Abstract 1387: Live cell visualization of RNA splicing dynamics upon APOBEC3 lentiviral transduction in aged bone marrow using a 3D biosensing nanobioreactor.

Abstract Background: During aging, hematopoietic stem and progenitor cells (HSPCs) undergo a progressive decline in regenerative capacity, exhibit skewed differentiation toward the myeloid lineage, show increased sensitivity to stressors, and accumulate somatic mutations, a process known as clonal hematopoiesis (CH), which increases susceptibility to hematological malignancies. Additional hallmarks of aging, include genomic instability, chronic inflammation, upregulation of inflammatory cytokines, and deregulated RNA splicing. Prior work demonstrated age-associated splice isoform expression in aged bone marrow (ABM) derived HSPCs (Crews⋯Jamieson, Cell Stem Cell, 2016). Here, we apply a 3D biosensing nanobioreactor (Pham⋯Jamieson, Cell Stem Cell, 2025) to track RNA splicing in aged bone marrow (ABM) derived HSPCs in real-time using confocal microscopy and evaluate age-related splice dysregulation linked to malignant evolution. Methods: Nanobioreactors were constructed from transparent, gas-permeable, 2-port fluoroethyl polymer (FEP) film bags with a porcine gelatin sponge matrix. Mononuclear ABM cells were isolated by Ficoll density centrifugation, and CD34+ cells were purified by magnetic bead selection. CD34+ cells were lentivirally transduced with a dual fluorescent splicing reporter (van der Werf et al., Cell Reports Medicine, 2023), cultured for 48 hours, and co-cultured with autologous CD34- stromal cells in the nanobioreactor at a 1:4 ratio. Results: In ABM-derived CD34+ cells, lentiviral transduction of the dual fluorescent splicing reporter resulted predominantly in red fluorescence protein (RFP) expression, indicating exon inclusion, as assessed by confocal microscopy. Based on preliminary whole-transcriptome sequencing analyses showing increased exon-skipping events, we hypothesize that APOBEC3C drives exon skipping in aged HSPCs. To test this, CD34+ selected cells from ABM aspirates were lentivirally transduced with the splicing reporter, followed by either APOBEC3C overexpression or pCDH vector control. Confocal imaging revealed a fluorescence shift consistent with increased exon skipping in APOBEC3C-transduced cells, confirming the reporter’s ability to capture dynamic splicing changes in response to APOBEC3C activity. Conclusion: We developed a 3D biosensing niche nanobioreactor system that enables live-cell visualization of RNA splicing dynamics in primary human HSPCs. Using this platform, we identified predominant exon inclusion in aged bone marrow-derived HSPCs and demonstrated that APOBEC3C overexpression induces exon skipping, a molecular signature associated with leukemic transformation. Future studies will compare aged and young bone marrow to delineate age-related splicing alterations and define RNA splicing-based biomarkers predictive of hematopoietic aging and malignant evolution. Citation Format: Emma Klacking, Inge van der Werf, Jane Isquith, Jessica Pham, Thomas Whisenant, Anna A. Khachatrian, Ludmil B. Alexandrov, Catriona H. M. Jamieson. Live cell visualization of RNA splicing dynamics upon APOBEC3 lentiviral transduction in aged bone marrow using a 3D biosensing nanobioreactor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1387.