Castruccio Castracani Carlo, Breda Laura, Papp Tyler E, Guerra Amaliris, Radaelli Enrico, Assenmacher Charles-Antoine, Finesso Giovanni, Mui Barbara L, Tam Ying K, Fontana Simona, Riganti Chiara, Fiorito Veronica, Petrillo Sara, Tolosano Emanuela, Parhiz Hamideh, Rivella Stefano
Department of Pediatrics, Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA.
Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.
Blood. 2025 Jan 2;145(1):98-113. doi: 10.1182/blood.2024025846.
X-linked sideroblastic anemia (XLSA) is a congenital anemia caused by mutations in ALAS2, a gene responsible for heme synthesis. Treatments are limited to pyridoxine supplements and blood transfusions, offering no definitive cure except for allogeneic hematopoietic stem cell transplantation, only accessible to a subset of patients. The absence of a suitable animal model has hindered the development of gene therapy research for this disease. We engineered a conditional Alas2-knockout (KO) mouse model using tamoxifen administration or treatment with lipid nanoparticles carrying Cre-mRNA and conjugated to an anti-CD117 antibody. Alas2-KOBM animals displayed a severe anemic phenotype characterized by ineffective erythropoiesis (IE), leading to low numbers of red blood cells, hemoglobin, and hematocrit. In particular, erythropoiesis in these animals showed expansion of polychromatic erythroid cells, characterized by reduced oxidative phosphorylation, mitochondria's function, and activity of key tricarboxylic acid cycle enzymes. In contrast, glycolysis was increased in the unsuccessful attempt to extend cell survival despite mitochondrial dysfunction. The IE was associated with marked splenomegaly and low hepcidin levels, leading to iron accumulation in the liver, spleen, and bone marrow and the formation of ring sideroblasts. To investigate the potential of a gene therapy approach for XLSA, we developed a lentiviral vector (X-ALAS2-LV) to direct ALAS2 expression in erythroid cells. Infusion of bone marrow (BM) cells with 0.6 to 1.4 copies of the X-ALAS2-LV in Alas2-KOBM mice improved complete blood cell levels, tissue iron accumulation, and survival rates. These findings suggest our vector could be curative in patients with XLSA.
X连锁铁粒幼细胞贫血（XLSA）是一种由负责血红素合成的基因ALAS2突变引起的先天性贫血。治疗方法仅限于补充吡哆醇和输血，除了异基因造血干细胞移植外没有根治方法，而只有一部分患者能够接受这种移植。缺乏合适的动物模型阻碍了该疾病基因治疗研究的发展。我们通过给予他莫昔芬或用携带Cre-mRNA并与抗CD117抗体偶联的脂质纳米颗粒处理，构建了一种条件性Alas2基因敲除（KO）小鼠模型。Alas2-KOBM动物表现出严重的贫血表型，其特征为无效红细胞生成（IE），导致红细胞、血红蛋白和血细胞比容数量减少。特别是，这些动物的红细胞生成显示多色红细胞增多，其特征为氧化磷酸化、线粒体功能以及关键三羧酸循环酶活性降低。相比之下，尽管存在线粒体功能障碍，但为延长细胞存活而进行的无效尝试中糖酵解增加。无效红细胞生成与明显的脾肿大和低铁调素水平相关，导致铁在肝脏、脾脏和骨髓中蓄积以及环形铁粒幼细胞形成。为了研究基因治疗方法对XLSA的潜力，我们开发了一种慢病毒载体（X-ALAS2-LV），以指导ALAS2在红细胞中的表达。向Alas2-KOBM小鼠输注含有0.6至1.4拷贝X-ALAS2-LV的骨髓（BM）细胞可改善全血细胞水平、组织铁蓄积和存活率。这些发现表明我们的载体可能对XLSA患者具有治愈作用。
