IDENTIFICATION OF SINGLE NUCLEOTIDE VARIANTS AND FUSION GENES THROUGH TRANSCRIPTOMICS OF PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA PATIENTS FROM A MULTICENTER CLINICAL STUDY IN ARGENTINA.

SHEILA CASTAÑEDA; FEDERICO ZABALEGUI; GUADALUPE AMÍN; CAROLINA BÁRBARA BELLI; ARIEL WAISMAN; ALEJANDRO LA GRECA; GUSTAVO SEVLEVER; SANTIAGO MIRIUKA; LUCÍA MORO

Simposio; LXVII REUNIÓN ANUAL DE LA SOCIEDAD ARGENTINA DE INVESTIGACIÓN CLÍNICA (SAIC); 2022

The FHL1 gene locates in the Xq26 region, encodes fourand a half LIM domain protein 1 and is expressed in skeletaland cardiac muscle. It has been related to cytoskeletalremodeling, myoblasts differentiation, sarcomereassembly and autophagy regulation. Mutations in FHL1are related to muscular dystrophy (MD) with a limited lifeexpectancy. The aim of this work was to generate andcharacterize an induced pluripotent stem cells (iPSCs)line derived from a patient with MD (MD-iPSC) that carriesa pathogenic heterozygous missense mutation inFHL1 (c.377G>A, p.C126T) for in vitro disease modelingand personalized therapy development. In silico analysisrevealed a misfolding of FHL1 p.C126T protein. To generateMD-iPSC, a blood sample was taken from the patientand erythroblasts were reprogrammed by transductionwith STEMCCA lentiviral vector. After iPSCs clonalmutation, STEMCCA silencing and normal karyotype ofMD-iPSCs. For pluripotency validation, alkaline phosphataseactivity and pluripotency genes expression wereassessed. Moreover, MD-iPSC was capable of differentiatinginto cells of the three germ layers by embryoid bodyformation. MD-iPSC derived cardiomyocytes were alsoobtained and characterized by observing high expressionof cardiac markers by RT-qPCR, immunohistochemistry,and western blot by day 23 of differentiation. A s geneediting could be a possible in vivo therapy, we correctedthe patient mutation in MD-iPSC by CRISPR-mediatedhomologous recombination. In summary, MD patient-deed to muscle cells (cardiomyocytes), and the pathogenicmutation was corrected by CRISPR. With these results,develop a personalized therapy. In this sense, we aregenerating associated adenovirus (AAV) particles as avector of the CRISPR system to transduce the patientcardiomyocytes simulating an in vivo therapy.