(T1030-01-04) Development of Polymer-Based miRNA Delivery System for CXCR4 Inhibition and miR155 Inhibition in Target Cells in the Liver to Treat Alcohol-Associated Liver Disease

Purpose: Alcohol-associated liver disease (AALD) have significantly risen over past two decades. 46% of the total 98,457 liver disease deaths in the US in 2022 ¹. High expression of microRNA 155 in Kupffer cells (KC) and binding of SDF-1 to the overexpressed chemokine receptor CXCR4 on hepatic stellate cells (HSC) induces the pathogenesis of AALD ^2-3. We established a microRNA delivery platform based on self-assembled polyplexes where therapeutic anti-miR155, is loaded into the CXCR4 antagonizing polymer PAMD (synthesized from AMD3100) ⁴ to target the specific cells in liver. We hypothesized that, in parallel to the CXCR4 inhibition on the HSC (with free polymer), the PAMD/anti-miR155 polyplexes will be delivered to the KC in liver and reduce the miR155 expression reversing the alcohol induced liver fibrosis. We used the Macrophage RAW 264.7 cell line as in vitro model to assess the cell uptake and miRNA transfection efficiency of the polyplexes. To overcome the challenge of bioavailability, we modified the delivery vector PAMD with cholesterol and or PEG conjugation.

Methods: PAMD was synthesized via Michael addition between AMD3100 and Hexamethylenebisacrylamide. PAMD was conjugated to cholesterol by 17% cholesterol substitution⁵. The PAMD-Ch was further PEGylated by nucleophilic substitution reaction with two different weight percentages (5 and 10) of polyethyleneglycol. The anti-miR155 encapsulation efficiency of polymer and copolymers were tested with agarose gel retardation assay to determine at what w/w ratio the polymer can fully complex to the therapeutic RNA. The percentage of RNA encapsulation efficiency are measured with RiboGreen assay. We measured the hydrodynamic size and surface charge of the polyplexes with dynamic light scattering (DLS). The colloidal stability assessment was performed by incubating the polyplexes into PBS. To assess the time-dependent usability, we stored the polyplexes at 4°C. The hydrodynamic size, and the PDI were monitored for about one month. The polyplex resistance to the plasma protein adsorption-induced dissociation and RNA release is simulated by FRET study by incubating them into human blood plasma at 37⁰C for 6h. The stability of the polyplexes against polyelectrolyte exchange in vivo is mimicked with the incubation of polyplexes into increased concentrations of the natural polyanion heparin for 30 min. FACS analysis and confocal imaging were performed to analyze the polyplex uptake. To test the transfection efficiency of anti-miR155, we performed real time PCR. Cytotoxic effect of the polyplexes and free polymers are evaluated with cell titer blue assay.

Results: All polymer and copolymers fully encapsulate anti-miR155 at a w/w ratio of 1.5 (Fig. 1a) with an encapsulation efficiency > 98% (Fig 1b) demonstrated by the nucleic acid gel retardation assay and riboGreen assay. Cholesterol modification of PAMD and or PEG conjugation to PAMD-Ch significantly reduces the hydrodynamic size of polyplexes < 100 nm with a zeta potential ranging from 8 to 10 mV, which is suitable for entry through liver fenestrae and facilitate endocytosis of the polyplexes (Fig.1c). The PAMD-Ch/ anti-miR155 (PCM) and PEG-PAMD-Ch/ anti-miR155 (PPCM) polyplexes have improved colloidal stability in PBS than PAMD/ anti-miR155 (PM) suggesting the capping effect of cholesterol and PEG content in preventing polyplex aggregation in biological system (Fig. 2c). In 4⁰C temperature, the maximum particle size of the PCM, and PPCMs were within 112 nm over a period of about one month (Fig. 2a). The minimized decrease in FRET ratio for PPCMs demonstrates better integrity and delivery efficiency of the system in vivo (Fig. 2d). In heparin release study, less than half of the RNA was released from PCM than PM at same heparin concentration whereas PPCMs with increasing PEG contents expresses the higher heparin resistance. Cholesterol modification of PAMD significantly improves hydrophobicity and cellular uptake of the polyplexes. However, PEG contents reduce the cellular uptake of the polyplexes due to their comparatively lower zeta potentials (Fig. 3a ). All polymer and copolymers retain microRNA transfection efficiency in vitro observed by the reduced miR155 gene expression in our in vitro cell model (Fig. 3d) with nominal cytotoxicity retaining over 95% cell viability (Fig. 3c )

Conclusion: We developed the miRNA delivery systems (PAMD-Ch/anti-miR155 and PEG-PAMD-Ch/anti-miR155) potential for efficient delivery of RNA interference (RNAi) therapeutics in the alcohol associated fibrotic liver. The copolymers PAMD-Ch and PEG-PAMD-Ch efficiently encapsulate therapeutic microRNA and forms cationic polyplexes ( < 100 nm, and 8 to 10 mV) improving their cellular uptake and accumulation in vitro. The improved colloidal stability, plasma stability, and heparin stability of the PCM and PPCM polyplexes indicates their higher delivery efficiency via systemic circulation and makes them promising drug delivery platform for the delivery of RNAi therapeutics in targeted liver. In the in vitro model of LPS-induced miR 155 overexpressing RAW 264.7 cells, the RNA transfection efficiency of the polyplexes indicates their miRNA 155 inhibition efficacy in the alcohol induced activates KCs.

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