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Nonhuman adenoviruses including bovine adenovirus serotype 3 (BAd3) and porcine adenovirus serotype 3 (PAd3) can circumvent pre-existing immunity against human adenovirus serotype 5 (HAd5) and are being developed as alternative vectors for gene delivery. To assess the usefulness of these vectors for in vivo gene delivery, we compared biodistribution, persistence, state of vector genome, and transgene and vector genes expression by replication-defective BAd3 and PAd3 vectors with those of HAd5 vector in a FVB/n mouse model following intravenous inoculation. BAd3 vector efficiently transduced the heart, kidney and lung in addition to the liver and spleen and persisted for a longer duration compared to PAd3 or HAd5 vectors. Biodistribution of PAd3 vector was comparable to that of HAd5 vector but showed more rapid vector clearance. Only linear episomal forms of BAd3, PAd3, and HAd5 vector genomes were detected. All three vectors efficiently expressed the green fluorescent protein (GFP) transgene proportionate to the vector genome copy number in various tissues. Furthermore, leaky expression of vector genes, both the early (E4) and the late (hexon) was observed in all three vectors and gradually declined with time. These results suggest that BAd3 and PAd3 vectors could serve as alternative or supplement to HAd5 for gene delivery applications.
Adenoviral (Ad) vectors have demonstrated great promise as therapeutic and prophylactic gene delivery systems (McConnell and Imperiale, 2004; Russell, 2000). Numerous advantages offered by Ad vectors include the ease of large scale production, ease of genetic manipulation, the lack of genomic integration, relatively nonpathogenic nature and their ability to transduce both dividing and non-dividing cells. Vectors based on human adenovirus (HAd) serotype 5 (HAd5) and HAd serotype 2 (HAd2) are currently most widely studied (Edelstein et al., 2004; Tatsis and Ertl, 2004). Attachment of HAd5 and HAd2 to a susceptible cell is mediated by the high-affinity binding of the Ad fiber knob to a primary receptor, coxsackievirus and Ad receptor (CAR) (Bergelson et al., 1997), followed by a secondary interaction of the penton base with integrins resulting in virus internalization into the cell (Wickham et al., 1993). This feature of Ad frequently either limits the clinical use of these vectors to primarily CAR expressing cells or poses challenge in targeting of specific tissues as a consequence of promiscuous tropism of Ad vector owing to wide distribution of CAR in a variety of cell types. Preferential sequestration of systemically administered Ad vectors to liver (hepatotropism) also leads to inefficient transduction of target organs other than liver and at high vector doses, poses serious adverse effects (Raper et al., 2003). Furthermore, due to the endemic nature of HAd5 and HAd2, pre-existing vector immunity may potentially inhibit the levels and duration of transgene expression following inoculation with an Ad vector. Vector-associated toxicity and induction of strong innate immunity are some of the other potential concerns for these vectors.
In order to expand the repertoire of Ad vectors, vectors based on less prevalent HAd serotypes such as HAd3, HAd11, and HAd35 or nonhuman adenoviruses such as bovine adenovirus type 3 (BAd3), porcine adenovirus type 3 (PAd3), ovine adenovirus, canine adenovirus, simian adenoviruses, and fowl adenovirus, are being investigated as alternative or supplement to HAd5 vectors (Bangari and Mittal, 2006b; Farina et al., 2001; Hofmann et al., 1999; Kanerva et al., 2002; Kremer et al., 2000; Michou et al., 1999; Reddy et al., 1999a; Reddy et al., 1999b; Seshidhar Reddy et al., 2003; Stone et al., 2005). Vectors based on nonhuman Ad or chimeric HAd vectors carrying the knob domain or entire fiber from nonhuman Ad have been developed that demonstrated novel and expanded tropism, in addition to evading HAd specific immunity (Bangari and Mittal, 2006a; Glasgow et al., 2004; Singh et al., 2008).
We have demonstrated that nonhuman Ad vectors based on PAd3 and BAd3 efficiently transduce several types of human and murine cells in culture (Bangari et al., 2005b). These vectors appeared to utilize distinct receptors for cell internalization (Bangari and Mittal, 2005; Bangari et al., 2005a) and cell entry was independent of CAR. Most importantly, we also showed that there were no pre-existing virus neutralizing antibodies against PAd3 or BAd3 in humans, and HAd5-neutralizing antibodies in human or raised in a rabbit or mice did not cross-neutralize PAd3 or BAd3 (Bangari et al., 2005b; Moffatt et al., 2000).
In the present study, we assessed the usefulness of replication-defective PAd3 and BAd3 vectors carrying the green fluorescent protein (GFP) gene as a reporter for in vivo gene delivery. Biodistribution, persistence, the state of the vector genome, transgene and vector genes expression of these nonhuman Ad vectors were compared with those of HAd5 vector in FVB/n mouse model following intravenous inoculation. The choice of the mouse strain was based on the fact that we have developed an immunocompetent FVB/n mouse model for breast cancer to investigate the role of EphA2 receptor tyrosine kinase in tumorigenesis (Noblitt et al., 2005). Interestingly, the genome copy numbers of BAd3 vector in the heart, kidney, lung and spleen were significantly (P < 0.05) higher than those of PAd3 and HAd5 vectors. BAd3 vector also persisted longer in all tissues examined compared to the other two vectors. The majority of vector genome was detected in the linear form for all three vectors. In addition, expression levels of the viral early region gene 4 (E4) and the late structural gene (hexon) for all three vectors were similar, indicating leaky vector gene expression and paralleled (reflected) vector biodistribution. The results of this study demonstrate that BAd3 and PAd3 vectors have the potential for in vivo gene delivery applications, as supplements or alternatives to the currently used HAd vectors.
The liver and spleen are the main target organs for E1-deleted Ad vector biodistribution following intravenous delivery (Ni et al., 2005; Stone et al., 2007) and the number of vector genomes decline drastically with time (Yang et al., 1994a). To investigate whether nonhuman Ad vectors based on PAd3 and BAd3 have significantly different biodistribution and persistence from that of HAd5, FVB/n mice were inoculated intravenously with 1010 vector particles (VP) of HAd-GFP (HAd5 with deleted E1 and the GFP transgene under the cytomegalovirus (CMV) promoter) (Bangari and Mittal, 2004), PAd-GFP (PAd3 with deleted E1A and the GFP transgene under CMV promoter) (Bangari and Mittal, 2004) or BAd-GFP (BAd3 with deleted E1A and the GFP transgene under CMV promoter) (Bangari et al., 2005b). At 0.25, 0.5, 1, 2, 4, 8, and 16 days post-inoculation, the mice were sacrificed and the liver, spleen, lung, kidney and heart were collected. Total cellular DNA extracted from various tissue samples were analyzed for quantification of vector genomes by real-time PCR specific to the GFP transgene as it was present in all three vectors. The sensitivity of this assay was 3 copies of the vector genome per 50 ng of total cellular DNA. The number of vector genomes and persistence of BAd-GFP in all tissues were significantly (P < 0.05) higher than those of either PAd-GFP or HAd-GFP except in the liver at 0.25 and 1 days post-inoculation where the levels were somewhat comparable in all three vector-inoculated mice (Fig. 1). It is interesting to note that especially in the lung, heart and kidney, significantly high levels of BAd-GFP genomes persisted even at 16 days post-inoculation (the last time point examined in the present study). In general, vector genome levels in PAd-GFP-transduced tissues were at comparable levels at earlier time points and significantly lower (P < 0.05) or even below the detection levels at later time points compared to those of HAd-GFP-inoculated tissues except the heart where the levels were comparable or even higher at some later time points (Fig. 1). No vector genome was detected in the mock-inoculated mice at any time point (data not shown).
The HAd5 vector genome usually persists in episomal form within the host cell nucleus (Ehrhardt et al., 2008; Ehrhardt et al., 2003). However, the vector genome may also get integrated into the chromosomal DNA at a very low frequency (10−3 to 10−5) (Harui et al., 1999). In order to address the safety aspect of PAd3 and BAd3 vectors, it was important to determine the state of their genomes in vivo. To assess the state of vector DNA in the liver of HAd-GFP, PAd-GFP or BAd-GFP inoculated mice, samples of total cellular DNA extracted from the livers collected at various time points post-inoculation were digested with KpnI (for HAd-GFP), XhoI (for PAd-GFP) or HpaI (for BAd) and subjected to Southern blot analysis. The probes for Southern blot assay were designed taking into consideration that they differentiate the linear episomal, circular episomal and integrated forms of HAd-GFP, PAd-GFP or BAd-GFP genome based on the fragment size of restriction endonuclease (RE)-digested DNA. The sensitivity of the Southern blot assay, as determined by the amount of vector genome used as a positive control, was found to be 10 pg of DNA. The expected fragment sizes for three forms of DNA (linear episomal, circular episomal, and integrated) were 2.3, 3.4, and >2.3 kb for HAd-GFP, 3, 5, and >3 kb for PAd-GFP, and 2, 6.8, and >2 kb for BAd-GFP (Fig. 2A). In HAd-GFP, PAd-GFP or BAd-GFP-inoculated mice, 2.3, 3, or 2 kb specific bands were detected up to 2, 1, or 8 days, respectively, suggesting that predominantly linear forms of the vector genomes were present in HAd-GFP, PAd-GFP, or BAd-GFP inoculated mice (Fig. 2B). Fragments of DNA that would correspond to circular or integrated form of the vector genomes were not observed even after overexposure of the Southern blots (data not shown). No signals were detected in samples from mock-inoculated mice (Fig. 2B). The presence of circular form of genomes was also investigated by PCR using specific primer sets for each vector, but none of the tissue samples yielded positive results (data not shown). Sensitivity of the PCR assay for detection of circular form of HAd5 genome, as determined by using serial dilutions of a plasmid (pDC-311; Microbix Biosystems, Toronto, ON, Canada) that contained HAd5 end fragments joint together, was 2.5 pg or 7 × 105 copies. However, the Southern blot was 25-fold more sensitive as compared to the PCR assay. The suboptimal sensitivity of PCR could be due to the presence of two long inverted terminal repeats and the GC-rich nature of the template resulting in complex secondary structures. Nevertheless, the results of Southern blot analyses and PCR indicated that the majority of HAd-GFP, PAd-GFP or BAd-GFP genomes remained as linear episomes.
In vivo efficacy of any gene delivery vector largely depends on the level and duration of transgene expression. In order to further substantiate that the persistence of significantly high levels of BAd-GFP genome in multiple organs actually has a positive impact on transgene expression levels, the total cellular RNA from various tissues of mice inoculated with HAd-GFP, PAd-GFP, or BAd-GFP were quantitated for GFP-specific mRNA transcript levels by real-time RT-PCR. Levels of transgene expression in BAd-GFP in the lung, heart and kidney were significantly (P < 0.05) higher than those of either with PAd-GFP or HAd-GFP (Fig. 3). However, transgene expression in the liver and spleen of BAd-GFP-inoculated mice were marginally better only at later time points. In the lung, heart and kidney from BAd-GFP inoculated mice, there was significantly (P < 0.05) higher levels of transgene expression that continued at least until 16 days post-inoculation, consistent with our vector biodistribution data (Fig. 1). In general, transgene expression levels in various tissues from PAd-GFP-inoculated mice were comparable to those from HAd-GFP-inoculated mice at early time points but were significantly lower (P < 0.05) or below detection levels at later time points except in the heart where the levels were comparable with both vectors (Fig. 3). No GFP expression was detected in mock-inoculated mice at any time point (data not shown). In order to detect the GFP protein in tissue sections, immunohistochemistry was performed. Positive cytoplasmic staining was observed in hepatocytes of HAd-GFP, PAd-GFP or BAd-GFP inoculated mice sacrificed at 0.25 and 0.5 day post-inoculation (Fig. 4) while no staining was detected in PBS-inoculated mice or controls that did not receive the anti-GFP antibody treatment. GFP expression by immunohistochemistry was also observed in other tissues (data not shown).
We were also able to detect substantial levels of anti-GFP ELISA antibodies (1 : 5120) in the serum samples of HAd-GFP, PAd-GFP or BAd-GFP inoculated mice at 16 day post-inoculation (Table 2). Similar levels of neutralizing antibodies against HAd5, PAd3, or BAd3 vector were also observed in serum samples collected at 16 days post-inoculation (Table 2) suggesting that various adenoviral vectors were equally capable of inducing vector-specific immunity.
Expression of vector genes at lower levels (“leaky gene expression”) has been reported in E1-deleted replication defective vectors (Elkon et al., 1997; Kafri et al., 1998; Yang et al., 1994b). Paradoxically, minimal levels of vector gene expression is better for vector survival or persistence as it helps to shield the vector-transduced cells from the host immune surveillance (Russell, 2000; Yang et al., 1994b). To investigate whether vector gene expression by nonhuman Ad vectors (BAd-GFP and PAd-GFP) would be comparable with those of HAd-GFP, the total cellular RNA from various tissues of mice infected with HAd-GFP, PAd-GFP, or BAd-GFP were quantitated for E4 or hexon mRNA transcript levels by real-time RT-PCR. We selected E4 (HAd5 E4 34 kDa open reading frame (orf), PAd3 E4 orf8 and BAd3 E4 orf3) as a representative of an early gene, and hexon as a representative of a late gene. BAd-GFP E4 expression levels in the lung, heart and kidney was significantly higher (P < 0.05) compared to levels obtained with PAd-GFP or HAd-GFP (Fig. 5). Whereas, in the liver and spleen, comparable E4 expression levels were observed with BAd-GFP or HAd-GFP. PAd-GFP E4 expression was comparable to that of HAd-GFP E4 in most tissues at least at early time points (Fig. 5). Similarly, BAd-GFP hexon expression levels in the lung, heart and kidney was significantly higher (P < 0.05) compared to hexon mRNA levels obtained with PAd-GFP or HAd-GFP (Fig. 6). However, in the liver and spleen, comparable levels of hexon mRNA expression were observed with BAd-GFP or HAd-GFP. The expression levels of hexon mRNA of PAd-GFP were comparable to those of HAd-GFP E4 in most tissues at least at early time points (Fig. 6). No expression of either E4 or hexon was observed in mock-inoculated mice (data not shown). Overall, it seemed that levels of the early and the late gene expression by HAd-GFP, PAd-GFP, or BAd-GFP were comparable taking into consideration the number of vector genomes in different tissues at various time points post-inoculation.
HAd vectors have demonstrated tremendous potential as a gene delivery vehicle for vaccine and gene therapy applications. However, the prevalence of pre-existing vector immunity in the majority of human population and their predominant hepatotropism may limit their utility. With the anticipation that nonhuman Ad vectors may circumvent these limitations, we have developed PAd3 and BAd3 vectors (Bangari and Mittal, 2004; Mittal et al., 1995b). In this study, we evaluated their biodistribution, persistence, state of vector genome, and transgene and vector genes expression to further explore the potential of these nonhuman Ad vectors for gene delivery.
In our study, biodistribution patterns of HAd-GFP in the liver, spleen, heart, lung and kidney were similar to the previous findings (Alemany and Curiel, 2001; Ni et al., 2005; Stone et al., 2007; Wood et al., 1999; Zinn et al., 1998). PAd-GFP localized predominantly in the liver and spleen similar to HAd-GFP. BAd-GFP showed distinct tropism as indicated by significantly (P < 0.05) higher levels of vector localization to the heart, kidney and lung compared to PAd3 and HAd5 vectors, whereas levels of BAd-GFP vector genome in the liver and spleen were at levels comparable to those of PAd-GFP and HAd-GFP at least at earlier time points. The distinct tissue tropism may partly be due to utilization of different receptor(s) by BAd3 (Bangari et al., 2005a). PAd3 and BAd3 vectors internalization is CAR-independent and there are differences in the transduction efficiency of various cell lines by these vectors compared to HAd5 (Bangari and Mittal, 2005; Bangari et al., 2005a; Bangari et al., 2005b). Recombinant fiber knob of HAd5, PAd3 or BAd3 inhibited cell entry of only homologous virus suggesting that these three viruses use distinct receptors for internalization (Bangari et al., 2005a). In addition to CAR, integrins play an important role as co-receptor in HAd5 entry and hence are likely to be a determinant in vector biodistribution. Integrin-binding motifs such as Arg-Gly-Asp (RGD) and Leu-Asp-Val (LDV) are also absent in the penton base of BAd3 and PAd3 (Reddy et al., 1998) and we have demonstrated earlier that BAd3 or PAd3 internalization was independent of αvβ3 or αvβ5 integrins (Bangari et al., 2005a).
Despite advancements in the understanding of Ad interactions with host cell receptors, the exact mechanism of tissue tropism remained unclear. Various hypotheses have been put forward to explain the predominant liver tropism exhibited by many Ad vectors. Some of the non-CAR binding Ad vectors have also been demonstrated to predominately localize to the liver, suggesting that factors other than the fiber knob-CAR interaction are also involved in mediating hepatotropism (Alemany and Curiel, 2001; Smith et al., 2002) CAR-independent uptake of CAR-binding Ad vectors by kupffer cells or hepatocytes has been shown (Shayakhmetov et al., 2004). Involvement of certain blood factors (such as coagulation factor IX, and complement components) has been proposed to explain Ad vector hepatotropism. These factors cross-link Ad vectors with heparan sulphate proteoglycans (HSPGs) on hepatocytes and thus leading to preferential hepatotropism. Therefore, modification of certain solvent-exposed loops in the fiber knob domain of the Ad vector has resulted in reduced vector uptake in the liver (Shayakhmetov et al., 2005). Recent studies have demonstrated that HAd5 uptake in the liver is mediated by binding of coagulation factor X (FX) to hexon (Kalyuzhniy et al., 2008; Waddington et al., 2008). However, there were significant variations among HAd serotypes to bind to FX, that correlated with their ability to transduce hepatocytes. Studies investigating interaction of FX and/or other blood factors with PAd3 or BAd3 capsid proteins would further clarify their role in tropism and biodistribution of these vectors. HSPG binding motif-KKTK in the fiber shaft of HAd5 has also been proposed to drive hepatotropism as deletion of this motif led to significant decline in hepatic tissue transduction (Nicol et al., 2004; Smith et al., 2003a; Smith et al., 2003b). Furthermore, the size and structure of fiber also determine tissue tropism. Ad vectors with short fibers, like HAd35 and HAd40, escape hepatic sequestration in contrast to vector with large fibers (Nakamura et al., 2003; Shayakhmetov et al., 2004). However, a chimeric HAd5 vector with ovine Ad-derived long shafted fiber demonstrated CAR-independence with no particular preference to the liver (Nakayama et al., 2006). Nicklin et al. also suggested that the Ad fiber, rather than knob-receptor interaction is a major determinant of hepatotropism (Nicklin et al., 2005). BAd3 fiber is unique in having a very long shaft with few kinks in its secondary structure and is devoid of KKTK motif (Ruigrok et al., 1994). Though the knob domain has been suggested to be the primary receptor-seeking moiety of most Mastadenovirus species (Nicklin et al., 2005), there is a possibility that fiber shaft may also play important role in BAd3 or PAd3 internalization.
It has been demonstrated that at low doses, Ad vectors were predominately sequestered by Kupffer cells resulting in suboptimal transduction of other cell types (Tao et al., 2001). However, the role of sequestration of Ad vectors by Kupffer cells on vector biodistribution can be evaluated effectively only at low vector doses. Since there was no information available on biodistribution of BAd3 and PAd3 vectors compared to HAd5, we chose a comparatively lower vector dose (1010 VP/mouse) for this study. Our results suggest that sequestration of BAd3 vector by Kupffer cells did not significantly influenced vector biodistribution. Subsequent studies with higher vector doses should further clarify the influence of reticuloendothelial system on transduction efficiencies of these vectors.
BAd-GFP persisted at significantly (P < 0.05) higher levels in all tissues even at 16 days post-inoculation compared to PAd-GFP or HAd-GFP. It is probably due to better survival of BAd-GFP genome in various tissues because of slow degradation and/or delayed removal of cells transduced with the vector genome. PAd-GFP had a similar biodistribution pattern as HAd-GFP but was present at lower levels in all tissues and its persistence in various tissues was for a shorter duration compared to HAd-GFP or BAd-GFP. Whether it is due to paucity of PAd3 receptors in FVB/n mouse tissues is not clear. Experiments to investigate the induction of a host innate and adaptive immune response following intravenous administration of these vectors are underway.
Because integration of the vector genome may lead to insertional mutagenesis, we examined the state of the vector genome in the liver of mice inoculated with HAd-GFP, PAd-GFP, or BAd-GFP. Vector genome could be present within the cell nucleus as a linear episomal form, circular episomal form, or integrated into the host genome (Ehrhardt et al., 2008). It has been reported that the majority of HAd5 genome in transduced cells persists in a episomal form (Ehrhardt et al., 2003). However, a fraction of the vector genome can also get integrated into the host chromosomal DNA (Harui et al., 1999). In addition, the genomes of some HAd serotypes such as HAd12 frequently integrate into the host cell genome mostly by random integration (Green et al., 1977; Knoblauch et al., 1996). Circularized vector genome may also replicate to some extent on its own leading to its enhanced persistence (Kreppel and Kochanek, 2004). In this study, only the linear episomal form of HAd-GFP, PAd-GFP, or BAd-GFP genome was detected suggesting that the majority of nonhuman Ad genome remained as linear episome similar to the HAd-GFP genome. BAd-GFP genome as identified by Southern blot assay persisted at higher levels up to 8-day-post-inoculation, further corroborating our results of vector biodistribution and persistence. Additional in vitro experiments are needed to further explore the state of nonhuman Ad vector genome.
Efficient gene delivery and robust expression of transgene is one of the attributes of first generation HAd5 vectors. It was expected from our vector biodistribution and persistence study that GFP transgene expression by BAd-GFP should be better than that of HAd-GFP or PAd-GFP. Comparable transgene expression, proportionate to the levels of vector genome present in the tissue, was observed. Significantly higher (P < 0.05) levels of transgene expression were detected in the heart, kidney and lung from BAd-GFP-inoculated mice and similar levels in the liver and spleen as compared to levels obtained with HAd-GFP.
Proteins encoded by the E1 region are required for the transactivation of other vector early and late genes (Russell, 2000), therefore, the first generation E1-deleted Ad vectors are replication defective. However, leaky expression of the early and late genes of such vectors has been reported even in the absence of E1 proteins expression (Elkon et al., 1997; Kafri et al., 1998; Yang et al., 1994b). The leaky vector gene expression is strong enough to induce vector-specific cytotoxic T lymphocyte response leading to the removal of vector- transduced cells. Unlike HAd-GFP in which the entire E1 region was deleted, only E1A regions were deleted in PAd-GFP and BAd-GFP (Bangari and Mittal, 2004; Bangari et al., 2005a). Partial deletion in the E1 region of PAd-GFP and BAd-GFP was found to be sufficient to render these vectors replication defective (Bangari and Mittal, 2004; Reddy et al., 1999a; van Olphen et al., 2002). In the present study, expression of both vector E4 (early) and hexon (late) genes was observed by all the three vectors at levels that correspond to the amount of vector present in various tissues.
Our results in FVB/n mouse model have strongly suggested that BAd3-based vectors have considerable potential as gene delivery vector for vaccine and gene therapy. Our recent study involving a BAd-vector based vaccine in BALB/c mice resulted in significantly higher levels of humoral and cell-mediated immune responses compared to the HAd5-based vaccine (Singh et al., 2008) suggesting that the results obtained with FVB/n mouse model are applicable to another mouse strain. The results presented here have prepared the foundation for additional studies (with higher vector dose and for longer duration post-inoculation) for evaluating vector toxicity, and innate and adaptive immune responses following systemic delivery of nonhuman Ad vectors.
Replication-defective HAd-GFP (Bangari and Mittal, 2004), PAd-GFP (Bangari and Mittal, 2004) and BAd-GFP (Bangari et al., 2005b) vectors with deletions in E1 or E1A region and carrying the GFP gene under the control of the cytomegalovirus (CMV) promoter were propagated as described previously. HAd-GFP, PAd-GFP and BAd-GFP vectors were grown and titrated in 293 (human embryonic kidney cells expressing Ad E1) (Graham et al., 1977), FPRT HE1 (fetal porcine retina cells expressing Ad E1) (Bangari and Mittal, 2004) and FBRT HE1 (fetal bovine retina cells expressing Ad E1) (van Olphen et al., 2002), respectively. The virus purification was done by cesium chloride-density gradient centrifugation as previously described (Bangari and Mittal, 2004). The physical particle counts of purified HAd-GFP, PAd-GFP and BAd-GFP vectors were estimated by spectrophotometry and expressed as vector particles (VP) per ml following a previously described protocol (Graham and Prevec, 1995). The VP/plaque forming units (p.f.u) ratio for HAd-GFP, PAd-GFP and BAd-GFP were 72, 100 and 608 respectively. Since plaque assays for these vectors were carried out in different cell lines as discernible plaques are not formed by all three vectors in a single cell type, efficiency of plaques formation may also vary with the virus and cell type combination. Therefore, VP was selected as the major criteria for vector quantification to maintain consistency with vector dosage.
Eight-to-ten-week-old female FVB/n mice were obtained from The Jackson Laboratory (Bar Harbor, ME). FVB/n mice were selected for the current study because of the availability of an immunocompetent tumor model using this strain (Addison et al., 1995; Noblitt et al., 2005). The use of this strain of mice would allow us to extend our study for investigation of HAd5, PAd3 and BAd3 vectors in cancer gene therapy. All animal inoculations were conducted in accordance with the guidelines and approval from Institutional Biosafety Committee and Institutional Animal Care and Use Committee. Mice were inoculated intravenously through tail vein with HAd-GFP, PAd-GFP, or BAd-GFP at a dose of 1010 VP per mouse in a volume of 100 µl of PBS++ (Phosphate buffer saline supplemented with 0.01 % MgCl2 and 0.01 % CaCl2). Mock-inoculated mice served as negative controls. Mice (3 animals per group) were sacrificed at various time points (0.25, 0.5, 1, 2, 4, 8 and 16 days) post-inoculation. The liver, spleen, lung, heart and kidney were collected and stored at −80°C. These tissue samples were used for total cellular DNA or RNA extraction for various assays. Serum samples were also collected for the analysis of Ad-specific neutralizing antibody.
A set of primer pair and a Taqman® probe specific to the GFP gene in HAd-GFP, PAd-GFP, or BAd-GFP were designed using Primer Express 2.0 software (Applied Biosystems, Foster city, CA). These primers and probe were used for determining the biodistribution of Ad vectors by quantification of vector genomes by real-time PCR, and analyzing transgene expression by determining expression levels of GFP mRNA by real-time RT-PCR.
Similarly, sets of primer pairs and Taqman® probes specific to the hexon gene or an E4 gene (HAd5 E4 34 kDa orf, PAd3 E4 orf 8 and BAd3 E4 orf 3) of each vector were designed for quantification of their hexon or E4 mRNA transcripts by real-time RT-PCR. All primer sets and Taqman® probes were synthesized by Applied Biosystems. The sequences of various primers and Taqman® probes are in Table 1. All probes were labeled with 6-carboxyfluorescein (FAM) at the 5' end and with minor groove binder (MGB) at the 3' end.
Total genomic DNA from 50 mg of tissue samples was isolated using DNeasy kit (Qiagen Inc., Valencia, CA) as per the manufacturer’s guidelines. Initially, standard curves were generated using 10-fold dilutions (from 3 to 3 × 108 copies) of purified genomic DNA of HAd-GFP, PAd-GFP, or BAd-GFP as a template for real-time PCR using GFP primers and probe. The copy number of each vector genome was calculated based on spectrophotometric quantification and molecular mass of vector genomic DNA. Subsequently, these dilutions of each vector were run as standards with each set of real-time PCR assay. DNA samples from mock-inoculated mice tissues served as negative controls. For quantification of the vector genome by real-time PCR using the GFP primers and probe, 50 ng of the total cellular DNA was used in a 25 µl reaction using Taqman® PCR core reagents (Applied Biosystems). The reaction mixture contained 10 × Taqman® buffer, 250 nM each of forward and reverse primers, and 100 nM of Taqman® probe along with other standard kit components. Each reaction was carried out in duplicate. The real-time PCR was performed using the Mx3000 Thermocycler (Stratagene, Cedar Creek, TX). The reaction conditions included incubation at 50°C for 2 min, followed by polymerase activation (95°C for 10 min), and 45 cycles of denaturation (95°C for 15 sec) and annealing/extension (60°C for 1 min). The threshold cycle (Ct) value for individual reactions was determined and data were analyzed with MxPro software (Stratagene) to obtain the absolute copy number of viral genome per 50 ng of total cellular DNA.
Total cellular RNA was isolated from 50 mg of each tissue sample using RNA miniprep kit (Stratagene). RNA samples were treated with DNase I to remove the residual DNA. Two hundred ng of total cellular RNA was processed for real-time RT-PCR using GFP-, E4-, or hexon-specific primers and probe and One-step Brilliant QRT-PCR Master Mix Kit (Stratagene). For normalization of the target gene expression, similar real-time RT-PCR reactions targeting the endogenous 18S rRNA were simultaneously carried out in separate tubes. Quantification of expression levels of GFP, E4, or hexon mRNA transcripts in various tissue samples was done by ΔΔCt method (Winer et al., 1999) and expressed in relation to the mean expression levels of respective genes observed by HAd-GFP at 0.25 day post inoculation (referred to as calibrator). Fold difference in expression levels in relation to calibrator was calculated as 2−ΔΔCt (where ΔΔCt = [Cttarget gene (unknown) − Ct18S rRNA (unknown)] − [Ct target gene (calibrator) − Ct18S rRNA (calibrator)], Ct is the cycle number at which fluorescence signal crosses the threshold). For calculation of arbitrary units, the mean expression levels of mRNA transcripts in HAd-GFP-inoculated mice tissues at 6 h post-inoculation were considered as 100 units. Reaction mixture consisted of 2 × QRT-PCR master mix, 250 nM each of respective forward and reverse primers, and 100 nM of Taqman® probe along with other standard kit components. Each reaction was carried out in duplicate. The real-time RT-PCR was performed using the Mx3000 Thermocycler. The reaction conditions included cDNA synthesis step at 50°C for 30 min, followed by polymerase activation (95°C for10 min), and 45 cycles of denaturation (95°C for 15 sec) and annealing/extension (60°C for 1 min). The Ct values for individual reactions were determined and data were analyzed with MxPro software to obtain the relative expression levels of GFP, E4, or hexon mRNA transcripts. All RNA samples were also used for PCR assay using GFP, E4, or hexon primers and probe to ensure the absence of the residual DNA.
Total cellular DNA from the livers of mice inoculated with HAd-GFP, PAd-GFP, BAd-GFP, or PBS was isolated by proteinase-K digestion and isopropanol precipitation as per previously described method (Sambrook and Russell, 2001). Twenty µg of the total cellular DNA was digested with KpnI (for HAd-GFP inoculated mice), XhoI (for PAd-GFP inoculated mice) or HpaI (for BAd-GFP inoculated mice) and loaded on to a 1% agarose gel. DNA from the liver of mock-inoculated mice and purified vector DNA were digested similarly and used as negative and positive controls, respectively. After agarose gel electrophoresis, the separated DNA fragments were transferred on to a positively charged nylon membrane (Hybond-N+, GE Healthcare Ltd., Little Chalfront, UK) by upward capillary transfer method (Sambrook and Russell, 2001). Following the transfer the DNA fragments were cross-linked to the membrane by baking at 80°C for 2 h. DNA hybridization was performed using HAd-GFP (nt 34,524–35,537), PAd-GFP (nt 32,353–33,582) or BAd-GFP (nt 32,520–33,615) specific probes which were chosen specifically from the area near the right terminus of the vector genome to aid in identification of the linear, circular, or integrated form of the vector genome. The probes were prepared by PCR amplification and radiolabeled using [alpha32P]dCTP (MP Biomedicals, Solon, OH) by random priming (DECAprime II kit, Applied Biosystems). The probes were column purified (NucAway spin columns, Applied Biosystems.) and used for DNA hybridization at 65°C overnight in Danhardt’s buffer (Sambrook and Russell, 2001). The membrane was washed twice each with 2 × SSC (0.3M sodium chloride, 0.03M sodium citrate), 0.1% SDS (Sodium dodecyl sulphate); 1 × SSC, 0.1% SDS and 0.1 × SSC, 0.1% SDS, wrapped with saran wrap and exposed to Cyclone Storage Phosphor Screen (Packard Instrument Company, Meridin, CT). After 2 h of exposure, signals were visualized by Cyclone Storage Phosphor System (Packard Instrument Company).
Formalin-fixed and paraffin-embedded tissues were deparaffinized in xylene and rehydrated using graded dilutions of ethanol according to standard immunohistochemistry procedures. The slides were then immersed in target retrieval solution (DakoCytomation, Carpinteria, CA) at 95°C for 15 min to retrieve antigens. Endogenous peroxidase activity was quenched by immersing slides in 3% hydrogen peroxide solution for 5 min. Endogenous biotin and avidin were blocked by incubating sections in avidin/biotin blocking reagents (Vector Laboratories, Burlingame, CA) for 15 min. The sections were then incubated in blocking solution (MOM immunodetection kit, Vector Laboratories) according to manufacturer’s instruction. The sections were then incubated with a monoclonal anti-GFP antibody (Millipore Corporation, Billerica, MA) at 1:500 dilution for 30 min at room temperature followed by a biotinylated anti-mouse secondary antibody (Vector Laboratories) for 10 min. This step was followed by incubation with streptavidine-horseradish peroxidase conjugate (DakoCytomation) for 30 min. Signal amplification was performed by Tyramide Signal Amplification Kit (PerkinElmer, Waltham, MA). The color development was performed by Aminoethyl Carbazole (Red) Substrate Kit (Zymed Laboratories Inc., San Francisco, CA). The sections were thoroughly rinsed with tris-buffered saline (100 mM Tris-HCl, pH 7.5 and 150 mM sodium chloride) in between each of the above mentioned steps. Counter staining was performed by dipping slides in hematoxylin solution. The slides were then thoroughly rinsed with water, mounted with Clearmount solution (Zymed Laboratories Inc.) and left to drying. The slides were then cover slipped with Permount (Fisher Scientific, Pittsburgh, PA).
Serum samples from vector-or PBS-inoculated mice sacrificed at 16 days post-inoculation were collected and tested for the presence of HAd5-, PAd3- or BAd3-neutralizing antibodies, as described previously (Moffatt et al., 2000). Similarly, the serum samples were also used to detect GFP-specific antibodies by ELISA as previously described (Mittal et al., 1995a). Briefly, 96-well ELISA plates (Immulon 2HB, Thermo Scientific, Asheville, NC) were coated with a purified preparation of GFP (1µg/ml) in carbonate bicarbonate buffer and then reacted with serial 2-fold dilutions of each of the test serum samples. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Bio-Rad, Hercules, CA) was used as secondary antibody. The optical density (OD) was measured at 450 nm or 490 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA). The reciprocal of the highest serum dilution with an OD reading of at least the mean + standard deviation (SD) above the PBS-inoculated mouse serum sample was taken as the ELISA antibody titer.
Student’s paired t-tests for multiple comparisons with adjustments in the P-value using Tukey or Bonferroni methods were used for determining the statistical significance. For all tests, P < 0.05 was considered significant.
This work was supported by Public Health Service grant CA110176 from the National Cancer Institute. We are thankful to Jane Kovach for her excellent secretarial assistance and Brian Denton for help with statistical analyses.