A NOVEL MODEL OF X-LINKED SIDEROBLASTIC ANEMIA (Alas2-KO, XLSA) WITH RING SIDEROBLAST, SEVERE ANEMIA

X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in Alas2, a gene responsible for heme synthesis. Most patients carry hypomorphic mutations, with variable phenotypic manifestations. Treatment options include pyridoxine supplements, transfusions, and allogeneic bone marrow (BM) transplantation. To study an inducible severe manifestation of XLSA, we engineered a conditional Alas2-KO mouse model using two approaches: tamoxifen administration and treatment with lipid nanoparticles (LNP) carrying Cre-mRNA and conjugated to the anti-CD117 antibody. Alas2-KO-BM animals displayed fetal anemia characterized by ineffective erythropoiesis (IE) and ring sideroblasts, the first model showing this phenotype. Erythropoiesis in these animals showed expansion of polychromatic erythroid cells, decreased activity in the electron transport chain and mitochondria's function, and reduced activity of crucial Tricarboxylic Acid (TCA) cycle enzymes. The IE was associated with marked splenomegaly, high erythroferrone, low hepcidin levels, and iron accumulation in the BM, liver, and spleen. To investigate a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) that exploits a globin promoter and enhancers to direct human ALAS2 expression in erythroid cells. Infusion of BM cells with 0.6-1.4 copies of the X-ALAS2-LV in Alas2-KO-BM mice rescued these mice by improving erythropoiesis and tissue iron accumulation. These findings suggest our vector could be curative in XLAS patients. Since X-ALAS2-LV rescues Alas2-KO-BM mice, we are now modifying X-ALAS2-LV with ALAS2 human mutations to study milder forms of XLAS and develop alternative therapeutic interventions, such as pharmacological treatments and in vivo gene editing.