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Preventing Progression of Hepatic Steatosis to Fibrosis with an AAV-Based Gene Strategy

Non-Alcoholic Fatty Liver Disease (NAFLD) is increasingly associated with Metabolic Syndrome, a group of metabolic malfunctions that promote cardiovascular disease. The NAFLD umbrella includes a range of chronic liver disease states, starting from steatosis and ultimately progressing to cirrhosis, which involves permanent liver tissue scarring (Engelmann and Tacke, 2022). It’s estimated that NAFLD has a global prevalence of over 30%, with an average of ~47 new cases per 1000 individuals each year. Overall, NAFLD’s yearly incidence is greater in men (~70/1000) than in women (30/1000) (Riazi et al. 2022).

NAFLD is the most common type of chronic liver disease globally. Although many clinical studies are currently evaluating various pharmacological candidates targeting pathogenic mechanisms, no drugs have yet been FDA approved for NAFLD. Therefore, treatment of NAFLD and nonalcoholic steatohepatitis (NASH) patients relies on diet, exercise, lifestyle improvements, and the use of drugs approved for diabetes, obesity, inflammation, and oxidative stress (Negi et al. 2021).

Disease progression in fatty liver disease. Obesity and metabolic disorders represent risk factors for NAFLD, which starts with fat accumulation in hepatocytes. Further disease progression is characterized by inflammation (NASH) and subsequent fibrogenesis leading to cirrhosis. NASH with and without cirrhosis bears the risk of developing hepatocellular carcinoma (HCC)”. Figure 1, retrieved without modifications from Engelmann and Tacke, 2022. https://creativecommons.org/licenses/by/4.0/

Pathogenic mechanisms underscoring NAFLD and progression to NASH

Pathogenesis in NAFLD has been proposed to arise from excessive lipid accumulation within hepatocytes, referred to as steatosis, which represents a hallmark of the disease. Hepatic steatosis occurs due to an imbalance in the liver's influx and use of fatty acids. Excessive fatty acid accumulation in the liver from three major sources, lipolysis, lipogenesis, and diet, leads to metabolic dysfunction. Once metabolic abnormalities are present, progression from NAFLD to NASH is precipitated by other insults, such as lipotoxicity and inflammation driving liver injury (Negi et al. 2021).

Biotherapeutics under clinical studies for NAFLD

Many investigational drugs, including small molecules and biotherapeutics, are undergoing clinical evaluation against key targets involved in NAFLD-NASH pathogenesis. Some of the biotherapeutics under evaluation include protein analogs (e.g., FGF19 and FGF21), bispecific antibodies (e.g., FGFR1/KLB), and peptide receptor agonists (e.g., GLP-1/glucagon). Overall, in preclinical and clinical studies, FGF19 and FGF21-based compounds have been shown to reduce bodyweight and improve lipid metabolism (Talukdar and Kharitonenkov 2021).

AAV gene delivery approach targets lipid oxidation

In a new preclinical approach, scientists at the University of Virginia School of Medicine have leveraged an AVV8 vector to drive the expression of a single-chain variable fragment of E06 (E06-scFv) in hepatocytes of a diet-induced NAFLD/NASH mouse model (Upchurch et al. 2022). Because excess radical oxygen species are generated in the liver under steatosis conditions, increased lipid oxidation products such as oxidized phosphatidylcholines (OxPCs) form part of the pathology of NAFLD/NASH.

“Increased oxidative stress causes lipid peroxidation, which can occur via enzymatic reactions, such as myeloperoxidase and 12/15-lipoxygenase, and non-enzymatic reactions, such as reactive oxygen species (ROS). Lipid peroxidation of membrane phospholipids results in their fragmentation and the generation of breakdown products which can further modify free amino groups of proteins and lipids, forming covalent adducts and creating oxidation-specific epitopes (OSEs), including malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), phosphocholine on oxidized phospholipids (PC-OxPL), and oxidized cardiolipin (OxCL). These epitopes are carried by oxidized low-density lipoproteins (OxLDL), modified proteins, microvesicles, and apoptotic cells, aspects that have been shown to be present during NAFLD.” Retrieved without modifications from Figure 1 Hendrikx and Binder 2020. https://creativecommons.org/licenses/by/3.0/

E06 is a naturally occurring IgM that targets and neutralizes oxidized phosphatidylcholines (OxPCs). Previous work by Joseph Witztum’s group at the University of California, San Diego, had demonstrated proof of principle for using E06-scFv to target OxPCs. In those studies, the Witztum team demonstrated that transgenic expression of E06-scFv in a mouse model of NASH effectively reduced steatosis, inflammation, fibrosis, cell death, and mitochondrial dysfunction and led to decreased accumulation of lipids in hepatocytes (Sun et al. 2019).

As such, work by Upchurch et al. leveraged a similar approach, but rather than constitutively expressing E06-scFv, the team relied on the use of an AAV8 vector to drive hepatic expression of the antibody fragment.

“We developed a cre-dependent adeno-associated viral construct for expression of scFv-E06. The E06 coding region was synthesized by GenScript from the publicly available, published sequence (Piscataway, NJ, USA), containing a C-terminal myc- and His-tag and flanking 5′ Mlu I and 3′ Nhe I restriction sites.” Upchurch et al. 2022

Overall, this strategy allowed them to elucidate that early AAV8-scFv-E06 expression can prevent lipid accumulation. In contrast, after steatosis is established, AAV8-scFv-E06 expression at a later disease stage helps reduce fibrosis and progression to NASH. Additionally, the team connected these positive outcomes to the reduction of specific OxPC species, providing an opportunity to understand further the molecular factors that may play a role in NAFLD initiation and progression to NASH.

Reference


Engelmann, C. & Tacke, F. The Potential Role of Cellular Senescence in Non-Alcoholic Fatty Liver Disease. Int. J. Mol. Sci. (2022) doi:10.3390/ijms23020652.

Hendrikx, T. & Binder, C. J. Oxidation-Specific Epitopes in Non-Alcoholic Fatty Liver Disease. Frontiers in Endocrinology (2020) doi:10.3389/fendo.2020.607011.

Negi, C. K., Babica, P., Bajard, L., Bienertova-Vasku, J. & Tarantino, G. Insights into the molecular targets and emerging pharmacotherapeutic interventions for nonalcoholic fatty liver disease. Metabolism: Clinical and Experimental (2022) doi:10.1016/j.metabol.2021.154925.

Riazi, K. et al. The prevalence and incidence of NAFLD worldwide: a systematic review and meta-analysis. The Lancet Gastroenterology and Hepatology (2022) doi.org/10.1016/S2468-1253(22)00165-0.

Sun, X. et al. Neutralization of Oxidized Phospholipids Ameliorates Non-alcoholic Steatohepatitis. Cell Metab. (2020) doi:10.1016/j.cmet.2019.10.014.

Talukdar, S. & Kharitonenkov, A. FGF19 and FGF21: In NASH we trust. Molecular Metabolism (2021) doi:10.1016/j.molmet.2020.101152.

Upchurch, C. M., et al. Targeting oxidized phospholipids by AAV-based gene therapy in mice with established hepatic steatosis prevents progression to fibrosis. Science DOI: 10.1126/sciadv.abn0050.

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