait variable previously shown to be modified in control and dystrophin-deficient adults was assessed using two-ways variance analysis (age, clinical status) with repeated measurements. A principal component analysis (PCA) was applied to perfect multivariate data interpretation.ResultsSpeed, stride length, total power and force significantly already decreased (p < 0.01) at the age of 2 months. The other gait variables (stride frequency, relative power distributions along the three axes) became modified at later stages. Using the PCA analysis, a global gait index taking into account the main gait variables was calculated, and was also consistent to detect the early changes in the GRMD gait patterns, as well as the progressive degradation of gait quality.ConclusionThe gait variables measured by the accelerometers were sensitive to early detect and follow the gait disorders and mirrored the heterogeneity of clinical presentations, giving sense to monitor gait in GRMD dogs during progression of the disease and pre-clinical therapeutic trials.

P.13.4 Diaphragm structural abnormalities revealed by NMR imaging in the dystrophic dog

In DMD patients, respiratory muscle weakness induces a severe respiratory failure in the course of the disease. Hence, treating also the dystrophic respiratory muscles is of paramount importance and one of the arguments in favor of systemic therapy. In parallel, there is a need for novel non-invasive outcome measures for the diaphragm muscle, complementary to the histologic evaluation and the radiographic studies previously reported. NMR imaging has proven very efficient to discriminate dystrophic muscles from healthy control ones and even more, successfully treated dystrophic muscles from untreated ones in a loco-regional therapeutic trial of GRMD dogs, the canine counterpart of DMD patients. We investigated the ability of NMR to evaluate the diaphragm, which was imaged with a 3D SPACE T1-weighted sequence in 5 healthy and 8 GRMD dogs (mean age: 13.6 and 14.6xa0months, respectively), using a 3-tesla NMR scanner. In addition, breath-hold T2-weighted HASTE sequences were performed in 3 healthy and 7 GRMD dogs. In all dogs, diaphragms were visualized, particularly the ventral segment, cranial to the liver. In healthy dogs, it appeared as a thin, homogeneous, T1- and T2-hypointense structure compared to the liver. In GRMD dogs, as early as at 3xa0months of age, the diaphragm was thickened (11.2xa0±xa06.0xa0mm vs 2.4xa0±xa00.7xa0mm in healthy dogs), when measured in the sagittal plan, cranially to the liver close to its insertion to the manubrium; pxa0 To conclude, this study demonstrated the feasibility of NMR imaging of the diaphragm muscle despite its thinness, its complex shape, and respiratory motion artefacts. We described for the first time the dystrophic diaphragm abnormalities, as they can be visualized by NMR imaging as early as at 3xa0months of age. NMRI is a promising outcome measure for the diaphragm evaluation in preclinical trials using GRMD dogs.

T.P.23 Quantitative evaluation of locoregional high venous pressure rAAV8-U7- ESE6-ESE8 exon-skipping therapy in the GRMD dog using NMR 1H imaging and 31P spectroscopy

Abstract Replacement therapy of dystrophinopathies has begun with promising results both in animals and in patients. Non-invasive quantitative tools are needed to evaluate its potential benefits or side effects but also to determine the optimal protocol. Fifteen GRMD dogs were treated by unilateral high-pressure high-volume injection into the cephalic vein with a rAAV8-U7-ESE6-ESE8 solution. They were divided into 6 groups according to the dose of viral particles and the volume injected. The lowest dose-lower volume combination was skipped. Two dogs were injected with saline only. After high dose injection, some muscles displayed up to 80% of dystrophin positive fibers. Three months after injection, we evaluated both forearms with spectroscopy at 4T and NMR imaging at 3T. Comparisons between injected and non-injected arm were blinded. All indices of mock dogs were within the reference range of the GRMD population. Both spectroscopy and imaging indices showed differences between the two arms at the highest dose irrespective of volume injected and at the intermediate dose-high volume combination. The putative identification of the treated arm proved systematically correct. Among the spectroscopy indices, Pi/PCr, PCr/ATP and PDE/(Pixa0+xa0PCr) were the most sensitive and 31P NMR showed changes proportional to the number of AAV particles injected. With regard to imaging, 31 indices were evaluated in the extensor carpi radialis and flexor carpi ulnaris. The most relevant were: muscle heterogeneity in T2w images, the T1w/T2w signal ratio (SR), the T2w/PDw SR and the maximal relative signal enhancement after injection of 0.5xa0mmol/kg gadoteric acid. When high or intermediate doses were injected, indices actually decreased towards the normal range in both arms, with a reduction that was more substantial in the muscles of the injected side. This study demonstrated NMR ability to detect changes in dystrophic muscle structure and metabolism in response to exon-skipping therapy.

Anatomical and mesoscopic characterization of the dystrophic diaphragm: An in vivo nuclear magnetic resonance imaging study in the Golden retriever muscular dystrophy dog

Because respiratory failure remains a major issue in Duchenne Muscular Dystrophy patients, respiratory muscles are a key target of systemic therapies. In the Golden Retriever Muscular Dystrophy (GRMD) dogs, the disease shows strong clinical and histological similarities with the human pathology, making it a valuable model for preclinical therapeutic trials. We report here the first nuclear magnetic resonance (NMR) imaging anatomical study of the diaphragm in GRMD dogs and healthy controls. Both T1- and T2-weighted images of the diaphragm of seven healthy and thirteen GRMD dogs, from 3 to 36 months of age, were acquired on a 3 tesla NMR scanner. Abnormalities of texture and shape were revealed and consisted of increases in signal intensity on T2-weighted images and in signal heterogeneity on both T1- and T2-weighted images of the dystrophic diaphragm. These abnormalities were associated with a significant thickening of the muscle and we identified a clear 8-mm-threshold distinguishing clinically preserved GRMD dogs from those more severely affected. In this study, we demonstrated the feasibility of NMR imaging of the diaphragm and depicted several anatomical and mesoscopic anomalies in the dystrophic diaphragm. NMR imaging of the diaphragm shows a promise as an outcome measure in preclinical trials using GRMD dogs.

G.P.92

We have previously demonstrated that a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping injected in GRMD by locoregional transvenous perfusion of the forelimb, restores up to 80% dystrophin expression and improves histological, Nuclear Magnetic Resonance (NMR) pathological indices and strength in a dose-dependent manner at 3xa0months post injection (PInj). A recent paper suggested that dystrophin-positive fibers and rAAV genome drastically decrease with time, especially between 3 and 6xa0months PInj. This may compromise the clinical development of an AAV-mediated gene transfer approach. To assess this point, we realized a 6 to 7xa0months follow-up in a group of 3 GRMD aged 3–4xa0months when injected in both forelimbs with 2.5E13xa0vg/kg of a rAAV8 U7snRNA vector promoting exon skipping. Dogs were injected, followed up and evaluated (NMR, strength assessment, Dystrophin expression, vector biodistribution) in the same way as the previously reported large cohort of dogs that were followed for 3xa0months. Muscle biopsies were performed at 3xa0months and complete autopsy at 6 to 7xa0months PInj. Compared to previously reported dogs injected with the same dose, evaluation of dystrophin expression, histological aspect, AAV genome, NMR parameters and strength showed not differences indicating that the treatment effect was fully maintained over 6xa0months. No loss in AAV genome quantification and dystrophin expression was observed between the 3xa0months muscle biopsy specimens and 6xa0months muscle autopsy in the 3 dogs. A decrement in transgene copy numbers at 6xa0months PInj was observed in non muscular tissues. These data show, for the first time, that AAV-mediated exon skipping fully maintains dystrophin expression and functional recovery over a six months period. We suggest that long-term rAAV genome stability is obtained in dystrophic muscles when a substantial reversion of the muscular pathological pattern is reached.

Early detection of contractile dysfunction in GRMD dogs by post-processing of standard cine FLASH-MRI

Duchenne muscular dystrophy (DMD) due to dystrophin deficiency leads to death by heart failure in nearly 30% of cases. An early detection of myocardial abnormalities in these patients would help in the optimization of their management. The GRMD is a canine model of DMD, that develops a severe cardiomyopathy.