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Access Type
WSU Access
Date of Award
January 2025
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Pharmaceutical Sciences
First Advisor
Wanqing W. Liu
Abstract
Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) now affects 30% of the global population. Without intervention, patients with liver steatosis may further develop inflammation and fibrosis, i.e., metabolic-associated steatohepatitis (MASH). Currently, there is only one FDA-approved drug in the treatment of MASH, with no drug targeting the causal factors of MASLD/MASH. Among several genes identified by genome-wide associations studies (GWAS), the role of patatin-like phospholipase domain containing 3 (PNPLA3) gene in MASLD and MASH is well established, with its single nucleotide polymorphism I148M being the single largest genetic risk factor for MASLD and MASH. The increased risk for MASLD attributed to the PNPLA3-148M allele relies on overrepresentation of the PNPLA3-148M isoform. The aim of our study is to validate a liver-specific, oligonucleotide-based therapeutic specifically targeting the PNPLA3-148M isoform as a potential treatment strategy for MASLD and MASH. To this end, both a prototype siRNA and antisense oligo (ASO) were to examine for their efficacy and safety profiles in vivo.Methods: Our group has designed, screened, and patented a few siRNAs that demonstrated highly selective knock-down activity to down-regulate the expression of PNPLA3-148M isoform in hepatocytes. The Liu lab have also successfully developed a transgenic human PNPLA3 mouse model carrying the human PNPLA3-148I or PNPLA3-148M isoform. We examined the effectiveness and safety profiles of our prototype siRNA and ASO in our transgenic mice (n=8 per genotype group) fed with a high sucrose diet for 15 weeks. The two candidates and scrambled control siRNA were conjugated with N-acetylgalactosamine (GalNAc) and subcutaneously (s.c.) injected at week 9 and continued for 6 weeks. The pharmacological and toxicological profiles were assessed. Results: Among mice received the scramble control siRNA, the transgenic PNPLA3-148M (Tg- PNPLA3-148M) mice exhibited significantly increased hepatic steatosis and triglycerides (TG) content compared to the transgenic PNPLA3-148I (Tg-PNPLA3-148I) mice or the non-transgenic wildtype (WT) mice. The prototype siRNA injection significantly reduced hepatic steatosis and hepatic TG content in the Tg-PNPLA3-148M group to the levels similar to that in the Tg-PNPLA3- 148I and WT groups. The ASO-injected Tg-PNPLA3-148M mice exhibited a trend of decreased hepatosteatosis and TG content but was not statistically significant. Both the siRNA and ASO treatment significantly reduced hepatic expression level of PNPLA3-148M but not PNPLA3-148I. The injection of siRNA was revealed to be safe based on the comprehensive evaluation of the physiological data (body weight, fat/lean composition, serum levels of glucose, TG, total cholesterol, ALT, AST, bilirubin, cytokines and chemokines, hepatic transcriptome, as well as hepatic and serum metabolomes) collected throughout the entire trial. The reduced hepatosteatosis and TG content in the siRNA injected Tg-PNPLA3-148M mice was associated with a metabolic reprograming e.g. increased fatty acids β-oxidation, increased cholesterol-bile acids conversion, etc. Also, the knockdown of PNPLA3-148M partially reversed the expression changes of a group of inflammatory genes altered by the phenotype. Although the RNA-seq data reveals that the prototype siRNA might have an off-target knockdown effect on the transcripts of Abgl5 gene in the mouse, it was further confirmed with no knockdown effect on the human ABGL5. In contrast, the ASO has a limited pharmacological effect and is revealed with concerns on its safety profile that is more likely associated with ASO itself regardless of the genotype. The ASO injection induced liver injuries as indicated by the elevated level of liver function markers AST and ALT, as well as the hepatic fibrosis marker αSMA. Moreover, it might be associated with the jeopardized mitochondrial function, which leads to the blockage of fatty acid oxidation and accumulated acylcarnitine. There is also evidence demonstrating over-breakdown of lean mass along with elevated serum creatinine level, suggesting a potential harm to the kidney function. Conclusion: Our PNPLA3-148M targeting siRNA demonstrated promising specificity, efficacy, and safety in treating MASLD, warranting further development for MASH treatment.
Recommended Citation
Peng, Zheyun, "A Sirna-Based Targeted Therapy For Treating Metabolic Dysfunction-Associated Steatotic Liver Disease: A Precision Medicine Approach" (2025). Wayne State University Dissertations. 4288.
https://digitalcommons.wayne.edu/oa_dissertations/4288