(M1030-05-26) Co-Administration of a Novel Hedgehog Inhibitor and Anti-miR-96 in a Lipid-Based Nanosystem for the Treatment of Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD)

Purpose: Metabolic Dysfunction Associated Steatotic Liver Disease (MASLD) is the most common liver disease worldwide and the leading cause of liver-related morbidity and mortality.¹ According to a study conducted on the NHANES database, 42% of patients in the US have steatotic liver disease, and almost all of them have metabolic dysfunction. Approximately 90% of these patients were diagnosed with MASLD, while 8% were diagnosed with Met-ALD (a combination of MASLD and moderate alcohol intake.² Obesity, type II diabetes, and adiposopathy are the leading causes of MASLD. Sonic hedgehog (SHH) proteins are released by injured hepatocytes into the surrounding tissue. Hh signaling aids in the regeneration and repair of hepatocytes following short-term liver damage. However, long-term dysregulation of Hh signaling promotes liver fibrosis. YAP1-dependent HSC activation is also enhanced by the overexpression of Hh ligands.³ Thus, inhibiting the Hh pathway is a potential approach to preventing increasing liver injury. Therefore, we synthesized a potent GDC-0449 analog MDB5 to inhibit smoothened (SMO) and improve Hh inhibition.⁴ Furthermore, miRNAs are deregulated in liver diseases, and based on our findings in samples from experimental mice and human patients have revealed that miR-96 is upregulated and targets anti-inflammation and anti-fibrotic genes such as SMAD7and FOXO1-3.⁵ Additionally, GLI induction can occur without Hh via the downstream consequences of TGF-β1 and RAS signaling. Hh and miR-96 are involved in the pathogenesis of hepatic insulin resistance and MASLD progression. Thus, we hypothesize that a combination of MDB5 and anti-miR-96 will synergistically prevent MASLD. Further, one of the significant obstacles in RNAi therapeutics is their poor in vivo stability, off-target effects, immunogenicity, and low cellular uptake. Thus, we developed a novel lipid nanoparticle system to co-deliver MDB5 and anti-miR-96 to improve delivery efficacy.

Methods: We assessed the in vitro efficacy of MDB5 and anti-miR-96 in AML12 and HSCs. Cells were treated with free fatty acids (FFA) [sodium oleate and palmitate (2:1)] on the second day to induce lipotoxicity. Subsequently, on the 3^rd day, cells were either treated with MDB5 (50 µM), a scrambled miRNA (as a negative control), anti-miR-96 (50 nM), or their combination to study their effect on gene expression. Intracellular RNA was extracted with the RNeasy Mini kit (Qiagen), followed by qRT-PCR to assess the expression of genes involved in MASLD progression, including ɑ-SMA, INSR, CPT1A, and Gli-1. We synthesized a novel ionizable lipid, MDC5 and further, we prepared ethanolic solutions containing MDC5, DOPE, cholesterol, and DSPE-mPEG at a particular molar ratio with a total lipid concentration of 25 mM. The RNA was added to a 50 mM acetate buffer (pH 4.2). LNPs were formed by mixing the lipids in ethanol and RNA in an aqueous solution using the NanoAssmblr Ignite Microfluidic system (Precision Nanosystem #NIN0001) at a total flow rate (TFR) of 3 mL/min. The resulting LNP solution was then dialyzed for 2 h against 20 mM acetate buffer (pH 6.0), followed by 1X PBS for 20 h at 4°C to remove ethanol. Particle size, PDI, and zeta potential were measured by DLS and Zeta Sizer. Encapsulation efficiency was determined using the RediplateTM 96 RiboGreen RNA quantification kit. The apparent pKa of the LNP was determined using a 2-(p-toluidino) naphthalene-6-sulfonic acid (TNS) assay.

Results: Synthesis of the novel MDC5 ionizable lipid was confirmed with H¹ NMR. Our real-time RT-PCR analysis showed that FFA treatment increased miR-96 expression, while transfection with anti-miR-96 decreased it significantly. Further, the combination of MDB5 and anti-miR-96 significantly reduced Gli-1 and ɑ-SMA expression in HSCs. CPT1a expression decreased in FFA-treated AML12 cells, indicating impaired mitochondrial β-oxidation and increased lipid accumulation, which the combination treatment significantly increased. The amino lipid to miRNA charge ratio was determined, corresponding to a nitrogen-to-phosphate (N/P) ratio of 16. LNPs exhibited a particle size of around 100 nm with PDI of 0.089, encapsulation efficiency of 80%, and showed no significant difference in particle size over 7 days, indicating the formulation’s stability. The apparent pKa of the LNP was evaluated and was found to be 6.15.

Conclusion: Our data has shown a significant reduction in the gene markers involved in the disease progression of steatotic liver disease in vitro. We also confirmed the formation of the novel ionizable lipid and explored its potentiality in encapsulating the nucleic acid and a small molecule. Thus, the LNP was tailored to obtain a uniform size and drug loading that will efficiently deliver the drugs and result in an enhanced treatment.

References: 1. Chan WK, Chuah KH, Rajaram RB, Lim LL, Ratnasingam J, Vethakkan SR. Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD): A State-of-the-Art Review. J Obes Metab Syndr. 2023 Sep 30;32(3):197–213.
2. Kalligeros M, Vassilopoulos A, Vassilopoulos S, Victor DW, Mylonakis E, Noureddin M. Prevalence of Steatotic Liver Disease (MASLD, MetALD, and ALD) in the United States: NHANES 2017-2020. Clin Gastroenterol Hepatol Off Clin Pract J Am Gastroenterol Assoc. 2023 Nov 8;S1542-3565(23)00914-X.
3. Tsuchida T, Friedman SL. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol. 2017 Jul;14(7):397–411.
4. Kumar V, Dong Y, Kumar V, Almawash S, Mahato RI. The use of micelles to deliver potential hedgehog pathway inhibitor for the treatment of liver fibrosis. Theranostics. 2019 Oct 12;9(25):7537–55.
5. Kumar V, Sethi B, Staller DW, Xin X, Ma J, Dong Y, et al. Anti-miR-96 and Hh pathway inhibitor MDB5 synergistically ameliorate alcohol-associated liver injury in mice. Biomaterials. 2023 Apr 1;295:122049.

Acknowledgements: Dr. Ram Mahato is funded by the NIH (R01DK135817-02) and we duly acknowledge NIH for providing financial support for this work.


Figure 1: Schematic summary of the proposed combination (ref. 2,3)


Figure 2: a. Effect of fatty acids on miR-96 expression
b. Effect of drug treatment on dysregulated genes CPTA, alpha-SMA and Gli 1



Figure 3: Characterization of the formulation for N/P ratio, size, PDI, and encapsulation efficiency