Deciphering Hematopoietic Reconstitution Dynamics of Lentiviral Vector HBBT87Q-Engineered Hematopoietic Stem Cells Gene Therapy for β-Thalassemia Patients

Transfusion-dependent β-thalassemia (β-TDT) is a monogenic hereditary disease caused by mutations in the hemoglobin β gene. Lentiviral vector-based gene-engineered hematopoietic stem/progenitor cells (GE-HSPCs) transplantation has made significant progress in treating β-TDT patients. However, no studies have yet explored the hematopoietic reconstitution dynamics and erythroid differentiation of lentiviral vector HBB^T87Q (LVV-HBB^T87Q) HSPCs populations at the single-cell transcriptomic/DNA level.

This study focuses on three β-TDT patients enrolled in the IIT clinical trial (NCT05860595), who received the autologous lentiviral vector gene therapy product KL003 carrying the codon-optimized HBB^T87Q gene. We performed 10X single-cell RNA sequencing (scRNA-seq) and genotyping on Bone marrow (BM) Lin^-CD34⁺ HSPCs and CD71⁺CD235a⁺ erythroblasts (EBs) samples at baseline and 1 month (M1), M3 and M6 after LVV-HBB^T87Q HSPCs infusion. Ligation-mediated target amplification (LTA-PCR) were done to monitor the integration site (IS) from GE-HSPCs to purified myeloid, lymphoid, erythroid cell lineages from peripheral blood (PB) and BM.

As of July 31, 2024, the median follow-up of 11.00 months (range, 11.00-11.96 months), all three patients achieve transfusion independence (TI). Engraftment of GE-HSPCs were successful and sustained in all patients. By integrating scRNA-seq and genotyping, we unveiled the biological properties and transcriptional features of LVV-HBB^T87Q HSPCs populations in contrast to their HBB^mut counterparts. Total 89,520 HSPCs cells and 40,512 EBs were included in analyzing. The expression of HBB^T87Q mRNA was significantly higher than that of endogenous HBB^mut mRNA, which is further validated by RT-qPCR. Notably, nearly 80% HBB^T87Q population was identified in primitive HSPCs and stay stable till M6. the most enriched stemness score was identified in the HBB^T87Q primitive HSPCs population, which exhibited an early phase erythroid differentiation bias and later balanced differentiation toward myeloid, lymphoid, erythroid cell lineages. Hemoglobin gene expression exhibited an almost complete switch from the HBB^mut to the HBB^T87Q gene in EBs. Strikingly, the proerythroblast (ProE)/basophilic erythroid subpopulation, highly enriched with HBB^T87Q expression, is also enriched in proliferation-related pathways. The IS hotspots in our Drug Product and follow-up samples are associated with the histone demethylase gene KDM2A (<30%). Based on well-established statistics model，human clonal tracking data by IS analysis from patients receiving GE-HSPCs further demonstrated balanced differentiation.

This work showed for the first time that in vivo dynamics and lineage commitment of LVV-HBB^T87Q HSPCs clusters in β-TDT, highlighting the enhanced fitness of LVV-HBB^T87Q HSPCs to compensate for demands in specific clinical temporal. Future studies should leverage these mechanistic insights to further improve lentiviral gene therapy.