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E - Disease Models and Clinical Applications -> Metabolic, Storage, Endocrine, Liver and Gastrointestinal Diseases

915: rAAV-Delivered Hepatocyte-Specific Expression of miR-375 Protects Against the Acetaminophen-Induced Acute Liver Failure in Mice

Type: Poster Session

Poster Board Number: 915
Presentation Details
Session Title: Thursday Poster Session
Start Time: 5/18/2023 12:00
End Time: 5/18/2023 14:00

Acetaminophen (APAP) overdose is the leading cause of acute liver failure (ALF) in many countries, including the United States. The high incidence of mortality associated with APAP-ALF can be attributed to its rapid onset. N-acetylcysteine (NAC) treatment, though effective, is time limited. Beyond ten hours after an over-dose, the liver cannot be rescued. Many studies have shown that various microRNAs (miR) can serve as potential biomarkers or therapeutic targets. Among these microRNAs, miR-375 levels correlate with the severity of APAP-challenge significantly in clinical samples, which makes it an appealing target for mitigating APAP-ALF. Using AAV8 capsid and Thyroxine Binding Globulin (TBG) promoter, miR375 was expressed in hepatocytes specifically. When challenged with APAP, ALF could be completely rescued in the miR-375 treated mice compared with control group. Liver APAP metabolites were measured with LC/MS-MS, increased levels of liver-protecting Glutathione (GSH) before APAP treatment and decreased cytotoxic byproduct NAPQI (N-acetyl-p-benzoquinone imine) protein adducts after APAP challenge were observed in miR-375 expressing liver. RNA-seq analysis identified three novel targets, Slc16a2, Acls5 and Cyb5b that were downregulated by miR-375. In vivo silencing using artificial miRNA (amiR) against these three genes via AAV8 could protect the liver from APAP-ALF. In addition, Cyp2E1, a key enzyme which was involved in the APAP metabolism into NAPQI, was downregulated by miR-375. To investigate if Cyp2E1 inhibition by miR-375 was required for the liver protection, we used a lower dose of miR-375 without changing the level of Cyp2E1, the liver could still be protected from APAP-ALF to a less degree. Partial/Incomplete protection of APAP-ALF could also be achieved by lowering Cyp2E1 to the level of which miR-375 treatment induced using fine-tuned dose of amiR against Cyp2E1. These results implied the protection by miR-375 was not fully Cyp2E1 dependent and other factors were involved. RNA-seq experiment using livers treated with miR-375 and amiRs against Slc16a2, Acls5 and Cyb5b was carried out. It revealed that the inhibition of Cyp2E1 was accompanied by upregulation of other key enzymes such as Gstm2, Gstm3 and Sult2A7, which were involved in APAP detoxification. Over-expression studies showed that Gstm3 could protect the liver from APAP-ALF in WT mice. Cyp2E1 heterozygous mice has been acquired and TBG-driven Gstm3 was delivered into the liver with AAV8, the results showed that APAP-ALF was well protected. Taken together, decreasing Cyp2E1 and the production of NAPQI along with increasing Gstm3 and liver glutathione were the two major pathways that miR-375 achieved the liver protection against APAP-ALF. We are exploring if miR-375 can serve as an alternative or better treatment than NAC in acetaminophen overdose and other liver diseases. J.L. and Y.W. are co-first authors; G.G. and J.X. are co-corresponding authors.

Jinghua Liu1, Yi Wang2, Guangping Gao1, Jun Xie1

1Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA,2Department of Pathophysiology, Sichuan University, Chengdu, China
 J. Liu: None.

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