LAUSR

β-thalassemia is one of the most prevalent genetic diseases worldwide. The current treatment for β-thalassemia is allogeneic hematopoietic stem cell transplantation (HSCT), which is limited due to lack of matched donors. Gene therapy has been developed as an alternative therapeutic option for transfusion-dependent β-thalassemia (TDT). However, successful gene therapy for β-thalassemia patients in China has not been reported. Here we present the results of preclinical studies of an optimally designed LV named LentiHBBT87Q in hematopoietic stem and progenitor cells (HSPCs) derived from Chinese TDT patients. LentiHBBT87Q was selected from a series of LVs with optimized backbone and de novo cloning strategy. It contains an exogenous T87Q β-globin gene (HBBT87Q) driven by a specific reconstituted locus control region (rLCR) and efficiently express HBB mRNA and HBB protein in erythroblasts derived from cord blood (CB) HSPCs. To facilitate clinical transformation, we manufactured clinical grade LentiHBBT87Q (cLentiHBBT87Q) and optimized its transduction procedure. Importantly, transduction of cLentiHBBT87Q restored expression of HBB monomer and adult hemoglobin (HbA) tetramer to relatively normal level in erythroblasts from bone marrow (BM) HSPCs of Chinese TDT patients, that carry the most common mutation types and cover various genotypes, including β0/β0. Furthermore, viral integration sites (VIS) of cLentiHBBT87Q were similar to other LVs safely used in previous clinical trials and gene-ontology (GO term) analysis of VIS targeted genes suggest no tumor-associated pathways were enriched in treated samples. Taken together, we have engineered the cLentiHBBT87Q that can restore β-globin expression in the HSPCs-derived erythroblasts of Chinese TDT patients with minimal risk on tumorigenesis, providing a favorable starting point for future clinical application.