P1521: A SEVERE MOUSE MODEL OF ALPHA-THALASSEMIA SHOWS ABNORMAL IRON METABOLISM, ERYTHROPOIESIS AND COAGULATION, AND CAN BE RESCUED BY A NOVEL GENE THERAPY APPROACH

Abstract

Background: Clinical presentation of deletional a-thal varies from an asymptomatic condition (one inactivated a-globin gene) to a complete knockout (Hb Bart’s Hydrops Fetalis). In patients with severe a-thal, a blood transfusion independent state is achievable through allogeneic bone marrow transplantation. Aims: The aims of this study are to develop a novel adult mouse model of a-thal and a gene therapy approach for this disease. Methods: We generated adult animals that do not produce a-globin chains (a-KO) through transplantation of homozygous B6.129S7-Hbatm1Paz/J fetal liver cells (FLC; isolated at E14.5) into WT recipient mice. To generate a gene therapy tool, we screened multiple lentiviral vectors to identify the variant capable of producing the highest human a-globin protein per copy. The selection was conducted in murine erythroleukemia cells and human umbilical cord derived erythroid progenitor (HUDEP) cells, modified by knocking out all the human a-globin genes. Results: The a-KO animals demonstrate a worsening phenotype, paradoxically showing elevated hematocrit, high reticulocyte count and a high number of red blood cells (RBC) which expressed only b-globin chains (HbH). RBC show aberrant morphology and aggregation of b-globin tetramers on RBC membranes. Due to severe inability of these RBC to deliver oxygen, the mice eventually succumb to anemia, showing splenomegaly and other organ pathologies, including vaso-occlusive events, associated with neutrophil infiltration, fibrinogen staining, von Willebrand factor (vWF) released, platelet recruitment and activation. These animals also show iron deposition in the liver and kidney, in agreement with very low levels of hepcidin expression in the liver, and elevated erythropoietin (EPO) in the kidney. We identified ALS20a, a vector where a-globin is under control of the b-globin promoter and its locus control region, as the most efficient vector. One copy of ALS20α produces exogenous a-globin at a level comparable to that produced by one endogenous a-globin gene. These results suggest that a relatively low VCN could result in dramatic therapeutic benefits. Use of ALS20a resulted in correction of the disease phenotype in a dose-dependent manner in a-KO mice. At VCN<1 we observe a delay in death proportional to the VCN value, while at VCN>1 we observe phenotypic normalization, including Hb, hepcidin and EPO levels. We tested ALS20a in CD34 cells isolated from four patients with both deletional and non-deletional HbH disease. We measured the change of b/a-globin mRNA ratio (b/aR) and protein level by HPLC in erythroblasts derived from these cultures. For the specimen with mutational HbH, the initial b/aR matches that of healthy controls, as the mutations do not eliminate the ability for the gene to produce aberrant mRNA transcripts, and decreased with increasing VCN. Erythroblasts with deletional HbH have a b/aR approximately 3x higher than normal cells, decreasing in a dose dependent manner with increasing VCN. HPLC detection of HbH (β4), a hallmark of HbH disease, is observed in hemolysis products from all non-transduced a-thal erythroblasts. A ~50% reduction of HbH is detected in the very same specimens upon integration of ALS20α (VCN between 1 and 2). Image:Summary/Conclusion: We generated an adult mouse model of lethal a-thal and, in preliminary experiments, we rescue it with ALS20a. Furthermore, ALS20a successfully improves a-globin levels in patient cells.