Abstract
Objective: This clinical trial explored the application of CRISPR-Cas9 technology for editing immune cells to specifically target tumor-associated macrophages (TAMs) in pediatric leukemia. The study aimed to determine whether engineered immune cells improved remission rates, reduced relapse, and minimized side effects when used alongside standard chemotherapy. This trial assessed the safety, tolerability, and clinical outcomesof this innovative approach in pediatric leukemia patients.
Methods: The trial was conducted at a tertiary care hospital of Islamabad, Pakistan between January 2024 and January 2025. A randomized controlled design was adopted, enrolling 120 pediatric patients aged 4–16 years with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML). Patients were assigned to either standard chemotherapy (control) or chemotherapy combined with CRISPR-Cas9–modified immune cell infusions (intervention). TAMs were isolated from bone marrow aspirates, edited ex vivo using CRISPR-Cas9 to disrupt CSF1R and PD-L1 genes, and reinfused. Safety outcomes included adverse events, cytokine release syndrome, and off-target mutations, while efficacy outcomes comprised remission rates, relapse-free survival (RFS), and minimal residual disease (MRD) negativity. Statistical analyses employed Kaplan–Meier survival curves and Cox regression modeling.
Results: The intervention group achieved higher complete remission rates at 12 months (82% vs. 64%, p < 0.05) and reduced relapse incidence at 24 months (18% vs. 34%). Relapse-free survival was significantly improved in the intervention arm (p = 0.032). MRD negativity was observed in 79% of the intervention cohort compared with 58% in controls. Adverse events were manageable; cytokine release syndrome occurred in 12% of intervention patients but was controlled with supportive therapy. Off-target mutations were detected in some cases but did not translate into clinically significant complications.
Conclusion: CRISPR-Cas9–modified immune cells targeting TAMs proved to be a safe, feasible, and effective adjunct to chemotherapy in pediatric leukemia patients. The therapy improved remission, reduced relapse, and demonstrated acceptable safety. These results supported the integration of CRISPR-based immunotherapy into pediatric oncology and warranted further validation in larger, multi-center trials.