XLSA breakthrough: gene therapy potential

In this issue of Blood, Castruccio Castracani et al¹ provide valuable information into the pathophysiology of X-linked sideroblastic anemia (XLSA) with a conditional Alas2 knockout (KO) mice model. Importantly, the authors indicate the potential effectiveness of gene therapy for XLSA.

Sideroblastic anemia is a group of congenital and acquired disorders defined by the presence of bone marrow ring sideroblasts, created by excess mitochondrial deposition of iron.² Congenital forms of sideroblastic anemia (congenital sideroblastic anemias [CSAs]) are rare and due to gene mutation involved in iron and heme metabolism.² CSAs are “iron-loading anemias,” characterized by systemic iron overload and ineffective erythropoiesis, due to erythropoietin-driven expansion of early-stage erythroid precursors and late-stage precursor apoptosis.³ The expanded pool of immature erythroid cells induces the release of erythroferrone, a hepcidin inhibitor, thus blocking the master regulator of systemic iron homeostasis that prevents intestinal iron absorption and macrophage iron recycling. This disruption of hepcidin causes secondary systemic iron overload.³ However, the detailed mechanism by which defects in iron/heme metabolism cause ineffective erythropoiesis remains unclear.

XLSA, the most common form of CSA, is caused by germ line mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene, which encodes the first and rate-limiting enzyme of heme biosynthesis. This pathway converts glycine and acetyl-coenzyme A to 5-aminolevulinic acid (ALA), requiring pyridoxal 5′-phosphate, the active form of vitamin B₆, as a necessary cofactor. Although vitamin B₆ has been used to treat XLSA, nearly half of the patients are refractory to treatment.² Additionally, oral ALA supplementation failed to improve anemia in vitamin B₆-refractory XLSA.⁴ Therefore, research of novel therapeutic strategies for XLSA is urgently needed.

Several attempts have been made to establish a disease model of XLSA. Murine models of XLSA, which involved disrupting the intronic enhancer region of ALAS2, were embryonically lethal.⁵ Recently, a series of viable ALAS2 knockin mice harboring common mutations (p.R170H, p.R411H, and p.R452H) have been reported.⁶ The current study generated a conditional Alas2 KO mice (Alas2^fl/fl/R26-CreER^T2) to deplete exon 5 of the ALAS2 gene.¹ These mice were treated with tamoxifen to induce Alas2 gene depletion. As an alternate approach, a lipid nanoparticle (LNP) carrying Cre-messenger RNA conjugated with an anti-CD117 antibody (LNP^CD117 Cre) was used to delete the Alas2 gene ex vivo. Irradiated wild-type mice were then infused with Lin⁻ hematopoietic stem cells (HSCs) from Alas2^fl/Y/R26-CreER^T2 mice treated with LNP^CD117 Cre. The Alas2-KO mice had severe microcytic anemia with the emergence of ring sideroblasts in bone marrow, recapitulating the human phenotype of XLSA, using both approaches (tamoxifen, LNP). Additionally, the Alas2-KO mice exhibited elevated serum erythroferrone, decreased serum hepcidin, and increased serum ferritin, indicative of systemic iron overload. The absence of obvious ring sideroblasts in other recent XLSA mice models⁶ may be due to the lack of systemic iron overload. Further investigation of the disparities between the 2 models is needed to understand the differing manifestations of disease. Intriguingly, the authors revealed that the Alas2-KO mice demonstrated an expansion of the polychromatic erythroid cell (P3) fraction, accompanied by reduced oxidative phosphorylation, increased lactate levels, and elevated proapoptotic gene expressions, indicating that heme deficiency could cause ineffective erythropoiesis. These metabolic changes in the P3 fraction appeared to hinder differentiation into orthochromatic cells, potentially contributing to progenitor cell accumulation with an impaired differentiation capacity. Additionally, enhanced glycolysis may compensate for mitochondrial dysfunction, further contributing to this expansion. Further assessment is needed to identify the similarity of expansion and metabolic changes in the polychromatic erythroid cell fraction in patients with XLSA.

The molecular mechanisms underlying the cell death of erythroid progenitors in the conditional Alas2-KO mice remain unclear. The authors revealed elevated proapoptotic gene expression, but the potential contribution of ferroptosis, which is a form of regulated cell death that depends on iron accumulation and lipid peroxidation, remains unknown. Recent studies demonstrated the involvement of ferroptosis in the anemic phenotype in human ex vivo XLSA models and zebrafish models of ALAS2 deficiency.^7,8 

The novelty of the current study lies in lentiviral gene therapy development for XLSA. To date, the long-term efficacy and safety of the lentiviral gene therapy have been demonstrated in hemoglobinopathies, such as sickle cell disease and transfusion-dependent β-thalassemia.⁹ The use of lentiviral vectors enables efficient transduction in nondividing cells, such as HSCs, and accommodates large gene inserts, including the ALAS2 gene. To achieve erythroid-specific expression of human ALAS2, the authors used the previously established GLOBE lentiviral vector backbone, which includes an erythroid-restricted promoter and enhancer elements (X-ALAS2-LV).¹⁰ The authors revealed that XALAS2-LV-treated mice with vector copy number (VCN) of >0.6 exhibited increased hemoglobin levels, elevated red blood cell counts, and an improved iron-overload phenotype, indicating the sufficiency of a relatively small number of integrations to rescue Alas2-KO mice.¹ Interestingly, the treated animals demonstrated a decreased P3 fraction expansion, along with improved glycolysis and mitochondrial activity. Although the long-term follow-up of the XALAS2-LV-treated mice remains necessary to evaluate hemoglobin level, iron parameters, and, perhaps, unexpected side effects, these results indicated that HSCs with a relatively low VCN of X-ALAS2-LV could effectively treat patients with XLSA.

In conclusion, lentiviral gene therapy demonstrates a promising therapeutic option for vitamin B₆-refractory severe XLSA. Additionally, curative treatment remains unavailable for CSAs other than XLSA; thus, studying this treatment approach to these diseases would be quite interesting.

Conflict-of-interest disclosure: The author declares no competing financial interests.