• 1xbet官方app

    Skip to main page content
    U.S. flag

    An official website of the United States government

    Dot gov

    The .gov means it’s official.
    Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

    Https

    The site is secure.
    The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

    Full text links

    Atypon full text link Atypon Free PMC article
    . 2023 Apr;34(7-8):289-302.
    doi: 10.1089/hum.2022.176.

    Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion

    Affiliations
    • PMID: 36950804
    • PMCID: PMC10125406
    • DOI: 10.1089/hum.2022.176

    Characterization of a Bioengineered AAV3B Capsid Variant with Enhanced Hepatocyte Tropism and Immune Evasion

    Jyoti Rana et al. Hum Gene Ther. 2023 Apr.

    Abstract

    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.

    Keywords: AAV3B; directed evolution; gene therapy; hepatocyte; huFNRG mice; humanized liver chimeric mice; neutralizing antibody; seroprevalence.

    PubMed Disclaimer

    Conflict of interest statement

    No competing financial interests exist.

    Figures

    Figure 1.
    Figure 1.
    Steps involved in isolating and evaluating AAV3B-V04 from a combinatorial AAV3B library. Step 1: sublibraries A, B, and C with mutations in VR-I, VR-IV, and VR-VII, respectively, are first assembled, combined, and then packaged. Step 2: Library is subjected to three rounds of replication in human hepatocarcinoma spheroid cultures. AAV3B-V04 is isolated and evaluated in vitro and in vivo. Step 3: Transduction efficiency is tested in mpPHH in vitro and in huFNRG mouse livers in vivo. Step 4: Evasion of NAb titers in IVIg and individual human serum samples is evaluated in vitro. AAV, adeno-associated virus; IVIg, intravenous immunoglobulin; mpPHH, mouse-passaged primary human hepatocytes; NAb, neutralizing antibodies; VR, variable region.
    Figure 2.
    Figure 2.
    Evolution of sequence frequencies. (A) Sequences were sorted by their highest frequency among the five samples (each sample corresponding to one round of selection in HUH-7 spheroid cultures). The top 25 sequences are displayed with a separate color (V1–V25). All other sequences are combined under a single color (Other). AAV3B-V04 is highlighted (light orange). AAV3B-DE5 is indicated as V01. (B) Alignment of parental capsid AAV3B and variants AAV3B-DE5, AAV3B-V04, and AAV3B-V05. Only diversified regions in VR-IV, VR-V, VR-VI, and VR-VII are shown. Numbers indicate amino acid position (VP1 numbering); sequence identity with AAV3B is represented by a dot. (C) Three-dimensional structural model of AAV3B showing the positions of the AAV3B-V04 mutations in red.
    Figure 3.
    Figure 3.
    Comparison of in vitro transduction efficiency. (A) Transduction efficiencies of AAV3B, AAV3B-DE5, AAV3B-V04, and AAV3B-V05 at varying MOIs on HUH-7 adherent cells, using RLuc relative luminescence units at 48 h as a readout. (B) Schema and fluorescence images of mpPHH cells transduced with 2.5 × 103 MOI of AAV3B, AAV3B-DE5, or AAV3B-V04 expressing the mScarlet reporter transgene product. AAV3B, AAV3B-DE5, and AAV3B-V04 MOIs corresponding to 50% mScarlet transduction in mpPHH cells as quantified on Cytation 7 are indicated. Data are an average of at least two replicates per group per test condition and are the best representative of at least three independent experiments. Statistical analysis is performed by two-way ANOVA with a Sidak's multiple comparison test for A. Data are represented as mean ± SEM. A p-value <0.05 is considered statistically significant and indicated as follows: *p < 0.05, **p < 0.01, and ****p < 0.0001. MOI, multiplicity of infection; RLuc, Renilla luciferase; SEM, standard error of the mean.
    Figure 4.
    Figure 4.
    In vivo transduction efficiency in mice. (A) Density plots showing frequencies of vehicle control, AAV3B-DE5, or AAV3B-V04 transduced human hepatocytes isolated from huFNRG mouse livers (n = 4–9/group). Transduced human hepatocytes are characterized as HLA-I+ mScarlet+ by flow cytometry. (B) Comparison of transduction frequencies of HLA-I+ mScarlet+ human hepatocytes. (C) Quantification of transduced hepatocytes from C57BL/6 mice (n = 3/group) intravenously injected with AAV8, AAV3B, or AAV3B-V04. (D) Quantification of vector genome copies/100 ng genomic DNA isolated from livers, kidneys, hearts, spleens, muscles, and brains of C57BL/6 mice (n = 3/group) intravenously injected with AAV8, AAV3B, or AAV3B-V04. Data are an average of at least two independent experiments. Statistical analysis is performed by one-way ANOVA with Tukey's multiple comparison test for (B). Data are represented as mean ± SEM. A p-value <0.05 is considered statistically significant and indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
    Figure 5.
    Figure 5.
    In vitro neutralization assays. (A) IVIg: Inhibition of HUH-7 adherent cell transduction by AAV3B, AAV3B-DE5, AAV3B-V04, or AAV3B-V05 using increasing concentrations of pooled IVIg (20–1,500 μg/mL). The IVIg concentration corresponding to 50% reduction in RLuc expression (RLU) for each group, compared to a no IVIg control, is indicated with dashed lines. (B) Determination of mean reciprocal NAb titers to AAV3B, AAV3B-DE5, and AAV3B-V04 for 30 seropositive samples from individual healthy donors using an in vitro RLuc-based assay. Samples that can inhibit transduction by 50% at ≥1:5 dilution are considered seropositive. (C) Distribution of NAb titers from (B). Frequencies of serum samples with NAb titers <1:20, <1:100, and >1:100 to AAV3B, AAV3B-DE5, and AAV3B-V04 are indicated. (D) Eight individual serum samples without detectable (<1:5) NAb to AAV3B are evaluated for NAb to AAV3B-DE5 and AAV3B-V04. Data are represented as mean ± SEM are an average of at least two replicates per group per test condition and are the best representative of at least two independent experiments. IC50 is determined for (A, B, D) using a four-parameter curve fit test. Statistical analysis is performed by two-way ANOVA with a Dunnett's comparison test for (A) and a one-way ANOVA with a Tukey's multiple comparison test for (B, D). A p-value <0.05 is considered statistically significant and indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. IC50, half-maximal inhibitory concentration.

    References

      1. Nathwani AC, Gray JT, Ng CY, et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 2006;107(7):2653–61; doi: 10.1182/blood-2005-10-4035 - DOI - PMC - PubMed
      1. Mingozzi F, Liu YL, Dobrzynski E, et al. Induction of immune tolerance to coagulation factor IX antigen by in vivo hepatic gene transfer. J Clin Invest 2003;111(9):1347–56; doi: 10.1172/JCI16887 - DOI - PMC - PubMed
      1. Colella P, Ronzitti G, Mingozzi F. Emerging issues in AAV-mediated in vivo gene therapy. Mol Ther Methods Clin Dev 2018;8:87–104; doi: 10.1016/j.omtm.2017.11.007 - DOI - PMC - PubMed
      1. Maestro S, Weber ND, Zabaleta N, et al. Novel vectors and approaches for gene therapy in liver diseases. JHEP Rep 2021;3(4):100300; doi: 10.1016/j.jhepr.2021.100300 - DOI - PMC - PubMed
      1. Grieger JC, Samulski RJ. Packaging capacity of adeno-associated virus serotypes: Impact of larger genomes on infectivity and postentry steps. J Virol 2005;79(15):9933–9944; doi: 10.1128/JVI.79.15.9933-9944.2005 - DOI - PMC - PubMed

    Publication types

    MeSH terms

    Substances

    LinkOut - more resources

    • Full Text Sources

    • Other Literature Sources