Vers le développement d'une thérapie génique pour la maladie de Pompe : Caractérisation du phénotype immunitaire de la maladie de Pompe et comparaison du potentiel thérapeutique de la thérapie génique avec le traitement standard actuel

La maladie de Pompe est une maladie lysosomale causée par des mutations sur l'enzyme α-glucosidase acide (GAA), responsable de la dégradation du glycogène lysosomal. Le déficit en GAA provoque l'accumulation de glycogène dans de multiples tissus, conduisant à une maladie neuromusculaire complexe avec une morbidité et mortalité élevées. En plus des symptômes connus, nos études montrent que le déficit en GAA affecte l’activation et le fonctionnement des cellules immunitaires chez les patients et le modèle murin de la maladie de Pompe. En particulier, nous avons observé une suractivation des cellules T effectrices, ainsi qu’un défaut dans l’induction et la fonction des cellules T régulatrices (Tregs). De plus, les souris atteintes de la maladie de Pompe présentent de l’inflammation tissulaire précoce, ce qui pourrait contribuer à la physiopathologie de la maladie. Ces résultats pourraient ouvrir des nouvelles voies pour étudier des stratégies qui retardent la progression de la maladie. Dans une deuxième série d'expériences, nous montrons qu'une thérapie génique ciblée au foie avec un transgène GAA sécrétable conduit à une meilleure correction de la maladie dans un modèle murin immunodéficient de la maladie, par rapport au traitement enzymatique substitutif existant. Étant donné que les Treg jouent un rôle essentiel dans la thérapie génique ciblée au foie, en induisant une tolérance immunitaire vers les transgènes hépatiques, de futures études devront évaluer l'impact des altérations immunitaires associées à cette maladie sur l'efficacité de la thérapie génique.Pompe disease is a lysosomal storage disease caused by mutations on the enzyme acid α-glucosidase (GAA), responsible for degrading lysosomal glycogen. GAA deficiency causes the accumulation of glycogen in multiple tissues, particularly in muscles and central nervous system, leading to a complex neuromuscular disease with high morbidity and mortality. In addition to the known symptoms, our studies in Pompe disease patients and mice show that GAA deficiency affects the activation and function of immune cells, particularly of T cells, leading to a higher activation of effector cells and impaired induction and suppressive function of regulatory T cells (Tregs). Moreover, Pompe disease mice present tissue inflammation at early stages of the disease, altogether suggesting that alterations in the immune system could contribute to the disease pathophysiology. These findings could open new venues to investigate strategies that delay the progression of the disease. In a second set of experiments we show that liver-directed gene therapy with a secretable GAA transgene results in superior disease rescue in an immunodefficient Pompe disease mouse model, when compared to the current enzyme replacement therapy. Because Tregs play an essential role in liver-directed gene therapy, by inducing immune tolerance towards hepatic transgenes, future studies will have to evaluate the potential impact of immune alterations associated to Pompe disease on the efficacy of liver-targeted gene transfer.La enfermedad de Pompe es una enfermedad lisosomal (lysosomal storage disease, LSD) causada por mutaciones en la enzima α-glucosidasa ácida (GAA), que hidroliza el glucógeno en glucosa en los lisosomas. La disfunción de esta enzima causa la acumulación de glucógeno en múltiples tejidos, principalmente en células musculares y neuronas. Como resultado, los pacientes desarrollan hipertrofia cardíaca, debilidad muscular, insuficiencia respiratoria, alteraciones cognitivas y muerte prematura por paro cardiorrespiratorio. Actualmente la enfermedad de Pompe se trata con terapia de reemplazo enzimático (Enzyme replacement therapy, ERT) con GAA recombinante humana (rhGAA). Este tratamiento ha demostrado corregir la patología cardíaca y extender la esperanza de vida de los pacientes. Sin embargo, la eficacia de la ERT en los músculos respiratorios y esqueléticos es parcial, y nula en el sistema nervioso central (SNC). Además, la proteína rhGAA es altamente inmunogénica, por lo que el tratamiento no es efectivo en ciertos pacientes. Otros inconvenientes son el elevado coste de la ERT y la necesidad de infusiones continuadas a lo largo de la vida del paciente. En este estudio mostramos que la terapia génica dirigida al hígado mediante vectores adeno-asociados (AAV) expresando una versión modificada de la GAA revierte de forma significativa la enfermedad a nivel de la musculatura esquelética y del SNC, en un modelo animal de la enfermedad de Pompe. Además, mostramos que este tratamiento es superior en eficacia al tratamiento estándar por ERT, incluso a dosis reducidas de vector AAV. Con tal de comprender mejor los mecanismos de acción de estos dos tratamientos, hemos llevado a cabo un estudio farmacocinético de los niveles de GAA en circulación y en múltiples tejidos en los dos casos. Dicho estudio muestra que niveles reducidos pero constantes de GAA en circulación proporcionados por el hígado permiten una mayor acumulación de GAA en los tejidos en comparación a la ERT, mejorando así la eficacia del tratamiento. Debido a que las reacciones inmunes contra el vector AAV y el transgén son un obstáculo importante en la aplicación clínica de la terapia génica, y a que alteraciones en los lisosomas han demostrado tener un impacto sobre el sistema inmune en diferentes modelos, también hemos estudiado el sistema inmune en el caso de la enfermedad de Pompe. Mediante estos estudios, hemos observado que la acumulación de glucógeno en los lisosomas de células inmunes está asociada a una mayor activación de estas células, tanto en pacientes como en ratones con enfermedad de Pompe, y particularmente en las células T. Además, ratones con enfermedad de Pompe presentan inflamación en los tejidos ya en las primeras etapas de la enfermedad. Por otra parte, mostramos que el mayor estado de activación de las células T podría deberse a alteraciones en el metabolismo de estas células, como resultado de las alteraciones lisosómicas. Finalmente, los ratones con enfermedad de Pompe presentan un defecto en la inducción de células T reguladoras Foxp3+ (Tregs), y estas células tienen un menor potencial inhibidor en comparación con Tregs de ratones sanos. Alteraciones en el sistema inmunitario podrían contribuir a la fisiopatología de la enfermedad. Por lo tanto, estos hallazgos podrían abrir nuevos caminos para investigar estrategias que retrasen la progresión de la enfermedad. Además, las Tregs juegan un papel esencial en la terapia génica dirigida al hígado, mediante la inducción de tolerancia inmune hacia transgenes expresados por hepatocitos. Por lo tanto, futuros estudios deberán evaluar el impacto de las alteraciones inmunitarias asociadas a la enfermedad de Pompe sobre la eficacia de la terapia génica.La malaltia de Pompe és una malaltia lisosomal (lysosomal storage disease, LSD) deguda a mutacions en l'enzim α-glucosidasa àcida (GAA), que hidrolitza el glicogen en glucosa als lisosomes. La disfunció d'aquest enzim causa l'acumulació de glicogen en múltiples teixits, principalment en cèl·lules musculars i neurones. Com a resultat, els pacients presenten hipertròfia cardíaca, debilitat muscular, insuficiència respiratòria, alteracions cognitives i mort prematura per aturada cardiorespiratòria. Actualment, la malaltia de Pompe és tractada amb teràpia de reemplaçament enzimàtic (Enzyme replacement therapy, ERT) amb GAA recombinant humana (rhGAA). Aquest tractament ha demostrat corregir la patologia cardíaca i estendre l'esperança de vida dels pacients. No obstant, l'eficàcia de l'ERT en els músculs respiratoris i esquelètics és parcial, i nul·la en el sistema nerviós central (SNC). A més, la proteïna rhGAA és altament immunogènica, de manera que el tractament no és efectiu en certs pacients. Altres inconvenients són l'elevat cost de l'ERT i la necessitat d'infusions continuades al llarg de la vida del pacient. En aquest estudi mostrem que la teràpia gènica dirigida al fetge mitjançant vectors adeno-associats (AAV) expressant una versió modificada de la GAA millora de forma significativa la malaltia a nivell de la musculatura esquelètica i del SNC, en un model animal de la malaltia de Pompe. A més, mostrem que aquest tractament és superior en eficàcia al tractament estàndard per ERT, fins i tot a dosis reduïdes de vector AAV. Per tal de comprendre millor els mecanismes d'acció d'aquests dos tractaments, hem dut a terme un estudi farmacocinètic dels nivells de GAA en circulació i en múltiples teixits en ambdós casos. Aquest estudi mostra que nivells reduïts però constants de GAA en circulació proporcionats pel fetge permeten una major acumulació de la GAA en els teixits en comparació a la ERT, millorant així l'eficàcia del tractament. Degut a que les reaccions immunes contra el vector AAV i el transgèn són un obstacle important en l'aplicació clínica de la teràpia gènica, i a que alteracions en els lisosomes han demostrat tenir un impacte sobre el sistema immune en differents models, també hem avaluat el sistema immune en el cas de la malaltia de Pompe. Mitjançant aquests estudis, hem observat que l'acumulació de glicogen en els lisosomes de les cèl·lules immunes està associada a una major activació d'aquestes cèl·lules en pacients i ratolins amb malaltia de Pompe, particularment en les cèl·lules T. A més, ratolins amb malaltia de Pompe presenten inflamació dels teixits ja en les primeres etapes de la malaltia. D’altra banda, hem observat que el major estat d'activació de les cèl·lules T podria ser degut a alteracions en el metabolisme d'aquestes cèl·lules, com a resultat de les alteracions lisosomals. Finalment, els ratolins amb malaltia de Pompe presenten un defecte en la inducció de cèl·lules T reguladores Foxp3+ (Tregs), i aquestes cèl·lules tenen un menor potencial inhibidor en comparació amb les Tregs de ratolins sans. Alteracions en el sistema immunitari podrien contribuir a la fisiopatologia de la malaltia. Per tant, aquestes observacions podrien obrir nous camins a l’hora d’investigar estratègies que retardin la progressió de la malaltia. A més, les Tregs juguen un paper essencial en la teràpia gènica dirigida al fetge, mitjançant la inducció de tolerància immune cap a transgens expressats per hepatòcits. Per tant, futurs estudis hauran d'avaluar l'impacte de les alteracions immunitàries associades a la malaltia de Pompe sobre l'eficàcia del tractament per teràpia gènica

Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis

Recent evidence has established that extracellular vesicles (EVs), including exosomes and microvesicles, form an endogenous transport system through which biomolecules, including proteins and RNA, are exchanged between cells. This endows EVs with immense potential for drug delivery and regenerative medicine applications. Understanding the biology underlying EV-based intercellular transfer of cargo is of great importance for the development of EV-based therapeutics. Here, we sought to characterize the cellular mechanisms involved in EV uptake. Internalization of fluorescently-labeled EVs was evaluated in HeLa cells, in 2D (monolayer) cell culture as well as 3D spheroids. Uptake was assessed using flow cytometry and confocal microscopy, using chemical as well as RNA interference-based inhibition of key proteins involved in individual endocytic pathways. Experiments with chemical inhibitors revealed that EV uptake depends on cholesterol and tyrosine kinase activity, which are implicated in clathrin-independent endocytosis, and on Na+/H+ exchange and phosphoinositide 3-kinase activity, which are important for macropinocytosis. Furthermore, EV internalization was inhibited by siRNA-mediated knockdown of caveolin-1, flotillin-1, RhoA, Rac1 and PAK1, but not clathrin heavy chain. Together, these results suggest that EVs enter cells predominantly via clathrin-independent endocytosis and macropinocytosis. Identification of EV components that promote their uptake via pathways that lead to functional cargo transfer might allow development of more efficient therapeutics through EV-inspired engineering

Cellular uptake of extracellular vesicles is mediated by clathrin-independent endocytosis and macropinocytosis

Recent evidence has established that extracellular vesicles (EVs), including exosomes and microvesicles, form an endogenous transport system through which biomolecules, including proteins and RNA, are exchanged between cells. This endows EVs with immense potential for drug delivery and regenerative medicine applications. Understanding the biology underlying EV-based intercellular transfer of cargo is of great importance for the development of EV-based therapeutics. Here, we sought to characterize the cellular mechanisms involved in EV uptake. Internalization of fluorescently-labeled EVs was evaluated in HeLa cells, in 2D (monolayer) cell culture as well as 3D spheroids. Uptake was assessed using flow cytometry and confocal microscopy, using chemical as well as RNA interference-based inhibition of key proteins involved in individual endocytic pathways. Experiments with chemical inhibitors revealed that EV uptake depends on cholesterol and tyrosine kinase activity, which are implicated in clathrin-independent endocytosis, and on Na+/H+ exchange and phosphoinositide 3-kinase activity, which are important for macropinocytosis. Furthermore, EV internalization was inhibited by siRNA-mediated knockdown of caveolin-1, flotillin-1, RhoA, Rac1 and PAK1, but not clathrin heavy chain. Together, these results suggest that EVs enter cells predominantly via clathrin-independent endocytosis and macropinocytosis. Identification of EV components that promote their uptake via pathways that lead to functional cargo transfer might allow development of more efficient therapeutics through EV-inspired engineering

Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle

International audienceThe pro-tolerogenic environment of the liver makes this tissue an ideal target for gene replacement strategies. In other peripheral tissues such as the skeletal muscle, anti-transgene immune response can result in partial or complete clearance of the transduced fibers. Here, we characterized liver-induced transgene tolerance after simultaneous transduction of liver and muscle. A clinically relevant transgene, α-sarcoglycan, mutated in limb-girdle muscular dystrophy type 2D, was fused with the SIINFEKL epitope (hSGCA-SIIN) and expressed with adeno-associated virus vectors (AAV-hSGCA-SIIN). Intramuscular delivery of AAV-hSGCA-SIIN resulted in a strong inflammatory response, which could be prevented and reversed by concomitant liver expression of the same antigen. Regulatory T cells and upregulation of checkpoint inhibitor receptors were required to establish and maintain liver-mediated peripheral tolerance. This study identifies the fundamental role of the synergy between Tregs and upregulation of checkpoint inhibitor receptors in the liver-mediated control of anti-transgene immunity triggered by muscle-directed gene transfer

Exposure to wild-type AAV drives distinct capsid immunity profiles in humans

International audienceRecombinant adeno-associated virus (AAV) vectors have been broadly adopted as a gene delivery tool in clinical trials, owing to their high efficiency of transduction of several host tissues and their low immunogenicity. However, a considerable proportion of the population is naturally exposed to the WT virus from which AAV vectors are derived, which leads to the acquisition of immunological memory that can directly determine the outcome of gene transfer. Here, we show that prior exposure to AAV drives distinct capsid immunity profiles in healthy subjects. In peripheral blood mononuclear cells (PBMCs) isolated from AAV-seropositive donors, recombinant AAV triggered TNF-α secretion in memory CD8+ T cells, B cell differentiation into antibody-secreting cells, and anti-capsid antibody production. Conversely, PBMCs isolated from AAV-seronegative individuals appeared to carry a population of NK cells reactive to AAV. Further, we demonstrated that the AAV capsid activates IL-1β and IL-6 cytokine secretion in monocyte-related dendritic cells (moDCs). IL-1β and IL-6 blockade inhibited the anti-capsid humoral response in vitro and in vivo. These results provide insights into immune responses to AAV in humans, define a possible role for moDCs and NK cells in capsid immunity, and open new avenues for the modulation of vector immunogenicity

AAV Gene Transfer with Tandem Promoter Design Prevents Anti-transgene Immunity and Provides Persistent Efficacy in Neonate Pompe Mice

International audienceHepatocyte-restricted, AAV-mediated gene transfer is being used to provide sustained, tolerogenic transgene expression in gene therapy. However, given the episomal status of the AAV genome, this approach cannot be applied to pediatric disorders when hepatocyte proliferation may result in significant loss of therapeutic efficacy over time. In addition, many multi-systemic diseases require widespread expression of the therapeutic transgene that, when provided with ubiquitous or tissue-specific non-hepatic promoters, often results in anti-transgene immunity. Here we have developed tandem promoter monocistronic expression cassettes that, packaged in a single AAV, provide combined hepatic and extra-hepatic tissue-specific transgene expression and prevent anti-transgene immunity. We validated our approach in infantile Pompe disease, a prototype disease caused by lack of the ubiquitous enzyme acid-alpha-glucosidase (GAA), presenting multi-systemic manifestations and detrimental anti-GAA immunity. We showed that the use of efficient tandem promoters prevents immune responses to GAA following systemic AAV gene transfer in immunocompetent Gaa-/- mice. Then we demonstrated that neonatal gene therapy with either AAV8 or AAV9 in Gaa-/- mice resulted in persistent therapeutic efficacy when using a tandem liver-muscle promoter (LiMP) that provided high and persistent transgene expression in non-dividing extra-hepatic tissues. In conclusion, the tandem promoter design overcomes important limitations of AAV-mediated gene transfer and can be beneficial when treating pediatric conditions requiring persistent multi-systemic transgene expression and prevention of anti-transgene immunity

Antigen-selective modulation of AAV immunogenicity with tolerogenic rapamycin nanoparticles enables successful vector re-administration

International audienceGene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration