The identification of the most efficient method for whole central nervous system targeting that is translatable to humans and the safest route of adeno-associated virus (AAV) administration is a major concern for future applications in clinics. Additionally, as many AAV serotypes were identified for gene introduction into the brain and the spinal cord, another key to human gene-therapy success is to determine the most efficient serotype. In this study, we compared lumbar intrathecal administration through catheter implantation and intracerebroventricular administration in the cynomolgus macaque. We also evaluated and compared two AAV serotypes that are currently used in clinical trials: AAV9 and AAVrh10. We demonstrated that AAV9 lumbar intrathecal delivery using a catheter achieved consistent transgene expression in the motor neurons of the spinal cord and in the neurons/glial cells of several brain regions, whereas AAV9 intracerebroventricular delivery led to a consistent transgene expression in the brain. In contrast, AAVrh10 lumbar intrathecal delivery led to rare motor neuron targeting. Finally, we found that AAV9 efficiently targets respiratory and skeletal muscles after injection into the cerebrospinal fluid (CSF), which represents an outstanding new property that can be useful for the treatment of diseases affecting both the central nervous system and muscle.

Pompe disease is a lysosomal storage disorder caused by acid-α-glucosidase (GAA) deficiency, leading to glycogen storage. The disease manifests as a fatal cardiomyopathy in infantile form. Enzyme replacement therapy (ERT) has recently prolonged the lifespan of these patients, revealing a new natural history. The neurologic phenotype and the persistence of selective muscular weakness in some patients could be attributed to the central nervous system (CNS) storage uncorrected by ERT. GAA-KO 6neo/6neo mice were treated with a single intrathecal administration of adeno-associated recombinant vector (AAV) mediated gene transfer of human GAA at 1 month and their neurologic, neuromuscular, and cardiac function was assessed for 1 year. We demonstrate a significant functional neurologic correction in treated animals from 4 months onward, a neuromuscular improvement from 9 months onward, and a correction of the hypertrophic cardiomyopathy at 12 months. The regions most affected by the disease i.e. the brainstem, spinal cord, and the left cardiac ventricular wall all show enzymatic, biochemical and histological correction. Muscle glycogen storage is not affected by the treatment, thus suggesting that the restoration of muscle functionality is directly related to the CNS correction. This unprecedented global and long-term CNS and cardiac cure offer new perspectives for the management of patients.

Pompe disease, which is due to acid alpha-glucosidase deficiency, is characterized by skeletal muscle dysfunction attributed to the accumulation of glycogen-filled lysosomes and autophagic buildup. Despite the extensive tissue damages, a failure of satellite cell (SC) activation and lack of muscle regeneration have been reported in patients. However, the origin of this defective program is unknown. Additionally, whether these deficits occur gradually over the disease course is unclear. Using a longitudinal pathophysiological study of two muscles in a Pompe mouse model, here, we report that the enzymatic defect results in a premature saturating glycogen overload and a high number of enlarged lysosomes. The muscles gradually display profound remodeling as the number of autophagic vesicles, centronucleated fibers, and split fibers increases and larger fibers are lost. Only a few regenerated fibers were observed regardless of age, although the SC pool was preserved. Except for the early age, during which higher numbers of activated SCs and myoblasts were observed, no myogenic commitment was observed in response to the damage. Following in vivo injury, we established that muscle retains regenerative potential, demonstrating that the failure of SC participation in repair is related to an activation signal defect. Altogether, our findings provide new insight into the pathophysiology of Pompe disease and highlight that the activation signal defect of SCs compromises muscle repair, which could be related to the abnormal energetic supply following autophagic flux impairment.

Pompe disease (glycogen storage disease type II) is a lysosomal storage disorder due to a mutation in the gene that encodes acid alpha-glucosidase (GAA). GAA deficiency causes the excessive storage of lysosomal glycogen in many cell types, leading to cell and, subsequently, tissue dysfunction. Cardiac, respiratory and skeletal muscles are the most severely affected. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA, Myozyme®, Genzyme, Cambridge) is the only approved treatment for Pompe disease. A new therapeutic strategy was developed consisting of delivering adeno-associated virus serotype 9 (AAV9) and serotype 10 (AAV10) vectors expressing human GAA into cerebrospinal fluid of GAA-KO 6neo/6neo Pompe mice. The purpose of this work was to investigate synchrotron Fourier transform infrared (sFT-IR) and deep ultraviolet (sDUV) microspectroscopies to detect new biomarkers of the disease and the AAV gene therapy in the cardiac muscle, one of the most affected organs in Pompe disease. Multivariate statistics applied to sFT-IR spectra between 4000 cm–1 and 950 cm–1 highlighted the potential of sFT-IR to discriminate Pompe (-/-), Wild type and AAV-treated animals from C–H stretching vibrations of CH3, from C–O, C–N and C–C stretching vibrations of amide I, II, III bands and from specific IR signature of the glycogen. Investigations performed by sDUV microscopy showed a significant increase of the tryptophan autofluorescent signal in the right ventricle for the AAV9-treated Pompe mice. The high-resolution sDUV microspectroscopy experiments suggested a correlation between the tryptophan-rich area and the GAA-rich area. These unprecedented results demonstrate that high-resolution UV microspectroscopy can be a complementary innovative approach to monitor the chemical change in label-free cardiac muscle section. Moreover, this non-destructive technology can be applied to a small amount of tissue allowing therapeutic assessment from biopsy of human patients.