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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 28, 2001 as doi:10.1096/fj.00-0460fje. |
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2
* Division of Pathophysiological and Experimental Pathology, Department of Pathology,
Department of Surgery and Science, and
Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan;
DNAVEC Research Inc., Tsukuba City, Ibaraki, Japan;
¶ Department of Viral Infection and Vaccine Control and
AIDS Research Center, National Institute for Infectious Diseases, Tokyo, Japan
2Correspondence: Division of Pathophysiological and Experimental Pathology, Department of Pathology, Graduate School of Medical Sciences, Kyushu University, 3–1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: yonemitu{at}pathol1.med.kyushu-u.ac.jp
SPECIFIC AIMS
Frustrations associated with currently available vectors in gene therapy for clinical restenosis involve either lower gene transfer efficiency (for lipid and adeno-associated virus) or the need for relatively longer exposure time, resulting in an inefficient therapeutic outcome and prolonged tissue ischemia. We recently developed a novel mononegavirus vector based on Sendai virus (SeV) that has shown high gene transfer ability to airways; the aim of this study was to clarify whether this novel vector might overcome these hurdles to extend its clinical availability to vascular system.
PRINCIPAL FINDINGS
1. SeV requires only a brief vector–cell interaction for
efficient gene transfer
Our previous study of gene transfer to mouse nose
demonstrated bolus injection of SeV was sufficient to show high
transgene expression obtained by perfusion, whereas lipid- or
adenovirus-mediated gene transfer was markedly enhanced by perfusion,
suggesting its rapid transfection ability. To assess this in the
vascular system, we tested the vector–cell contact time-dependent
transgene expression level. In SeV-lacZ transfected bovine smooth
muscle cells (BSMCs), ß-Galactosidase activity was not markedly
affected by vector–cell interaction time, and only 1 min exposure was
sufficient to a level of enzymatic activity similar to that of 48 h exposure (Fig. 1A
, n=6, respectively). Similar findings were seen
in both human saphenous veins (Fig. 1B
) and BSMCs
transfected with SeV-luciferase (data not shown). In contrast,
adenovirus encoding lacZ exhibited a vector incubation time-dependent
increase in BSMCs (Fig. 1C
), as previously reported.
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2. SeV-mediated transgene expression is stable in both
proliferating and arrested smooth muscle cells
Paramyxoviridae, including SeV, exhibit genome
replication and express their genes in a cytoplasmic manner, suggesting
stable reporter gene expression in daughter cells. In fact, high level
of luciferase gene expression persisted in both logarithmically
proliferating or arrested BSMCs, at least 1 month at MOI (multiplicity
of infections) = 0.1, 1, and 10.
3. Gene transfer efficiency to human vessels
SeV-NLS-lacZ (SeV-encoding nuclear localized
lacZ gene) was infused into a 3–4 cm cut vein at an
infusion pressure of 0,150, 300 mmHg, or 760 mmHg for 10 min. Some
vessels were injured with 4F Fogarty balloon catheter, and the vector
solution was infused at the same pressure. A gross observation of
vessels exposed to the SeV-NLS-lacZ revealed diffuse and frequent blue
spots on the luminal surface and adventitia (Fig. 2A
, B
). Histological examination indicated that these cells
were largely composed of endothelial cells at the luminal surface, vasa
vasorum, and adventitial fibroblasts (Fig. 2C
, D
).
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In balloon-injured vessels, blue spots were markedly decreased in number on the luminal surface, and histological examination confirmed that only scattered cells with blue nuclei were observed in vessels with thin neointima; almost no blue cells could be seen in vessels with thick neointima. We suggest these did not always imply low transduction efficiency to vascular SMCs, because relatively frequent medial cells were positive for X-Gal in some vessels with a tear in the neointima.
SeV-mediated gene transfer efficiency of uninjured vessels was similar to that of simple floating samples at any infusion pressure. An apparent increase in X-gal-positive cells was detected in both the media and adventitia of veins with neointimal tears. Even in the torn samples, however, gene transfer efficiency was still low in the neointima, suggesting low vector permeability to the neointimal area.
CONCLUSIONS
Key aspects of this study are that 1) a brief exposure of vessel wall to vector solution was enough to efficient gene transfer and 2) viral genome and exogenous gene expressions were relatively stable in both proliferating and arrested cells at least 1 month in vitro. These results are in clear contrast to reported findings of adenovirus vectors, commonly used in the field of vascular gene transfer studies. In addition, we demonstrated that several important advantages of adenoviral vectors, such as high level transgene expression and high transfection ability independent on cell cycle, were common to recombinant SeV, suggesting its utility in the clinical setting.
Our preclinical results using liposomes coated with SeV envelope proteins (HVJ liposomes), which could achieve high gene transfer in rabbit vessels, indicated low efficiency in diseased human vessels similar to both present data using SeV and previous published data using adenoviruses. These results suggest that a biological barrier in diseased vessels may be common to virus-based vectors.
Regarding the persistence of transgene expression, inasmuch as recombinant SeV is a cytoplasmic transcription system, our data indicate that not only exogenous gene expression, but also its genome replication, is stable and inherited by daughter cells. We also showed that a serious reduction in gene expression could not be seen by 1 month, suggesting the possibility of persistent exogenous gene expression. One limitation of this study is that we have not yet assessed the induction of host immune response in vivo, especially cytotoxic T lymphocyte activation, which has been a major hurdle to limit the exogenous gene expression via adenovirus-mediated gene transfer. An extensive study is required to clarify this.
Other important factors should be examined with regard to use of SeV in a clinical setting: 1) exogenous gene expression is driven by its own RNA polymerase in cytoplasmic manner, suggesting loss of safety issue such as tumorigenesis; 2) SeV is well known to be pathogenic for rodents, but not for human beings. This has been supported by a previous report indicating no serious adverse effect in nonhuman primate via nasal administration of wild-type SeV. Although extensive safety and toxicity studies should be carried out before clinic use, these characteristics of SeV may suggest its feasibility for use in human studies.
Overall, although the reservation regarding the low rate of vector
uptake to neointima and media is common to the available vascular gene
transfer vectors, recombinant SeV seems to overcome several other
important issues in the field of vascular gene delivery. We conclude
that SeV vector should be an important alternative for gene therapy in
vessel
wall.FIGURE 3
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0460fje ; to cite this
article, use FASEB J. (March 28, 2001) 10.1096/fj.00-0460fje 
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40%) through the neointimal
tear (C). Blue nuclei indicate NLS-lacZ-positive
cells.