Restoration of hematopoiesis following gene correction in beta-thal

Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. Recently, purification strategies based on differential expression of CD49f and CD90 surface molecules enrich for long-term (CD49f+) and short-term (CD49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid potential. Recent work has proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD49f− cells. A general perturbed and stress condition is present in the thalassemic BM microenvironment, which is expected to have impact non only on erythropoiesis but also on hematopoiesis. Thus, to address which model of hematopoiesis/erythropoiesis occurs in Bthal, we defined by immunophenotype analysis the lineage commitment in patients affected by the pathology compared to healthy donors. Furthermore, in patients treated in the therapy protocol TIGET BTHAL (#NCT02453477) this type of analysis allowed to study the features of reconstituted hemato/erythropoiesis by gene-modified transplanted CD34+ cells.  Initial results showed differences in the primitive compartment with an increased proportion of multipotent progenitors in Bthal patients compared to healthy donors. We were also able to unveil age-related differences, thanks to the availability of adult and pediatric subjects’ samples. By subjecting the classically defined progenitor subsets to a new cell sorting scheme, that efficiently resolved My, Ery, and Mk lineage fates, we quantified the newly identified My and Ery subsets within the CD34+CD38+ compartment and found a reduction of Ery subsets in Bthal samples. Moreover, in gene therapy treated patients we found fluctuations in the contribution of HSC and MPP to the hematopoiesis during follow up.  Future RNA-seq analysis, performed on HSC and derived progenitors, will delineate the transcriptional networks governing hematopoiesis in thalassemia.