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Item Betaine chemistry, roles, and potential use in liver disease(Elsevier, 2016-06) Day, Christopher R.; Kempson, Stephen A.; Department of Cellular & Integrative Physiology, IU School of MedicineBackground Betaine is the trimethyl derivative of glycine and is normally present in human plasma due to dietary intake and endogenous synthesis in liver and kidney. Betaine is utilized in the kidney primarily as an osmoprotectant, whereas in the liver its primary role is in metabolism as a methyl group donor. In both organs, a specific betaine transporter mediates cellular uptake of betaine from plasma. The abundance of both betaine and the betaine transporter in liver greatly exceeds that of other organs. Scope of review The remarkable contributions of betaine to normal human and animal health are summarized together with a discussion of the mechanisms and potential beneficial effects of dietary betaine supplements on liver disease. Major conclusions A significant amount of data from animal models of liver disease indicates that administration of betaine can halt and even reverse progression of the disruption of liver function. Betaine is well-tolerated, inexpensive, effective over a wide range of doses, and is already used in livestock feeding practices. General significance The accumulated data indicate that carefully controlled additional investigations in humans are merited. The focus should be on the long-term use of betaine in large patient populations with liver diseases characterized by development of fatty liver, especially non-alcoholic fatty liver disease and alcoholic liver disease.Item Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion(Liebert, 2023-04) Rana, Jyoti; Marsic, Damien; Zou, Chenhui; Muñoz-Melero, Maite; Li, Xin; Kondratov, Oleksandr; Li, Ning; de Jong, Ype P.; Zolotukhin, Sergei; Biswas, Moanaro; Pediatrics, School of MedicineCapsid engineering of adeno-associated virus (AAV) can surmount current limitations to gene therapy such as broad tissue tropism, low transduction efficiency, or pre-existing neutralizing antibodies (NAb) that restrict patient eligibility. We previously generated an AAV3B combinatorial capsid library by integrating rational design and directed evolution with the aim of improving hepatotropism. A potential isolate, AAV3B-DE5, gained a selective proliferative advantage over five rounds of iterative selection in hepatocyte spheroid cultures. In this study, we reanalyzed our original dataset derived from the AAV3B combinatorial library and isolated variants from earlier (one to three) rounds of selection, with the assumption that variants with faster replication kinetics are not necessarily the most efficient transducers. We identified a potential candidate, AAV3B-V04, which demonstrated significantly enhanced transduction in mouse-passaged primary human hepatocytes as well as in humanized liver chimeric mice, compared to the parental AAV3B or the previously described isolate, AAV3B-DE5. Interestingly, the AAV3B-V04 capsid variant exhibited significantly reduced seroreactivity to pooled or individual human serum samples. Forty-four percent of serum samples with pre-existing NAbs to AAV3B had 5- to 20-fold lower reciprocal NAb titers to AAV3B-V04. AAV3B-V04 has only nine amino acid substitutions, clustered in variable region IV compared to AAV3B, indicating the importance of the loops at the top of the three-fold protrusions in determining both transduction efficiency and immunogenicity. This study highlights the effectiveness of rational design combined with targeted selection for enhanced AAV transduction via molecular evolution approaches. Our findings support the concept of limiting selection rounds to isolate the best transducing AAV3B variant without outgrowth of faster replicating candidates. We conclude that AAV3B-V04 provides advantages such as improved human hepatocyte tropism and immune evasion and propose its utility as a superior candidate for liver gene therapy.Item Engineering and In Vitro Selection of a Novel AAV3B Variant with High Hepatocyte Tropism and Reduced Seroreactivity(Elsevier, 2020-10) Biswas, Moanaro; Marsic, Damien; Li, Ning; Zou, Chenhui; Gonzalez-Aseguinolaza, Gloria; Zolotukhin, Irene; Kumar, Sandeep R.P.; Rana, Jyoti; Butterfield, John S.S.; Kondratov, Oleksandr; de Jong, Ype P.; Herzog, Roland W.; Zolotukhin, Sergei; Pediatrics, School of MedicineLimitations to successful gene therapy with adeno-associated virus (AAV) can comprise pre-existing neutralizing antibodies to the vector capsid that can block cellular entry, or inefficient transduction of target cells that can lead to sub-optimal expression of the therapeutic transgene. Recombinant serotype 3 AAV (AAV3) is an emerging candidate for liver-directed gene therapy. In this study, we integrated rational design by using a combinatorial library derived from AAV3B capsids with directed evolution by in vitro selection for liver-targeted AAV variants. The AAV3B-DE5 variant described herein was undetectable in the original viral library but gained a selective advantage upon in vitro passaging in human hepatocarcinoma spheroid cultures. AAV3B-DE5 contains 24 capsid amino acid substitutions compared with AAV3B, distributed among all five variable regions, with strong selective pressure on VR-IV, VR-V, and VR-VII. In vivo, AAV3B-DE5 demonstrated improved human hepatocyte tropism in a liver chimeric mouse model. Importantly, this variant exhibited reduced seroreactivity to human intravenous immunoglobulin (i.v. Ig), as well as individual serum samples from 100 healthy human donors. Therefore, molecular evolution using a combinatorial library platform generated a viral capsid with high hepatocyte tropism and enhanced evasion of pre-existing AAV neutralizing antibodies.Item Modular crosslinking of gelatin based thiol-norbornene hydrogels for in vitro 3D culture of hepatic cells(ACS Biomaterials Science and Engineering, 2015-10-21) Greene, Tanja L.; Lin, Chien-Chi; Xie, Dong; Dai, Guoli; Yoshida, KenAs liver disease becomes more prevalent, the development of an in vitro culture system to study disease progression and its repair mechanisms is essential. Typically, 2D cultures are used to investigate liver cell (e.g., hepatocyte) function in vitro; however, hepatocytes lose function rapidly when they were isolated from the liver. This has promoted researchers to develop 3D scaffolds to recreate the natural microenvironment of hepatic cells. For example, gelatin-based hydrogels have been increasingly used to promote cell fate processes in 3D. Most gelatin-based systems require the use of physical gelation or non-specific chemical crosslinking. Both of these methods yield gelatin hydrogels with highly interdependent material properties (e.g., bioactivity and matrix stiffness). The purpose of this thesis research was to prepare modularly crosslinked gelatin-based hydrogels for studying the influence of independent matrix properties on hepatic cell fate in 3D. The first objective was to establish tunable gelatin-based thiol-norbornene hydrogels and to demonstrate that the mechanical and biological properties of gelatin hydrogels can be independently adjusted. Furthermore, norbornene and heparin dual-functionalized gelatin (i.e., GelNB-Hep) was prepared and used to sequester and slowly release hepatocyte growth factor (HGF). The second objective was to investigate the viability and functions of hepatocytes encapsulated in gelatin-based hydrogels. Hepatocellular carcinoma cells, Huh7, were used as a model cell type to demonstrate the cytocompatibility of the system. The properties of GelNB hydrogels were modularly tuned to systematically evaluate the effects of matrix properties on cell viability and functions, including CYP3A4 activity and urea secretion. The last objective was to examine the effect of heparin immobilization on hepatocyte viability and functions. The conjugation of heparin onto GelNB led to suppressed Huh7 cell metabolic activity and improved hepatocellular functions. This hybrid hydrogel system should provide a promising 3D cell culture platform for studying cell fate processes.