DEVELOPMENT OF A CELLULAR MODEL TO EXPLORE NEURONAL PATHOLOGY IN SANFILIPPO DISEASE BY CRISPR/CAS9 GENOME EDITING

SOLEDAD PORTE ALARCON; MONICA LIDIA KOTLER; ADRIANA MARIA BELEN ABIUSO; ROXANA MAYRA GOROJOD; MARCOS GABRIEL FRANCIA; ALEJANDRA GUBERMAN

buenos aires

Congreso; LXVI Reunión Científica Anual de la Sociedad Argentina de Investigación Clínica (SAIC).; 2021

Sanfilippo syndrome type IIIA (Mucopolysaccharidosis IIIA; MPSIIIA)is a rare intractable disease characterized by an early-onset, severe,progressive neurodegeneration. It is caused by mutations inthe gene encoding for the lysosomal hydrolase N-SulfoglucosamineSulfohydrolase (SGSH), which is crucial in the stepwise degradationof the sulfated glycosaminoglycan (GAG) heparan sulfate (HS).Nowadays, there is a lack of cellular models to explore the mechanismsof disease in the central nervous system (CNS). In this work,we aimed to develop a novel neuronal model by employing CRISPR/Cas9 technology. We designed two sgRNAs targeting exon 1 of themouse sgsh gene and cloned these sequences in the pX330 vector.Insert-containing plasmids were amplified and checked by PCR andsequencing. HT-22 hippocampal neurons were transfected with PEI,and transfected cells were selected with puromycin. Subsequently,clones were isolated and amplified. We determined the lack of enzymeactivity by employing the fluorogenic substrate 4-MU-GlcNSand obtained five putative knock-out cell lines where we tested lysosomaland mitochondrial integrity. Once confirmed the successfulknockout by sequencing, these cell lines will constitute reliable reportersof the cellular context of disease and great models to studycell-type-specific damage. The strategy used is versatile and will beemployed in other cell lineages to study cell-cell interactions of differentcell types within the CNS. Further research may lead to theidentification of relevant signaling pathways to investigate candidatedrugs for novel therapies.