© 1997 by European Society of Cardiology
Copyright © 1997, European Society of Cardiology
Gene transfer into vascular cells using adeno-associated virus (AAV) vectors
aDepartment of Cardiology, Jichi Medical School, Minamikawachi-machi, Tochigi, Japan
bDepartment of Molecular Biology, Institute of Hematology, Jichi Medical School, Minamikawachi-machi, Tochigi, Japan
cDepartment of Anatomy, Jichi Medical School, Minamikawachi-machi, Tochigi, Japan
dAvigen Inc., Alameda, CA, USA
* Corresponding author. Department of Molecular Biology, Institute of Hematology, Jichi Medical School, Minamikawachi-machi, Tochigi 329-04, Japan. Tel.: +81 (285) 44-2111; fax: +81 (285) 44-8675; e-mail: kozawa@jichi.ac.jp
Received 26 February 1997; accepted 3 June 1997
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Abstract |
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Objectives: Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features for gene therapy, including the ability to transduce non-dividing cells and its long-term transgene expression. In this study, an AAV vector containing bacterial β-galactosidase gene (lacZ) was used to transduce cultured rat vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo. Methods: VSMC were transduced with AAV-lacZ at multiplicities of infection (MOI) ranging from 5.0x105 to 1.0x107. Expression of β-galactosidase (β-gal) in VSMC was evaluated by X-gal staining and a β-gal ELISA method. Excised rat aortas were incubated with medium containing AAV-lacZ. Expression of β-gal in the aortic segments was evaluated by X-gal staining. Results: With increasing MOI, up to 50% of cultured VSMC were positive by X-gal staining and the β-gal expression increased up to 15 ng/mg protein. The expression gradually decreased during the culture but was detectable for at least 1 month. In the ex vivo study, AAV vectors transduced endothelial and adventitial cells in rat aortic segments, while no expression was seen in medial VSMC. Conclusions: AAV vectors can efficiently transduce rat VSMC in vitro. AAV-mediated ex vivo gene transfer into the normal aorta resulted in efficient gene transfer into endothelial and adventitial cells but not into medial VSMC. These findings suggest that AAV-based vectors are promising for use in cardiovascular gene therapy.
KEYWORDS Adeno-associated virus vector; Gene transfer; Smooth muscle cell; Endothelial cell; Aorta
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Transferring foreign DNA into the arterial wall offers an experimental approach to investigate the roles of individual genes in vascular pathophysiology [1]. Furthermore, local expression of therapeutic transgenes at specific sites will have beneficial effects in an autocrine/paracrine manner. The efficient delivery and long-term expression of therapeutic genes are necessary for genetic manipulation strategies in the cardiovascular system. Successful gene transfer into the vascular structures has been reported using retroviral [2–7]and adenoviral vectors [8–17]. Retroviral vectors transduce vascular cells stably without adverse effects, but with lower transduction efficiency [4]. Moreover, target cell replication is required for retroviral integration, but the rate of cell proliferation in the vascular wall dose not exceed a few percent [18, 19]. Adenoviral vectors are highly efficient, but gene expression is transient due to its episomal feature [9, 11, 14]. In addition, adenoviral vectors may have some direct cytopathic effects and induce immunological responses to transduced cells [13], because leaky expression of adenoviral genes cannot be completely eliminated.
Recombinant viral vectors based on the nonpathogenic parvovirus, adeno-associated virus (AAV), have a number of attractive features, including lack of cytotoxicity, ability to transduce both dividing and non-dividing cells [20, 21], and long-term transgene expression [22–24]. AAV vectors have been evaluated in preclinical models for cystic fibrosis [22], Parkinson's disease [23], anemia [24], and brain tumors [25]. However, to our knowledge, there have been no reports regarding application of AAV-based vectors to the cardiovascular system. Thus, in this study, we constructed recombinant AAV vectors containing Escherichia coli β-galactosidase gene (lacZ) under the control of the cytomegalovirus (CMV) immediate early promoter, and transduced cultured vascular smooth muscle cells (VSMC) in vitro and rat thoracic aortas ex vivo with these vectors.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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2.1 Construction of AAV-lacZ
Fig. 1 demonstrates the structure of wild-type AAV genome and AAV-lacZ. In AAV-lacZ, rep and cap sequences of the wild-type AVV genome were deleted and replaced with CMV immediate early promoter-lacZ cassette.
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2.2 Production of AAV vectors
We produced AAV vectors without the use of helper virus [26]. Trypsinized human 293 cells were plated in 100-mm culture dishes at 1x106 cells containing 10 ml of DME/F12 medium (GIBCO BRL) plus 10% fetal bovine serum (CSL Ltd.), 1% penicillin/streptomycin and 1% glutamine and incubated at 37°C under 5% CO2 over the next 24–48 h. After cells reached 50% confluence, 293 cells were co-transfected by the calcium-phosphate co-precipitation method with 10 µg of pWee1909 lacZ (ITR-CMV-lacZ-SV40 polyA-ITR and the AAV rep/cap sequences) and 10 µg of pladeno1 (containing the adenovirus early genes; E2a, E4 and VA). Medium was changed after incubation at 37°C for 6 h. The cells were incubated further for approximately 72 h.
Transfected 293 cells were collected, cell suspensions were centrifuged at 300xg for 5 min and resuspended in 1 ml of Tris-buffered saline (TBS). Cell suspensions in TBS were subjected to 3 cycles of freezing and thawing. To remove tissue debris, suspensions were centrifuged at 10 000xg for 10 min, and the supernatants were collected. These supernatants were then used for AAV-lacZ vector solution.
2.3 Particle titer determination
The vector particle titer was determined by measuring encapsidated vector genomes resistant to DNase I treatment. 50 µl of vector solution was incubated with 5 U DNase I (Boehringer Mannheim Corp.) in buffer (10 mmol/l Tris, pH 7.5, 5 mmol/l MgCl2) at 37°C for 1 h to digest plasmid DNA that was not encapsidated. DNase-resistant viral DNA was collected after proteinase K (Boehringer Mannheim Corp.) digestion at 37°C for 1 h (10 mmol/l Tris, pH 8.0, 10 mmol/l EDTA pH 8.0, proteinase K 37.5 µg/ml). Serial dilutions of virion DNA were applied to a nylon membrane (Hybond N+, Amersham) using the Schleicher & Schuell slot-blot manifold, and standard curves were obtained using serial dilutions of plasmid DNA fragments. Membranes were subjected to UV cross-linking and hybridized with 32P-labeled lacZ probe. The hybridization signals were quantified by image analyzer (BAS 2000 image analyzer system, Fujix). Particle titers were calculated as the number of virion DNA in dilutions, and expressed as the number of particle per milliliter.
2.4 Cell culture
Primary cultures of VSMC were isolated from the media of thoracic aortas of Sprague-Dawley rats (200–250 g) by enzymatic digestion as previously described [27]. Cells were maintained in DME/F12 medium plus 10% fetal bovine serum, 1% penicillin/streptomycin and 1% glutamine, and incubated at 37°C under 5% CO2. Cells were routinely passaged just before reaching confluence by brief exposure to 0.125% trypsin and 0.5 mmol/l EDTA and were passaged at a ratio of 1:3 in 100-mm culture dishes. Experiments were performed with cultured cells at passage 5–10.
All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by US National Institute of Health (NIH Publication No. 85-23, revised 1985).
2.5 In vitro gene transfer into cultured VSMC
The percentage of cultured VSMC transduced with the AAV-lacZ vectors was estimated by staining with chromogenic substrate, 5-bromo-4-chloro-3-iodolyl-β-D-galactopyranoside (X-gal), by a modification of the procedure described previously [28]. Briefly, the cells were fixed with phosphate-buffered saline (PBS) containing 0.05% glutaraldehyde for 15 min at room temperature. After fixation, lacZ expression was evaluated by histochemical staining with X-gal in PBS containing 5 mmol/l K3Fe(CN)6, 5 mmol/l K4Fe(CN)63H2O, 1–2 mmol/l MgCl2, and 1 mg/ml X-gal at 37°C for 6 h. Levels of β-galactosidase protein were measured using β-gal ELISA kit (Boehringer Mannheim Corp.), as directed by the manufacturer. In brief, transduced VSMC were washed with PBS twice and lysed by MOPS-buffered saline containing Triton X-100. 100 µl of these cell lysates were applied to microtiter plates which were precoated with a monoclonal antibody to β-galactosidase. After a 1 h incubation period at 37°C, these microtiter plates were washed with washing buffer (PBS containing Tween 20) three times and then incubated with anti-β-galactosidase antibody conjugated with digoxigenin (0.5 µg/ml) for 1 h, anti-digoxigenin antibody conjugated to peroxidase (150 mU/ml) for 1 h, and peroxidase substrates at room temperature for 30 min. The absorbance of the samples at 405 nm (reference wavelength 490 nm) was measured and β-galactosidase protein levels were determined by calibration curve.
2.6 Ex vivo gene transfer into thoracic aorta
AAV-mediated gene transfer was evaluated in rat aortic segments. Rats were killed by overdose of ether. Thoracic aortic segments were harvested and put into DMEM/F12 plus 10% fetal bovine serum, 1% penicillin/streptomycin and 1% glutamine, incubated at 37°C under 5% CO2 with viral solution diluted to 2x1012 particles/ml. After 24-h incubation with the vector-containing medium, aortic segments were fixed with a mixture of 2% paraformaldehyde and 0.2% glutaraldehyde in PBS for 5 min at 4°C, rinsed in PBS and then frozen in liquid nitrogen. Cryostat sections (10 µm thick) were made with a Reichert-Jung Frigocut 2800E. Histochemical staining for β-galactosidase activity was performed in the sections, as described above. Control experiments were also performed in which the vector was omitted from the incubation medium.
2.7 Statistical analysis
Values are expressed as means±S.D. Differences of β-galactosidase expression were assessed by Fisher's protected least significant difference test. A value of p<0.05 was considered statistically significant.
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3 Results |
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3.1 In vitro transduction and expression of β-galactosidase
Cultured rat VSMC were transduced with dilutions of the AAV-lacZ vector with TBS (total volume of vector solution was 10% of that of culture medium), at MOI ranging from 5.0x105 to 1.0x107. Cells were exposed to AAV-lacZ for 48 h and stained with X-gal (Fig. 2A). Based on visual assessment of the percentage of X-gal-stained cells, transduction efficiency increased with increasing MOI over this entire range, with over 50% of the cells at a MOI of 1.0x107. No X-gal staining was observed in VSMC which were not transduced (Fig. 2B).
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To obtain a more quantitative assessment of gene transfer, β-galactosidase protein levels were determined by the ELISA. Cells were exposed to AAV-lacZ for 48 h and the cell lysates were harvested. Fig. 3 shows the MOI–response relationship of β-galactosidase expression in cultured rat aortic VSMC. With increasing MOI, β-galactosidase expression increased to about 15 ng β-galactosidase per mg protein at a MOI of 1.0x107.
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Fig. 4 shows the time–response relationship of β-galactosidase expression in cultured rat aortic VSMC. VSMC were exposed to AAV lacZ-containing media (at a MOI of 5.0x106) for the indicated periods, washed twice with PBS, and returned to normal media. At 7 days after initiation of transduction, β-galactosidase expression was measured. Gene expression reached a plateau level after 2-day incubation.
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To determine the duration of β-galactosidase expression after in vitro gene transfer, AAV-lacZ-transduced VSMC (MOI of 1.0x107) were harvested at 0, 3, 7, 14, 21, and 28 days after transduction. During this period, VSMC were passaged twice a week. The expression of β-galactosidase showed a peak at day 3 post-transduction (26.5±5.76 ng/mg protein), and was reduced to approximately 1% of the maximum expression level by 28 days after transduction (0.235±0.06 ng/mg protein) (Fig. 5).
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3.2 Ex vivo gene transfer into aortic segments
We next investigated AAV-mediated gene transfer into ex vivo rat thoracic aortas. As shown in Fig. 6, endothelial and adventitial cells of aortic segments were stained blue with X-gal when assayed 24 h after transduction. No medial VSMC were stained positive for β-gal activity. We observed no X-gal staining in mock-infected aortic segments (data not shown). We investigated whether VSMC could be transduced by the same methods when endothelia of aortas were scratched. However, medial VSMC were still not stained positively with X-gal (data not shown).
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4 Discussion |
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We demonstrated here that AAV vectors can efficiently transduce cultured rat aortic VSMC in dose- and time-dependent manners. Maximum transgene expression with AAV vectors occurred at 2–3 days after transduction in our experiments. In ex vivo experiments, endothelial and adventitial cells of the rat aorta were also efficiently transduced with the AAV vector.
Wild-type AAV integrates into the host cell genomic DNA upon infection in the absence of helper virus. This integration is targeted to a region defined as AAVS1 which maps to q13.2–13.4 on human chromosome 19 [29–31]. This site-specific integration is at least mediated by AAV-encoded rep proteins [32]. As recombinant AAV vectors do not have rep proteins, site specificity of integration is lost. Flotte et al. [22]reported that CFTR mRNA and protein were detected in the airway epithelium of the infected lung lobes for up to 6 months after vector administration. Kaplitt et al. [23]reported stable AAV-mediated tyrosine hydroxylase gene transfer and long-term transgene expression in neurons of adult rats for 3 months. Kessler et al. [24]also demonstrated the secretion of erythropoietin and corresponding increases in red blood cell production that persisted for up to 40 weeks with intramuscular administration of an AAV vector containing the human erythropoietin gene. Our results demonstrated that about 1% of maximum β-galactosidase expression persisted at 28 days after transduction. As integration is unnecessary for transgene expression, the transduced cells may have carried episomal, double-stranded copies of the vector, some of which may eventually be lost. Most vascular cells were nonproliferating in vivo like bronchial epithelial cells, neurons, and skeletal muscles. Therefore, expression of the transgene may be sustained at a higher level in vivo than that in our in vitro model system. This long-term expression may be therapeutically sufficient for some cardiovascular diseases.
Our ex vivo experiments revealed that AAV-lacZ could transduce both endothelial and adventitial cells but failed to transduce medial VSMC of the aortas. One possible explanation for this result is barrier functions of endothelium and internal elastic lamina. Rome et al. [33]evaluated the extent to which anatomical barriers to vector penetration might influence successful in vivo gene transfer into the normal arterial wall and revealed that the anatomy of the normal elastic arterial wall imposes significant limitations on the penetration of particles in the size range of most gene-transfer vectors. Yao et al. [8]reported adenovirus-mediated gene transfer into both endothelial and adventitial cells but not in the muscular medial layers of ex vivo rat aorta. Lemarchand et al. [11]also reported that replication-defective adenovirus vectors can transfer genes only to the endothelium of uninjured arteries both ex vivo and in vivo. These findings are compatible with our results and suggest that endothelia prevent AAV vector penetration to medial VSMC. Therefore, we investigated whether VSMC could be transduced by the same methods when endothelia of aortas were scratched. However, medial VSMC were still not stained positively with X-gal (data not shown). This finding suggests that not only endothelium but also internal elastic lamina, which might be not removed by the scratching, might prevent vector penetration into medial layer of the vessels.
Steg et al. [14]reported differences in transduction efficiency into rabbit iliac arteries with adenoviral vectors between a double-balloon catheter (DBC) and an angioplasty balloon catheter coated with hydrogel (HBC). In the DBC group, transgene expression was limited to endothelial cells when the endothelium was left intact. Rare VSMC (<2.2%) were transduced even when endothelium was removed. In contrast, HBC delivery resulted in transduction of up to 9.6% of medial VSMC. They demonstrated that transduction efficiency into medial VSMC was higher by HBC delivery than by DBC delivery. Therefore, some modifications of vector delivery systems, such as pressure loading, may improve the efficiency of AAV-mediated gene transfer into the vascular tissue.
The difference in transduction efficiencies into cultured VSMC and medial smooth muscle cells of the aorta in the present study might also be due to differences in cell cycle status or changes in VSMC phenotype. Gutzman et al. [9]exposed rat carotid arteries to adenoviral vectors at 0, 3, 7, or 12 days after injury. In these segments, β-galactosidase expression was minimal at 0 or 3 days after injury but maximal when infection was delayed until 7 or 12 days after injury. Neointimal cells constituted the dominant target of adenovirus gene transfer. Medial VSMC, whether covered or uncovered by neointimal cells, were minimally transduced. In the same way, AAV-mediated gene transfer into medial VSMC might be more efficient in their proliferating state like neointimal VSMC. In vivo experiments are needed to confirm this hypothesis.
Russel et al. [34]transduced stationary and dividing primary human fibroblast cultures with AAV vectors encoding alkaline phosphatase and neomycin phosphotransferase. They reported that the transduction frequency of S phase cells was about 200-fold higher than that of non-S phase cells. On the other hand, Podsakoff et al. [21]showed the ability of an AAV vector to transduce nonproliferating cell populations. They introduced 293 cells or fibroblasts into nonproliferative state by treatment with the DNA synthesis inhibitors, fluorodeoxyuridine and aphidicolin, or by contact inhibition induced by confluence and serum starvation. They reported that transgene expression in nondividing cells was equivalent to that in dividing cells. Therefore, it is controversial whether the transduction efficiency using AAV vectors is influenced by cell cycle status.
Despite cultured VSMC were efficiently transduced with AAV-lacZ in vitro, medial VSMC of aortas failed to be transduced in ex vivo organ culture. This finding suggests that VSMC may not be a good target for AAV vector-mediated gene transfer. However, the present study gives us important information regarding the application of AAV vectors to genetic manipulation of vascular structures.
Time for primary review 39 days.
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Acknowledgements |
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This work was supported in part by Grants-in-Aid from the Ministry of Health and Welfare of Japan, by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, by CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation (JST), by a grant from the Japan Heart Foundation, a Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease, and by a grant from the Uehara Memorial Foundation.
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