Bone Marrow Transplant

Mucopolysaccharidosis type I-H (MPS I-H) is a rare lysosomal storage disorder caused by α-L-Iduronidase deficiency. Early haematopoietic stem cell transplantation (HSCT) is the sole available therapeutic option to preserve neurocognitive functions. We report long-term follow-up (median 9 years, interquartile range 8-16.5) for 51 MPS I-H patients who underwent HSCT between 1986 and 2018 in France. 4 patients died from complications of HSCT and one from disease progression. Complete chimerism and normal α-L-Iduronidase activity were obtained in 84% and 71% of patients respectively. No difference of outcomes was observed between bone marrow and cord blood stem cell sources. All patients acquired independent walking and 91% and 78% acquired intelligible language or reading and writing. Intelligence Quotient evaluation (n = 23) showed that 69% had IQ ≥ 70 at last follow-up. 58% of patients had normal or remedial schooling and 62% of the 13 adults had good socio-professional insertion. Skeletal dysplasia as well as vision and hearing impairments progressed despite HSCT, with significant disability. These results provide a long-term assessment of HSCT efficacy in MPS I-H and could be useful in the evaluation of novel promising treatments such as gene therapy

Optimisation d'une approche de thérapie génique pour la glycogénose de type III basée sur des vecteurs viraux de type adéno-associés

La glycogénose de type III (GSDIII) est une maladie rare du métabolisme liée à des mutations du gène AGL codant l'enzyme débranchante du glycogène (GDE). Elle s'accompagne d'une accumulation de glycogène dans le foie, le cœur et les muscles. La grande taille de l'ADN complémentaire de GDE rend difficile l'utilisation d'une stratégie de thérapie génique basée sur un vecteur adéno-associé recombinant (rAAV) unique.Dans la première partie de ce travail, nous avons développé une approche utilisant un vecteur rAAV unique en générant une forme tronquée fonctionnelle de GDE, afin de permettre la correction de l'atteinte musculaire et cardiaque dans plusieurs modèles de GSDIII. En utilisant la structure tridimensionnelle de GDE et la position des variants faux-sens décrits dans cette pathologie, nous avons généré plusieurs GDE tronquées conservant une activité enzymatique. Une des GDE ayant une délétion dans le domaine N-terminal, ∆Nter2-GDE, conserve une activité équivalente à celle de la GDE de taille complète in vivo. Un vecteur rAAV exprimant ∆Nter2-GDE, injecté par voie intraveineuse à des souris Agl-/-, a permis la correction complète de l'accumulation de glycogène dans le cœur et les muscles squelettiques trois mois après injection et une normalisation de l'histologie et de la force musculaire des souris. Une correction équivalente a aussi été obtenue dans le cœur et les muscles de rats Agl-/- injectés avec ce même vecteur. Enfin, l'expression de ∆Nter2-GDE dans des cellules musculaires humaines invalidées pour le gène AGL permet aussi de corriger l'accumulation de glycogène, sans toxicité spécifique.Ces résultats démontrent la possibilité de corriger l'atteinte musculaire dans plusieurs modèles de GSDIII en utilisant une GDE tronquée et sont prometteurs pour une éventuelle utilisation clinique de ce vecteur pour traiter des patients atteints de GSDIII.Dans la seconde partie de ce travail, nous avons optimisé un vecteur rAAV unique afin de cibler le foie et de corriger l'atteinte hépatique des souris Agl-/-. Le vecteur développé dans la première partie ne permet pas une correction du foie du fait de l'utilisation d'un promoteur et d'une capside virale sans ciblage ou expression dans le foie. L'injection d'un vecteur rAAV optimisé pour le ciblage du foie dans des souris Agl-/- de 4-5 mois, ayant déjà une fibrose hépatique avancée, permet une correction transitoire des paramètres biochimiques (glycémie, ASAT, ALAT), avec une perte de correction progressive 6 mois après injection. Une injection plus précoce, dans des souris Agl-/- de 6 semaines sans fibrose lors de l'injection, permet une correction du phénotype hépatique chez la moitié des animaux, persistant 9 mois après injection. L'autre moitié des animaux présentent néanmoins une perte progressive de correction à partir de 6 mois post-injection, comme chez les souris injectées à un âge plus avancé. Les raisons de ce phénomène sont en cours de caractérisation.Une poursuite de l'optimisation reste donc nécessaire afin d'améliorer la correction du phénotype hépatique et surtout afin de pouvoir corriger la totalité des atteintes de la GSDIII, hépatique, musculaire et cardiaque, en utilisant un seul vecteur rAAV.Glycogen storage disease type III (GSDIII) is a rare inborn error of metabolism affecting liver, skeletal muscle, and heart, due to mutations of the AGL gene encoding for the glycogen debranching enzyme (GDE). The 4.6 kb GDE cDNA represents the major technical challenge toward the development of a single recombinant adeno-associated virus (rAAV)-derived vector strategy and alternative approaches using bacterial GDE orthologs or dual vectors have been proposed.In the first part of this thesis, we developed a single rAAV vector strategy expressing a truncated GDE protein in order to correct the heart and muscle impairment in multiple GSDIII models. Using molecular modelling and the existing information on AGL missense variants, we generated multiple truncated GDEs retaining activity. Among them, the N-terminal-truncated mutant ∆Nter2-GDE had a similar efficacy in vivo compared to the full-size enzyme. A rAAV vector expressing ∆Nter2-GDE allowed complete glycogen clearance in the heart and muscle of Agl-/- mice, three months after intravenous injection, as well as normalization of histology features and restoration of muscle strength. Similarly, glycogen accumulation and histological features were corrected in a recently generated Agl-/- rat model. Finally, ∆Nter2-GDE corrected glycogen accumulation in an in vitro human skeletal muscle cellular model of GSDIII, without specific toxicity. In conclusion, our results demonstrate the ability of a functional truncated GDE transgene to correct the muscle and heart phenotype in multiple models of GSDIII, supporting its clinical translation to GSDIII patients.In the second part of this thesis, we optimized a single vector rAAV approach to correct the liver phenotype, which could not be achieved with our ∆Nter2-GDE expressing rAAV vector because of the use of a promoter and a capsid both with liver-detargeting features. We designed an rAAV with a capsid with high liver tropism expressing the full-length GDE under the control of a liver promoter. Intravenous administration of high dose of this vector to 4-5-month old Agl-/- could only achieve transient correction of the glycemia and liver enzymes 2 months after injection, with progressive loss of correction during the follow-up. Because, at this age, Agl-/- mice already display significant liver fibrosis, we then administered the rAAV vector intravenously to younger 6-week-old Agl-/- mice. Glycemia normalized quickly after injection. Long-term correction of the liver phenotype, up to 9 months post-injection, was only achieved in half of the injected mice, with normalization of both glycogen content and histologic features of the disease. In the other half of the cohort, a progressive loss of correction was observed from 6 months post-injection, similar to Agl-/- mice injected at an older age. The mechanism behind the progressive loss of correction observed is unclear and currently under investigations.Further optimization is required to improve liver targeting and GDE expression, especially with the use of the ∆Nter2-GDE mutant, as well as to achieve the correction of both the liver and muscle diseases

Approche de thérapie génique utilisant des vecteurs AAV pour la glycogénose de type III (GSDIII)

International audienc

AAV gene therapy for liver diseases: from the bedside to the bench

International audienc

Jugements d'évaluation : des constituants détachés à d'autres formes régulières

National audienc

Parametric imagin for assessing tumor response follow-up with PET/CT during radiotherapy in patients with non-small cell lung cancer.

International audienc

Pathological modeling of glycogen storage disease type III with CRISPR/Cas9 edited human pluripotent stem cells

International audienceIntroduction: Glycogen storage disease type III (GSDIII) is a rare genetic disease caused by mutations in the AGL gene encoding the glycogen debranching enzyme (GDE). The deficiency of this enzyme, involved in cytosolic glycogen degradation, leads to pathological glycogen accumulation in liver, skeletal muscles and heart. Although the disease manifests with hypoglycemia and liver metabolism impairment, the progressive myopathy is the major disease burden in adult GSDIII patients, without any curative treatment currently available. Methods: Here, we combined the self-renewal and differentiation capabilities of human induced pluripotent stem cells (hiPSCs) with cutting edge CRISPR/Cas9 gene editing technology to establish a stable AGL knockout cell line and to explore glycogen metabolism in GSDIII. Results: Following skeletal muscle cells differentiation of the edited and control hiPSC lines, our study reports that the insertion of a frameshift mutation in AGL gene results in the loss of GDE expression and persistent glycogen accumulation under glucose starvation conditions. Phenotypically, we demonstrated that the edited skeletal muscle cells faithfully recapitulate the phenotype of differentiated skeletal muscle cells of hiPSCs derived from a GSDIII patient. We also demonstrated that treatment with recombinant AAV vectors expressing the human GDE cleared the accumulated glycogen. Discussion: This study describes the first skeletal muscle cell model of GSDIII derived from hiPSCs and establishes a platform to study the mechanisms that contribute to muscle impairments in GSDIII and to assess the therapeutic potential of pharmacological inducers of glycogen degradation or gene therapy approaches

Outcomes of 38 patients with PFIC3: Impact of genotype and of response to ursodeoxycholic acid therapy

Background & Aims: Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare liver disease caused by biallelic variations in ABCB4. Data reporting on the impact of genotype and of response to ursodeoxycholic acid (UDCA) therapy on long-term outcomes are scarce. Methods: We retrospectively describe a cohort of 38 patients with PFIC3 with a median age at last follow-up of 19.5 years (range 3.8–53.8). Results: Twenty patients presented with symptoms before 1 year of age. Thirty-one patients received ursodeoxycholic acid (UDCA) therapy resulting in serum liver test improvement in 20. Twenty-seven patients had cirrhosis at a median age of 8.1 years of whom 18 received a liver transplant at a median age of 8.5 years. Patients carrying at least one missense variation were more likely to present with positive (normal or decreased) canalicular MDR3 expression in the native liver and had prolonged native liver survival (NLS; median 12.4 years [range 3.8-53.8]). In contrast, in patients with severe genotypes (no missense variation), there was no detectable canalicular MDR3 expression, symptom onset and cirrhosis occurred earlier, and all underwent liver transplantation (at a median age of 6.7 years [range 2.3–10.3]). The latter group was refractory to UDCA treatment, whereas 87% of patients with at least one missense variation displayed an improvement in liver biochemistry in response to UDCA. Biliary phospholipid levels over 6.9% of total biliary lipid levels predicted response to UDCA. Response to UDCA predicted NLS. Conclusions: Patients carrying at least one missense variation, with positive canalicular expression of MDR3 and a biliary phospholipid level over 6.9% of total biliary lipid levels were more likely to respond to UDCA and to exhibit prolonged NLS. Impact and implications: In this study, data show that genotype and response to ursodeoxycholic acid therapy predicted native liver survival in patients with PFIC3 (progressive familial intrahepatic cholestasis type 3). Patients carrying at least one missense variation, with positive (decreased or normal) immuno-staining for canalicular MDR3, and a biliary phospholipid level over 6.9% of total biliary lipids were more likely to respond to ursodeoxycholic acid therapy and to exhibit prolonged native liver survival

A functional mini-GDE transgene corrects impairment in models of glycogen storage disease type III

International audienc