Background <p>B-cell acute lymphoblastic leukemia (B-ALL) is characterized by the malignant burgeoning of abnormal B-cell lymphoblasts. In recent years, the use of chimeric antigen receptor T-cell (CAR-T) therapy which targets CD19 antigen present on the surface of B-cells, has gained significant attention as a treatment option against aggressive and refractory forms of B-ALL. However, the loss of CD19 antigen on B-cell surface due to aberrant splicing under therapy pressure has been suggested as one of the main factors for the emerging CAR-T therapy resistance. The primary aim of this study was to elucidate the presence and characteristics of aberrant CD19 splicing patterns in pediatric B-ALL patients at the time of initial diagnosis stage.</p> Methodology <p>Herein, using RT-PCR based splice assays we have examined CD19 splicing patterns in 43 primary pediatric B-ALL patient samples spread across various subtypes, and investigated underlying mechanisms harboring aberrant splicing. </p> Results <p>We observe that CD19 isoform lacking exon 5-6 is present in ~ 55% of pediatric patients at the initial diagnosis stage itself. Our in-silico analysis identified splicing regulator MBNL1 as a potential modulator of CD19 exon 5-6 splicing. Subsequent qRT-PCR analysis in patient samples revealed that MBNL1 is overexpressed in patient samples exhibiting exon 5-6 skipping. Furthermore, our functional studies demonstrate that loss of MBNL1 in B-ALL cell line induces exon 5-6 skipping, thereby confirming its mechanistic role in CD19 splicing regulation.</p> Conclusions <p>Taken together, we for the first time report the existence of aberrantly spliced CD19 isoform lacking exon 5-6 in primary pediatric patients at the diagnosis stage. Our results suggest that this MBNL1 dysregulation contributes to this splicing event, potentially predisposing patients to resistance against CD19-directed immunotherapies.</p>