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Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2246-2253

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:2246-2253.)
© 1995 American Heart Association, Inc.

Articles

Quantitative Analysis of Repeat Adenovirus-Mediated Gene Transfer Into Injured Canine Femoral Arteries

Hikaru Ueno; Jian-Jun Li; Hideharu Tomita; Hiroaki Yamamoto; Yan Pan; Yumi Kanegae; Izumu Saito; Akira Takeshita

From the Molecular Cardiology Unit (H.U., J.-J.L., H.T., H.Y., Y.P., A.T.), Research Institute of Angiocardiology and Cardiovascular Clinic, Kyushu University School of Medicine, Fukuoka, and the Laboratory of Molecular Genetics (Y.K., I.S.), Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Correspondence to Hikaru Ueno, MD, PhD, Senior Assistant Professor of Medicine, Kyushu University School of Medicine, 3-1-1, Maidashi, Higashi-ku, Fukuoka 812-82 Japan.


*    Abstract
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 *Abstract
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Abstract We quantitatively evaluated the effectiveness of a repeat administration of a recombinant adenoviral vector expressing bacterial Escherichia coli lacZ into the same arterial site of a relatively large animal, the dog. The replication-defective adenoviral vector was introduced percutaneously into balloon-injured femoral arteries through a double-balloon catheter. After a single dose of adenoviral vector, up to 90% of surface (73±16%, n=7) and smooth muscle cells in multiple layers of the media showed transgene expression as evaluated by 5-bromo-4-chloro-3-indoyl ß-D-galactopyranoside histostaining without extralocal expression, as assessed by polymerase chain reaction. High-level expression (measured as ß-galactosidase activity) peaked 7 days after transfer and was transient, although it was retained for a month. Second doses of the same adenovirus to the same arterial site were given 1, 2, 5, or 8 weeks after the first administration. At 1 week the second dose significantly enhanced lacZ expression. At 2, 5, or 8 weeks the second dose reinduced lacZ expression at 25% to 30% of the full expression. lacZ expression was also detected in preimmuned dogs, although the expression levels correlated inversely to the titer of neutralizing antibodies in their serum. These results demonstrate that arterial gene expression can be enhanced by a second administration of the same adenovirus after a short interval and that a repeat dose after a long interval partially but significantly reinduces gene expression despite the presence of an immune response. These data may provide an additional scientific foundation for the use of adenovirus-mediated arterial gene transfer in future clinical practice.


Key Words: in vivo arterial gene transfer • repeat gene transfer • recombinant adenovirus • balloon injury • gene therapy


*    Introduction
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 *Introduction
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In vivo gene transfer into the cells of the vascular wall can elicit site-specific production of recombinant protein for a prolonged period of time. Use of this technique could facilitate understanding of the molecular pathophysiology of progressive proliferative vascular disorders and have beneficial therapeutic effects if genes encoding growth-regulatory molecules could be delivered to the cells of diseased vascular walls. Reports have accumulated that show the usefulness of recombinant adenoviruses as vectors for efficient gene transfer into a wide variety of cell types and tissues, including vascular wall cells, either in vitro or in vivo.1 2 3 4 5 6 7 8 9 10 11 12 13 14 The higher and more homogeneous the level of gene expression is, the more effective it should be. One simple way to achieve this goal may be a repeat application of the vectors. Repetitive administration of adenovirus may also be required for long-term expression, since an adenovirus-transferred gene is not integrated into the host's genome, and gene expression is thus transient. A second administration of adenovirus may not be effective in reinducing gene expression because the host's immune responses against the viral proteins would affect gene transfer and/or disrupt cells via activation of cytotoxic lymphocytes. In fact, a second dose of adenovirus applied intraperitoneally in rats within 1 month of the first infection induces only minimal gene expression.15 If this is true in humans, and since most people have at some time been infected with adenoviruses, this could limit the usefulness of such gene transfer. Thus, the effectiveness of repeat applications of the same adenovirus to the same arterial site and the induced level of gene expression in the arteries of preimmuned animals need to be examined in a quantitative manner before the value of repeated applications of adenvirus as a clinical tool can be fully assessed.

In this study, we introduced a replication-defective adenovirus expressing Escherichia coli lacZ by the percutaneous transluminal method by inserting a double-balloon catheter into the balloon-injured femoral artery of a relatively large animal model, the dog, and quantitatively assessed the effectiveness of a repeat administration with the same adenoviral vector. We observed that a second dose of the same adenovirus given within 1 week of the first enhanced gene expression at the same arterial site. If the second application was performed more than 2 weeks after the first, it reinduced gene expression at a lower but significant level, despite the presence of neutralizing antibodies in the serum. We also quantified expression levels in preimmuned animals and found that gene expression could be induced, although the expression level was inversely correlated with the titer of neutralizing antibodies in the serum. These data may help to provide an additional scientific basis for the clinical use of adenovirus-mediated arterial gene transfer.


*    Methods
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 *Methods
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Preparation of Adenovirus
Replication-defective recombinant adenoviral vectors expressing E. coli lacZ were prepared.14 16 Briefly, lacZ placed between a CA promoter that was composed of cytomegalovirus enhancer and chicken ß-actin promoter17 was inserted into a cassette cosmid vector that contained an entire adenovirus type 5 genome except for the E1a, E1b, and E3 regions (pAdexCALacZL). A recombinant adenovirus was constructed by in vitro homologous recombination in 293 cells18 by using pAdexCALacZL and the adenovirus DNA terminal–protein complex with a modification of a method previously described.19 The desired recombinant adenovirus, designated as AdexCALacZL, was purified by ultracentrifugation through a CsCl2 gradient followed by extensive dialysis. The titer of the virus stock was assessed by a plaque formation assay that used the 293 cell. In some experiments we used AdexSRLacZL,16 in which lacZ was driven by the SR{alpha} promoter.20 We also prepared a control adenovirus, Adex1w, which did not contain any exogenous gene to be expressed.14 16

In Vivo Gene Transfer Into Injured Artery by the Percutaneous Transluminal Method
Adult mongrel dogs (male or female; weight, 10 to 15 kg) were used for in vivo gene transfer. All animals were treated according to the protocols approved by Kyushu University animal care committees at the center animal care facility. For in vivo gene transfer, dogs were anesthetized with sodium pentobarbital (25 mg/kg IV), intubated, and ventilated with room air by a volume-cycled respirator. The right carotid artery was exposed under sterile conditions, and a 5F double-balloon catheter (Clinical Supply Co) was placed in the femoral artery. The endothelium was removed by balloon abrasion. The two balloons were then inflated to create a closed space (about 25 mm long) for gene transfer. The space was filled through side holes in the catheter with either AdexCALacZL or AdexSRLacZL (final titer, 3.0x108 pfu in 0.15 mL sorbitol-added lactated Ringer's saline; Otsuka Pharmaceutical), and incubation was continued for 30 minutes. Intra-arterial filling pressure was less than 150 mm Hg as measured by a pressure transducer. As controls, some of the balloon-injured femoral arteries were incubated with either the control virus Adex1w or vehicle only. After incubation the solution was retrieved through the catheter, the space was washed several times with saline before the balloons were deflated, and blood circulation was restored. The vessels were harvested 3 to 42 days later and fixed in phosphate-buffered saline, pH 7.4, containing 2% formaldehyde and 0.2% glutaraldehyde for 2 hours at 4°C. After fixation the vessels were evaluated for lacZ expression by histostaining with a chromogenic substrate, X-Gal (Wako Chemicals) in phosphate-buffered saline containing 5 mmol/L K3[Fe(CN)6], 5 mmol/L K4[Fe(CN)6], 1 mmol/L MgCl2, and 1 mg/mL X-Gal at 37°C for 5 hours. The area of the inner blue-stained surface was morphometrically measured from photographs by using an automated computer-based image analyzer (Digitizer KD4600, Graphtec Corp). Samples were embedded in paraffin after X-Gal staining, and 5-µm sections were cut and counterstained with nuclear fast red. After gene transfer, all dogs were fed and monitored in a separate room according to institutional regulations.

Quantification of ß-Gal Activity
Expression of lacZ was quantified by measuring ß-gal (Sigma Chemical Co) activity as determined by colorimetric assay by using chlorophenol red-3-D-galactopyranoside (Boehringer Mannheim Biochemica)21 as described.14 16 For preparation of protein extracts, adventitia of the arteries were stripped off in ice-cold buffer under a microscope. Purified bacterial ß-gal was used to depict the standard curve for quantitative analysis. By definition, 1 unit is the amount of enzyme that hydrolyzes 1 µmol chlorophenol red-3-D-galactopyranoside/min at 37°C. The activity of ß-gal was normalized with respect to the protein content as determined by a dye-binding assay.22

PCR Detection of AdexSRLacZL After Arterial Gene Transfer
Dogs were subjected to AdexSRLacZL administration on both sides of the femoral arteries for 45 minutes. Five days later, DNA was extracted from various tissues including brain, heart, lung, kidney, liver, spleen, testis, and the targeted artery by a proteinase K digestion method.23 Viral DNA was also prepared from AdexSRLacZL as a control. To amplify the AdexSRLacZL specific sequence of 280 bp, a sense primer (5'-CTCGAGGAACTGAAAAACCA-3') was chosen from the SR{alpha} sequence and an antisense primer (5'-GGCGAAAGGGGGATGTGCTG-3') from the lacZ coding sequence. A PCR of 33 cycles, each consisting of denaturation at 93°C for 1 minute, annealing at 67°C for 1 minute, and extension at 72°C for 1 minute, was performed by using 10 µg genomic DNA with Taq DNA polymerase (2.5 U/sample; Wako Chemicals) in a program-controlled thermal cycler (PC-700; Astec). A 487-bp DNA of canine Ran/TC4 gene24 was amplified as a control by using the primers 5'-AGCCCCAAGTCCAGTTCAAGC-3' and 5'-AGTTTTCTAGCAAGCCACAGG-3'. Aliquots of PCR products were analyzed on a 2% agarose gel with DNA markers of 100-bp ladders (GIBCO-BRL).

Neutralizing Antibodies Against Human Adenovirus in Serum
Serum samples were assayed simultaneously for the presence of anti-adenovirus neutralizing antibodies according to the routine procedures at the SRL, Tokyo, Japan. Neutralization values are presented as the maximum dilution of serum required to prevent Hep-2 cells from becoming infected with the type 5 wild-type human adenovirus.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
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Adenovirus-Mediated In Vivo Gene Transfer Into Injured Artery by the Percutaneous Transluminal Method
Using a double-balloon catheter, we percutaneously applied AdexCALacZL (3.0x108 pfu) into canine femoral arteries that had been balloon-injured just before the gene transfer. The lacZ expression in the arterial wall was evaluated 3 days to 6 weeks later by X-Gal histostaining and quantified by measuring ß-gal activity in protein extracts. The interior wall of the injured artery showed a deep blue coloration (Fig 1BDown). The percentage of the inner surface area stained blue was up to 90% (73±16%, mean±SD; n=7). The peripheral portion of the injured arterial wall adjacent to the infected area revealed no coloration, indicating that gene transfer occurred in a site-specific manner (Fig 1BDown). We had filled the isolated space with virus-containing solution at a pressure of <150 mm Hg; at these pressures, the virion did not penetrate into the adventitia. Occasional scattered staining of the adventitia was observed that was probably a result of infection through the vasa vasorum.10 11 Vessels subjected to the same catheterization procedures but incubated with vehicle alone or with the control adenovirus Adex1w showed minimal, if any, coloration after X-Gal staining (Fig 1ADown). Microscopic examination demonstrated that not only cells directly exposed to the lumen but cells several layers deep within the media were stained (Fig 1CDown). Stained cells were identified as smooth muscle cells by immunohistochemical staining with an anti–smooth muscle {alpha}-actin antibody25 (Boehringer Mannheim Biochemica; data not shown).



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  		Figure 1. Photomicrographs showing adenovirus-mediated in vivo gene transfer into injured canine femoral arteries with the double-balloon catheter technique. Balloon-injured femoral arteries were incubated for 30 minutes with either (A) Adex1w (control virus) or (B) AdexCALacZL. Arteries were evaluated after 3 days for lacZ expression by X-Gal histostaining. A and B show gross specimens; C shows a section counterstained with nuclear fast red. M and O indicate the medial and outer layers of the artery, respectively (C, original magnification x100).

We next examined the time course of lacZ expression in the arteries. The level of ß-gal activity was at its maximum on day 7 after infection (153.0±38.9 mU/mg protein, mean±SD; n=6), and it then decreased gradually (Fig 2Down). A high level of lacZ expression was sustained for a month. Levels of ß-gal activity that were less than 5% of the maximum, although still significant, were detected 6 weeks after gene transfer.



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  		Figure 2. Line graph showing duration of lacZ expression in femoral arteries in vivo. Balloon-injured canine femoral arteries were exposed to either AdexCALacZL or the control virus Adex1w. At the indicated number of days after gene transfer, ß-gal activity was determined and is expressed in milliunits per milligram of total protein of the tissue extract. Values are mean±SD (n=5-7). The arteries exposed to either Adex1w or vehicle alone showed only nominal activity.

We monitored the general condition, appetite, body weight, and electrocardiogram of the dogs, and biochemical parameters in the blood were measured before and up to a month after gene transfer. We found no significant alterations attributable to the adenovirus administration (data not shown).

PCR Analysis of lacZ Expression in Other Organs After Arterial Gene Transfer
The use of a delivery system involving a double-balloon catheter enabled us to minimize the amount of virus used and to retrieve most of the adenovirus through the catheter after incubation. The amount of virus that could escape to other organs via the blood stream should have been minimal. To confirm this, tissue sections from various organs including brain, heart, lung, liver, spleen, kidney, skeletal muscle, and testis were examined for lacZ expression by X-Gal histostaining. We found no coloration in any of these specimens (data not shown). To confirm that only the restricted arterial segment was infected with the adenovirus, we extracted DNA from various tissues 5 days after AdexSRLacZL administration to the injured artery and amplified the DNA by PCR with primers specific for AdexSRLacZL. While a 280-bp band appeared in the samples from the arterial segments that had been directly exposed to AdexSRLacZL, no corresponding 280-bp band appeared in DNA samples extracted from the other tissues (Fig 3ADown). Bands of 487 bp corresponding to canine Ran/TC4 sequences24 were amplified in all samples, indicating that intact DNA was extracted from the tissues and that PCR was performed properly (Fig 3BDown).



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  		Figure 3. PCR analysis of adenoviral vector sequences in various tissues. Five days after gene transfer in vivo using AdexSRLacZL, DNA was extracted from various tissues including brain, heart, lung, liver, kidney, spleen, testis, and the femoral artery that was directly exposed to the adenovirus (Artery). DNA (10 µg) was amplified by using specific primes for (A) AdexSRLacZL and (B) the canine Ran/TC4 gene. PCR products were analyzed on a 2% agarose gel. Arrows indicate a 280-bp band specific for AdexSRLacZL in A and a 487-bp band specific for Ran/TC4 in B. The DNA of AdexSRLacZL was also analyzed as a positive control in A.

Enhanced Gene Expression After a Repeat Gene Transfer
The catheter-mediated gene-delivery system allows for a repeat gene transfer into the same arterial segment. We examined whether a repeat application of the same adenoviral vector could enhance the expression of the gene. Both femoral arteries in the dogs were exposed to AdexCALacZL (4.2x107 pfu) immediately after balloon injury; 7 days later the right artery alone was subjected to a second exposure to AdexCALacZL (3.0x108 pfu), and the left artery was exposed to the control virus Adex1w (3.0x108 pfu). No further balloon injury was inflicted at the time of the second infection. ß-Gal activity in the arteries was determined after an additional 7 days. The ß-gal activity was 152±31 mU/mg protein in the right artery, which was exposed twice, but only 45±12 mU/mg protein in the left artery, which was exposed only once (mean±SD, n=4) (Fig 4Down). This result demonstrates that a repeat administration of the same adenoviral vector to the same arterial site within 7 days of the first inoculation leads to enhanced lacZ expression. When a higher titer of adenovirus (3.0x108 pfu) was applied the first time, the enhancement of gene expression was not as prominent as described above (Fig 4Down), suggesting a limited capacity of arterial wall cells for lacZ expression. The result may also suggest that the higher dose (3.0x108 pfu) is sufficient to induce submaximal gene expression. On day 7 after the first administration of AdexCALacZL (3.0x108 pfu), neutralizing antibodies against human adenovirus were not detected in 7 of 10 dogs, but their level was 1:16 in 2 dogs and 1:32 in 1 dog.



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  		Figure 4. Bar graph showing enhancement of ß-gal activity in arteries exposed twice to AdexCALacZL. Right and left balloon-injured femoral arteries were exposed to AdexCALacZL (4.2x107 or 3.0x108 pfu; see below); at the indicated time after the first exposure, the right artery alone was subjected to a second dose of AdexCALacZL (3x108 pfu), and the left artery was exposed to the control adenovirus Adex1w (3x108 pfu). Seven days after the second virus application, ß-gal activity in tissue extracts was determined. In some experiments, AdexCALacZL was applied to the injured arteries at 4.2x107 pfu the first time when the second administration was to be performed 7 days after the first (indicated as 1 WK-L) (see "Methods"). Data are mean±SD (n=4). S and D indicate a single or double exposure, respectively, to AdexCALacZL.

We next examined whether a repeat dose after a long interval, during which the immune response should have fully developed, would also be effective in inducing gene expression. To this end, 2 to 8 weeks after the first exposure to AdexCALacZL (3.0x108 pfu), a second administration of the same adenovirus at the same dose was performed without additional balloon injury. A significant induction of ß-gal activity was detected in the arteries exposed twice to AdexCALacZL (Fig 4Up). The expression levels after a second dose 2, 5, or 8 weeks after the first were essentially the same, each being about 25% to 30% of that achieved at the first infection. In all dogs (n=12) tested, significant levels of neutralizing antibodies (mean titer, 1:256) were detected in the serum collected at the time of the second infection.

Adenovirus-Mediated Arterial Gene Transfer in Preimmuned Dogs
We quantified the titer of neutralizing antibodies in serum taken at different times after injections of large amounts of AdexCALacZL (2x1010 pfu IV each). A significant level of antibodies was detected in all animals tested within 2 to 3 weeks of the first injection, although both the magnitude and the temporal changes in the antibody titer varied among animals (Fig 5Down). The titer tended to decrease with time and was enhanced within the first week after the repeat injection of the adenovirus. Although a considerable humoral antibody response was observed, no significant alteration was detected in the biochemical parameters tested (data not shown).



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  		Figure 5. Line graph showing anti-adenovirus response to AdexCALacZL in serum. Three dogs were injected with large amounts of AdexCALacZL (2x1010 pfu IV each) on several occasions (indicated by arrows), and serum was collected at the times shown to enable measurement of neutralizing antibodies against human adenovirus.

The ß-gal activity in injured arteries 7 days after infection with AdexCALacZL (3.0x108 pfu) was quantified in preimmuned dogs that had been injected with AdexCALacZL (2x1010 pfu IV each) at least twice to raise cogent immune responses against the adenovirus. Expression levels in the injured arteries were inversely correlated with the titer of antibodies in the serum (correlation coefficient=.79) (Fig 6Down). However, it should be noted that a significant level of expression, about 25% of that obtained in naive animals, was still elicited even in the presence of a titer of antibodies as high as 1:512.



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  		Figure 6. Plot showing correlation between lacZ expression and the titer of neutralizing antibodies in serum. Dogs were injected with AdexCALacZL (2x1010 pfu IV each) two or three times at 3- to 4-week intervals. Adenovirus-mediated arterial gene transfer using AdexCALacZL (3x108 pfu) was performed 7 to 10 days after the last systemic injection of AdexCALacZL, and ß-gal activity was determined 7 days after the gene transfer. The titer of neutralizing antibodies was determined in the serum collected at the time of gene transfer.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
We found that delivery of a replication-defective adenovirus through a catheter achieved efficient gene transduction into an injured artery in a site-specific manner without apparent local cytotoxicity or systemic side effects and without extralocal expression of the transferred gene (Figs 1 through 3UpUpUp). The duration of gene expression in our dogs was longer than that reported in rats, in which ß-gal activity in injured carotid arteries declined precipitously 10 to 14 days after infection.3 4 Although only medium-term gene expression could be expected, expression that remains at a significant level for a month or so may be sufficient to alter pathophysiology in some settings.

The adenoviral vector was made replication-defective by deletion of the E1 region from the viral genome. However, this mutation does not completely block transcription from the remainder of the genome,26 27 which results in the raising of antibodies and a cellular immune response against the adenoviral proteins (compare Fig 5Up). Activated cytotoxic T cells may disrupt the transfected cells, leading to a truncation of gene expression.4 7 28 29 In our study we neither assessed cytotoxic T-cell activities against the infected cells nor confirmed histologically whether or where T lymphocytes had attacked the lacZ-expressing cells, partly due to a lack of antibodies that recognize the canine CD8+ T lymphocyte. However, a variety of findings support the above notion and suggest that use of less antigenic adenoviral vectors could lengthen the period of gene expression. The level of gene expression was suppressed in the preimmuned animals,15 30 31 and an inverse correlation was observed between the titer of neutralizing antibodies in serum and the level of gene expression (Fig 6Up),31 although the relation between neutralizing antibodies and the cellular immune response is not clear. An immunosuppressant, cyclosporine, prevents the time-dependent reduction in gene expression.32 Gene expression persists longer both in the T-cell–deficient nude mouse26 and in neonatal animals, in which the immune system is less well developed than in adults.32 33 34 Finally, an adenoviral vector in which a temperature-sensitive mutation was engineered in the E2 region of the viral genome both reduced inflammatory responses and prolonged the duration of gene expression.35 36 37 However, the immune response–mediated direct lysis of infected cells may not be the only factor involved in the reduction of gene expression, since expression can be prolonged for 6 to 18 weeks (compare Fig 2Up),38 and induction of gene expression can occur in preimmuned animals (Fig 6Up).15 16 30 31 Moreover, cyclosporine treatment could not completely prevent the decline in expression in the ventricle and had no effect on expression in the liver.32 Further investigation may be required for a full understanding of reductions in gene expression and the fate of adenovirus-transferred DNA; such studies may uncover a way to prolong gene expression.

An important finding of this study is that a second application to the same arterial site within 1 week of the first introduction of the adenoviral vector can enhance lacZ expression (Fig 4Up). This may be valuable information when a higher and more homogeneous level of expression of a nonsecreted protein is required. For example, expression of a growth-regulatory molecule should be enhanced by reapplication of the adenoviral vector on the day after angioplasty in addition to its transfer at the time of arterial intervention. Furthermore, 2, 5, or even 8 weeks after the first exposure, a second application of the same adenovirus induced lacZ expression at significant levels (Fig 4Up). Moreover, in preimmuned dogs, significant levels of gene expression were achieved, as assessed by the titer of neutralizing antibodies in the serum, even in the presence of substantial immune responses (Fig 6Up). Although caution is required in applying the data obtained in the present study in dogs to humans (the dog is not a permissive host for human adenoviruses, and its immune system may differ from ours), the results may suggest that adenovirus-mediated arterial gene transfer is likely to be at least partially successful in humans, most of whom have been infected at some time with adenoviruses and therefore should have some level of immunity against them. These observations may imply the possibility that, depending on the clinical situation, the same gene can be transferred repeatedly, or alternatively, multiple different genes can be transferred at different times into the same arterial site. For the treatment of restenosis of an artery after balloon angioplasty, a combined gene transfer might be more effective. For example, an antiproliferative gene might be transferred just after angioplasty, followed later by transfer of a molecule that inhibits extracellular matrix production.

There have been four reports of quantitative evaluation of repeat adenovirus administration in vivo.15 30 31 32 Two of them involved studies of the respiratory tissues of cotton rats. In one, the adenovirus was applied directly to the respiratory tract, and a repeat dose was applied 120 days after the first; this resulted in successful gene transfer at a 25% level compared with that in naive rats.31 In the other, the adenovirus was given systemically by intraperitoneal injection; a repeat injection 1 week or 1 month later failed to express the gene, and 3 months after the first exposure only a 10% level of expression was achieved.15 In mice infected once with an adenovirus expressing luciferase, 0% and 4% luciferase activity was detected in lung and liver, respectively, when the second inoculation was given 8 weeks after the first.30 Others have reported only an insignificant increase in chloramphenicol acetyltransferase activity after a second dose of virus in adult rats, although admittedly a relatively small amount of virus (6x107 pfu/rat) was administered.32 We observed a considerably better expression of lacZ on repeat infection in dog femoral arteries despite the presence of neutralizing antibodies at a similar or even higher titer than those detected in the previous studies.15 30 31 32 We have confirmed that adenovirus-mediated arterial gene transfer is not enhanced by prior injury,14 which does enhance gene expression after liposome-mediated gene transfer if it is performed 3 to 7 days after injury.39 40 Thus, the differences between the results of different studies may be a function of animal species, route of administration, or viral titer used. Studies are now under way to try to determine whether third or subsequent applications can induce gene expression and whether the duration of gene expression after a repeat administration is the same as or shorter than that achieved by the first application. It should be noted that, in the present study, the expression levels were measured 7 days, not 2 to 4 days as in some studies,15 31 after the second application of the adenoviral vector.

Another concern regarding the use of viral vectors is the localized inflammation that can result from direct toxicity or from the immune response to viral proteins. Administration of high doses (109 to 1011 pfu) of E1- and E3-deleted adenoviral vectors in vivo is associated with the presence of pronounced inflammatory cellular infiltrates composed of T lymphocytes, macrophages, and histiocytes.7 28 29 41 42 43 However, no detectable inflammation or major histological difference from control has been observed in other studies.13 16 38 44 45 46 The extent of the inflammatory response is dose dependent. Nasal administration of a small amount (107 pfu) of a wild-type adenovirus (type 5) evokes only minimal cellular inflammation in the lung of cotton rats despite a strong humoral immunologic response.31 The exposure of humans to low doses of adenovirus results in serological evidence of infection but no accompanying pulmonary infiltrates.47 Zabner et al46 observed neither inflammatory histopathologic changes nor altered efficacy of gene transduction in the airway epithelium of cotton rats after a second transbronchial administration of adenovirus (2.1 to 3.2x108 pfu) given 2 weeks after the first infection. This was despite the presence of neutralizing antibodies at the time of readministration. However, they did not quantify the level of expression, nor did they evaluate gene expression when the second dose was administered more than 2 weeks after the first infection. It should be noted that the adenoviral vector used in their study preserved the E3 region of the viral genome. In our study, the inflammatory cell infiltration that was observed within the first 3 days after arterial gene transfer might be largely attributable to the balloon injury itself, since there was no major or consistent histological difference between arteries subjected to adenovirus and control arteries injected with saline (data not shown), as we also observed when we injected canine myocardium with AdexCALacZL.16 Moreover, in intact arteries in which injury effects were avoided we did not observe a significant difference between arteries exposed to AdexCALacZL and those exposed to saline or Adex1w (data not shown). It may be significant that we used 3x108 pfu adenovirus/artery, a relatively small amount, and retrieved most of the virus through a catheter after a 30-minute incubation. In this study we did not rigorously investigate the effect of viral concentration on gene expression and/or on local inflammatory response. However, the finding that a repeat dose given 7 days after the first infection (both 3x108 pfu) did not enhance gene expression may well suggest that the dose we used is sufficient to induce submaximal gene expression without causing adverse local inflammation. The immune response may also vary between species: in rabbit coronary arteries inflammation has not been detected,44 whereas a prominent inflammatory response has been noted periadventitially in porcine coronary arteries7 and myocardium.29 Mice also develop pneumonia after intranasal inoculation of the type 5 wild-type adenovirus, although higher doses of the virus are required than in the cotton rat, which is permissive for human adenoviruses,31 48 49 suggesting that the dose of adenoviral vector used in this study might induce an inflammatory response in human arteries. Again, differences between studies may be due not only to the species used but to differences in route of administration, viral titer, or purity of the vector. Although an inflammatory response was detected shortly after gene transfer, it is significant that we observed neither a prolonged inflammatory response nor any adverse histological change such as massive fibrosis in the infected arteries 2 or 5 weeks after adenoviral infection.

Our study demonstrates the effectiveness of both a repeat gene transfer by the same adenoviral vector into the same arterial site and arterial gene transfer in preimmuned animals. These results may help to further secure the basis for the use of adenovirus-mediated arterial gene transfer in the clinical setting. The data also suggest that the duration of gene expression and the extent of inflammatory responses might be improved by further modifications to the adenoviral vectors, such as an additional mutation in the E2 region,35 36 37 preservation of the E3 region,46 50 or additional deletion of other regions of the viral genome.


*    Selected Abbreviations and Acronyms
 
ß-gal = ß-galactosidase
X-Gal = 5-bromo-4-chloro-3-indoyl ß-D-galactopyranoside
pfu = plaque formation unit
PCR = polymerase chain reaction


*    Acknowledgments
 
This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan to Drs Ueno and Saito and by grants from the Japan Heart Foundation, the Research Foundation for Cancer and Cardiovascular Disease (Osaka, Japan), the Sandoz Foundation for Gerontological Research (Switzerland), and the Study Group of Molecular Cardiology (Tokyo, Japan) to Dr Ueno.

Received July 7, 1995; accepted September 15, 1995.


*    References
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 up arrowIntroduction
 up arrowMethods
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 *References
 

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