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*Liver Diseases
The Journal of Immunology, 2007, 178: 1767-1773.
Copyright © 2007 by The American Association of Immunologists, Inc.

Fiber-Modified Adenovirus Vectors Decrease Liver Toxicity through Reduced IL-6 Production1

Naoya Koizumi*,{dagger}, Tomoko Yamaguchi*, Kenji Kawabata*, Fuminori Sakurai*, Tomomi Sasaki*, Yoshiteru Watanabe{dagger}, Takao Hayakawa{ddagger} and Hiroyuki Mizuguchi2,*,§

* Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Osaka, Japan; {dagger} Department of Pharmaceutics and Biopharmaceutics, Showa Pharmaceutical University, Tokyo, Japan; {ddagger} Pharmaceuticals and Medical Devices Agency, Tokyo, Japan; and § Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Adenovirus (Ad) vectors are one of the most commonly used viral vectors in gene therapy clinical trials. However, they elicit a robust innate immune response and inflammatory responses. Improvement of the therapeutic index of Ad vector gene therapy requires elucidation of the mechanism of Ad vector-induced inflammation and cytokine/chemokine production as well as development of the safer vector. In the present study, we found that the fiber-modified Ad vector containing poly-lysine peptides in the fiber knob showed much lower serum IL-6 and aspartate aminotransferase levels (as a maker of liver toxicity) than the conventional Ad vector after i.v. administration, although the modified Ad vector showed higher transgene production in the liver than the conventional Ad vector. RT-PCR analysis showed that spleen, not liver, is the major site of cytokine, chemokine, and IFN expression. Splenic CD11c+ cells were found to secret cytokines. The tissue distribution of Ad vector DNA showed that spleen distribution was much reduced in this modified Ad vector, reflecting reduced IL-6 levels in serum. Liver toxicity by the conventional Ad vector was reduced by anti-IL-6R Ab, suggesting that IL-6 signaling is involved in liver toxicity and that decreased liver toxicity of the modified Ad vector was due in part to the reduced IL-6 production. This study contributes to an understanding of the biological mechanism in innate immune host responses and liver toxicity toward systemically administered Ad vectors and will help in designing safer gene therapy methods that can reduce robust innate immunity and inflammatory responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Recombinant adenovirus (Ad)3 vectors are widely used for gene therapy experiments and clinical gene therapy trials. One of the limitations of Ad vector-mediated gene transfer is the immune response after systemic administration of the Ad vector (1, 2). The immune response to the Ad vector and Ad vector-transduced cells dramatically affects the kinetics of the Ad vector-delivered genes and the gene products. The potent immunogenic toxicities and consequent short-lived transgene expression of Ad vectors are undesirable properties if Ad vectors are to be more broadly applied. The immunogenic toxicities associated with the use of Ad vectors involve both innate and adaptive immune responses.

In the first generation Ad vector lacking the E1 gene, leaky expression of viral genes from the vector stimulates an immune response against the Ad vector-transduced cells (3, 4, 5). The CTL response can be elicited against viral gene products and/or transgene products expressed by transduced cells. The molecular mechanism of this toxicity has been studied extensively, and the helper-dependent (gutted) Ad vector, which deletes all of the viral protein-coding sequences, has been developed to overcome this limitation (6, 7, 8). The humoral virus-neutralizing Ab responses against the Ad capsid itself are another limitation, preventing transgene expression upon the subsequent administration of vectors of the same serotype. Because hexons are mainly targeted by neutralizing Abs, hexon modification has been reported to allow for escape from neutralizing Abs (9). The Ad vectors belonging to types of the subgroup other than Ad type 5, including an Ad type 11- or 35-based vector, or to species other than human have also been developed (10, 11, 12, 13).

Regarding the innate immune response, shortly after systemic injection of the Ad vector cytokines/chemokines are produced and an inflammatory response occurs in response to the Ad vector and Ad vector-transduced cells. It has been reported that activated Kupffer cells (and monocytes and resident macrophages) and dendritic cells (DC) release proinflammatory cytokines/chemokines such as IL-6, TNF-{alpha}, IP-10, and RANTES, causing the activation of an innate immune response (14, 15). NF-{kappa}B activation is likely to play a central role in inflammatory cytokine/chemokine production (16, 17). Although many papers regarding the innate immune response to the Ad vector have been published thus far, the biological mechanism has not been clearly elucidated. Even the cell types responsible for the innate immune response have not been identified. Understanding the mechanism of and identifying the cell types responsible for the innate immune response and liver inflammation are crucial to the construction of new vectors that are safer and efficiently transduce target tissue. Modification of the Ad vector with polyethylene glycol (PEG) reduces the innate immune response and also prolongs persistence in the blood and circumvents neutralization of the Ad vectors by Abs (18, 19, 20, 21). We have previously reported that the mutant Ad vector ablating coxsackievirus and Ad receptor (CAR) (the first receptor) binding, {alpha}v integrin (the secondary receptor) binding, and heparan sulfate glycosaminoglycan (HSG) (the third receptor) binding reduced (or blunted) liver toxicity and IL-6 production (22). However, these two Ad vectors mediate significantly lower tissue transduction due to steric hindrance by PEG chains and a loss of binding activity to the receptor, respectively (20, 21, 22). An Ad vector showing efficient transduction and reduced innate immune response has not yet been developed.

In the present study, we elucidate the molecular mechanism of the innate immune response by the Ad vector and characterize the safer Ad vector, which reduces the innate immune response and liver toxicity. We found that the fiber-modified Ad vector containing a stretch of lysine residues (K7 (KKKKKKK) peptide) (23, 24, 25) that target heparan sulfates on the cellular surface greatly reduced IL-6 and liver toxicity after i.v. injection into mice compared with the conventional Ad vector. IL-6 and the other immune cytokines, chemokines, and IFNs were mainly produced from the spleen and especially from conventional DC (CD11c+B220– cells), not the liver. The spleen distribution of the K7-modified Ad vector was reduced compared with the conventional Ad vector. The K7-modified Ad vector decreased the liver toxicity (aspartate aminotransferase (AST) levels), at least in part due to the reduced serum IL-6 levels. Importantly, this K7-modified Ad vector maintained high transduction efficiency in vivo and showed somewhat higher transgene production in the liver than a conventional Ad vector.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Ad vector

Two luciferase-expressing Ad vectors, Ad-L2 and AdK7-L2, have been constructed previously (25, 26). The CMV promoter-driven luciferase gene derived from the pGL3-Control was inserted into the E1 deletion region of the Ad genome. Ad-L2 contains wild-type fiber, whereas AdK7-L2 contains the polylysine peptide KKKKKKK in the C-terminal of the fiber knob (25). Viruses (Ad-L2 and AdK7-L2) were prepared as described previously (25) and purified by CsCl2 step gradient ultracentrifugation. Determination of virus particle titers was accomplished spectrophotometrically by the method of Maizel et al. (27).

Ad-mediated transduction in vivo

Ad-L2 or AdK7-L2 were i.v. administered to C57BL6 mice (1.0 x 1010 virus particles (VP)) (6-wk-old males obtained from Nippon SLC). Forty-eight hours later, the heart, lung, liver, kidney, and spleen were isolated and homogenated as previously described (28). Luciferase production was determined using a luciferase assay system (PicaGene 5500; Toyo Inki). Protein content was measured with a Bio-Rad assay kit using BSA as a standard.

The amounts of Ad genomic DNA in the each organ were quantified with the TaqMan fluorogenic detection system (ABI Prism 7700 sequence detector; PerkinElmer Applied Biosystems). Samples were prepared with DNA templates isolated from each organ (25 ng) by an automatic nucleic acid isolation system (NA-2000; Kurabo Industries). The amounts of Ad DNA were quantified with the TaqMan fluorogenic detection system (PerkinElmer Applied Biosystems) as described in our previous report (22).

To analyze the involvement of IL-6 signaling in liver toxicity in response to Ad vector administration, 100 µg per mouse of an anti-IL-6R Ab (clone D7715A7; BioLegend) that specifically blocks IL-6 signaling was i.p. administered to C57BL6 mice 1.5 h before Ad-L2 administration (3.0 x 1010 VP). Rabbit IgG (clone R3-34; BD Biosciences) was administered as a control. Serum samples and liver tissue were collected 48 h later, and AST levels in the serum and luciferase production in the liver were determined.

Liver serum enzymes and cytokine levels after systemic administration

Blood samples were collected by the inferior vena cave at the indicated times (3 or 48 h) after i.v. administration of Ad-L2 or AdK7-L2 (3.0 x 1010 and 1.0 x 1011 VP, respectively). IL-6 and IL-12 levels in serum samples collected at 3 h after Ad injection were measured by an ELISA kit (BioSource International). The levels of AST in serum samples collected at 24 and 48 h were measured with the Transaminase-CII kit (Wako Pure Chemical). Forty-eight hours after the Ad vector injection, the mice were killed and their livers were collected. The liver was washed, fixed in 10% formalin, and embedded in paraffin. After sectioning, the tissue was dewaxed in ethanol, rehydrated, and stained with H&E. This process was commissioned to the Applied Medical Research Laboratory (Osaka, Japan).

Cytokines and chemokines mRNA levels in tissue after systemic administration

Total tissue RNA samples were isolated by the reagent ISOGEN (Wako Pure Chemical) 3 h after the i.v. administration of Ad-L2 or AdK7-L2 (1.0 x 1011 VP). Reverse transcription was performed using the SuperScript first-strand synthesis system for first-strand cDNA synthesis (Invitrogen Life Technologies) according to the instructions of the manufacturer. IL-6 and IL-12 mRNA in the liver and spleen were quantified with the TaqMan fluorogenic detection system (PerkinElmer Applied Biosystems). Semiquantified RT-PCR analysis was also performed to determine mRNA levels of the cytokines, chemokines, and IFNs (total eight mRNA). The primer sequences and probes were as follows: IL-6 forward, 5'-GAG GAT ACC ACT CCC AAC AGA CC-3'; IL-6 reverse, 5'-AAG TGC ATC ATC GTT GTT CAT ACA-3' (reverse); IL-6 probe, 5'-CAG AAT TGC CAT TGC ACA ACT CTT TTC TCA-3'; IL-12p40 forward, 5'-GGA AGC ACG GCA GCA GAA TA-3'; IL-12p40 reverse, 5'-AAC TTG AGG GAG AAG TAG GAA TGG-3'; IL-12p40 probe, 5'-CAT CAT CAA ACC AGA CCC GCC CAA-3'; TNF-{alpha} forward, 5'-CCT GTA GCC CAC GTC GTA GC-3'; TNF-{alpha} reverse, 5'-TTG ACC TCA GCG CTG AGT TG-3'; RANTES forward, 5'-ATG AAG ATC TCT GCA GCT GCC CTC ACC-3'; RANTES reverse, 5'-CTA GCT CAT CTC CAA ATA GTT GAT G-3'; MIP-2 forward, 5'-ACC TGC CGG CTC CTC AGT GCT GC-3'; MIP-2 reverse, 5'-GGC TTC AGG GTC AAG GCA AAC-3'; IFN-{alpha} forward, 5'-AGG CTC AAG CCA TCC CTG T-3'; IFN-{alpha} reverse, 5'-AGG CAC AGG GGC TGT CTT TCT TCT-3'; IFN-beta forward, 5'-TTC CTG CTG TGC TTC TCC AC-3'; IFN-beta reverse, 5'-GAT TCA CTA CCA GTC CCA GAG TC-3'; IFN-{gamma} forward, 5'-GAG GAT ACC ACT CCC AAC AGA CC-3'; IFN-{gamma} reverse, 5'-AAG TGC ATC ATC GTT GTT CAT ACA-3'; GAPDH forward, 5'-TTC ACC ACC ATG GAG AAG GC-3'; and GAPDH reverse, 5'-GGC ATG GAC TGT GGT CAT GA-3'. The expected sizes of the PCR products are as follows: IL-6, 193 bp; IL-12p40, 155 bp; TNF-{alpha}, 374 bp; RANTES, 252 bp; MIP-2, 221 bp; IFN{alpha}, 272 bp; IFNbeta, 607 bp; IFN-{gamma}, 306 bp; and GAPDH, 237 bp.

Cell sorting of splenic cells

Splenic conventional DC, plasmacytoid DC, and B cells, which were CD11c+B220–, CD11c+B220+, and CD11c–B220+ cells, respectively, were sorted by FACS Aria (BD Biosciences). Total RNA samples were isolated from each cell by the reagent ISOGEN, and RT-PCR analysis was then performed as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
This study was undertaken to elucidate the biological mechanism in the innate immune host responses toward i.v. administered Ad vector. The relationship between the innate immune response and liver toxicity by systemic administration of the Ad vectors was also examined.

Gene transduction and Ad vector accumulation in vivo

In this study we used the conventional Ad vector (Ad-L2) and a fiber-modified Ad vector containing a polylysine (K7) peptide (AdK7-L2), both of which express luciferase under the control of the CMV promoter. First, we examined luciferase production in the organ and the biodistribution of viral DNA after i.v. administration of AdK7-L2 (1.0 x 1010 VP) into mice compared with Ad-L2 (see Fig. 3). The vector dose of 1.0 x 1010 VP was selected because this dose did not induce any apparent toxicity (IL-6 and AST production) with either Ad-L2 or AdK7-L2. When a higher dose (3.0 x 1010 or 1.0 x 1011 VP) was used, only Ad-L2 and not AdK7-L2 showed toxicity (described later), which does not reflect an exact comparison of the transduction efficiency. The Ad type 5-based vector delivers the foreign gene predominantly in the liver after i.v. injection into mice (29, 30). Interestingly, AdK7-L2 mediated ~6-fold higher liver transduction than Ad-L2 (Fig. 1A). In contrast, the luciferase production in the heart, lung, kidney, and spleen in response to AdK7-L2 was similar to that in response to Ad-L2. To examine the biodistribution of Ad-L2 and AdK7-L2 in mice, the amounts of Ad DNA in each organ 48 h after the injection of Ad vectors were measured with the TaqMan fluorogenic detection system. More AdK7-L2 DNA accumulated in the liver than Ad-L2 DNA (Fig. 1B), although the amounts of AdK7-L2 DNA in the heart, lung, kidney, and spleen were less than those of Ad-L2 DNA. In particular, the amounts of AdK7-L2 DNA in the spleen were ~56-fold less than those of Ad-L2 DNA. The data regarding luciferase production (Fig. 1A) and the amounts of Ad DNA in most organs (Fig. 1B) showed discrepancies. Luciferase production in the liver was >2 log order higher than that in other organs, while the amounts of Ad DNA in liver were not as striking among the organs compared with luciferase production. This difference is likely due to the difference in the amount of nonspecific viral uptake among the organs. Reduced spleen accumulation of AdK7-L2 DNA, compared with Ad-L2 DNA, was also observed at a dose of 1.0 x 1011 VP (data not shown).


Figure 3
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  		FIGURE 3. Cytokine, chemokine, and IFN mRNA levels in liver and spleen after the systemic administration of Ad-L2 or AdK7-L2 into mice. Total mRNA samples were isolated from liver and spleen at 3 h after i.v. administration of Ad-L2 or AdK7-L2 (1.0 x 1011 VP). After the reverse transcriptase reaction, IL-6 and IL-12 cDNA were measured with the quantitative TaqMan PCR assay (A). The expression of TNF-{alpha}, RANTES, MIP-2, IFN-{alpha}, IFN-beta, and IFN-{gamma} was measured by semiquantitative RT-PCR assay (B). All data represent the means ± SD of four mice. Cycle number is given in parentheses.

 

Figure 1
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  		FIGURE 1. Luciferase production and biodistribution of viral DNA after the i.v. administration of Ad-L2 or AdK7-L2 into mice. Ad-L2 or AdK7-L2 (1.0 x 1010 VP) was i.v. injected into the mice. Forty-eight hours later, the heart, lung, liver, kidney, and spleen were harvested, and luciferase production (A) and Ad vector DNA (B) in each organ were measured by a luciferase assay system or the quantitative TaqMan PCR assay, respectively. All data represent the means ± SD of 4–6 mice.

 
Serum cytokines and AST levels

The systemic administration of Ad vectors results in the initiation of strong innate immune responses and inflammation in animals and humans (1), and this toxicity limits the utility of Ad vectors for gene therapy. To evaluate the innate immune response and liver toxicity of each Ad vector, we measured the levels of IL-6, IL-12, and AST in serum. Because IL-6 in the serum and hepatic toxicity analysis was detected at a dose of >1.0 x 1011 or 3.0 x 1010 VP, respectively, these doses were used.

IL-6 levels in response to AdK7-L2 were one-fourth of those with Ad-L2 (Fig. 2A). In contrast, there was no difference in serum IL-12 levels between Ad-L2 and AdK7-L2. Thus, IL-6 and IL-12 appear to be produced by a different mechanism. TNF-{alpha} in the serum after the injection of Ad-L2 or AdK7-L2 could not be detected (data not shown). Ad-L2 led to high levels of serum AST at 48 h after injection, while AdK7-L2 did not induce AST (Fig. 2B). At 24 h, neither Ad-L2 nor AdK7-L2 induced AST. In histological analysis, degranulation or denucleation occurred in hepatocytes from Ad-L2, while AdK7-L2 did not induce hepatocyte toxicity (Fig. 2C). The results using AdK7-L2 were similar to those in the untreated mice (Fig. 2, B and C), suggesting that AdK7-L2 does not show any liver toxicity. These results suggest that AdK7-L2 shows less IL-6 production and almost no liver toxicity.


Figure 2
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  		FIGURE 2. Cytokines and liver enzyme levels in serum after the systemic administration of Ad-L2 or AdK7-L2 into mice. Blood samples were collected by inferior vena cave at 3 h (A) or 24 and 48 h (B) after i.v. administration of Ad-L2 or AdK7-L2 (1.0 x 1011 VP for A or 3.0 x 1010 VP for B). The livers were collected after 48 h following the injection (3.0 x 1010 VP) (C). A, IL-6 and IL-12 levels in the serum were measured by ELISA. B, AST levels in the serum were measured using a Transaminase-CII kit. C, Paraffin sections of the livers were prepared. Each section was stained with H & E. Data represent the means ± SD of four mice.

 
Cytokines mRNA levels in liver and spleen cells

Ad vectors induce the expression of various cytokines and chemokines in the innate immune responses by effector cells such as macrophages and DC (15, 17, 31, 32, 33). Liver and spleen are two major organs responsible for the location of immune cells. We attempted to determine which organ (liver or spleen) produces cytokines, chemokines, and IFNs (IL-6, IL-12, TNF-{alpha}, RANTES, MIP-2, IFN{alpha}, IFNbeta, and IFN-{gamma}) by quantitative real-time RT-PCR or semiquantitative RT-PCR analysis. IL-6 and IL-12 mRNA levels were not induced in the liver after i.v. administration of Ad vectors (Fig. 3A). This result was also checked by the result that specific IL-6 and IL-12 mRNA bands were not detected in the liver by RT-PCR analysis (data not shown). Expression of TNF-{alpha}, RANTES, MIP-2, IFN-{alpha}, IFN-beta, and IFN-{gamma} mRNA was also detected mainly in the spleen, not the liver (Fig. 3B). IL-6, MIP-2, and IFN-{gamma} mRNA levels in the spleen in response to AdK7-L2 were lower than those in response to Ad-L2. In the liver, TNF-{alpha}, RANTES, MIP-2, and IFN-{gamma} mRNA were detected by a high cycle number of PCR after Ad (Ad-L2 or AdK7-L2) injection, whereas IFN-{alpha} and IFN-beta could be not detected (Fig. 3B).

We next identified the cell types responsible for the IL-6 and IL-12 expression in the spleen after i.v. administration of the Ad vector (Ad-L2). Spleen cells were sorted by FACS Aria based on the expression of CD11c and B220 in conventional DC (CD11c+B220–), plasmacytoid DC (CD11c+B220+), and B cells (CD11c–B220+ cells). IL-6 and IL-12 mRNA were mainly detected in the splenic conventional DC. Only a faint band of IL-12 mRNA was also detected in the splenic plasmacytoid DC (CD11c+B220+) (Fig. 4). These results suggest that splenic conventional DC are major effector cells of innate immune response (at least IL-6 and IL-12 production) against systemically administered Ad vectors.


Figure 4
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  		FIGURE 4. IL-6 and IL-12 mRNA levels in splenic CD11c-positive cells after the systemic administration of Ad-L2 into mice. Total mRNA samples were isolated from sorted splenic cells 3 h after i.v. administration of Ad-L2 (1.0 x 1011 VP). The expression levels of IL-6 and IL-12 mRNA were measured by RT-PCR assay. Lane 1, B cell (B220+CD11c–); lane 2, conventional DC (B220–CD11c+); lane 3, plasmacytoid DC (B220+ CD11c+); lane 4, other cells (B220–CD11c–). Cycle number is given in parentheses.

 
Elimination of IL-6 signaling reduces liver toxicity

It has previously been shown that TNF-{alpha} is likely to be involved in host responses to Ad vectors in vitro and in vivo (34). Recently, Shayakhmetov et al. (35) have reported that IL-1 signaling, not TNF-{alpha} signaling, is involved in Ad vector-associated liver toxicity after i.v. administration. However, the mechanism of liver toxicity after i.v. Ad administration is poorly understood. In the present study, although AdK7-L2 mediated higher luciferase expression and a higher accumulation of viral DNA in the liver than Ad-L2, it remains unclear why AdK7-L2 showed almost background levels of liver toxicity while Ad-L2 showed high toxicity. As reported previously, inflammatory cytokines, chemokines, and IFNs could be the mediators responsible for liver toxicity (2). IL-6 levels in the serum were the most strikingly different between AdK7-L2 and Ad-L2. Furthermore, IL-6 stimulated acute phase protein (serum amyloid A, fibrinogen, {alpha}1-anti-trypsin, and {alpha}1-acid glycoprotein) in rat and human hepatocytes (36, 37). Therefore, we next examined the effects of serum IL-6 on liver toxicity (Fig. 5). To do this, we used an anti-IL-6R Ab that inhibits the signal through the IL-6 receptor. The IL-6 receptor system consists of two functional molecules, an 80-kDa ligand-binding chain (IL-6R) and a 130-kDa nonligand-binding but signal-transducing chain (gp130). The anti-IL-6R Ab blocks the binding of IL-6 to the IL-6R (38, 39). The anti-IL-6R Ab or the control Ab was i.p. injected 1.5 h before the injection of Ad-L2. The AST levels in the serum and luciferase production in the liver were determined 48 h later. Administration of anti-IL-6R Ab significantly (~2-fold) reduced Ad vector-mediated AST levels in the serum compared with PBS or the control Ab (Fig. 5A). Importantly, anti-IL-6R Ab injection did not interfere with luciferase production in the liver (Fig. 5B). These results suggest that IL-6 signaling is involved in liver toxicity after i.v. administration of an Ad vector.


Figure 5
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  		FIGURE 5. Effects of serum IL-6 on serum AST levels and liver luciferase production after the systemic administration of Ad-L2 into mice. C57BL/6 mice were i.p. administered 100 µg per mouse of anti-IL-6R Ab (clone D7715A7), which was specific for blocking IL-6 signaling, or rabbit IgG as a control (clone; R3-34). Ad-L2 or AdK7-L2 (3.0 x 1010 VP) was i.v. injected into the mice 1.5 h later. Blood samples and liver tissue were collected 48 h after the injection of Ad-L2. The AST levels in the serum were measured using a Transaminase-CII kit. Luciferase production in the liver was measured by a luciferase assay system. All data represent the means ± SD of three to four mice. *, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we found that the fiber-modified Ad vector containing the K7 peptide, which has high affinity with heparin sulfate, shows much lower serum IL-6 and liver toxicity than the conventional Ad vector. This improved characteristic is likely involved with the reduced biodistribution of the vector to the spleen compared with that of the conventional Ad vector. RT-PCR analysis showed that the spleen, not the liver, is the major site of cytokine, chemokine, and IFN (IL-6, IL-12, TNF-{alpha}, RANTES, MIP-2, IFN-{alpha}, IFN-beta, and IFN-{gamma}) production and that splenic conventional DC are the major effector cells of the innate immune response (at least IL-6 and IL-12 production) after i.v. administration of Ad vectors. We also showed that IL-6 signaling is involved in part with liver toxicity in response to Ad vectors. Importantly, this fiber-modified Ad vector containing the K7 peptide maintained higher transduction efficiency in all the organs examined, and the liver transduction was higher than that of the conventional Ad vector. Although there have been some reports that modified Ad vectors such as the pegylated Ad vector (18, 19, 20, 21), the Ad vector containing the Ad type 35 fiber shaft and knob (40), and the triple mutant Ad vector with ablation of CAR, {alpha}v integrin, and HSG binding (22) show decreased innate immune response and liver toxicity, these types of vector lose their transduction activity in vivo. To our knowledge, this is the first report of an Ad vector that maintains high transduction efficiency in vivo with reduced toxicity.

The fiber-modified Ad vector containing the K7 peptide has been developed to overcome the limitations imposed by the CAR dependence of Ad infection. Expanded and efficient gene transfer has been reported based on the use of mutant fiber proteins containing a stretch of lysine residues (23, 24, 25). However, there has been no report on the difference in gene transfer activity and toxicity in vivo between the conventional Ad vector and the fiber-modified Ad vector containing the K7 peptide. We have demonstrated that the fiber-modified Ad vector containing the K7 peptide mediates ~6-fold higher mouse liver transduction in response to i.v. administration than the conventional Ad vector (Fig. 1A). The amounts of fiber-modified Ad vector DNA in the liver after i.v. administration were also 5-fold higher than those with the conventional Ad vector (Fig. 1B). It has been reported that the interaction between the Ad type 5 fiber and the HSG of a hepatocyte is involved in the accumulation in the mouse liver and the cynomolgus monkey liver of systemically administered Ad vectors (41, 42). This fiber-modified Ad vector might mediate more efficient gene transduction through a much higher affinity for HSG. In contrast, the amounts of fiber-modified Ad vector DNA in the spleen after i.v. administration were 56-fold lower than those of the conventional Ad vector (Fig. 1B). Biodistribution of viral DNA reflects the total of receptor-mediated uptake and nonspecific uptake. Luciferase production in the cells mainly reflects receptor-mediated uptake. We previously reported that most Ad DNAs are taken up in the liver nonparenchymal cells, not parenchymal cells, after i.v. administration (22). In this study, the conventional Ad vector would also be taken up in the macrophages and DC by nonspecific uptake, resulting in significantly higher Ad DNA and lower luciferase production in the spleen. In contrast, the fiber-modified Ad vector would be taken up more in the liver via receptor-mediated uptake and nonspecific uptake, resulting in significantly lower Ad DNA in the other organs, especially the spleen. Even though the amount of AdK7-L2 uptake in the spleen, heart, lung, and kidney was less than that of Ad-L2 uptake, the amount of receptor-mediated uptake in these organs would be similar between Ad-L2 and AdK7-L2, suggesting that these vectors showed similar levels of luciferase production in the organs other than the liver.

The initiation of inflammatory innate immune responses occurs after the systemic administration of Ad vectors to animals and humans, and this toxicity limits the utility of Ad vectors for gene therapy. Increased cytokine/chemokine production after the injection of Ad vectors has been reported to be due to the introduction of input Ad vectors to Kupffer cells in the liver and DC (15, 17, 43, 44, 45, 46). Detailed analysis of the organs responsible for the expression of cytokines, chemokines, and IFNs by RT-PCR suggests that their production can mainly be attributed to spleen cells (especially splenic conventional DC), not liver cells (Figs. 3 and 4), which is consistent with the recent report of Bart et al. (47). Therefore, interference with spleen distribution of the Ad vector should provide a useful method for safer gene therapy.

TLRs, which are crucial to the recognition of pathogen-associated molecular patterns, are expressed on various types of immune cells including macrophages, DC, B cells, splenic types of T cells, and even on nonimmune cells such as fibroblasts and epithelial cells (48). For example, HSV and CMV (dsDNA virus) activate inflammatory cytokines and type I IFN secretion by the stimulation of TLR9 (49, 50, 51, 52, 53). The innate immune receptor to the Ad has not yet been identified. It has not even been determined whether TLRs are involved in Ad-mediated innate immune response in vivo, although it has been reported that TLR signals are not involved in the DC maturation induced by the Ad vector (46). As shown in Fig. 3B, cytokine production against the Ad vector occurred mainly in conventional DC. It is noted that the TLR9-mediated innate immunity responses to DNA virus are cell type-specific and limited to plasmacytoid DC (50). The unidentified sensor receptor(s) for double-stranded Ad DNA or Ad capsid protein in conventional DC might play a critical role in the expression of inflammatory cytokines/chemokines and type I IFN. Although we have previously reported that large amounts of conventional Ad vector accumulate in nonparenchymal cells, including Kupffer cells and liver sinusoidal (endothelial) cells (22, 54), the expression of mRNA of cytokines, chemokines, and IFNs in the liver was weak after administration of the Ad vector (Fig. 3B). A lack of putative sensor receptor(s) against Ad or the inability of sensor receptor(s) to recognize Ad due to the specific cellular disposition of Ad in Kupffer cells might result in a reduced production of cytokines/chemokines/IFNs in the liver.

Another interesting finding is that the fiber-modified Ad vector containing the K7 peptide showed almost background levels of AST activity, which reflects liver toxicity (Fig. 2B). Histological analysis supported this finding (Fig. 2C). Because the K7-modified Ad vector showed higher transgene activity and a higher accumulation of viral DNA into the liver (Fig. 1), the transduction and distribution of the vector into the liver did not participate in liver toxicity. The cytokines/chemokines play a major causative role in liver damage associated with systemic Ad infusion as well as in the induction of an antiviral immune response (2). Ad-induced cytokines/chemokines recruit immune effector cells (neutrophils, monocyte/macrophages, and NK cells) to Ad-transduced cells (mainly liver), resulting in acute hepatic toxicity. Shayakhmetov et al. (35) have reported that hepatocytes and Kupffer cells trigger IL-1 transcription in liver tissue after i.v. administration of Ad vectors and that interference of IL-1-signaling reduces liver toxicity. We speculated that IL-6 could be the main mediator for hepatic toxicity because IL-6 is one of the main cytokines in the early stages of inflammation, IL-6 production by the fiber-modified Ad vector was much reduced (approximately a quarter) compared with that by the conventional Ad vector, and all of the cytokines/chemokines/IFNs we examined (including IL-6) were mainly produced by the spleen, not the liver. Treatment of the anti-IL-6R Ab decreased liver toxicity (Fig. 5), suggesting that IL-6 plays at least some role in liver toxicity induced by systemic injection of the Ad vector. Because the AST levels were only partially reduced by the treatment with the anti-IL-6R Ab, another mechanism such as IL-1 signaling, rapid Kupffer cell death (55, 56), activation of the liver endothelium (55), or other factors might be involved in the liver toxicity. Nevertheless, it is attractive that the K7-modified Ad vector did not show liver toxicity despite the higher transduction efficiency and higher accumulation of the vector into the liver (probably Kupffer cells).

Our present study provides new insight into the cellular biological mechanism related to the innate immune response and liver toxicity against the systemically administered Ad vector. Modification of vector tropism should contribute to safe gene therapy procedures.


   Acknowledgments
 
We thank Misae Nishijima and Haiying Huang for their technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Ministry of Health, Labor, and Welfare of Japan. Back

2 Address correspondence and reprint requests to Dr. Hiroyuki Mizuguchi, Laboratory of Gene Transfer and Regulation, National Institute of Biomedical Innovation, Asagi 7-6-8, Saito, Ibaraki, Osaka 567-0085, Japan. E-mail address: mizuguch{at}nibio.go.jp Back

3 Abbreviations used in this paper: Ad, adenovirus; AST, aspartate aminotransferase; CAR, coxsackievirus and Ad receptor; DC, dendritic cell; HSG, heparan sulfate glycosaminoglycan; PEG, polyethylene glycol; VP, virus particle. Back

Received for publication August 29, 2006. Accepted for publication November 10, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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