开发基于RT-dPCR的功能性放行检测用于Diamond-Blackfan贫血的RPS19基因治疗

Diamond-Blackfan Anemia (DBA) is a congenital ribosomopathy primarily caused by heterozygous loss-of-function mutations in RPS19 , a key component of the 40S ribosomal subunit. RPS19 haploinsufficiency disrupts ribosome biogenesis, triggers p53-dependent cellular stress, and impairs the survival of erythroid progenitor cells. Beyond severe anemia, DBA patients exhibit a slightly elevated risk of hematologic and solid malignancies, highlighting a direct link between ribosomal dysfunction and cancer predisposition. Currently, the only curative approach for DBA is allogeneic bone marrow transplantation which is associated with risk of graft failure and graft-versus-host disease. Gene addition using a third-generation self-inactivating lentiviral vector encoding RPS19 (SJEFS-S19 LV) offers a promising strategy to restore functional RPS19 expression in patient hematopoietic stem and progenitor cells (HSPCs) while maintaining genomic safety and avoiding immune toxicities. Preclinical studies demonstrate that SJEFS-S19 LV effectively corrects erythropoietic defects, restores pre-rRNA processing, and generates a polyclonal, genomically stable population of HSPCs. LVs predominantly integrate into open chromatin regions associated with active transcription. To ensure product quality and functional integrity, we developed a reverse transcription digital PCR (RT-dPCR)-based release assay designed to detect and quantify RPS19 transcript originating specifically from the integrated lentiviral vector following transduction. This approach partitions nucleic acid samples into thousands of micro-reactions, enabling absolute quantification with minimal amplification bias. For determining the RPS19 LV functionality, HEK293 RPS19 -heterozygous knockout cells were transduced at varying multiplicities of infection (MOI), and total RNA was extracted for one-step RT-dPCR targeting the LV-derived codon-optimized RPS19 sequence. Increasing MOI correlated with higher normalized RPS19 transcript copies per ng genomic DNA, confirming successful vector integration and expression. This assay provides a robust, quantitative measure of functional gene expression, supporting critical quality attribute assessment of SJEFS-S19 LV. This functional assay concept is being translated into a critical release assay for the RPS19 Gene Therapy Drug Product in transduced CD34+ cells. Overall, this assay platform enables precise assessment of vector performance and provides a framework for translating LV gene therapy toward clinical applications in DBA patients, with potential implications for understanding how restoration of ribosomal protein function may impact oncogenic susceptibility.