Oncogene activation by lentiviral vectors drives senescence linked clonal haematopoiesis and mutation accumulation in HSPC gene therapy

Hematopoietic stem and progenitor cell gene therapy (HSPC-GT) harnesses the regenerative capacity of HSPCs by inserting therapeutic genes via integrative vectors, notably lentiviral vectors (LVs). Once reinfused, these modified cells sustain lifelong multilineage hematopoiesis. However, vector integration can inadvertently activate proto-oncogenes, with potential consequences ranging from clonal dominance to leukemogenesis, as highlighted by adverse events in clinical trials. Before transformation, ectopic oncogene activation in cells may trigger oncogene-induced senescence (OIS), a tumor-suppressive response characterized by permanent proliferation arrest and the senescence-associated secretory phenotype (SASP), which promotes chronic inflammation and disrupts tissue homeostasis. To investigate the long-term consequences of random oncogene activation by insertional mutagenesis, we transplanted wild-type (WT) or Cdkn2a-/- mouse HSPCs, lacking the key senescence regulators p16^INK4a and p19^ARF, transduced with either a genotoxic LV carrying the strong SFFV promoter (LV.SF) or a clinically relevant self-inactivating (SIN) LV harboring the weaker PGK promoter (LV.PGK). Peripheral blood composition was monitored over 2.5 years, and clonal dynamics were assessed through high-throughput vector integration site (IS) retrieval from purified B, T, and myeloid cells. Mice receiving Cdkn2a-/- LV.SF-transduced cells developed aggressive myeloid malignancies with significantly reduced latency (p < 0.0001). Although WT/LV.SF mice did not succumb for malignancy, they exhibited extreme myeloid skewing and progressive loss of clonal diversity across all lineages, when compared to LV.PGK. This signature is consistent with features of accelerated hematopoietic aging likely caused by the genotoxicity of LV.SF insertions. IS analysis revealed recurrent activation of the Braf oncogene in Cdkn2a-/- tumors and, to a lesser extent, in WT recipients, implicating insertional activation as a key driver of transformation and accelerated aging. To address if genotoxic vector integrations could impact cellular stress and trigger somatic mutations, we developed VISMA (Vector Integration Site Mutation Analysis) where we analyzed the genomic sequences flanking ISs (N>200,000 IS, corresponding >9 Gb of genomic sequence) and developed a novel Mutation Index (MI), where the number of somatic mutation identified in IS-flanking regions is normalized by clone number and sequencing coverage. VISMA revealed significantly elevated MI in LV.SF-treated mice relative to LV.PGK controls (p < 0.001), with myeloid clones accumulating the highest mutation burden. This effect was exacerbated in Cdkn2a-/- mice, indicating that disruption of senescence barriers enhances mutation accumulation. Whole-genome sequencing confirmed the elevated mutation burden in LV.SF groups, ruling out sequencing artifacts or integration bias. Finally, we applied a Bayesian inference model integrating IS dynamics and somatic mutations to infer clonal evolution trajectories. This revealed increased mutational complexity, subclonal diversity, and faster clonal expansion in the LV.SF condition compared to LV.PGK. Collectively, our data reveal that insertional activation of oncogenes in HSPC-GT induces a cascade of biological events such as chronic inflammation, clonal restriction, and somatic evolution that compromise long-term hematopoietic integrity. Importantly, by coupling vector IS analysis with quantitative mutation profiling, we establish a novel framework to detect and interpret somatic mutagenesis directly from sequencing data. This approach unveils an underrecognized layer of vector genotoxicity and opens new avenues for dissecting mutation dynamics in gene therapy contexts.