B8 - Genetic Disorders of the Blood and Immune System (Including hemoglobinopathies, hematopoietic lineages, and inherited immune deficiencies such as ADA, X-SCID, and WAS, etc.)
172: A Novel Model of X-Linked Sideroblastic Anemia (Alas2-KO, XLSA) with Ring Sideroblasts, Severe Anemia, and Iron Overload is Rescued by Lentiviral Gene Transfer
Type: Oral Abstract Session
Presentation Details
Session Title: Correction of Genetic Disorders of the Blood and Immune System
X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia caused by mutations in Alas2, a gene responsible for heme synthesis in erythroid cells. 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 and increased ERFE, the erythroid suppressor of hepcidin. Since hepcidin is responsible for limiting dietary iron absorption, decreased hepcidin levels were associated with increased serum iron, transferrin saturation levels, and iron overload. 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. We also included an insulator in the 3’ self-inactivating long terminal repeat (SIN-LTR) to enhance safety. 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. While the hemoglobin and RBC levels were improved or corrected in the LV-treated group, the non-treated group showed fatal anemia, with low RBC levels. Accordingly, the untreated group showed increased erythropoietin levels and reticulocyte counts because of IE and severe anemia. The ineffective erythropoiesis was characterized by extra-medullary erythropoiesis and severe splenomegaly, while these parameters were improved or normalized in the X-ALAS2-LV-treated group. These data show that the fatal anemia in Alas2-KO animals can be improved or corrected by X-ALAS2-LV. Notably, animals rescued by X-ALAS2-LV showed vector copy numbers (VCN) in the range of 0.6-1.4, indicating that a small number of integrations were sufficient to rescue Alas2-KO animals. As XLSA patients still exhibit hypomorphic or residual expression of the ALAS2 gene (as the complete absence is incompatible with survival), we hypothesize minimal gene transfer or mini-transplants with HSCs treated with XALAS2-LV could be curative in this disease. Moreover, 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.
Plain Language Summary
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. We generated an inducible severe model of XLSA. Using this model, we investigated 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.
Carlo Castruccio Castracani1, Amaliris Guerra1, Laura Breda1, Tyler Papp2, Enrico Radaelli2, Charles-Antoine Assenmacher2, Giovanni Finesso2, Yink Tam3, Barbara Mui3, Simona Fontana4, Chiara Riganti4, Veronica Fiorito4, Sara Petrillo4, Emanuela Tolosano4, Hamideh Parhiz5, Stefano Rivella1,2
1Children's Hospital of Philadelphia, Philadelphia, PA,2University of Pennsylvania, Philadelphia, Philadelphia, PA,3Acuitas Therapeutics, Vancouver, BC, Canada,4University of Turin, Turin, Italy,5University of Pennsylvania, Philadelphia, PA"
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