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Molecular characterisation of a novel knock-out mouse model in the context of MPSIVA ex-vivo gene therapy development

M Berti(1,2) S Crippa(2) R Jofra-Hernandez(2,3) S Ceriotti(1) G Alberti(2) E O Savoia(2) L Santi(2) G De ponti(2) I Visigalli(3) G Zambonini(3) F Sanvito(3,7) R Norata(3,7) M Rocchi(3,7) S Bolamperti(9) I Villa(9) P D'Adamo(8) C Forni(2) A Spinelli(4) A Aiuti(2,5,6) M E Bernardo(2,5,6)

1:Universita Vita-Salute San Raffaele; 2:San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET); 3:Good Laboratory Practice (GLP) test facility, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget); 4:Preclinical imaging Facility, San Raffaele Scientific Institute, Milan, Italy; 5:Paediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientific Institute, Milan, Italy.; 6:University Vita-Salute San Raffaele, Faculty of Medicine, Milan, Italy; 7:Pathology Unit, Department of Experimental Oncology, IRCCS San Raffaele Scientific Institute, Milan, Italy; 8:Mouse Behaviour Facility, San Raffaele Scientific Institute, Milan, Italy; 9:Osteoporosis and bone and mineral metabolism Unit, San Raffaele Scientific Institute, Milan, Italy

Mucopolysaccaridosis type IVA (MPSIVA), caused by deficient N-acetylgalactosamine-6-sulphate sulphatase (GALNS), presents with skeletal, cardiac, and joint disorders, normally treated with enzyme replacement therapy. This limits GAGs accumulation without correcting the skeletal disease. Based on recent studies showing safety of the procedure and a beneficial effect on skeletal manifestations of ex-vivo haematopoietic stem progenitor cell gene therapy (HSPC-GT) in MPS type I-Hurler patients, we propose to develop a lentiviral-based HSPC-GT approach for MPSIVA. The disease, however, still lacks an animal model representative of patients’ skeletal symptoms. Thus, we have generated Galns-/- mice by CRISPR-Cas9, using two gRNAs targeting Galns’ intron1 and exon2, in C57BL6/J strain. After biochemical and histopathological characterisation, we selected the genotype inducing the most evident skeletal symptomatology, without off-target alterations. Our selected model appears smaller than wild-type controls, with shorter femurs and tibiae on CT-scan and decreased cortical and trabecular areas. Vacuolation in cartilaginous sections were highlighted by histomorphometry and immunohistochemistry. Molecularly, our selcted mutation induces the translation of a protein from an alternative open reading frame, which appears inactive, contrarily to previously described enzyme-null mice. Furthermore, we highlighted different composition and GAGs distribution among mice, rats, and patients’ cartilage, which we are evaluating by immunohistochemistry. Overall, the development of an MPSIVA murine model provides a novel means to assess the safety and efficacy of innovative therapies, which we are currently testing. Also, this model represents an alternative to the available rat model, easier to compare to previously acquired preclinical HSPC-GT data.

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