Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies Aguilar González, Araceli González Correa, Juan Elías Barriocanal Casado, Eliana Ramos Hernández, Iris Greco, Sara Rodríguez Sevilla, Juan José Molina Estévez, Francisco Javier Sánchez Martín, Rosario María Martín Molina, Francisco Muñoz Fernández, Pilar Pompe disease Lentiviral vectors Cellular disease models Optimised GAA (acid alphaglucosidase) CRISPR/Cas9 technology Adeno-associated virus Pompe disease (PD) is a rare disorder caused by mutations in the acid alpha-glucosidase (GAA) gene. Most gene therapies (GT) partially rely on the cross-correction of unmodified cells through the uptake of the GAA enzyme secreted by corrected cells. In the present study, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All of the generated GAA-KO cells lacked GAA activity and presented an increased autophagy and increased glycogen content by means of myotube differentiation as well as the downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Additionally, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalising the autophagic marker p62 and surface levels of CI-MPRs. Interestingly, in vivo administration of liver-directed AAVs expressing the chimeras further confirmed the good behaviour of IFG, achieving cross-correction in heart tissue. In summary, we generated different isogenic murine muscle cell lines mimicking the severe PD phenotype, as well as validating their applicability as preclinical models in order to reduce animal experimentation. 2022-07-01T08:00:24Z 2022-07-01T08:00:24Z 2022-06-04 journal article Aguilar-González, A... [et al.]. Isogenic GAA-KO Murine Muscle Cell Lines Mimicking Severe Pompe Mutations as Preclinical Models for the Screening of Potential Gene Therapy Strategies. Int. J. Mol. Sci. 2022, 23, 6298. [https://doi.org/10.3390/ijms23116298] http://hdl.handle.net/10481/75770 10.3390/ijms23116298 eng http://creativecommons.org/licenses/by/4.0/ open access Atribución 4.0 Internacional MDPI