Hepatitis B virus infection enhances susceptibility toward adeno-associated viral vector transduction in vitro and in vivo

Authors

  • Marianna Hösel,

    1. Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
    2. German Center for Infection Research, Partner sites Bonn-Cologne and Munich, Germany
    3. Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany
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    • These authors contributed equally.

  • Julie Lucifora,

    1. German Center for Infection Research, Partner sites Bonn-Cologne and Munich, Germany
    2. Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
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    • These authors contributed equally.

  • Thomas Michler,

    1. Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
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  • Gisela Holz,

    1. Department of Gastroenterology and Hepatology, University Hospital of Cologne, Cologne, Germany
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  • Marion Gruffaz,

    1. INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon, Lyon, France
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  • Stephanie Stahnke,

    1. Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
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  • Fabien Zoulim,

    1. INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon, Lyon, France
    2. Hospices Civil of Lyon (HCL), Liver Unit of Croix-Rousse Hospital, Lyon, France
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  • David Durantel,

    1. INSERM, U1052, Cancer Research Center of Lyon (CRCL), University of Lyon, Lyon, France
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  • Mathias Heikenwalder,

    1. German Center for Infection Research, Partner sites Bonn-Cologne and Munich, Germany
    2. Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
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  • Dirk Nierhoff,

    1. Department of Gastroenterology and Hepatology, University Hospital of Cologne, Cologne, Germany
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  • Rachel Millet,

    1. Centre International de Recherche en Infectiologie (CIRI, International Center for Infectiology Research), Inserm U1111, CNRS UMR5308, Ecole Normale Supérieure de Lyon, Université de Lyon, Lyon, France
    2. LabEx Ecofect, Université de Lyon, Lyon, France
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  • Anna Salvetti,

    1. Centre International de Recherche en Infectiologie (CIRI, International Center for Infectiology Research), Inserm U1111, CNRS UMR5308, Ecole Normale Supérieure de Lyon, Université de Lyon, Lyon, France
    2. LabEx Ecofect, Université de Lyon, Lyon, France
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  • Ulrike Protzer,

    Corresponding author
    1. German Center for Infection Research, Partner sites Bonn-Cologne and Munich, Germany
    2. Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany
    • Address reprint requests to: Ulrike Protzer, M.D., Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstr. 30, 81675 Munich, Germany. E-mail: protzer@helmholtz-muenchen.de; fax: +49-89-41406821; and Hildegard Büning, Ph.D., Center for Molecular Medicine Cologne, University of Cologne, Robert-Koch-Str. 21, 50931 Cologne, Germany. E-mail: hildegard.buening@uk-koeln.de; fax: +49-221-478-97332.

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  • Hildegard Büning

    Corresponding author
    1. Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
    2. German Center for Infection Research, Partner sites Bonn-Cologne and Munich, Germany
    3. Department I of Internal Medicine, University Hospital Cologne, Cologne, Germany
    • Address reprint requests to: Ulrike Protzer, M.D., Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstr. 30, 81675 Munich, Germany. E-mail: protzer@helmholtz-muenchen.de; fax: +49-89-41406821; and Hildegard Büning, Ph.D., Center for Molecular Medicine Cologne, University of Cologne, Robert-Koch-Str. 21, 50931 Cologne, Germany. E-mail: hildegard.buening@uk-koeln.de; fax: +49-221-478-97332.

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  • Potential conflict of interest: Dr. Nierhoff is on the speakers' bureau for Bristol-Myers Squibb.

  • This work was supported by grants from the German Research Foundation (SFB670, TP8; to H.B.), the Center for Molecular Medicine Cologne (B1; to H.B.), the Helmholtz Alliance for Immunotherapy (HAIT; to U.P.), and the Institut National de la Santé et de la Recherche Médicale (INSERM), Université Claude Bernard Lyon-1 and Ecole Normale Supérieure de Lyon (to A.S. and R.M.).

Abstract

Gene therapy has become an accepted concept for the treatment of a variety of different diseases. In contrast to preclinical models, subjects enrolled in clinical trials, including gene therapy, possess a history of infection with microbes that may influence its safety and efficacy. Especially, viruses that establish chronic infections in the liver, one of the main targets for in vivo gene therapy, raise important concerns. Among them is the hepatitis B virus (HBV), which has chronically infected more than 350 million people worldwide. Here, we investigated the effect of HBV on adeno-associated viral (AAV) vectors, the most frequently applied gene transfer vehicles for in vivo gene therapy. Unexpectedly, we found that HBV greatly improved AAV transduction in cells replicating HBV and identified HBV protein x (HBx) as a key factor. Whereas HBV-positive and -negative cells were indistinguishable with respect to cell-entry efficiency, significantly higher numbers of AAV vector genomes were successfully delivered to the nucleus in the presence of HBV. The HBV-promoting effect was abolished by inhibitors of phosphatidylinositol 3-kinase (PI3K). PI3K was required for efficient trafficking of AAV to the nucleus and was enhanced in HBV-replicating cells and upon HBx expression. Enhancement of AAV transduction was confirmed in vivo using HBV transgenic mice and could successfully be applied to inhibit HBV progeny release. Conclusion: Our results demonstrate that acute, as well as chronic, infections with unrelated viruses change the intracellular milieu, thereby likely influencing gene therapy outcomes. In the case of HBV, HBx-mediated enhancement of AAV transduction is an advantage that could be exploited for development of novel treatments of HBV infection. (Hepatology 2014;59:2110-2120)

Abbreviations
AAV

adeno-associated virus

Abs

antibodies

Akt

protein kinase B

cccDNA

covalently closed circular DNA

cDNA

complementary DNA

CMV

cytomegalovirus

DMEM

Dulbecco's modified Eagle's medium

FAK

focal adhesion kinase

FCS

fetal calf serum

GFP

enhanced green fluorescent protein

GOI

genomic particle per cell ratio

HBcAg

HBV core antigen

HBeAg

HBV early antigen

HBsAg

HBV surface antigen

HBV

hepatitis B virus

HBx

HBV protein x

HSPG

heparan sulfate proteoglycan

IFN-γ

interferon-gamma

IP10

10 kDa IFN-γ-induced protein

IV

intravenously

LC

LightCycler

mRNA

messenger RNA

PBS

phosphate-buffered saline

PHH

primary human hepatocytes

p.i.

postinoculation

PI3K

phosphatidylinositol 3-kinase

qPCR

quantitative polymerase chain reaction

rcDNA

relaxed circular DNA

RSV

Rous sarcoma virus

shRNA

short hairpin RNA

Tet

tetracyclin

Tg

transgenic

TTR

transthyretin

WT

wild type

Vectors based on the adeno-associated virus (AAV), a nonpathogenic member of the parvoviridae, have become increasingly popular as a gene transfer vehicle, in particular, for in vivo gene therapy.[1] They deliver a DNA genome within a tightly packaged nonenveloped protein capsid that mediates cell transduction. The latter is initiated by binding an attachment receptor, which is heparan sulfate proteoglycan (HSPG),[2] in the case of AAV serotype 2 (AAV2), the prototype AAV vector. Cell attachment is followed by binding to coreceptors, which either support the initial contact or—as with αvβ5 and α5β1 integrins—initiate vector internalization through clathrin-dependent endocytosis.[3, 4] In addition, integrin-binding activates phosphatidylinositol 3-kinase (PI3K) through focal adhesion kinase (FAK), a step required for trafficking of AAV-containing endosomes along the cytoskeleton to the nucleus, where transgene expression takes place.[5, 6]

In contrast to results obtained in preclinical models, host immune responses have limited therapeutic efficacy of liver-directed clinical trials. Specifically, T-cell responses against AAV capsid epitopes were elicited, which resulted in an asymptomatic hepatitis and destroyed vector-modified hepatocytes after intraportal AAV2 or systemic application of liver-tropic AAV8 vectors.[7] While on one hand, species-specific differences on the sensing, as well as on the effector, side of the immune system do exist,[8, 9] experimental animals and humans differ in that humans do have a history of previous infection with microbes, part of which may have become chronic.[10]

With approximately 350 million virus carriers, hepatitis B virus (HBV) is a prototype example of a liver-specific virus with a striking tropism for humans that causes chronic liver infection.[11] HBV belongs to the family of hepadnaviridae delivering a relaxed circular (rc)DNA genome that, upon conversion into a covalently closed circular DNA (cccDNA), serves as a transcription template for the pregenomic/precore RNA and three subgenomic RNAs.[11] Pregenomic RNA is reverse transcribed into new rcDNA and functions as messenger RNA (mRNA) for viral capsid and polymerase proteins.[11] Precore RNA encodes a nonstructural protein, which is processed and secreted as HBV e antigen (HBeAg),[12] whereas two of the three subgenomic RNAs encode three viral envelope proteins (the large [L] protein, the middle [M] protein, and the small [S] protein). The third subgenomic RNA encodes the regulatory protein, HBV protein x (HBx), which is a multifunctional protein interacting with various cellular processes and is essential for HBV replication and for establishing HBV infection.[13] Besides virions, HBV-infected cells secrete empty envelopes detectable as HBV surface antigen (HBsAg).

Prompted by the hypothesis that chronic virus infections may cause important changes in the hepatic microenvironment,[10] which may impact on the host-AAV interaction, here we investigated AAV transduction in HBV-replicating cell lines, in HBV-infected primary human hepatocytes (PHH), and in HBV transgenic (Tg) mice. We observed an unexpected increase in AAV transduction efficiency in the presence of HBV that was independent of the promoter used to drive the transgene expression, the transgene itself, and the AAV serotype applied, but depended on the HBV-specific HBx protein. In an effort to exploit this “helper effect” therapeutically, we exchanged the marker gene for interferon-gamma (IFN-γ) or an anti-HBV short hairpin RNA (shRNA). The vectors impaired HBV progeny production to a significantly higher degree in HBV-replicating cells in cell culture and in vivo, revealing the potential of this unique HBV-AAV interaction for development of novel AAV-based anti-HBV treatment strategies.

Material and Methods

Reagents and Plasmids

Wortmannin and LY294002 were purchased from Sigma-Aldrich (St. Louis, MO) and Cell Signaling Technologies (Danvers, MA), respectively. The plasmid, pscTTR-IFN-γ, was produced by digestion of pscTTR-luc,[14] with KpnI and HindIII and replacing the human IFN-γ gene amplified from splenocyte complementary DNA (cDNA) for the luciferase gene.

AAV Vector Production and Detection

AAV vectors were produced in HEK293 cells (ATCC no.: CRL-1573) by triple transfection of pXX6,[15] a vector plasmid (pscAAV/EGFP,[16] pscTTR-luc, pscCMVluc,[14] or pscTTR-IFN-γ), and an AAV helper plasmid (pRC[16] or pXR817), as previously described.[16] Genomic particle titers were determined by real-time LightCycler (LC) polymerase chain reaction (PCR; Roche Diagnostics, Mannheim, Germany) using transgene-specific primers (Supporting Table 1). For transduction, cells were preincubated on ice for 30 minutes, incubated with AAV vectors at a genomic particle-per-cell ratio (GOI) of 1 × 104 or—in the case of rAAV2-TTR-IFN-γ—of 1 × 105 for a further 45 minutes on ice, followed by incubation at 37°C for 2 hours. Cells were washed with phosphate-buffered saline (PBS) and further incubated at 37°C for the indicated time. After a 10-minute treatment of cells with 0.05% trypsin followed by washing steps with PBS, total cellular DNA or RNA were extracted by the DNeasy Tissue Kit or by the RNeasy Kit (both from Qiagen, Hilden, Germany). Nuclear fraction was isolated by the Qproteome Cell Compartment Kit (Qiagen).[18] Vector genomes were quantified using the LightCycler (LC) System and normalized to plasminogen activator (PLAT) gene using Relative Quantification Software (Roche Diagnostics). To monitor gene expression, total cellular RNA was reverse-transcribed into cDNA and analyzed by LC quantitative PCR (qPCR) using the aminolevulinate synthase 1 as a reference gene.[14] Luciferase expression was determined using the Renilla luciferase assay system (Promega, Mannheim, Germany) and normalized to the total protein concentrations determined by bicinchoninic acid protein assay (Pierce, Rockford, IL).

Cell Cultures and HBV Infection

PHHs were obtained within the framework of the nonprofit foundation HTCR, including the informed patient's consent,[19] and prepared from surgical human liver biopsies by a standard two-step collagenase perfusion and serial differential centrifugation. PHHs were maintained in PHH medium, a Williams-based medium supplemented as previously described.[20] HepG2 (ATCC no.: HB-8065TM), HepG2.2.15, and HepG2 H1.321 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS). After achieving confluency, cells were maintained in PHH/DMEM medium (1:1) containing 1% FCS.

HBV inocula preparations, HepaRG cell culture, differentiation, and HBV infection were performed as previously described.[13, 22] HBeAg levels were determined using immunoassay (HBeAg 2.0; Abbott Laboratories, Wiesbaden, Germany). Progeny HBV DNA was isolated from cell supernatants using the DNeasy Tissue Kit and quantified by LC PCR using HBV rcDNA-specific primers.

Mouse Experiments

HBV1.3 mice replicate HBV in hepatocytes from an integrated wild-type (wt) 1.3 overlength genome, whereas HBVxfs mice replicate HBV from an integrated 1.3 overlength genome carrying a frameshift mutation resulting in a premature stop codon, thus expressing a truncated version of HBx.[23] Animals received human care and were maintained under specific pathogen-free conditions. Animal experiments were approved by the Regierung von Oberbayern. Female (Fig. 2) and male (Fig. 6) mice from 3 to 5 months of age were injected into the tail vein (intravenously; IV) with 2 × 10e[9] genomic particles/g body weight of AAV8-H1-shHBVS1.1 (Michler and Protzer, unpublished data) or of rAAV8-H1-sh-hAAT19-RSV-GFP (short name: rAAV8-RSV-GFP, kindly provided by Dirk Grimm[24]), respectively. To quantify AAV vector genomes in the liver, DNA was extracted from liver lysates using the NucleoSpin Tissue kit (Macherey-Nagel, Mountain View, CA) and analyzed by qPCR using primers for enhanced green fluorescent protein (GFP) and—as a reference—for mouse prion protein. To quantify HBV progeny, DNA was extracted from serum by the QIAamp MinElute Virus Spin Kit (Qiagen) and analyzed by qPCR using HBV rcDNA-specific primers.

Immunohistological Stainings

Livers of test animals were fixed in 4% paraformaldehyde, and immunohistological costainings were performed using mouse anti-HBV core antigen (HBcAg; Leica, Wetzlar, Germany) and rabbit anti-GFP (Fitzgerald Industries International, Acton, MA) as primary antibodies (Abs). HBcAg was visualized using mouse horseradish-peroxidase–coupled Abs and staining with diaminobenzidin, whereas GFP was stained with alkaline-phosphatase–coupled anti-rabbit Abs and Fast Red (Invitrogen).

Statistical Analysis

Statistical calculation was performed by t test for dependent samples. A P value <0.05 was considered statistically significant.

Results

HBV Promotes AAV Transduction

We first compared expression from AAV vectors encoding for luciferase under the control of the ubiquitously active cytomegalovirus (CMV) or the liver-specific transthyretin (TTR) promoter in cells replicating HBV or not. For that, we exploited HepG2.2.15 and HepG2H1.3, two HepG2 derivatives stably transfected with HBV genomes, and the parental HepG2 cell line (Fig. 1A). Level of HBV replication was monitored by secretion of HBeAg (Fig. 1A, right panel). Independent of the promoter, we observed a positive correlation between luciferase activity and the presence of HBV (Fig. 1A, left panel).

Figure 1.

HBV infection enhances efficiency of AAV vectors. (A) HepG2, HepG2H1.3wt, HepG2.2.15 at day 5 after seeding, (B) HBV-infected PHH at day 4 postinoculation (p.i.), (B-D) HBV-infected HepaRG at day 4 p.i., or noninfected cells (mock) were transduced with (A) rAAV2-CMV-luc, (A-C) rAAV2-TTR-luc, or (D) rAAV8-CMV-luc. (A and B) Forty-eight hours later or (C and D) at the indicated time points, luciferase activity (left panels) and HBeAg (right panels) were determined. For better comparison, luciferase levels in the following samples were set to 1: (A) HepG2 cells infected with rAAV-CMV-luc; (B) mock-infected HepaRG cells or PHH; and (C and D) HBV-infected HepaRG cells at day 4 p.i. (beginning of detectable HBV replication). Mean values from three independent experiments are given.

To confirm these results in the context of a natural HBV infection, HBV-infected HepaRG cells and HBV-infected PHH were infected with rAAV2-TTR-luc and luciferase activity was measured in comparison to non-HBV-infected cells. In cell culture, HBV infects only a subfraction of cells, which usually does not exceed 30% (Gripon et al.[22] and unpublished result)—reflecting the maximum number of infected cells in chronic HBV infection.[25] Nevertheless, again, for both HepaRG cells and PHH, we observed a significant increase of luciferase activity (Fig. 1B, left panel) in those cells infected with HBV (Fig. 1B, right panel). This promoting effect increased in parallel to HBV replication activity and was independent of the AAV serotype used for transduction (Fig. 1C,D), but correlated with HBV gene expression levels (Fig. 1 A-D).

During the initial hepatocyte infection and upon progeny production, HBV produces a number of proteins, some of which are released.[11] When incubating a mixed culture of HepG2 (HBV) and HepG2.2.15 (HBV+) cells with AAV2 vectors encoding for GFP, we clearly observed stronger GFP signals in those cells that stained positive for HBcAg (Fig. 2A). This result suggests that the HBV-specific factor/s has to be present in the cell in which it enhances AAV transduction.

Figure 2.

Intracellular HBV-specific factor promotes AAV-mediated cell transduction and enhances transgene expression in vivo. (A) HepG2 and HepG2.2.15 cells were cocultivated for 24 hours, followed by incubation with rAAV2-CMV-GFP. Twenty-four hours posttransduction (p.t.), cells were stained for HBcAg and analyzed for GFP expression. Three representative examples (#) are shown. Panels from left to right: anti-HBcAg staining (red); transgene (GFP)-expressing cells (green); overlay; and phase contrast. Quantification of GFP signals in HBcAg-positive (HepG2.2.15) and in HBcAg-negative cells is shown below. (B) HBV1.3 Tg mice were i.v. injected with rAAV8-RSV-GFP. Hepatocytes were counted and divided into negative or positive for HBcAg, and GFP signal was determined for each group. Mean values from six independent measurements are given.

In addition, injection of an AAV8 vector encoding for GFP under the control of the Rous sarcoma virus (RSV) promoter into HBV1.3 Tg mice (Fig. 2B), followed by staining of liver tissue for HBcAg to identify HBV replicating cells, showed the strongest GFP signal in those hepatocytes that were actively producing HBV particles—although, in these mice, all cells contain the HBV transgene. Thus, HBV promotes AAV transduction also in vivo.

HBV Enhances AAV Transduction at a Postentry Step

HBV may enhance AAV2 transduction either by increasing the efficiency of vector internalization and/or by influencing one or more postentry steps. To test whether HBV increases endocytosis of AAV, intracellular vector genomes and luciferase activity were measured in HBV-infected PHH, compared to controls (Fig. 3A). Results from these experiments demonstrated that, despite significantly higher levels of luciferase activity in HBV-infected PHH, the quantity of total intracellular AAV vector genomes was not altered by HBV.

Figure 3.

HBV promotes nuclear delivery of AAV vectors. (A) HBV-infected PHH were transduced with rAAV2-TTR-luc. Luciferase activity and quantity of intracellular AAV vectors was measured 48 hours later. (B) HepG2 and HepG2H1.3wt were transduced with rAAV2-TTR-luc. Twenty-four hours p.t., AAV genomes were determined in whole-cell extracts and in nuclear fraction by qPCR. Luciferase expression was determined by reverse-transcription qPCR at the mRNA level and by luciferase assay at the protein level. Mean values from three and independent experiments are given.

Using a second approach, we measured intranuclear AAV DNA and transgene-specific mRNA to assay whether HBV enhances nuclear import and/or transcription of AAV vector genomes (Fig. 3B). Again, we observed a comparable number of total intracellular AAV vector genomes in cells replicating HBV (HepG2-H1.3wt), compared to HBV-negative cells (HepG2). However, intranuclear AAV DNA was increased almost 3-fold in the HBV-replicating cells, correlating with significantly higher levels of transgene-specific mRNA and luciferase activity (Fig. 3B). In addition, we calculated transcript/intranuclear vector genome ratios for both cell lines and observed a slightly enhanced efficacy of transcription in HepG2H1.3wt cells, for which we calculated 1.2 transcripts per intranuclear DNA genome, compared to 0.7 in HepG2 cells (Supporting Table 2).

Together, these results strongly suggest that HBV infection enhances trafficking of AAV into the nucleus and, in addition, slightly increases transcription from AAV genomes.

HBx Protein Is Sufficient to Enhance AAV Transduction

To determine which HBV protein is responsible for enhancing AAV transductions, we used HepaRG cells expressing HBV core (HepaRG-TR-core), HBeAg (HepaRG-TR-precore), HBx (HepaRG-TR-X), or HBsAg (HepaRG-TR-S) in an inducible manner (Supporting Fig. 1). The different cell lines as well as controls (HepaRG and HepaRG-TR) were transduced with rAAV2-TTR-luc in the presence or absence of tetracyclin (Tet), and luciferase activity was determined. Only HepaRG-TR-X cells, and only when expressing HBx, enhanced AAV transduction, as indicated by the almost 10-fold increase in luciferase activity (Fig. 4A).

Figure 4.

HBx protein alone can enhance efficiency of AAV vectors. (A) Indicated HepaRG cell lines were treated with or without 5 µg/mL of Tet for 24 hours, followed by incubation with rAAV2-TTR-luc. (B) Differentiated HepaRG cells were infected with HBV(wt) or HBV(x-) before incubation with rAAV2-TTR-luc. Luciferase activity was measured at 48 hours p.t. Values are given as median ± standard deviation of three (A) or six (B) independent experiments.

To confirm the involvement of HBx, we infected HepaRG cells with wt HBV (HBV[wt]) or an HBx-deficient HBV (HBV[x-]) before transduction with rAAV2-TTR-luc and observed a significantly lower luciferase expression when cells were infected with HBV(x-), compared to those infected with HBV(wt) (Fig. 4B).

Thus, although we cannot exclude that HBV polymerase or M or L envelope proteins may contribute to the HBV helper effect, our results allow for concluding that HBx alone is sufficient to enhance AAV transduction.

HBV and HBx Protein Enhance AAV Transduction Through PI3K Activation

Both FAK and its effector, PI3K, which remodulate the actin cytoskeleton and enable trafficking and nuclear import of AAV,[5, 6] can be targets of HBx.[26, 27] Therefore, we measured PI3K activity using protein kinase B (Akt) phosphorylation as an indicator. We found an increased Akt phosphorylation if cells replicated HBV (HepG2.2.15, HepG2.H1.3wt) or expressed HBx (HepaRG-TR-X; Fig. 5A). Prolonged treatment (72 hours) with PI3K inhibitors Wortmannin or LY-294002 inhibited PI3K-dependent phosphorylation of Akt (Fig. 5A and data not shown, respectively) without detectable toxicity (Supporting Fig. 2). In line with our hypothesis, treatment with Wortmannin or LY-294002 reduced luciferase expression in a dose-dependent manner in HBV-replicating or in HBx-expressing cells, thereby abolishing the helper effect of HBV for AAV transduction (Fig. 5B,C). In addition, transduction of HepG2H1.3wt with rAAV2-TTR-luc in the presence of Wortmannin (24 hours) had no influence on the quantity of total intracellular vector genomes, but was sufficient to significantly reduce both the quantity of intranuclear AAV genomes and of luciferase gene transcription, compared to that in cells not treated with Wortmannin (Supporting Fig. 3).

Figure 5.

HBV activates the PI3K pathway. (A) HepG2 and HepG2H1.3wt were treated with the indicated concentrations of Wortmannin for 1 hour or were left untreated. HepaRG-TR-X cells were treated with 2 µM of Wortmannin for 1 hour followed by transduction with rAAV2-TTR-luc. Seventy-two hours p.t., levels of Akt and pAkt were analyzed by western blotting. (B) HepG2, HepG2H1.3wt (left panel), or HepaRG-TR-X cells (right panel) were treated with Wortmannin and incubated with rAAV2-TTR-luc 1 hour later. Luciferase activity was measured at 72 hours p.t. (A and B) HBx expression in HepaRG-TR-X cells was induced by Tet treatment 24 hours before transduction. (C) HepG2 and HepG2H1.3wt cells were treated with LY294002 for 1 hour. Luciferase activity was measured at 72 hours p.t. with rAAV2-TTR-luc. Mean values of three (B [left panel] and C) or four (B, right panel) independent experiments are given.

Exploiting AAV Vectors in Anti-HBV Strategies

Enhanced efficiency of transgene expression in cells chronically infected with HBV, compared to noninfected cells, is a feature that turns AAV into a promising tool for gene-based anti-HBV strategies. As proof of principle, here we exploited AAV-mediated overexpression of IFN-γ and of an anti-HBV shRNA, respectively. We decided to use IFN-γ because of its known inhibitory activity on HBV infection[23, 28] and compared efficacy of AAV-mediated IFN-γ expression in HepG2H1.3wt and HepG2 cells. We measured a 5-fold increased IFN-γ expression as well as a 15-fold up-regulation of its target 10 kDa interferon gamma-induced protein (IP-10) gene, in HBV-replicating cells (Fig. 6A). Concomitantly, we detected a 50% reduction in HBV progeny release in HepG2H1.3wt cells transduced with rAAV2-TTR-IFN-γ, in comparison to untreated cells or cells transduced with a control vector (rAAV2-TTR-luc; Fig. 6B).

Figure 6.

HBV infection enhances therapeutic efficiency of AAV vectors in vitro and in vivo. (A). HepG2 and HepG2H1.3 were transduced with rAAV2-TTR-IFN-γ. Expression of IFN-γ and IP10 genes was analyzed by reverse-transcription qPCR at 24 hours p.t. Values are given as median ± standard deviation of three independent experiments. (B) HepG2H1.3 were either left untreated (mock) or transduced with rAAV-TTR-IFN-γ (AAV-IFN-g) or with rAAV2-TTR-luc (AAV control). HBV progeny DNA was determined at 72 hours after transduction (C). HBV1.3 and HBVxfs Tg mice (n = 3) were injected with rAAV8-shHBV1.1. Four weeks later, AAV genomes in mouse liver (left panel) and progeny HBV rcDNA from serum (right panel) were measured in duplicates by qPCR. Amounts of AAV vector in the liver were determined relative to the reference gene. Levels of progeny HBV DNA are given in percent (%) of respective control groups (mice before AAV treatment, 100%).

The HBV-promoting effect is restricted to those cells that possess an actively replicating HBV and is therefore ideally suited for cis-acting therapeutic strategies. Therefore, we applied, in our second approach, an AAV8 vector expressing an H1-promoter-driven shRNA against HBV (rAAV8-shHBV1.1.) to HBV1.3 Tg mice. To further confirm the key function of HBx, we applied rAAV8-shHBV1.1 in parallel to HBVxfs Tg mice, which replicate HBV with a frameshift in the HBx-coding region. Blood was collected for monitoring levels of HBV progeny, whereas liver tissue was harvested to quantify AAV vector genomes. Whereas the same amount of AAV targeted the liver in both mouse strains (Fig. 6C, left panel), qPCR analysis of sera revealed a ≈50% reduction in HBV viremia in H1.3-Tg mice, demonstrating the efficacy of this cis-acting anti-HBV strategy. In addition, a clear evidence for the role of HBx in promoting AAV transduction also in the in vivo setting is indicated by the significant higher level of HBV progeny detected in AAV-treated HBVxfs-, compared to HBV1.3 Tg, mice (Fig. 6C, right panel).

Taken together, these results highlight the potential of AAV-based gene therapy, in particular, in view of the here-identified HBx-mediated helper effect as an attractive strategy for treatment of HBV infection.

Discussion

In the present study, we tested the hypothesis that previous history of viral infection impacts on AAV transduction. Specifically, we report more efficient transduction of hepatocytes infected with HBV by AAV vectors—independent of the serotype, the transgene, and the promoter. Expression of HBx, a multifunctional HBV protein, was sufficient to exert this effect that was mainly caused by a significantly improved nuclear delivery of vector genomes. In addition, accessibility of vector genomes was slightly increased, as indicated by the moderately enhanced transcription efficiency. Finally, we were able to confirm the relevance of our findings for AAV-based therapeutic applications aiming to control HBV infection.

AAV vectors are frequently applied in gene therapy, in particular, in vivo.[1] However, in spite of their successful usage as gene therapy vectors, high AAV particle-per-cell ratios are required for cell transduction. In addition, in contrast to preclinical results, in some patients, host responses toward AAV limit therapeutic efficacy.[10]

Both caveats are tackled by investigating host-AAV interactions, including the very early steps of cell transduction. Thereby, pre- and/or postentry barriers, such as lack of receptor expression, proteasomal degradation, inefficient nuclear delivery, and/or inhibition or inability of second-strand synthesis, were unraveled. While incorporation of receptor-binding ligands into the AAV capsid or usage of adapter molecules shows promise in overcoming pre-entry barriers,[29] a variety of physical, chemical, and biological agents enhance AAV transduction at postentry steps. Gamma irradiation or other DNA-damaging agents, for example, induce a DNA damage response as a result of which conversion of the single-stranded AAV genome into a transcriptionally active double-stranded DNA is promoted.[30, 31] Furthermore, inhibition of degradation and/or improved nuclear delivery are postulated as mechanisms by which substitution of tyrosine residues in the viral capsid or proteasome inhibitors enhance AAV transduction,[32, 33] whereas overexpression of T-cell protein tyrosine phosphatase and protein phosphatase was reported to resolve the FKBP52-mediated blockage of second-strand synthesis.[34]

Here, we observed that HBV and, more specifically, the HBx protein promotes AAV transduction. The significant improvement in transgene expression observed in HBV-expressing hepatoma cell lines, in HBV-infected PHH and in hepatocytes of HBV Tg mice, was caused by improved nuclear delivery of vector genomes, which we attributed to the enhanced activity of the PI3K pathway in HBV-positive cells.[27, 35-37] In addition, HBV improved transcription efficacy by a thus far not identified mechanism.

Our findings on the consequences of chronic HBV infection on AAV transduction is the first example demonstrating that chronic infection with an unrelated virus changes the intracellular environment for incoming viral vector particles, thereby influencing the host-vector interaction. Here, HBV or, more specifically, HBx enhances activity of intracellular signaling pathways promoting the intracellular processing of AAV. This effect is functioning within HBV-replicating, but not in neighboring, cells, a valuable feature for a potential clinical use of AAV vectors for treating HBV infection. Exemplary, we impaired HBV progeny release by AAV vectors either expressing IFN-γ or an anti-HBV specific shRNA and demonstrated (1) a significant inhibition of HBV progeny production in cell culture and in Tg mice, compared to controls, and (2) a significant enhanced therapeutic efficacy in cell culture and in vivo in the presence of HBx.

In summary, we provide the first example that HBV influences the efficacy of AAV transduction in human hepatocytes ex vivo and—which is of utmost importance for clinical approaches—also in vivo. Furthermore, we identified that the HBV-specific HBx protein enhances AAV transduction and report on enhanced PI3K-driven nuclear delivery of AAV vectors as the underlying mechanism. Besides paving the way for the development of novel anti-HBV therapeutic strategies, our findings highlight the hitherto unrecognized effect of chronic infection with an unrelated virus on the host-vector interaction and point toward the need to investigate host-vector interaction under conditions that resemble situations found in the human population.

Acknowledgment

The authors thank Hanna Janicki (University Hospital Cologne), Laura Escalona-Espinosa (University Hospital Cologne), Nadine Winkler (University Hospital Cologne), Theresa Asen (Technical University Munich), and Romina Bester (Technical University Munich) for their excellent technical supports and Siemens Healthcare Diagnostics and Dirk Grimm (DKFZ) for providing reagents, as well as Jude Samulski (University of North Carolina at Chapel Hill) and Jim Wilson (University of Pennsylvania) for providing pXX6 and pXR8, respectively. The authors acknowledge the support of the nonprofit foundation HTCR, which holds human tissue on trust, making it broadly available for research on an ethical and legal basis.

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