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Isogenic GAA-KO murine muscle cell lines mimicking severe Pompe mutations as preclinical models for screening of potential gene therapy strategies

A Aguilar-González(1,2) J E González-Correa(1) E Barriocanal-Casado(3) I Ramos-Hernández(1) M A Lerma-Juárez(4) S Greco(5) J J Rodríguez-Sevilla(1,6) F J Molina-Estévez(1) V Montalvo-Romeral(7) G Ronzitti(7) F Martin(1,8) P Muñoz(1,9)

1:GENyO- Centro de Genomica e Investigacion Oncologica: Pfizer / Universidad de Granada / Junta de Andalucia; 2:Department of Medicinal and Organic Chemistry, Faculty of Pharmacy, University of Granada, Campus Cartuja, 18071 Granada, Spain; 3:Department of Neurology, Columbia University Medical Center, New York, NY, U.S.A.; 4:Instituto de Investigación del Hospital Universitario La Paz, IdiPAZ, 28029, Madrid, Spain; 5:Comecer. Via Maestri del Lavoro, 90, 48014, Castel Bolognese RA, Italy; 6:Hospital del Mar- Parc de Salut MAR, Barcelona, Spain.; 7:Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE; 8:Department of Immunology, Faculty of Medicine. University of Granada. Avda. de la Investigación 11, 18071, Granada, Spain.; 9:Department of Cellular Biology, Faculty of Sciences. University of Granada. Campus Fuentenueva, 18071, Granada, Spain

Pompe disease (PD) is a rare disorder caused by mutations in the acid alphaglucosidase (GAA) gene. Most gene therapies (GT) partially rely on cross-correction of unmodified cells through the uptake of GAA enzyme secreted by corrected cells. In the present study, different chimeric murine GAA proteins (IFG, IFLG and 2G) were designed with the aim to improve their therapeutic activity. Additionally, we generated isogenic murine GAA-KO cell lines resembling severe mutations from Pompe patients. All GAA-KO cells generated lacked of GAA activity, presented increased autophagy and glycogen content upon myotube differentiation as well as downregulation of mannose 6-phosphate receptors (CI-MPRs), validating them as models for PD. Phenotypic rescue analyses using lentiviral vectors point to IFG chimera as the best candidate in restoring GAA activity, normalizing 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 crosscorrection in heart tissue. In summary, we have generated different isogenic murine muscle cell lines mimicking severe PD phenotype and validated their applicability as preclinical models in order to reduce animal experimentation.

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