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

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.

Efficient Delivery of Globotriaosylceramide Synthase siRNA using Polyhistidine‐Incorporated Lipid Nanoparticles

“…The encapsulation efficiency was calculated using the following equation: ((total amount of siRNA) – (unloaded siRNA) / (total amount of siRNA)) × 100 (%). [ 38,39 ]…”

Systematic In Vitro Evaluation of a Library of Approved and Pharmacologically Active Compounds for the Identification of Novel Candidate Drugs for KMT2A-Rearranged Leukemia

“…Lipid-based nanoparticles encapsulating siRNA targeting NR5A1 and scrambled siRNA (LNP-siRNA) were prepared using a NanoAssemblr™ Spark ® instrument (Precision Nanosystems) as previously described ( 11 ). Prior to addition to cell cultures, LNP-siRNA suspensions were further diluted in PBS (pH 7.4) or culture media to lessen traces of ethanol to non-toxic levels (<0.5% vol/vol).…”

The tumor therapeutic potential of long non-coding RNA delivery and targeting

“…In acute myeloid leukemia (AML), Connorty et al. 60 identified lncRNA LINC01257 as having an oncogenic role by promoting the proliferation of leukemia. By mixing siRNA with a lipid mixture (D-Lin-MC3, DSPC, cholesterol, DMG-PEG, DOPE-Rho), they targeted LINC01257 in AML cells by NxGen microfluidics systems ( Fig.…”