Targeted Delivery of MicroRNA-29b by Nanoparticles Provides Antileukemic Activity and Increases Sensitivity to the Hypomethylating Agent Decitabine (DAC) in Acute Myeloid Leukemia (AML)
Authors: Huang Xiaomeng , Schwind Sebastian , Yu Bo , Pang Jiuxia , Santhanam Ramasamy , Chan Kenneth K. , Garzon Ramiro , Blum William , Bloomfield Clara D. , Liu Shujun , Perrotti Danilo , Lee Robert J. , Byrd John C. , Muthusamy Natarajan , Lee L. James , Marcucci Guido 
Published: October 2019

Abstract
Abstract 81
Despite advances in our understanding of disease mechanisms the outcome of the majority of AML patients (pts) remains poor. Epigenetic gene silencing by DNA hypermethylation has been shown to contribute to AML. Recently we demonstrated promising results of treatment with the hypomethylating agent DAC in older AML pts. Pts with higher miR-29b expression were more likely to respond to DAC. MiR-29b targets DNA methyltransferases (DNMTs; i.e. DNMT1, DNMT3A & DNMT3B) and has hypomethylating and tumor suppressor activity in AML. Thus uncovering a method to increase intracellular miR-29b and sensitize AML blasts to DAC may be of therapeutic value. Free synthetic miRs are degraded in bio-fluid and have very limited cellular uptake. To overcome these limitations, we developed a novel non-viral delivery system for synthetic miRs. Polyethylenimine was used to capture synthetic miRs & form a polyplex core. An outside layer of lipid components, 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine, Linoleic acid & 1,2-Dimyristoyl-sn-glycerol, methoxypolyethylene Glycol (MW=2,000) was used to protect the miRs from degradation & clearance and facilitate their uptake. Using this approach we produced nanoparticles (NPs) encapsulating 0.4 μM synthetic miR-29b precursors (pM29b) or scramble controls (sc) [both Ambion]. Since AML blasts and cell lines overexpress the transferrin receptor, we conjugated the NPs with transferrin (Tf) to increase the uptake by targeted delivery. To evaluate the efficiency of NP uptake we encapsulated FAM-fluorescent labeled-pM29b and treated AML cell lines (i.e. Kasumi-1 & MV4-11). Tf-conjugated NP (TfNP) treated cells showed the highest uptake by confocal microscopy and flow-cytometry (mean fluorescence intensity (MFI): 21.0 & 22.9 for Kasumi-1 & MV4-11 respectively) compared to non-Tf-conjugated NPs (NP; MFI: 8.3 & 13.0). To evaluate if pM29b was processed into its mature form we measured levels of mature miR-29b in MV4-11 & Kasumi-1 by qPCR after treatment with TfNP encapsulating pM29b (TfNP29b) or NP encapsulating pM29b (NP29b). At 48h mature miR-29b increased 2000 and 2100 fold after treatment with TfNP29b but only 20 and 50 fold following treatment with NP29b in MV4-11 & Kasumi-1 respectively, compared to untreated or TfNPsc treated cells. Thus TfNPs were 40–100 times more efficient than NPs in increasing levels of miR-29b. Our observations were validated in primary blasts from 3 AML pts. At 48h miR-29b levels increase on average 400 fold in TfNP29b vs TfNPsc treated blasts. The levels of an unrelated miR (i.e. miR-140) remained unchanged in all our experiments. These results demonstrate that pM29b is efficiently delivered and processed into its mature form after TfNP delivery, leading to a specific increase of miR-29b in AML cell lines & primary blasts with otherwise low endogenous miR-29b expression. Downregulation of the miR-29b targets DNMT1, DNMT3A, and DNMT3B on RNA & protein level, determined by qPCR and western blotting respectively, was observed in both cell lines & primary AML samples at 48 hours following TfNP29b compared to TfNPsc treatment. The observed fold reduction on RNA level for DNMT1 was 0.63, 0.53 & 0.63, for DNMT3A 0.48, 0.53 & 0.58, and for DNMT3B 0.60, 0.73 & 0.53 in treated MV4-11, Kasumi-1 cells & pts blasts (mean, n=3), respectively. Increased miR-29b, following TfNP29b treatment resulted in antileukemic activity. Treatment with TfNP29b compared to TfNPsc led to 30% & 35% reduction in proliferation at day 5 and 50% & 64% decreased colony forming ability (scored after two weeks) in MV4-11 & Kasumi-1 cells, respectively. To investigate a possible sensitizing effect of miR-29b to DAC treatment, we treated MV4-11 cells and primary AML blasts with 2.5 uM DAC, which is comparable to concentrations achievable in treated AML pts. 72 hours after DAC treatment, decreased viability (measured by MTS assays) was observed in cells treated with TfNP29b (for 48h) followed by DAC compared with those treated with TfNPsc followed by DAC: 36% vs 53% (p<0.001) for MV4-11 cells and 63% vs 73% (p<0.01) for primary pts blasts. In conclusion, we report an effective nanoparticle-based delivery system for miRs. Our delivery system for pM29b showed in vitro antileukemic activity and improved response to DAC in AML cells, demonstrating that miR-29b is not only a predictor for clinical response to DAC, but may also functions as a novel therapeutic, sensitizer to hypomethylating agents in AML.

Disclosures:
No relevant conflicts of interest to declare.