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; Ernest Moles; Charles E de Bock; Nisitha Jayatilleke; Jenny L Smith; Soheil Meshinchi; Chelsea Mayoh; Maria Kavallaris; Richard B Lock

doi:10.3390/pharmaceutics13101681

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

Pharmaceutics. 2021 Oct 14;13(10):1681. doi: 10.3390/pharmaceutics13101681.

Authors

Patrick Connerty^{1

2

3}, Ernest Moles^{1

2

3

4}, Charles E de Bock^{1

2

3}, Nisitha Jayatilleke¹, Jenny L Smith^{5

6}, Soheil Meshinchi^{5

6}, Chelsea Mayoh^{1

2}, Maria Kavallaris^{1

2

3

4}, Richard B Lock^{1

2

3}

Affiliations

¹ Children's Cancer Institute, Lowy Cancer Research Centre, UNSW Sydney, Sydney, NSW 2052, Australia.
² School of Women's and Children's Health, UNSW Sydney, Sydney, NSW 2052, Australia.
³ University of New South Wales Centre for Childhood Cancer Research, UNSW Sydney, Sydney, NSW 2052, Australia.
⁴ Australian Centre for Nanomedicine, ARC Centre of Excellence in Bio-Nano Science and Technology, UNSW Sydney, Sydney, NSW 2052, Australia.
⁵ Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
⁶ Division of Pediatric Hematology/Oncology, University of Washington, Seattle, WA 98109, USA.

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.

Keywords: RNA interference; lipid-based nanoparticles; long non-coding RNA; nanomedicine; nanoparticle-assisted siRNA delivery.

Abstract

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