Establishing a Patient-Derived Immortalized Erythroid Progenitor Cell Model to Investigate Disease Mechanisms and Lentiviral Gene Therapy Vector in Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemias (CDAs) are rare inherited disorders characterized by ineffective erythropoiesis. Type II CDA (CDA II) is the most prevalent, caused by mutations in the SEC23B gene. Clinically, CDA II is manifest by mild to severe anemia, relative reticulocytopenia, jaundice, hepatosplenomegaly, and cholelithiasis. Bone marrow erythroblasts in these patients show erythroid hyperplasia and 10-30% binucleation or occasionally multinucleation. Previous in-vivo models using mice and zebrafish have not accurately replicated the disease phenotypes observed in humans.

In this study, we aimed to establish a patient-derived immortalized erythroid progenitor cell (iEPC) model to investigate disease mechanisms. We isolated peripheral blood mononuclear cells (PBMNCs) from two patients with clinical and pathological phenotypes consistent with CDA II, both of whom exhibited SEC23B mutations as identified by next-generation sequencing. One patient, compound heterozygous for mutations NM_006363.4:c.1043A>G and NM_006363.4:c.1898delC, displayed more severe symptoms and was transfusion-dependent, unlike the other patient, who was compound heterozygous for mutations NM_006363.4:c.1589G>A, and NM_006363.4:c.1905+3G>T and did not require transfusions.

Using a lentiviral vector, we transduced the PBMNCs with HPV E6/E7 genes and cultured them in erythroid progenitor expansion medium, successfully generating immortalized erythroid progenitors that were maintained for over 90 days. These iEPCs expressed erythroid markers CD71 and CD235a and were predominantly in the proerythroblast or basophilic erythroblast stages. The iEPCs could be differentiated into later stages of erythropoiesis using a cytokine-enriched medium. Despite typical expression kinetics of CD71, CD105, and CD235a during differentiation, the CDA II iEPCs showed reduced enucleation compared to a wild-type iEPC line, indicative of ineffective erythropoiesis.

Morphological analysis using Giemsa stain revealed binucleation in 20 to 30% of cells at the orthochromatic stage. Transmission electron microscopy showed a discontinuous double membrane in mature erythroblasts of both CDA lines. Western blot analysis confirmed reduced SEC23B expression in the CDA II lines. RNA sequencing of iEPCs pre- and post-differentiation identified novel pathways involved in disease pathogenesis. After transducing the iEPCs with a lentiviral vector expressing SEC23B, we observed a significant decrease in binucleated cells and an increase in terminally differentiated cells.

Our results demonstrate that iEPCs can be successfully generated from patients with rare red cell diseases and that CDA II disease modeling using iEPCs is viable for studying disease mechanisms and evaluating gene therapy vectors.