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Introduction: Standard of care therapies for children with acute myeloid leukemia (AML) cause potent off-target toxicity to healthy cells, highlighting the need to develop new therapeutic approaches that are safe and specific for leukemia cells. Long non-coding RNAs (lncRNAs) are an emerging and highly attractive therapeutic target in the treatment of cancer due to their oncogenic functions and selective expression in cancer cells. However, lncRNAs have historically been considered ‘undruggable’ targets because they do not encode for a protein product. Therefore, the discovery of novel lncRNA targets and the development of efficient methods to silence them is critical for developing new therapies for AML. In this study, we describe the development of an siRNA-loaded lipid nanoparticle (LNP) for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Materials and Methods: Transcriptomic analysis of children with AML from both the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative and AAML1031 study was performed to identify AML specific lncRNAs. The subsequently identified lncRNA LINC01257 was knocked down in AML Kasumi-1 cells via electroporation of siRNA targeting LINC01257 (si-LINC01257). Changes in proliferation and cell viability were assessed via cell count and Annexin V/7AAD staining 48 hours post-transfection. LNPs incorporating non-targeting si-SCR or si-LINC01257 were synthesized through NxGen non-turbulent microfluidics. LNPs were formulated reproducing the FDA-approved Onpattro® siRNA delivery vector, D-Lin-MC3-DMA:DSPC:cholesterol:PEG-DMG. LNP-si-LINC01257 uptake was measured by confocal microscopy and flow cytometry. Kasumi-1 cells and healthy peripheral blood mononuclear cells (PBMCs) were treated with 250nM LNP-si-SCR or LNP-si-LINC01257 for 48 hours. Changes in proliferation and cell viability were assessed with cell counts and Annexin V/7AAD staining 48 hours post-treatment. Results: LINC01257 was significantly overexpressed in patients carrying the t(8;21) translocation compared to healthy controls ( P = 85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment successfully ablated LINC01257 expression, which was accompanied by a 55% reduction in total cell count following 48 hours of treatment (P= 0.02). In contrast, healthy PBMCs, which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. Conclusion: Our data demonstrate the effective use of LNP-based RNA interference modalities for the silencing of cancer-driving lncRNAs and highlight their potential as a viable therapeutic and non-toxic approach in the management of AML. Citation Format: Patrick Connerty, Ernest Moles, Charles deBock, Nisitha Jayatilleke, Jenny L. Smith, Soheil Meshinchi, Chelsea Mayoh, Maria Kavallaris, Richard B. Lock. Development of siRNA-loaded lipid nanoparticles targeting long non-coding RNA LINC01257 as a novel and safe therapeutic approach for paediatric acute myeloid leukaemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P143.