MPS II models for the study of joint and bone pathophysiology using CRISPR/Cas9 technology

VAENA EMILIO; ORMAZABAL MAXIMILIANO; CECI R; PROCOPIO D; ROZENFELD PA; DARDIS A

Simposio; 18th World Symposium; 2024

Mucopolysaccharidosis type II (MPS II) is an X-linked lysosomal storage disease characterized for the deficiency of the lysosomal enzyme iduronato 2 sulfatase activity, affecting different organs and tissues as liver, spleen, bones, joints, heart, lungs and central nervous system (CNS). The available treatment to date don’t solved completely the inflammatory processes in CNS characterized by increased levels of inflammatory cytokines, oxidative stress, white matter lesions and brain atrophy, neither in bone and joint tissue, where the patients present morphological alterations in the architecture of the cartilaginous growth plate and less bone matrix formation by osteoblasts. Currents in vitro and in vivo models of the disease provide knowledge about the cellular and molecular basis of the pathology observed in bones and joint, showing increased levels of TNFα, defects in organic matrix content of articular cartilage, alterations in apoptosis levels and nitric oxide (NO) production. This models, in fact, don’t fully explain the specific pathophysiological mechanisms involved in the MPS II pathogenesis, without knowing specific aspects of the different tissues involved, and how may are affected different cellular processes as the inflammatory state, apoptosis, secondary substrates accumulation, signaling pathways, and the possibility of evaluate new therapeutic approaches. We developed in vitro cellular models for MPS II using continuous cells lines of joint and bone tissue, the SW982 (human synovial fibroblast) and SaOS-2 (human osteosarcoma) edited by using CRISPR/Cas9 technology. Using two RNA guides targeting exon 2 and 4 of the IDS gene, we generated clones with deficient IDS enzyme activity, checked the presence of pathogenic variants in the IDS sequence and finally we used this new MPSII cells models to further characterization studying the accumulation of GAGs, measuring pro inflammatory cytokines and chemokines as IL-1β, TNFα, IL-6 and IL-8 and specific functional assays for the different cell lines.