Browsing by Author "de Jong, Ype P."

Now showing 1 - 9 of 9
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    AAV and hepatitis: Cause or coincidence?
    (Elsevier, 2022) de Jong, Ype P.; Herzog, Roland W.; Pediatrics, School of Medicine
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    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 Medicine
    Capsid 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.
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    Ectopic clotting factor VIII expression and misfolding in hepatocytes as a cause for hepatocellular carcinoma
    (Elsevier, 2022-12-07) Kapelanski-Lamoureux, Audrey; Chen, Zhouji; Gao, Zu-Hua; Deng, Ruishu; Lazaris, Anthoula; Lebeaupin, Cynthia; Giles, Lisa; Malhotra, Jyoti; Yong, Jing; Zou, Chenhui; de Jong, Ype P.; Metrakos, Peter; Herzog, Roland W.; Kaufman, Randal J.; Pediatrics, School of Medicine
    Hemophilia A gene therapy targets hepatocytes to express B domain deleted (BDD) clotting factor VIII (FVIII) to permit viral encapsidation. Since BDD is prone to misfolding in the endoplasmic reticulum (ER) and ER protein misfolding in hepatocytes followed by high-fat diet (HFD) can cause hepatocellular carcinoma (HCC), we studied how FVIII misfolding impacts HCC development using hepatocyte DNA delivery to express three proteins from the same parental vector: (1) well-folded cytosolic dihydrofolate reductase (DHFR); (2) BDD-FVIII, which is prone to misfolding in the ER; and (3) N6-FVIII, which folds more efficiently than BDD-FVIII. One week after DNA delivery, when FVIII expression was undetectable, mice were fed HFD for 65 weeks. Remarkably, all mice that received BDD-FVIII vector developed liver tumors, whereas only 58% of mice that received N6 and no mice that received DHFR vector developed liver tumors, suggesting that the degree of protein misfolding in the ER increases predisposition to HCC in the context of an HFD and in the absence of viral transduction. Our findings raise concerns of ectopic BDD-FVIII expression in hepatocytes in the clinic, which poses risks independent of viral vector integration. Limited expression per hepatocyte and/or use of proteins that avoid misfolding may enhance safety.
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    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 Medicine
    Limitations 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.
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    Expansion, in vivo–ex vivo cycling, and genetic manipulation of primary human hepatocytes
    (National Academy of Sciences, 2020-01-08) Michailidis, Eleftherios; Vercauteren, Koen; Mancio-Silva, Liliana; Andrus, Linda; Jahan, Cyprien; Ricardo-Lax, Inna; Zou, Chenhui; Kabbani, Mohammad; Park, Paul; Quirk, Corrine; Pyrgaki, Christina; Razooky, Brandon; Verhoye, Lieven; Zoluthkin, Irene; Lu, Wei-Yu; Forbes, Stuart J.; Chiriboga, Luis; Theise, Neil D.; Herzog, Roland W.; Suemizu, Hiroshi; Schneider, William M.; Shlomai, Amir; Meuleman, Philip; Bhatia, Sangeeta N.; Rice, Charles M.; de Jong, Ype P.; Pediatrics, School of Medicine
    Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum. mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.
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    IL-15 blockade and rapamycin rescue multifactorial loss of factor VIII from AAV-transduced hepatocytes in hemophilia A mice
    (Elsevier, 2022-12-07) Butterfield, John S. S.; Yamada, Kentaro; Bertolini, Thais B.; Syed, Farooq; Kumar, Sandeep R. P.; Li, Xin; Arisa, Sreevani; Piñeros, Annie R.; Tapia, Alejandro; Rogers, Christopher A.; Li, Ning; Rana, Jyoti; Biswas, Moanaro; Terhorst, Cox; Kaufman, Randal J.; de Jong, Ype P.; Herzog, Roland W.; Pediatrics, School of Medicine
    Hepatic adeno-associated viral (AAV) gene transfer has the potential to cure the X-linked bleeding disorder hemophilia A. However, declining therapeutic coagulation factor VIII (FVIII) expression has plagued clinical trials. To assess the mechanistic underpinnings of this loss of FVIII expression, we developed a hemophilia A mouse model that shares key features observed in clinical trials. Following liver-directed AAV8 gene transfer in the presence of rapamycin, initial FVIII protein expression declines over time in the absence of antibody formation. Surprisingly, loss of FVIII protein production occurs despite persistence of transgene and mRNA, suggesting a translational shutdown rather than a loss of transduced hepatocytes. Some of the animals develop ER stress, which may be linked to hepatic inflammatory cytokine expression. FVIII protein expression is preserved by interleukin-15/interleukin-15 receptor blockade, which suppresses CD8+ T and natural killer cell responses. Interestingly, mice with initial FVIII levels >100% of normal had diminishing expression while still under immune suppression. Taken together, our findings of interanimal variability of the response, and the ability of the immune system to shut down transgene expression without utilizing cytolytic or antibody-mediated mechanisms, illustrate the challenges associated with FVIII gene transfer. Our protocols based upon cytokine blockade should help to maintain efficient FVIII expression.
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    Immune Responses to Viral Gene Therapy Vectors
    (Elsevier, 2020-03-04) Shirley, Jamie L.; de Jong, Ype P.; Terhorst, Cox; Herzog, Roland W.; Pediatrics, School of Medicine
    Several viral vector-based gene therapy drugs have now received marketing approval. A much larger number of additional viral vectors are in various stages of clinical trials for the treatment of genetic and acquired diseases, with many more in pre-clinical testing. Efficiency of gene transfer and ability to provide long-term therapy make these vector systems very attractive. In fact, viral vector gene therapy has been able to treat or even cure diseases for which there had been no or only suboptimal treatments. However, innate and adaptive immune responses to these vectors and their transgene products constitute substantial hurdles to clinical development and wider use in patients. This review provides an overview of the type of immune responses that have been documented in animal models and in humans who received gene transfer with one of three widely tested vector systems, namely adenoviral, lentiviral, or adeno-associated viral vectors. Particular emphasis is given to mechanisms leading to immune responses, efforts to reduce vector immunogenicity, and potential solutions to the problems. At the same time, we point out gaps in our knowledge that should to be filled and problems that need to be addressed going forward.
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    Liver gene therapy and hepatocellular carcinoma: A complex web
    (Elsevier, 2021) de Jong, Ype P.; Herzog, Roland W.; Pediatrics, School of Medicine
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    Optimizing liver health before and after gene therapy for hemophilia A
    (American Society of Hematology, 2024) Ragni, Margaret V.; Mead, Henry; de Jong, Ype P.; Kaczmarek, Radoslaw; Leavitt, Andrew D.; Long, Brian; Nugent, Diane J.; Sabatino, Denise E.; Fong, Sylvia; von Drygalski, Annette; Walsh, Christopher E.; Luxon, Bruce A.; Pediatrics, School of Medicine
    Gene therapy for severe hemophilia A uses an adeno-associated virus (AAV) vector and liver-specific promoters that depend on healthy hepatocyte function to achieve safe and long-lasting increases in factor VIII (FVIII) activity. Thus, hepatocyte health is an essential aspect of safe and successful gene therapy. Many people living with hemophilia A have current or past chronic hepatitis C virus infection, metabolic dysfunction-associated steatosis or steatohepatitis, or other conditions that may compromise the efficacy and safety of AAV-mediated gene therapy. In addition, gene therapy may induce an immune response to transduced hepatocytes, leading to liver inflammation and reduced FVIII activity. The immune response can be treated with immunosuppression, but close monitoring of liver function tests and factor levels is necessary. The long-term risk of hepatocellular carcinoma associated with gene therapy is unknown. Routine screening by imaging for hepatocellular carcinoma, preferable every 6 months, is essential in patients at high risk and recommended in all recipients of hemophilia A gene therapy. This paper describes our current understanding of the biologic underpinnings of how liver health affects hemophilia A gene therapy, and provides practical clinical guidance for assessing, monitoring, and managing liver health both before and after gene therapy.