Assistant Professor Rowan University, United States
Introduction: Pediatric acute myeloid leukemia (AML) is the second most prevalent cancer in children, with a 68% survival rate and a 25-35% chance of relapse [1]. Current standard-of-care includes chemotherapy and bone marrow transplantations, but these approaches have limited efficacy and severe side effects [1]. More recently, immunotherapies have emerged for pediatric AML, but these are met with severe toxicities that often limit their success [2, 3]. Therefore, there is a need for pediatric AML therapeutics that are effective and safer than current options. I am developing a lipid nanoparticle (LNP) platform designed to deliver therapeutic antibodies and nucleic acids to inhibit AML progression. LNPs have shown efficacy in treating and vaccinating against multiple cancers, including adult AML, as well as diseases such as SARS-CoV-2 [4-9]. LNPs encapsulate therapeutic nucleic acids, such as small interfering RNA (siRNA), for increased stability and cellular uptake. I engineered LNPs with encapsulated siRNA to enable siRNA-mediated knockdown of Wilms Tumor 1 (WT1). WT1 drives cell proliferation and chemotherapy resistance and is overexpressed in pediatric AML [10]. In addition to WT1 inhibition, LNPs are surface-coated with PD-L1, an immune checkpoint overexpressed on AML cells compared to healthy cells [11]. I hypothesized that this LNP platform would enable a multifaceted approach to AML treatment through WT1 knockdown, PD-L1 targeting, and checkpoint inhibition [12]. Ultimately, this work could provide a better understanding of potential targets for AML therapies while establishing LNPs to treat pediatric AML.
Materials and
Methods: Baseline WT1 and PD-L1 expression in Kasumi-1 cells, a pediatric AML cell line, was established using flow cytometry. Kasumi-1 cells were permeabilized and stained for WT1 protein before analysis. Separately, cells were pre-treated with IFN-γ for 24 hours prior to staining with PD-L1 antibodies to assess expression. LNPs were formulated by combining an ethanol phase with lipid components and an aqueous phase containing siRNA and citrate buffer, followed by purification by dialysis [13]. Hydrodynamic diameter and polydispersity indices were obtained by Dynamic Light Scattering (DLS). Encapsulation efficiencies of siRNA in LNPs were calculated using Ribogreen Assays. I created a library of 26 chemically unique Cy5 siRNA-loaded LNPs to assess how LNP formulation influences siRNA delivery to AML cells. Cells were treated for 4 hours with each of the 26 LNPs with encapsulated Cy5-labelled siRNA. We used flow cytometry to assess delivery of each LNP to AML cells by Cy5 signal. LNPs that yielded high uptake (>60%) were selected to deliver WT1 siRNA. Cells were treated with LNPs with WT1 siRNA, or LNPs with GFP siRNA as the control, for 24 hr. Cells were stained with WT1 antibodies and evaluated by flow cytometry to assess WT1 knockdown. Next, we conjugated Fab regions of PD-L1 or isotype control antibodies onto LNPs using maleimide chemistry and validated successful conjugation using BCA Assays, gel electrophoresis, and DLS. Following LNP characterization, we treated Kasumi-1 cells with LNPs (500 ng siRNA/well) and assessed mRNA levels and protein expression by qRT-PCR and flow cytometry, respectively.
Results, Conclusions, and Discussions:
Results: LNPs are monodisperse and hydrodynamic diameter ranged from 115-185 nm. siRNA encapsulation efficiency ranged from 55-86%. Antibody conjugation was verified by BCA assays and DLS, which showed an increase in hydrodynamic diameter of ~20 nm. We treated Kasumi-1 cells with each of the 26 LNPs, which identified LNP20 (40% ionizable lipid, 10% phospholipid, 45% cholesterol, 5% PEG) and LNP22 (34% ionizable lipid, 30% phospholipid, 34% cholesterol, 2% PEG) as top platforms for high Cy5 delivery to over 70% of treated cells. We used these formulations to encapsulate WT1 siRNA. As a baseline, we found that 90.37% of Kasumi-1 cells express high levels of WT1, validating their use for these studies. Following treatment, we assessed WT1 expression by flow cytometry, which revealed that LNPs decreased WT1 expression ~50% compared to controls. Further, a cell proliferation assay decreased proliferation 40% compared to controls. We have conjugated PD-L1 antibodies to LNPs, and ongoing studies are evaluating the ability for LNPs to target these cells and mediate checkpoint blockade.
Discussion: Our data demonstrates LNP-mediated knockdown of WT1 leading to decreased proliferation, establishing LNPs as a promising means to halt AML progression. Ongoing studies are incorporating the active targeting approach for a multifaceted approach to AML treatment. This study provides a better understanding of potential targets for AML while establishing an LNP platform to treat this disease. Ultimately, this platform could provide a multifaceted approach to treat pediatric AML through targeting, checkpoint blockade, and WT1 inhibition to improve patient outcomes.
References [1] A. Lonetti, A, Front Pediatr 7 (2019).[2] A.J. Lamble, Blood Adv 3 (2019). [3] J.B. Koedijk, Cancers (Basel) 13 (2021). [4] T.M. Allen, Adv Drug Deliv Rev 65 (2013). [5] A. Miller, Journal of drug delivery (2013). [6] K.J. Kauffman, Nano Lett 15 (2015. [7] R.S. Riley, Nat Rev Drug Discov 18 (2019). [8] D.T. Johnson, Leukemia 36 (2022). [9] J. Gumbrecht, CNN (2020). [10] R. Rampal, Haematologica 101 (2016). [11] M. Silva, Onco 2 (2022). [12] B. Zhou, J Transl Med 18 (2020). [13] R.E. Young, Bioactive Materials 34 (2024).
