Development of siRNA-Loaded Lipid Nanoparticles Targeting Long Non-Coding RNA LINC01257 as a Novel and Safe Therapeutic Approach for t(8;21) Pediatric Acute Myeloid Leukemia

Patrick Connerty,Richard B Lock,Ernest Moles,Soheil Meshinchi,Maria Kavallaris,Charles E De Bock,Jenny L Smith,Nisitha Jayatilleke,Chelsea Mayoh

doi:10.3390/pharmaceutics13101681

Abstract

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. Here, we describe the development of a new siRNA-loaded lipid nanoparticle for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Transcriptomic analysis of children with AML identified LINC01257 as specifically expressed in t(8;21) AML and absent in healthy patients. Using NxGen microfluidic technology, we efficiently and reproducibly packaged anti-LINC01257 siRNA (LNP-si-LINC01257) into lipid nanoparticles based on the FDA-approved Patisiran (Onpattro®) formulation. LNP-si-LINC01257 size and ζ-potential were determined by dynamic light scattering using a Malvern Zetasizer Ultra. LNP-si-LINC01257 internalization and siRNA delivery were verified by fluorescence microscopy and flow cytometry analysis. lncRNA knockdown was determined by RT-qPCR and cell viability was characterized by flow cytometry-based apoptosis assay. LNP-siRNA production yielded a mean LNP size of ~65 nm with PDI ≤ 0.22 along with a >85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment was able to successfully ablate LINC01257 expression which was accompanied by a significant 55% reduction in total cell count following 48 h of treatment. In contrast, healthy peripheral blood mononuclear cells (PBMCs), which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. This is the first report demonstrating the use of LNP-assisted RNA interference modalities for the silencing of cancer-driving lncRNAs as a therapeutically viable and non-toxic approach in the management of AML.

Highlights

Acute myeloid leukemia (AML) is a molecularly heterogeneous disease that is defined by the uncontrolled rapid expansion of immature myeloid blast cells
We explored whether the presence of chromosomal translocations in children with AML can lead to over-expression of oncogenic Long non-coding RNAs (lncRNAs) and, if so, which lncRNAs correlate with poorer outcomes
Treatments (TARGET) study, we identified 15 lncRNAs that are differentially expressed in children with t(8;21)-positive compared to those with wild type (WT) AML (Figure 1A)

Summary

Introduction

Acute myeloid leukemia (AML) is a molecularly heterogeneous disease that is defined by the uncontrolled rapid expansion of immature myeloid blast cells. Multiple rounds of chemotherapy remain the gold standard treatment option for AML, current therapies lack specificity against leukaemic cells and severely affect healthy tissues [2]. CAR-T cell therapy and small molecule inhibitors (SMIs) have emerged as efficacious approaches to target AML cells and specific proteins that drive the disease [3]. These therapies suffer from off-target toxicity and their use is limited to only a small fraction of AML patients [3,4]. To improve treatment outcomes in children with AML, it is necessary to explore new treatments that are specific to leukemia cells and thereby safe for healthy cells

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Conclusion