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Adenovirus serotype 35 vector-mediated transduction following direct administration into organs of nonhuman primates

Abstract

Adenovirus (Ad) serotype 35 (Ad35) vectors have attracted remarkable attention as alternatives to conventional Ad serotype 5 (Ad5) vectors. In a previous study, we showed that intravenously administered Ad35 vectors exhibited a safer profile than Ad5 vectors in cynomolgus monkeys, which ubiquitously express CD46, an Ad35 receptor, in a pattern similar to that in humans. However, the Ad35 vectors poorly transduced the organs. In this study, we examined the transduction properties of Ad35 vectors after local administration into organs of cynomolgus monkeys. The vectors transduced different types of cells depending on the organ. Hepatocytes and microglia were mainly transduced after the vectors were injected into the liver and cerebrum, respectively. Injection of the vectors into the femoral muscle resulted in the transduction of cells that appeared to be fibroblasts and/or macrophages. Conjunctival epithelial cells showed transgene expression following infusion into the vitreous body of the eyeball. Transgene expression was limited to areas around the injection points in most of the organs. In contrast, Ad35 vector-mediated transgene expression was not detected in any of the organs not injected with Ad35 vectors. These results suggest that Ad35 vectors are suitable for gene delivery by direct administration to organs.

Main

Adenoviruses (Ads) are nonenveloped, double-stranded DNA viruses with icosahedral symmetry. To date, 51 human adenovirus (Ad) serotypes have been identified and classified into six species.1, 2 Among these serotypes, Ad serotype 5 (Ad5), which belongs to species C, is the basis of almost all the Ad vectors commonly used, including those used in clinical trials. Conventional Ad5 vectors have several advantages as gene delivery vehicles. However, it is now well established that the hurdles to Ad5 vector-mediated gene therapy are the high seroprevalence to Ad5 in adults and the refractoriness of cells lacking the expression of coxsackievirus-adenovirus receptor, which is a primary receptor for Ad5, to Ad5 vectors. Pre-existing anti-Ad5 immunity significantly decreases the transduction efficiencies of Ad5 vectors. Even when an Ad5 vector-based vaccine was administered locally into muscle, pre-existing anti-Ad5 antibodies reduced its efficacy.3, 4 A lack of coxsackievirus-adenovirus receptor expression renders the cells unsusceptible to Ad5 vectors at least in vitro. Important target cells for gene therapy, including hematopoietic stem cells and dendritic cells, often poorly express coxsackievirus-adenovirus receptor. In addition to these drawbacks, Ad5 vectors have high hepatic tropism. Even when Ad5 vectors are locally injected into a diseased area (for example, a tumor), they are drained from the injection sites into the systemic circulation and primarily transduce hepatocytes because of their high hepatic tropism; on the other hand, efficient transduction is obtained around the injection points. When Ad vectors carry a transgene that exerts cytotoxic effects on transduced cells, Ad vector-mediated hepatic transduction leads to severe hepatotoxicity.5, 6, 7

In contrast, human species B Ad serotype 35 (Ad35) vectors, which our group and several others have developed,8, 9, 10, 11 possess attractive properties that can overcome the drawbacks of conventional Ad5 vectors. First, Ad35 vector-mediated transduction is not hampered by anti-Ad5 antibodies, because Ad35 belongs to a different species (species B) than Ad5 (species C). Second, Ad35 vectors bind to human CD46 as a receptor. Human CD46 is expressed on almost all human cells, leading to broad tropism of Ad35 vectors in human cells, including coxsackievirus-adenovirus receptor-negative cells.8, 12 However, intravenous administration of Ad35 vectors resulted in inefficient transduction in the organs of human CD46-transgenic (CD46TG) mice and cynomolgus monkeys, which express CD46 in a pattern similar to that of humans.13, 14, 15 These results indicate that CD46 does not successfully serve as a receptor for intravascularly injected Ad35 vectors and that Ad35 vectors are unsuitable for intravascular transduction. However, this property of Ad35 vectors would suggest a potential advantage, in that unwanted transduction would not occur in organs other than the organs targeted following direct injection of Ad35 vectors when draining from injected sites into the bloodstream. These properties suggest that Ad35 vectors would be suitable for gene transfer by local administration into the organs. In this study, we examined the transduction properties of Ad35 vectors following intraorgan administration in nonhuman primates, that is, cynomologus monkeys.

A previously constructed Ad35 vector expressing β-galactosidase (Ad35LacZ)15 was locally administered at a dose of 1.5 × 1011 vector particles (VP) per point (high dose) or 3 × 1010 VP per point (low dose) in the following eight organs of two cynomolgus monkeys (designated no. 8 and no. 9; no. 8 received the high dose of Ad35LacZ and no. 9 received the low dose): liver, cerebrum, eyeball (vitreous body), quadriceps femoris muscle, pancreas, kidney, spleen and nasal cavity. Four days after administration, the tissues around the injection sites (approximately 40 × 40 × 10 mm3 with a central focus at the injection point) were collected and subjected to an analysis of β-galactosidase expression and histological pathology. The health condition of the monkeys was also monitored until necropsy.

Overall, both monkeys did well during the experiment. There were no apparent abnormalities in body temperature or heart rate, although no. 8, the high-dose monkey, exhibited slight reductions in blood pressure and body weight. Both monkeys apparently exhibited increased serum levels of aspartate aminotransferase and creatine phosphokinase on days 0–2 after injection. Mild decreases in hemoglobin levels and increases in levels of lactate dehydrogenase and C-reactive protein were also found in both animals. However, these changes were probably due to the operation. The levels of alanine aminotransferase, alkaline phosphatase, albumin, glucose, calcium, chloride and sodium in the serum were mostly within the normal ranges.

After the direct injection of the Ad35 vectors, the transduction profiles were assessed by immunostaining of β-galactosidase in the tissue sections; Table 1 summarizes the results. A detailed transduction profile in each organ is described below.

Table 1 β-galactosidase expression in the organs following direct injection of Ad35LacZ into organs

Liver

Direct injection of Ad35LacZ to the liver caused tissue damage around the injection site (Figures 1a and b). Infiltration of inflammatory cells, necrotic focus and regenerated bile duct epithelial cells were observed. Immunostaining of the liver sections revealed that hepatocytes were mainly transduced with Ad35LacZ in both no. 8 and no. 9 monkeys (Figures 2a and b). A higher level of β-galactosidase was expressed in the liver of no. 8 than in that of no. 9. The transduced cells were predominantly distributed around the injection point (approximately 1 × 1 mm2) and were not found outside the periphery of the injection site. β-galactosidase was not expressed in the liver lobes, which were not injected with Ad35LacZ. β-galactosidase-expressing cells were mainly found on the border region between the normal and damaged areas. Direct injection of naked plasmid DNA or Ad5 vectors into mouse liver also resulted in the localized distribution of transgene-expressing cells around the injection points.16, 17 The liver would not allow dispersion of locally injected Ad vectors in the tissue.

Figure 1
figure1

Tissue histology in the organs of cynomolgus monkeys 4 days after intraorgan injection of Ad35LacZ. (a and b) The liver, (c and d) cerebrum, (e and f) eyeball, (g and h) skeletal muscle, (i and j) pancreas, (k and l) kidney and (m and n) spleen. Young male cynomolgus monkeys (Macaca fascicularis) were housed and handled in accordance with the rules for animal care and management of the Tsukuba Primate Center and with the guiding principles for animal experiments using nonhuman primates formulated by the Primate Society of Japan. The animals (approximately 3 years of age, 1.9 and 2.2 kg) were certified free of intestinal parasites and seronegative for simian type-D retrovirus, herpesvirus B, varicella-zoster-like virus and measles virus. The protocol of the experimental procedures was approved by the Animal Welfare and Animal Care Committee of the National Institute of Biomedical Innovation (Osaka, Japan). The liver, cerebrum, eyeball, nasal cavity, pancreas, kidney, skeletal muscle and spleen of cynomolgus monkeys were each injected with Ad35LacZ suspended in 200 μl (100 μl for eyeball) of phosphate-buffered saline at a dose of 1.5 × 1011 vector particles (VP) per point (monkey no. 8) or 3 × 1010 VP per point (monkey no. 9). Four days after injection, tissue sections were hematoxylin–eosin stained by a routine method. Dotted-line circles in (b) and (c) indicate the necrotic area in the liver and the softening area in the cerebrum, respectively.

Figure 2
figure2

β-galactosidase expression in the organs of cynomolgus monkeys 4 days after intraorgan injection of Ad35LacZ. (a and b) The liver, (c and d) cerebrum, (e and f) eyeball, (g and h) skeletal muscle, (i and j) pancreas, (k and l) kidney and (m and n) spleen. Ad35LacZ was locally administered in the organs of cynomolgus monkeys at the low (3 × 1010 vector particles (VP) per point) or high dose (1.5 × 1011 VP per points) as described in Figure 1. Four days after injection, the tissues were collected for analysis of β-galactosidase expression and histological pathology. Immunostaining of β-galactosidase was performed using anti-β-galactosidase antibody (Abcam, Cambridge, UK).

Cerebrum

Ad35LacZ was stereotaxically injected into the left frontal lobe of the cerebrum. After infusion of the high dose of Ad35LacZ, softening of the tissue, which appeared necrotic, was widely observed in the left basal ganglia (Figure 1c). Neutrophils were infiltrated into the necrotic area. In contrast, injection of a low dose of Ad35LacZ resulted in no apparent toxicity, although slight bleeding was found around the artery (Figure 1d). Transduced cells, which appeared to be microglia, were found around the softening regions of both no. 8 and no. 9 animals, although the latter had fewer transduced microglia (Figures 2c and d). There were no β-galactosidase-expressing cells in the right hemisphere of the brain, which was infused with phosphate-buffered saline buffer (data not shown).

Eye

Ad35LacZ was infused into the vitreous body for inoculation into the eyeball. The high dose induced invasion by inflammatory cells, including macrophages and neutrophils, into the ciliary body, iris and retina (Figure 1e). Necrotic changes were also found in all layers of the retina. The low dose caused similar damage to the eyeball. The high dose mediated transduction in the conjunctival epithelial cells (Figure 2e). β-galactosidase expression was not observed in other areas. After injection into the vitreous body, Ad35LacZ might be drained from it and transduce the conjunctival epithelial cells. Bora et al.18 demonstrated that human CD46 was hardly expressed in eye tissues, suggesting that these tissues are refractory to Ad35 vectors. We did not find β-galactosidase expression in the eye of no. 9 animal. Phosphate-buffered saline injection did not result in transgene expression or apparent abnormality in the eyeball (data not shown).

Femoral muscle

Severe inflammation did not occur after intramuscular injection of the high dose, although we found slight damage to the muscle fibers (Figure 1g). In contrast, the low dose induced more severe inflammation (Figure 1h). Infiltration of neutrophils and macrophages was seen in the muscle of no. 9. It is currently unclear why the low dose induced higher levels of damage. A slight difference in the injection point might affect Ad35 vector-induced inflammatory responses in the muscle. β-galactosidase expression was found only in the cells that appeared to be macrophages and/or fibroblasts located among the muscle fibers in both monkeys (Figures 2g and h). No muscle fibers expressed β-galactosidase in either monkey. It remains to be elucidated why intramuscular injection of Ad35 vectors mediated poor transduction in muscle fibers of cynomolgus monkeys. Ad35 vectors transduced the muscle following intramuscular injection in wild-type mice and in CD46TG mice.12, 14 The transduction mechanism and efficiencies of Ad35 vectors in muscle fibers might differ among species, and the muscle of nonhuman primates might be more refractory to transduction than that of rodents. Thirion et al.19 demonstrated that Ad vectors would transduce human, rat and mouse primary muscle cells through different pathways. Danko et al.20 reported that transgene expression levels by intramuscular injection of naked DNA were lower in dogs and nonhuman primates than in rodents. On the other hand, several studies demonstrated the utility of Ad35 vectors as vaccine vectors that express antigen by intramuscular administration in mice and nonhuman primates.3, 4 Macrophages and/or dendritic cells transduced with Ad35 vectors might play important roles in transgene-specific immune responses by intramuscular injection of Ad35 vectors.

Pancreas

Injection into the pancreas caused no severe damage to that organ in either monkey (Figures 1i and j). We did not find transduced cells in the pancreas of no. 8; in contrast, β-galactosidase was apparently expressed in exocrine acinar cells of no. 9 in the pancreatic lobules (Figures 2i and j). Chemiluminescence assay of β-galactosidase also revealed significant levels of β-galactosidase expression in the pancreas of no. 9 but not in that of no. 8 (data not shown). Wang et al.21 also demonstrated that direct injection of conventional Ad vectors and adenoassociated virus vectors into murine pancreas achieved efficient transduction in acinar cells. Pancreatic acinar cells would be susceptible to Ad vectors.

Kidney

Ad35LacZ injection to the left kidney induced infiltration by inflammatory cells, including lymphocytes, into the interstitial tissue of the kidney (Figures 1k and l). The right kidney, which was injected with phosphate-buffered saline, did not exhibit β-galactosidase expression or inflammatory responses (data not shown). The high dose did not mediate β-galactosidase expression, but the low dose led to apparent transduction (Figures 2k and l). The renal tubular epithelial cells were mainly transduced with Ad35LacZ. In the kidney, compared with the other organs, transduced cells were more widely spread around the injection points. Refractoriness to the high dose and massive β-galactosidase expression by the low dose in the pancreas and kidney together form a major conundrum in this study. The differences in transduction efficiencies might be due to the slight differences in injection sites. Especially, Ad35LacZ may have been drained into the renal tubule of no. 9 following injection into the kidney, leading to efficient transduction in the renal tubule epithelial cells. Ad35 was originally identified in the kidney and causes cystitis,22 indicating the tropism of Ad35 for renal epithelial cells.

Spleen and nasal cavity

Unexpectedly, direct injection of Ad35LacZ to the spleen did not induce inflammatory responses such as hyperplasia (Figures 1m and n). There was no β-galactosidase expression in the spleen of either monkey (Figures 2m and n). For transduction in the mucosal membrane of the nasal cavity, Ad35 vector suspensions were instilled into the nasal cavity of each monkey, but neither one showed β-galactosidase expression or cellular damage in the mucosal membrane of the nasal cavity (data not shown).

Other organs

β-galactosidase production in the lung, heart, thymus, bone marrow, lymph node, bladder and testis, which were not injected with Ad35LacZ, were examined by chemiluminescence assay. None of these organs showed detectable β-galactosidase expression (data not shown).

Next, we determined the blood concentrations of Ad35LacZ genome DNA in the blood using quantitative real-time PCR to examine whether or not Ad35LacZ locally injected to the organs was drained from the injection site into the bloodstream. The Ad35 vector DNA was detected in the blood as soon as 6 h post-injection, then gradually decreased (Figure 3). However, the blood-clearance kinetics of Ad35LacZ following intraorgan injection were slower than those following intravenous administration, which were previously reported,23 although the total amounts of Ad35 vector doses in this study (no. 8: 1.5 × 1011 VP × 8 points; no. 9: 3 × 1010 VP × 8 points) were comparable to or lower than those in the previous study in which Ad35LacZ was intravenously infused in cynomolgus monkeys (0.4–2 × 1012 VP per kg, 1.88–2.96 kg).23 Ad35 vector DNA was still detectable 4 days after injection. These results suggest that Ad35 vectors or Ad35 vector DNA remaining in the injection sites might be gradually released from the injection sites and drained into the bloodstream.

Figure 3
figure3

Blood concentrations of Ad35 vectors in cynomolgus monkeys following intraorgan administration. Ad35LacZ was locally administered in the organs of cynomolgus monkeys at the low (3 × 1010 vector particles (VP) per point, closed square) or high dose (1.5 × 1011 VP per points, open triangle) as described in Figure 1. Blood was collected at the indicated post-injection time points (6, 24, 48, 72 and 96 h post-injection). Total DNA, including Ad vector DNA, was isolated from the blood, and the Ad vector DNA contents were measured by quantitative TaqMan PCR assay, as previously described.23

Furthermore, to examine whether or not Ad35LacZ draining into the bloodstream was accumulated in the organs, we determined the Ad35 DNA contents in the portions of the liver and spleen that were away from the respective injection sites. The liver and spleen play crucial roles in the clearance of systemically injected Ad vectors. The Ad35 vector DNA was not detected in those portions of the liver in no. 9, but was detected in the portions of the liver in no. 8 and in those of the spleen in both monkeys (data not shown). These results suggest that Ad35LacZ or the Ad35 vector DNA draining into the systemic circulation would be taken up by the liver and spleen. We further assessed the Ad35 DNA contents in the lungs, heart, thymus and bone marrow, in which Ad35 vectors were not directly infused. Ad35 vector DNA was detected in the lungs and heart of no. 8 but not in those of no. 9 (data not shown). We did not detect Ad35 vector DNA in the thymus or bone marrow of either monkey. Considering that intravascularly injected Ad35 vectors did not efficiently transduce organs,15 organs must not be transduced with Ad35LacZ, which is drained into the bloodstream and taken up by the organs.

In most cases of cancer gene therapy using Ad vectors, the vectors are administered directly to the tumor regions.24, 25, 26 When used as vaccine vectors, on the other hand, Ad vectors are intramuscularly injected.27, 28 In addition, Ad vectors are intramyocardially injected in angiogenic gene therapy.29, 30 Thus, direct infusion of Ad vectors to organs is one of the most frequent application methods in clinical settings. However, there has been little information about the transduction properties of these vectors following direct injection into organs. This study demonstrated that different types of cells were transduced with Ad35 vectors depending on the organ after direct infusion into the organ. The differences in the histological structures and cell types comprising the organs would explain the differences in transduction properties of locally injected Ad35 vectors. This study provides important information for clinical study by intraorgan injection of Ad35 vectors, although the characteristics of the organs (structure, cell types and so on) differ different between normal tissue and diseased areas.

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Acknowledgements

We thank Fumiko Ono and Chieko Ohno (The Corporation for Production and Research of Laboratory Primates, Ibaraki, Japan) for their help. This study was supported by grants from the Ministry of Health, Labour, and Welfare of Japan and by a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.

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Sakurai, F., Nakamura, Si., Akitomo, K. et al. Adenovirus serotype 35 vector-mediated transduction following direct administration into organs of nonhuman primates. Gene Ther 16, 297–302 (2009). https://doi.org/10.1038/gt.2008.154

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Keywords

  • adenovirus serotype 35 vector
  • local administration
  • nonhuman primate
  • CD46

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