An Erythroid-Specific Lentiviral Vector Improves Anemia and Iron Metabolism in a New Model of Xlsa

X-linked sideroblastic anemia (XLSA) is caused by germline mutations in the erythroid-specific 5-aminolevulinate synthase (Alas2) gene, encoding the enzyme ALAS2. Typically, XLSA affects hemizygous males with hypochromic microcytic anemia, systemic iron overload, and ring sideroblasts. Treatments are limited to pyridoxine supplements and blood transfusions, with allogeneic hematopoietic stem cell (HSC) transplantation as the only definitive cure, accessible to very few patients. The absence of a suitable animal model has hindered preclinical studies. We have engineered a Cre recombinase LoxP XLSA mouse model by inducing Alas2 knock-out using either tamoxifen or ex-vivo HSC recombination via lipid nanoparticles (LNP) carrying Cre-mRNA conjugated to an anti-CD117 antibody. The resulting Alas2-KO HSCs are transplanted into lethally irradiated recipient mice, generating - chimeras that are Alas2 deficient only in the bone marrow compartment (Alas2-KOBM). Alas2 -KOBM animals display a severe anemic phenotype characterized by ineffective erythropoiesis (IE), with low numbers of red blood cells (RBC) (3.0±0.3), hemoglobin (Hb) (3.4±0.4), and hematocrit (HCT) (16.3±1.6).In these animals, IE is reflected by an expansion of polychromatic erythroid (PolyE) cells and concomitant reduction of orthochromatic/reticulocyte and RBC populations. Alas2-KOBM animals show splenomegaly with extramedullary hematopoiesis, lymphoid hyperplasia, and erythroid hyperplasia in the BM. Notably, the iron staining of BM tissues and smears confirms the presence of ring sideroblasts. Electron microscopy (EM) analysis of the isolated PolyE population revealed iron accumulation in the mitochondria, corroborating the presence of iron overload and the inability of this population to differentiate due to the lack of Alas2. To confirm the dysregulation of iron homeostasis, we evaluated erythroferrone (ERFE) and hepatic hepcidin (HAMP) serum levels. Diseases characterized by IE are generally associated with high levels of erythropoietin (EPO) and ERFE and low levels of HAMP, leading to iron overload. Alas2-KOBManimals showed increased ERFE and decreased HAMP levels, consistent with increased EPO, serum iron levels, and organ iron overload. We also observed increased ferritin levels and transferrin saturation. Metabolic analysis of PolyE cells showed reduced oxidative phosphorylation, mitochondria's function, and key Tricarboxylic Acid (TCA) cycle enzyme activity. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. To alleviate the Alas2-KOBM phenotype, we developed a lentiviral vector (X-ALAS2-LV) to promote human Alas2 expression in erythroid cells. The infusion of BM cells with 0.6-1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice rescued the phenotype and significantly improved CBC levels, tissue iron accumulation, and survival rates. Treated animals showed remarkable improvements in Hb (10.3±0.5), RBC (6.5±0.3), and HCT (33.6±1.0) levels. The splenomegaly observed in Alas2-KOBM mice was also markedly reduced. Iron levels in the liver, spleen, and BM were normalized, and the formation of ring sideroblasts was prevented. Additionally, no iron accumulation was detected in the mitochondria. Treated animals exhibited improved mitochondrial and glycolytic functions, with the expression of glycolytic enzymes and electron transport chain components trending toward normal levels . Most importantly, there was a significant reduction in pro-apoptotic and inflammatory gene expression, indicating anti-apoptotic and anti-inflammatory effects conferred by reintroducing functional Alas2. Secondary BM transplants confirmed the integration stability and efficacy of treatment with X-ALAS2-LV. Secondary recipients showed vector copy numbers and effective erythropoiesis comparable to primary chimeras . Our study established a robust anemic phenotype in mice through Alas2 gene deletion, including the first case of ring sideroblasts formation. Notably, animals rescued by X-ALAS2-LV showed VCN in the range of 0.6-1.4, suggesting that HSCs carrying relatively low VCN could be curative in XLSA patients with hypomorphic or residual Alas2 expression. This potential for a curative treatment provides a solid foundation for further clinical development and instills confidence in the vector's potential.

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