Experimental Variables that Affect Human Hepatocyte AAV Transduction in Liver Chimeric Mice.
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ABSTRACT:
SUBMITTER: Zou C
PROVIDER: S-EPMC7326722 | biostudies-literature | 2020 Sep
REPOSITORIES: biostudies-literature
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Project description:Existing recombinant adeno-associated virus (rAAV) serotypes for delivering in vivo gene therapy treatments for human liver diseases have not yielded combined high-level human hepatocyte transduction and favorable humoral neutralization properties in diverse patient groups. Yet, these combined properties are important for therapeutic efficacy. To bioengineer capsids that exhibit both unique seroreactivity profiles and functionally transduce human hepatocytes at therapeutically relevant levels, we performed multiplexed sequential directed evolution screens using diverse capsid libraries in both primary human hepatocytes in vivo and with pooled human sera from thousands of patients. AAV libraries were subjected to five rounds of in vivo selection in xenografted mice with human livers to isolate an enriched human-hepatotropic library that was then used as input for a sequential on-bead screen against pooled human immunoglobulins. Evolved variants were vectorized and validated against existing hepatotropic serotypes. Two of the evolved AAV serotypes, NP40 and NP59, exhibited dramatically improved functional human hepatocyte transduction in vivo in xenografted mice with human livers, along with favorable human seroreactivity profiles, compared with existing serotypes. These novel capsids represent enhanced vector delivery systems for future human liver gene therapy applications.
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Project description:Background & aimsPorphyrias result from anomalies of heme biosynthetic enzymes and can lead to cirrhosis and hepatocellular cancer. In mice, these diseases can be modeled by administration of a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), which causes accumulation of porphyrin intermediates, resulting in hepatobiliary injury. Wnt/β-catenin signaling has been shown to be a modulatable target in models of biliary injury; thus, we investigated its role in DDC-driven injury.Methodsβ-Catenin (Ctnnb1) knockout (KO) mice, Wnt co-receptor KO mice, and littermate controls were fed a DDC diet for 2 weeks. β-Catenin was exogenously inhibited in hepatocytes by administering β-catenin dicer-substrate RNA (DsiRNA), conjugated to a lipid nanoparticle, to mice after DDC diet and then weekly for 4 weeks. In all experiments, serum and livers were collected; livers were analyzed by histology, western blotting, and real-time PCR. Porphyrin was measured by fluorescence, quantification of polarized light images, and liquid chromatography-mass spectrometry.ResultsDDC-fed mice lacking β-catenin or Wnt signaling had decreased liver injury compared to controls. Exogenous mice that underwent β-catenin suppression by DsiRNA during DDC feeding also showed less injury compared to control mice receiving lipid nanoparticles. Control livers contained extensive porphyrin deposits which were largely absent in mice lacking β-catenin signaling. Notably, we identified a network of key heme biosynthesis enzymes that are suppressed in the absence of β-catenin, preventing accumulation of toxic protoporphyrins. Additionally, mice lacking β-catenin exhibited fewer protein aggregates, improved proteasomal activity, and reduced induction of autophagy, all contributing to protection from injury.Conclusionsβ-Catenin inhibition, through its pleiotropic effects on metabolism, cell stress, and autophagy, represents a novel therapeutic approach for patients with porphyria.Lay summaryPorphyrias are disorders resulting from abnormalities in the steps that lead to heme production, which cause build-up of toxic by-products called porphyrins. Liver is commonly either a source or a target of excess porphyrins, and complications can range from minor abnormalities to liver failure. In this report, we inhibited Wnt/β-catenin signaling in an experimental model of porphyria, which resulted in decreased liver injury. Targeting β-catenin affected multiple components of the heme biosynthesis pathway, thus preventing build-up of porphyrin intermediates. Our study suggests that drugs inhibiting β-catenin activity could reduce the amount of porphyrin accumulation and help alleviate symptoms in patients with porphyria.
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Project description:Recent successes in clinical gene therapy applications have intensified the interest in using adeno-associated viruses (AAVs) as vectors for gene delivery into human liver. An inherent intriguing characteristic of AAVs is that vector variants vary substantially in their ability to transduce hepatocytes from different species. This has historically limited the value of preclinical studies using rodent models for predicting the efficiency of AAV vectors in liver-targeted gene therapy clinical studies. In this work, we aimed to investigate the key determinants of the observed differential interspecies transduction abilities among AAV variants. We took advantage of domain swapping strategies between AAV-KP1, a newly identified variant with enhanced murine liver tropism, and AAV3b, which functions poorly in mice. The systematic in vivo comparison of AAV3b/AAV-KP1 chimeric variants allowed us to identify a threonine insertion at position 265 within variable region I (VR-I) as the key residue that confers murine hepatic transduction to human-derived clade B (AAV2-like) and clade C (AAV3b-like) variants. We propose to use this insertion to generate phylogenetically related AAV surrogates in support of toxicology and dosing studies in the murine liver model.
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Project description:We report how human hepatocytes with PNPLA3 148I and 148M variants (rs738409) engrafted in the livers of chimeric mice respond to different durations of hypercaloric western style diet (WD). To determine how WD affected human and mouse gene expression we performed RNA-sequencing (RNA-seq) on livers from fasting mice.
2021-02-13 | GSE166693 | GEO