(M1030-02-08) A Novel Liposomal Delivery for MicroRNA-497 and Venetoclax in Acute Myeloid Leukemia

Purpose: Acute Myeloid Leukemia (AML) is one of the most common and lethal hematologic malignancies in older adults. Currently, treatments are still challenging in older AML patients with a low 5-year survival rate. Thus, new therapeutic methods are urgently required. In this study, a novel miRNA candidate, miR497 was identified to synergistically treat AML with venetoclax, which is an FDA approved BCL-2 inhibitor. Due to the instability of miRNA in body fluids and the non-specific distribution of both venetoclax and miRNA, a liposomal delivery system, HSPC-MDC2 LP was developed to increase the drug stability as well as their accumulation at the target tumor site. With this delivery system, efficient treatment in the AML mouse model is expected to be achieved.

Methods: Liposomal formulation preparation. Liposomes were prepared using Hydro Soy Phosphatidylcholine (HSPC), 1,3-bis(4,6-dimethyl-2-tetradecylpyridinium-1-yl)propoane dihexafluorophosphate (MDC2), Cholesterol, and DSPE-PEG2000. HSPC, MDC2, cholesterol, DSPE-PEG2000, and venetoclax were dissolved in ethanol/acetone and sonicated to form a homogeneous solution. The lipid solution was injected into 5% dextrose solution dropwise under vigorous stirring. After continuous stirring for 5 min, the ethanol/acetone was removed by rotary evaporation. The final HSPC-MDC2 LP solution was obtained by centrifuging at 10000 rpm to remove the unloaded venetoclax. MiRNA was then added into the solution and incubated for 40 min to obtain the miRNA-CLL1-HSPC-MDC2 LP. The size and zeta potential were determined by Zetasizer. The drug loading was determined by HPLC.
In vitro transfection efficiency of miRNA-HSPC-MDC2 LP. Two human AML cell lines, OCI-AML2 and SKM-1 were used to examine the transfection efficiency of miRNA-HSPC-MDC2 LP. Cells treated with miR-497 were divided into three groups: control, lipofectamine 2000, and miRNA-HSPC-MDC2 LP. Cells were treated for 24h, and the total RNA was extracted for measuring miRNA expression by qPCR. Four genes were selected to be examined: MAP2K1, STAT3, MCL-1 and BCL-2.
AML targeting site identification. An AML targeting site was identified in three AML cell lines, OCI-AML2, SKM-1 and U937 using flow cytometry. Briefly, cells were lysed and incubated with two antibodies, Brilliant Violet CD123 and PE-CLL-1. After fixing, the fluorescent signals were determined by flow cytometry to figure out the expression on AML cells.
AML mouse model generation. NSG mice are conditioned with a sublethal dose of irradiation 12 to 24 hours before transplantation. Luciferase stably expressing OCI-AML2 cells (1×10⁶) were intravenously injected to NSG mice.

Results: The mean particle size of HSPC-MDC2 LP was 106.7±5 nm with the PDI of 0.213, and the zeta potential was 18.9 mV. The drug loading was 8.62% w/w. miR-497 was fully complexed with HSPC-MDC2 LP at the N/P ratio of 16:1. Since the miR-497 targets p38-MAPK signaling pathway, several related genes were evaluated to examine the transfection efficiency of our formulation. Cells transfected with miRNA-HSPC-MDC2 LP showed lowest disease-related gene expression, indicating higher delivery efficiency of miRNA-HSPC-MDC2 LP. CD123 and CLL-1 are two relatively new target site of AML cells, and by flow cytometry we figured out that CLL-1 was expressed significantly in our AML cells rather than CD123. Also, CLL-1 was not expressed in normal hematopoietic stem cells, which made it a promising target in further studies.

Conclusion: We have demonstrated that venetoclax and miR-497 combination has synergic therapeutic effect on AML cells and our liposomal delivery system, miRNA-HSPC-MDC2 LP has a higher in vitro delivery efficacy. Furthermore, a promising AML target, CLL-1 was identified for further improvement of the delivery system to obtain a better AML treatment.

Characterization of HSPC-MDC2 LP. (a) Size distribution of HSPC-MDC2 LP. (b) HSPC-MDC2 LP complexation with miR-497.

Transfection efficiency of HSPC-MDC2 LP. (a) OCI-AML2. (b) SKM-1.

Target identification of AML cells.