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Gene Transfer Strategies to Inhibit Neointima Formation
Gene Transfer Strategies to Inhibit Neointima Formation Martin F. Crook and Levent M. Akyürek*
Vascular smooth muscle cell (VSMC) proliferation after arterial injury results in neointima formation and plays an important role in the pathogenesis of restenosis after angioplasty, in-stent restenosis, vascular bypass graft occlusion, and allograft vasculopathy. Major progress has been made recently in elucidating the cellular and molecular mechanisms underlying neointima formation. However, no known curative treatment currently exists. In cases in which pharmacologic and surgical interventions have had limited success, gene therapy remains a potential strategy for the treatment of such vascular proliferative diseases. To date, recombinant adenoviral vectors continue to be the most efficient methods of gene transfer into the arterial wall. However, concerns over the safety of using viral vectors in a clinical situation have inspired the considerable progress that has been made in improving both viral and nonviral modes of gene transfer. This review discusses some of the recent insights and outstanding progress in vascular gene therapeutic approaches to inhibit neointima both from a biologic and therapeutic perspective. (Trends Cardiovasc Med 2003;13:102–106) © 2003, Elsevier Science Inc.
A number of tools for vascular gene therapy are available, each with different challenges. Therefore, considerable effort has been focused on improving current methods of gene delivery, which has resulted in a variety of gene transfer techniques being developed, each with differing transduction efficiencies and limitations. Generally, these methods can be categorized as to whether they employ viral or nonviral vectors of gene delivery (Table 1).
Martin F. Crook is at the National Heart, Lung, and Blood Institute, Cardiovascular Biology Branch, Bethesda, Maryland, USA. Levent M. Akyürek is at the Department of Anatomy & Cell Biology, and Wallenberg Laboratory, Göteborg University, Göteborg, Sweden. * Address correspondence to: Levent M. Akyürek, Department of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3-5, Box 420, SE-405 30, Göteborg, Sweden. Tel.: (46) 31-77-33331; fax: (46) 31-7733350; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
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• Nonviral Gene Transfer Probably the simplest method of gene transfer available today is the local delivery of naked DNA. In fact, one of the first approved human protocols for vascular gene transfer involved the local delivery of naked DNA encoding the vascular endothelial growth factor protein to stimulate angiogenesis (Isner et al. 1996). Fortunately, the naked DNA is not normally integrated into the host genome, thereby avoiding potential insertional mutagenesis; however, it results in shortterm transient gene expression, because the transgene is diluted with cell replication. Such methods have very low transfection efficiency (1%), but transgene expression has been shown to be detectable in skeletal muscle and cardiac myocytes (Leclerc et al. 1992). To enhance the low efficiency of naked DNA gene transfer, cationic liposome–DNA complexes have been developed that have been shown to improve gene delivery (Gao and Huang 1995). Additionally, gene delivery of DNA–liposome complexes have been
improved by incorporation of proteins derived from the hemagglutinating virus of Japan (HVJ-liposomes), which promotes receptor-mediated endocytosis (Morishita et al. 1993b). More recently, Kaneda et al. (2002) have devised a method whereby naked DNA is incorporated directly into the HVJ envelope protein, thereby facilitating its cellular uptake. Other newly developed nonviral techniques of gene transfer include those that use ultrasound (Taniyama et al. 2002) or intense near-infrared femtosecond laser pulses (Tirlapur and Konig 2002). Several groups have developed methods that promote the local delivery of genes to the arterial wall (reviewed by Nabel 1995). Recently, in vivo studies using stents coated with naked DNA or viral vectors also have been shown to be effective in promoting gene transfer into the vessel wall (Klugherz et al. 2000 and 2002). Alternatively, tissue-engineered stents have been utilized for implanting genetically modified cells into the arterial wall (Panetta et al. 2002). These methods will provide a significant advantage in terms of site-specific gene expression in the vasculature. • Viral Gene Transfer Recombinant viral vectors have been developed that exploit their natural receptormediated mechanisms of cell entry and thus are more effective at gene transfer. Several families of viruses have been utilized for gene transfer, including retroviruses, adenoviruses, and adeno-associated viruses (AAV) (Akyürek and Nabel 2002). Generally, these genetically modified viruses are replication defective and should, therefore, be nonpathogenic. However, their use is limited still by their immunogenicity and tissue toxicity in major organs, thus raising concerns about safety in a clinical setting. Retroviruses By the end of the 1980s, one of the first vector systems used for gene transfer in the arterial wall utilized replicationdefective retroviruses. This system allowed for the integration of the transgene into the host genome, providing long-term transgene expression, but raised concerns regarding insertional mutagenesis. Cultured endothelial (Nabel et al. 1989) or smooth muscle cells (SMCs) (Plautz et TCM Vol. 13, No. 3, 2003
Table 1. Tools for vascular gene therapy Nonviral gene transfer Naked plasmid DNA Plasmid DNA coupled with cationic liposomes Plasmid DNA coupled with HVJ-liposomes Ultrashort laser pulses Ultrasound-mediated enhancement of transfection Stents coated with naked DNA Viral gene transfer Retroviruses Lentiviruses Adenoviruses Gutted viruses Adenovirus-associated viruses Other viruses
al. 1991) have been infected ex vivo and seeded back in vivo. Furthermore, these genetically modified cells retained a normal life span in the vessel wall. However, the tropism of retroviruses is limited to replicating cells, thus reducing its applicability in normal arteries, where both endothelial and VSMC turnover is low. Recent progress has been made in the design of the lentivirus genus of retroviruses (Amado and Chen 1999). These vectors are able to infect cells regardless of their replication status, because their preintegration complex can get through the intact nuclear membrane of the target cell (Amado and Chen 1999). In addition, up to 6 months of transgene expression can be detected. Recently, these vectors have been used successfully, following catheter-based gene transfer, to monitor gene transfer efficiency by magnetic resonance imaging (Yang et al. 2001). In addition, pseudotype retroviral particles from the vesicular stomatitis virus have been developed (Kasahara et al. 1994). Adenoviruses In recent years, replication-defective adenoviral vectors have been used widely for experimental and clinical gene therapy studies (Akyürek and Nabel 2002). To date, adenoviral vectors remain the most efficient vectors to perform in vivo arterial gene transfer. They infect both replicating and nonreplicating mammalian cells and can be produced at titers as high as 1013 particles per milliliter, allowing for sufficient gene transfer in vivo. Adenoviral vectors do not integrate TCM Vol. 13, No. 3, 2003
into the host genome and therefore are unlikely to cause insertional mutagenesis. However, this limits transgene expression to only 2 to 3 weeks and may therefore not be applicable for longterm treatment. In addition, the use of first-generation adenoviral vectors has been limited by host inflammatory and immune responses that impair the duration of transgene expression and preclude repeat vector application (Nabel 1995). Nonetheless, because the majority of VSMC proliferation takes place early after vascular injury, short-term transient transgene expression may be sufficient to reduce neointima formation (Nabel 1995). Some of the limitations of first-generation adenoviral vectors discussed above have been overcome by the development of the so-called “gutless” or third-generation adenoviral systems, which lack much of the viral genome and are therefore far less immunogenic, which in turn promotes longer transgene expression. In one study (Kim et al. 2001), the phenotypic correction of hyperlipidemia caused by apolipoprotein E deficiency in mice was shown to last the natural life span without toxicity. Recently, progress has been made in the development of tissue-specific adenoviral vectors for arterial gene therapy. Transgene expression with viral heterologous promoters such as cytomegalovirus (CMV) and rous sarcoma virus (RSV) results in ubiquitous expression in many cells and tissues, including the liver and lung, whereas tissue-specific transcriptional gene regulation generally limits recombinant gene expression to specific cells and tissues (Kim et al. 1997). Using reporter genes, adenoviral vectors programmed by VSMC-specific promoters have been demonstrated to drive transgene expression exclusively in the arterial and venous SMCs both in vitro and in vivo (Kim et al. 1997, Akyürek et al. 2000). In an animal model of vascular injury, reporter gene expression driven by a smooth muscle-specific promoter, SM22, has been shown to be restricted to SMCs, whereas viral RSV promoter programmed unrestricted transgene expression in the vasculature following adenoviral gene transfer (Figure 1). Similar results have been demonstrated by specifically targeting vascular endothelial cells (Nicklin et al. 2001). Because not all intimal cells need or are intended to be transduced in vivo to reach a ther-
apeutic benefit, their low level of expression compared with viral promoters may be circumvented by a therapeutic approach that enhances the efficacy of gene transfer in the arterial wall (Akyürek et al. 2001). By limiting transgene expression, tissue-specific adenoviral gene therapy may provide added safety for human studies. AVVs AAVs are nonpathogenic parvoviruses that have attracted the interest of gene therapists because of their ability to integrate specifically into the host genome. This allows for long-term transgene expression with less of a chance to cause insertional mutagenesis compared with retroviral vectors. However, these vectors are limited by their packaging constraints and are difficult to generate at a titer high enough for clinical application. Other Viruses Herpes and vaccinia viruses are being developed (Clowes 1997); however, their applicability in the cardiovascular system has not been established. • Choice of Candidate Genes for Vascular Gene Transfer to Prevent Neointimal Hyperplasia Although substantial progress has been made in improving the technology for modifying gene expression in vivo, the question still remains as to which genes should be modified. VSMC proliferation plays an important role in restenosis and atherosclerosis, and the molecular mechanisms that regulate this process are only now being defined. Because restenosis has many components, including proliferation of SMCs, angiogenesis, recruitment of inflammatory and hematopoietic stem cells, apoptosis, platelet adherence and aggregation, thrombus formation, and vascular remodeling, all of these processes could be potential targets of a gene therapy approach (Table 2). Several studies have described numerous genes that tightly control transit through the cell cycle (reviewed in Nabel 2002); we limit our discussion to a variety of candidate genes that play important roles in cell-cycle regulation, emphasizing their utility for inhibiting neointima formation via gene therapy. In addition,
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Figure 1. Local intra-arterial administration of replication-deficient recombinant adenovirus in the iliofemoral arteries of Yorkshire domestic pigs. Three days after arterial injury induced by a double-balloon catheter and infection with either AdSM22-hpAP or AdRSV-hpAP, the injured arteries were harvested and stained for either alkaline phosphatase (AP) or cell-surface markers, such as smooth muscle-specific -actin and von Willebrand (vWF) factor detecting VSMC and endothelial cells, respectively. Arrowheads denote internal elastic laminae. In identical areas of sequential sections, black arrows indicate AP-expressing intimal and medial VSMC after infection with AdSM22-hpAP, whereas blue arrows point to endothelial and medial vascular SMCs after infection with AdRSV-hpAP. Sections were counterstained with methyl green. HpAP, human placental alkaline phosphatase. Original magnification 200.
we discuss the potential use of RNA interference (RNAi) in knocking down some of the candidate genes as an alternative to antisense technology. It has been known for some time that
Table2. Targetstopreventneointima formation in vascular diseases Proliferation of smooth muscle cells Angiogenesis Adhesion of inflammatory cells Endothelial dysfunction Apoptosis Unstable plaque disruption Thrombosis Progenitor hematopoietic stem cell differentiation Intimal lipid accumulation
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mitogens such as platelet-derived growth factor play an important role in stimulating VSMCs to proliferate once they have dedifferentiated from a contractile to a synthetic phenotype (Ross 1993). This has led to the understanding of the signal transduction pathways that ultimately activate the transcription of a variety of genes that are necessary for mitosis. Several studies have shown that overexpressing genes that negatively regulate these signal transduction pathways inhibit cellular proliferation in vitro and, thus, neointimal hyperplasia in vivo (Tanner et al. 1998, Tanner et al. 2000). The cyclin-dependent kinase inhibitor p27Kip1 is constitutively expressed at high levels in quiescent medial and intimal VSMCs of healthy arteries and negatively regulates cellular proliferation, primarily by inhibiting cyclin E-cyclin-
dependent kinase 2 activity (Tanner et al. 1998). However, upon vascular injury, p27Kip1 protein levels are reduced rapidly when mitogen-induced VSMC proliferation proceeds (Tanner et al. 1998). In turn, as the wound repair process progresses, the deposition of collagen and other extracellular matrix components leads to activation of autocrine and paracrine feedback loops, resulting in an increase in p27Kip1 expression and a reduction in cyclin E levels, thus promoting exit from the cell cycle (Tanner et al. 1998). These observations led to the hypothesis that the overexpression of p27Kip1 in the arteries after vascular injury may attenuate the proliferative response, which was subsequently demonstrated in balloonedinjured porcine arteries (Tanner et al. 2000). Likewise, other studies showed that the overexpression of any of the TCM Vol. 13, No. 3, 2003
cyclin-dependent kinase inhibitor (p16Ink4, p21Cip1, or p57Kip2) in various models of restenosis also led to a reduction in neointimal hyperplasia (reviewed in Nabel 2002). In addition, the overexpression of a fusion protein containing active portions of both p27Kip1 and p16Ink4 significantly reduced neointima formation in the porcine coronary arteries after balloon injury (Tsui et al. 2001). Many of the signal transduction pathways that are activated by mitogenic stimuli result in the activation of E2Fresponsive genes, such as cyclin E and cyclin A, that are essential for cell-cycle progression. These genes are negatively regulated by members of the Rb family, which, when hyperphosphorylated by cyclin D–cdk4/6 and cyclin E–cdk2 complexes, results in the release of the transcription factor E2F and activation of transcription (reviewed in Nabel 2002). Thus, several strategies have sought to reduce VSMC proliferation by inhibiting the transcriptional activity of the E2F transcription factor. Early work in the vascular gene therapy field showed that the overexpression of a nonphosphorylatable, constitutively active form of Rb lead to a reduction in neointima formation in rat and porcine models of restenosis (Chang et al. 1995). More recently, the overexpression of a related family member Rb2 (p130) also has been shown to inhibit VSMC proliferation and neointima formation by sequestering E2F (Claudio et al. 1999). In addition, another study (Wills et al. 2001) created a potent transcriptional repressor of E2F-dependent transcription by fusing the C-terminal fragment of Rb (p56) to the DNA and DP1-binding domains of E2F, which was shown to be effective in limiting neointima formation. In an alternative strategy, the delivery of decoy oligonucleotides that contain multiple E2F binding sites was effective in inhibiting neointima formation in animal studies and improved the patency of conduits used in coronary artery bypass graft (Mann et al. 1999). Likewise, recent studies (Ahn et al. 2002, Kume et al. 2002) demonstrated the benefit of using decoy oligonucleotides in inhibiting the activity of other transcription factors involved in cellular proliferation, such as AP-1. In contrast, the use of antisense technology has allowed for the inhibition of genes that promote cell division and thus has also been found to be effective TCM Vol. 13, No. 3, 2003
in reducing neointimal hyperplasia in various models of arterial injury. In addition, a recent study (Morishita et al. 1993a) has looked at the combined effect of inhibiting multiple cell-cycle genes such as proliferating-cell nuclear antigen and cdc2. Whereas these approaches were shown to be effective in inhibiting neointima formation, their efficacy may be improved with the advent of RNAi. This conserved process involves the silencing of specific genes by double-stranded RNA (Fire et al. 1998). Recently, the use of short doublestranded RNA oligonucleotides or small interfering RNA (siRNA) to specifically “knock-down” genes of interest was shown to be an effective tool in mammalian cells in vitro (Hannon 2002, Paddison et al. 2002). Furthermore, the generation of constructs that utilize a truncated RNA polymerase II promoter or an RNA polymerase III promoter to overexpress short-hairpin RNA species makes the prospect of combining these constructs with recombinant adenoviral technology realistic (Paddison et al. 2002, Xia et al. 2002). Recently, viral-mediated delivery of siRNA was shown to specifically reduce expression of targeted genes in various cells, both in vitro and in vivo (Xia et al. 2002). The merging of this RNAi with current modes of gene transfer may allow for the “knock-down” of any particular gene in vivo. This may be particularly useful in cases in which current clinical methods of gene expression inhibition have been ineffective. Thus, the choice of candidate genes for modification has become broader, and therefore might lead to further developments in the understanding and treatment of vascular diseases.
References Ahn JD, Morishita R, Kaneda Y, et al.: 2002. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 90:1325–1332. Akyürek LM, Nabel EG: 2002. Gene therapy of thrombotic and hemorrhagic disorders. In Loscalzo J, Schafer AI, eds. Thrombosis and Hemorrhage, 3rd ed. Philadelphia, Lippincott, Williams & Wilkins, pp 1096–1108. Akyürek LM, Nallamshetty S, Aoki K, et al.: 2001. Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo. Mol Ther 3:779–786.
Akyürek LM, Yang ZY, Aoki K, et al.: 2000. SM22 promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo. Mol Med 6:983–991. Amado RG, Chen ISY: 1999. Lentiviral vectors—the promise of the gene therapy within reach? Science 285:674–676. Chang MW, Barr E, Lu MM, Barton K, et al.: 1995. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest 96:2260–2268. Claudio PP, Fratta L, Farina F, et al.: 1999. Adenoviral RB2/p130 gene transfer inhibits smooth muscle cell proliferation and prevents restenosis after angioplasty. Circ Res 85:1032–1039 Clowes WA: 1997. Vascular gene transfer using smooth cells. Ann NY Acad Sci 811:293–297. Fire A, Xu S, Montgomery MK, et al.: 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. Gao X, Huang L: 1995. Cationic liposomemediated gene transfer. Gene Ther 2:710–722. Hannon GJ: 2002. RNA interference. Nature 418:244–251. Isner JM, Walsh K, Symes J, et al.: 1996. Arterial gene transfer for therapeutic angiogenesis in patients with peripheral artery disease. Hum Gene Ther 7:959–988. Kaneda Y, Nakajima T, Nishikawa T, et al.: 2002. Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system. Mol Ther 6:219–226. Kasahara N, Dozy AM, Kan YW: 1994. Tissue-specific targeting of retroviral vectors through ligand-receptor interactions. Science 266:1373–1376. Kim I-H, Jockowicz, Piedra PA, et al.: 2001. Lifetime correction of genetic deficiency in mice with a single injection of helperdependent adenoviral vector. Proc Natl Acad Sci USA 89:13,282–13,287. Kim S, Lin H, Barr E, et al.: 1997. Transcriptional targeting of replication-defective adenovirus transgene expression to smooth muscle cells in vivo. J Clin Invest 100:1006–1014. Klugherz BD, Jones PL, Cui X, et al.: 2000. Gene delivery from a DNA controlledrelease stent in porcine coronary arteries. Nat Biotechnol 18:1181–1184. Klugherz BD, Song C, DeFelice S, et al.: 2002. Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Hum Gene Ther 10:443-454. Kume M, Komori K, Matsumoto T, et al.: 2002. Administration of a decoy against the activator protein-1 binding site suppresses neointimal thickening in rabbit balloon-injured arteries. Circulation 105: 1226–1232.
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Leclerc G, Gal D, Takeshita S, et al.: 1992. Percutaneous arterial gene transfer in a rabbit model. Efficiency in normal and balloon-dilated atherosclerotic arteries. J Clin Invest 90:936–944. Mann MJ, Whittemore AD, Donaldson MC, et al.: 1999. Ex vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet 354:1493–1498. Morishita R, Gibbons GH, Ellison KE, et al.: 1993a. Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 90:8474–8478. Morishita R, Gibbons GH, Kaneda Y, et al.: 1993b. Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells. Hypertension 21:894– 899. Nabel EG: 1995. Gene therapy for cardiovascular disease. Circulation 91:541–548. Nabel EG: 2002. CDKs and CKIs: Molecular targets for tissue remodeling. Nat Rev Drug Discov 1:587–598. Nabel EG, Plautz G, Boyce FM, et al.: 1989. Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science 244:1342–1344. Nicklin SA, Reynolds PN, Brosnan MJ, et al.: 2001. Analysis of cell-specific promoters for viral gene therapy targeted at the vascular endothelium. Hypertension 2001:38:65–70. Paddison PJ, Caudy AA, Bernstein E, et al.: 2002. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958. Panetta CJ, Miyauchi K, Berry D, et al.: 2002. A tissue-engineered stent for cell-based vascular gene transfer. Hum Gene Ther 13:433–441. Plautz G, Nabel EG, Nabel GJ: 1991. Introduction of vascular smooth muscle cells expressing recombinant genes in vivo. Circulation 83:578–583. Ross R: 1993. The pathogenesis of athersclerosis: a perspective for the 1990s. Nature 362:801–809. Taniyama Y, Tachibana K, Hiraoka K, et al.: 2002. Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 9:372–380. Tanner FC, Boehm M, Akyürek LM, et al.: 2000. Differential effects of the cyclindependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation. Circulation 101: 2022–2025. Tanner FC, Yang ZY, Duckers E, et al.: 1998. Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res 82: 396–403.
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Tirlapur UK, Konig K: 2002. Targeted transfection by femtosecond laser. Nature 418: 290.
and neointima formation. Gene Ther 8: 1847–1854.
Tsui LV, Camrud A, Mondesire J, et al.: 2001. p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasi and coronary artery occlusion. Circ Res 89:323–328.
Xia H, Mao Q, Paulson HL, et al.: 2002. siRNA-mediated gene silencing in vitro and in vivo. Nature Biotech 20:1006–1010.
Wills KN, Mano T, Avanzini JB, et al.: 2001. Tissue-specific expression of an anti-proliferative hybrid transgene from the human smooth muscle alpha-actin promoter suppresses smooth muscle cell proliferation
Yang X, Atalar E, Li D, et al: 2001. Magnetic resonance imaging permits in vivo monitoring of catheter-based vascular gene delivery. Circulation 104:1588–1590. PII S1050-1738(02)00255-4
TCM
Genetic Manipulation of Primate Embryonic and Hematopoietic Stem Cells with Simian Lentivirus Vectors Yutaka Hanazono,* Takayuki Asano, Yasuji Ueda, and Keiya Ozawa
During the past several years, many articles have described how human embryonic stem (ES) cells and adult hematopoietic stem cells (HSCs) can differentiate into cardiac muscle, blood vessels, and various other types of cells. The articles raised the expectation that these stem cells may become useful for the treatment of a variety of diseases, including cardiovascular diseases. Genetic manipulation of ES cells and HSCs would be important for such future applications of the cells. Until now, retroviral vectors have been used primarily for stable expression of transgenes in murine ES cells and HSCs. Because murine models may not predict reliably the biology of ES cells and HSCs in humans, we have utilized primate ES cells and HSCs as targets of gene transfer. We have shown that primate ES cells and HSCs can be transduced efficiently with lentiviral vectors derived from the simian immunodeficiency virus, and that the high transgene expression persists without transcriptional silencing. This highly efficient gene transfer method allows for safe and faithful gene delivery to primate ES cells and HSCs to test potential research and therapeutic applications. (Trends Cardiovasc Med 2003;13:106–110) © 2003, Elsevier Science Inc.
Yutaka Hanazono, Takayuki Asano, and Keiya Ozawa are at the Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan. Yasuji Ueda is at the DNAVEC Research Inc., Ibaraki, Japan. * Address correspondence to: Yutaka Hanazono, MD, PhD, Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. Tel.: (81) 285-58-7402; fax: (81) 285-448675; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
• Stem Cell Therapy and Animal Models With the establishment of embryonic stem (ES) and embryonic germ (EG) cell lines (Reubinoff et al. 2000, Shamblott et al. 1998, Thomson et al. 1998), great attention has been given to the therapeutic potential of these cells from both a biologic and medical perspective. This potential is based on their remarkable ability to differentiate into most of the specialized cells of the three EG layers and their ability TCM Vol. 13, No. 3, 2003
Vascular smooth muscle cell (VSMC) proliferation after arterial injury results in neointima formation and plays an important role in the pathogenesis of restenosis after angioplasty, in-stent restenosis, vascular bypass graft occlusion, and allograft vasculopathy. Major progress has been made recently in elucidating the cellular and molecular mechanisms underlying neointima formation. However, no known curative treatment currently exists. In cases in which pharmacologic and surgical interventions have had limited success, gene therapy remains a potential strategy for the treatment of such vascular proliferative diseases. To date, recombinant adenoviral vectors continue to be the most efficient methods of gene transfer into the arterial wall. However, concerns over the safety of using viral vectors in a clinical situation have inspired the considerable progress that has been made in improving both viral and nonviral modes of gene transfer. This review discusses some of the recent insights and outstanding progress in vascular gene therapeutic approaches to inhibit neointima both from a biologic and therapeutic perspective. (Trends Cardiovasc Med 2003;13:102–106) © 2003, Elsevier Science Inc.
A number of tools for vascular gene therapy are available, each with different challenges. Therefore, considerable effort has been focused on improving current methods of gene delivery, which has resulted in a variety of gene transfer techniques being developed, each with differing transduction efficiencies and limitations. Generally, these methods can be categorized as to whether they employ viral or nonviral vectors of gene delivery (Table 1).
Martin F. Crook is at the National Heart, Lung, and Blood Institute, Cardiovascular Biology Branch, Bethesda, Maryland, USA. Levent M. Akyürek is at the Department of Anatomy & Cell Biology, and Wallenberg Laboratory, Göteborg University, Göteborg, Sweden. * Address correspondence to: Levent M. Akyürek, Department of Anatomy and Cell Biology, Göteborg University, Medicinaregatan 3-5, Box 420, SE-405 30, Göteborg, Sweden. Tel.: (46) 31-77-33331; fax: (46) 31-7733350; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
102
• Nonviral Gene Transfer Probably the simplest method of gene transfer available today is the local delivery of naked DNA. In fact, one of the first approved human protocols for vascular gene transfer involved the local delivery of naked DNA encoding the vascular endothelial growth factor protein to stimulate angiogenesis (Isner et al. 1996). Fortunately, the naked DNA is not normally integrated into the host genome, thereby avoiding potential insertional mutagenesis; however, it results in shortterm transient gene expression, because the transgene is diluted with cell replication. Such methods have very low transfection efficiency (1%), but transgene expression has been shown to be detectable in skeletal muscle and cardiac myocytes (Leclerc et al. 1992). To enhance the low efficiency of naked DNA gene transfer, cationic liposome–DNA complexes have been developed that have been shown to improve gene delivery (Gao and Huang 1995). Additionally, gene delivery of DNA–liposome complexes have been
improved by incorporation of proteins derived from the hemagglutinating virus of Japan (HVJ-liposomes), which promotes receptor-mediated endocytosis (Morishita et al. 1993b). More recently, Kaneda et al. (2002) have devised a method whereby naked DNA is incorporated directly into the HVJ envelope protein, thereby facilitating its cellular uptake. Other newly developed nonviral techniques of gene transfer include those that use ultrasound (Taniyama et al. 2002) or intense near-infrared femtosecond laser pulses (Tirlapur and Konig 2002). Several groups have developed methods that promote the local delivery of genes to the arterial wall (reviewed by Nabel 1995). Recently, in vivo studies using stents coated with naked DNA or viral vectors also have been shown to be effective in promoting gene transfer into the vessel wall (Klugherz et al. 2000 and 2002). Alternatively, tissue-engineered stents have been utilized for implanting genetically modified cells into the arterial wall (Panetta et al. 2002). These methods will provide a significant advantage in terms of site-specific gene expression in the vasculature. • Viral Gene Transfer Recombinant viral vectors have been developed that exploit their natural receptormediated mechanisms of cell entry and thus are more effective at gene transfer. Several families of viruses have been utilized for gene transfer, including retroviruses, adenoviruses, and adeno-associated viruses (AAV) (Akyürek and Nabel 2002). Generally, these genetically modified viruses are replication defective and should, therefore, be nonpathogenic. However, their use is limited still by their immunogenicity and tissue toxicity in major organs, thus raising concerns about safety in a clinical setting. Retroviruses By the end of the 1980s, one of the first vector systems used for gene transfer in the arterial wall utilized replicationdefective retroviruses. This system allowed for the integration of the transgene into the host genome, providing long-term transgene expression, but raised concerns regarding insertional mutagenesis. Cultured endothelial (Nabel et al. 1989) or smooth muscle cells (SMCs) (Plautz et TCM Vol. 13, No. 3, 2003
Table 1. Tools for vascular gene therapy Nonviral gene transfer Naked plasmid DNA Plasmid DNA coupled with cationic liposomes Plasmid DNA coupled with HVJ-liposomes Ultrashort laser pulses Ultrasound-mediated enhancement of transfection Stents coated with naked DNA Viral gene transfer Retroviruses Lentiviruses Adenoviruses Gutted viruses Adenovirus-associated viruses Other viruses
al. 1991) have been infected ex vivo and seeded back in vivo. Furthermore, these genetically modified cells retained a normal life span in the vessel wall. However, the tropism of retroviruses is limited to replicating cells, thus reducing its applicability in normal arteries, where both endothelial and VSMC turnover is low. Recent progress has been made in the design of the lentivirus genus of retroviruses (Amado and Chen 1999). These vectors are able to infect cells regardless of their replication status, because their preintegration complex can get through the intact nuclear membrane of the target cell (Amado and Chen 1999). In addition, up to 6 months of transgene expression can be detected. Recently, these vectors have been used successfully, following catheter-based gene transfer, to monitor gene transfer efficiency by magnetic resonance imaging (Yang et al. 2001). In addition, pseudotype retroviral particles from the vesicular stomatitis virus have been developed (Kasahara et al. 1994). Adenoviruses In recent years, replication-defective adenoviral vectors have been used widely for experimental and clinical gene therapy studies (Akyürek and Nabel 2002). To date, adenoviral vectors remain the most efficient vectors to perform in vivo arterial gene transfer. They infect both replicating and nonreplicating mammalian cells and can be produced at titers as high as 1013 particles per milliliter, allowing for sufficient gene transfer in vivo. Adenoviral vectors do not integrate TCM Vol. 13, No. 3, 2003
into the host genome and therefore are unlikely to cause insertional mutagenesis. However, this limits transgene expression to only 2 to 3 weeks and may therefore not be applicable for longterm treatment. In addition, the use of first-generation adenoviral vectors has been limited by host inflammatory and immune responses that impair the duration of transgene expression and preclude repeat vector application (Nabel 1995). Nonetheless, because the majority of VSMC proliferation takes place early after vascular injury, short-term transient transgene expression may be sufficient to reduce neointima formation (Nabel 1995). Some of the limitations of first-generation adenoviral vectors discussed above have been overcome by the development of the so-called “gutless” or third-generation adenoviral systems, which lack much of the viral genome and are therefore far less immunogenic, which in turn promotes longer transgene expression. In one study (Kim et al. 2001), the phenotypic correction of hyperlipidemia caused by apolipoprotein E deficiency in mice was shown to last the natural life span without toxicity. Recently, progress has been made in the development of tissue-specific adenoviral vectors for arterial gene therapy. Transgene expression with viral heterologous promoters such as cytomegalovirus (CMV) and rous sarcoma virus (RSV) results in ubiquitous expression in many cells and tissues, including the liver and lung, whereas tissue-specific transcriptional gene regulation generally limits recombinant gene expression to specific cells and tissues (Kim et al. 1997). Using reporter genes, adenoviral vectors programmed by VSMC-specific promoters have been demonstrated to drive transgene expression exclusively in the arterial and venous SMCs both in vitro and in vivo (Kim et al. 1997, Akyürek et al. 2000). In an animal model of vascular injury, reporter gene expression driven by a smooth muscle-specific promoter, SM22, has been shown to be restricted to SMCs, whereas viral RSV promoter programmed unrestricted transgene expression in the vasculature following adenoviral gene transfer (Figure 1). Similar results have been demonstrated by specifically targeting vascular endothelial cells (Nicklin et al. 2001). Because not all intimal cells need or are intended to be transduced in vivo to reach a ther-
apeutic benefit, their low level of expression compared with viral promoters may be circumvented by a therapeutic approach that enhances the efficacy of gene transfer in the arterial wall (Akyürek et al. 2001). By limiting transgene expression, tissue-specific adenoviral gene therapy may provide added safety for human studies. AVVs AAVs are nonpathogenic parvoviruses that have attracted the interest of gene therapists because of their ability to integrate specifically into the host genome. This allows for long-term transgene expression with less of a chance to cause insertional mutagenesis compared with retroviral vectors. However, these vectors are limited by their packaging constraints and are difficult to generate at a titer high enough for clinical application. Other Viruses Herpes and vaccinia viruses are being developed (Clowes 1997); however, their applicability in the cardiovascular system has not been established. • Choice of Candidate Genes for Vascular Gene Transfer to Prevent Neointimal Hyperplasia Although substantial progress has been made in improving the technology for modifying gene expression in vivo, the question still remains as to which genes should be modified. VSMC proliferation plays an important role in restenosis and atherosclerosis, and the molecular mechanisms that regulate this process are only now being defined. Because restenosis has many components, including proliferation of SMCs, angiogenesis, recruitment of inflammatory and hematopoietic stem cells, apoptosis, platelet adherence and aggregation, thrombus formation, and vascular remodeling, all of these processes could be potential targets of a gene therapy approach (Table 2). Several studies have described numerous genes that tightly control transit through the cell cycle (reviewed in Nabel 2002); we limit our discussion to a variety of candidate genes that play important roles in cell-cycle regulation, emphasizing their utility for inhibiting neointima formation via gene therapy. In addition,
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Figure 1. Local intra-arterial administration of replication-deficient recombinant adenovirus in the iliofemoral arteries of Yorkshire domestic pigs. Three days after arterial injury induced by a double-balloon catheter and infection with either AdSM22-hpAP or AdRSV-hpAP, the injured arteries were harvested and stained for either alkaline phosphatase (AP) or cell-surface markers, such as smooth muscle-specific -actin and von Willebrand (vWF) factor detecting VSMC and endothelial cells, respectively. Arrowheads denote internal elastic laminae. In identical areas of sequential sections, black arrows indicate AP-expressing intimal and medial VSMC after infection with AdSM22-hpAP, whereas blue arrows point to endothelial and medial vascular SMCs after infection with AdRSV-hpAP. Sections were counterstained with methyl green. HpAP, human placental alkaline phosphatase. Original magnification 200.
we discuss the potential use of RNA interference (RNAi) in knocking down some of the candidate genes as an alternative to antisense technology. It has been known for some time that
Table2. Targetstopreventneointima formation in vascular diseases Proliferation of smooth muscle cells Angiogenesis Adhesion of inflammatory cells Endothelial dysfunction Apoptosis Unstable plaque disruption Thrombosis Progenitor hematopoietic stem cell differentiation Intimal lipid accumulation
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mitogens such as platelet-derived growth factor play an important role in stimulating VSMCs to proliferate once they have dedifferentiated from a contractile to a synthetic phenotype (Ross 1993). This has led to the understanding of the signal transduction pathways that ultimately activate the transcription of a variety of genes that are necessary for mitosis. Several studies have shown that overexpressing genes that negatively regulate these signal transduction pathways inhibit cellular proliferation in vitro and, thus, neointimal hyperplasia in vivo (Tanner et al. 1998, Tanner et al. 2000). The cyclin-dependent kinase inhibitor p27Kip1 is constitutively expressed at high levels in quiescent medial and intimal VSMCs of healthy arteries and negatively regulates cellular proliferation, primarily by inhibiting cyclin E-cyclin-
dependent kinase 2 activity (Tanner et al. 1998). However, upon vascular injury, p27Kip1 protein levels are reduced rapidly when mitogen-induced VSMC proliferation proceeds (Tanner et al. 1998). In turn, as the wound repair process progresses, the deposition of collagen and other extracellular matrix components leads to activation of autocrine and paracrine feedback loops, resulting in an increase in p27Kip1 expression and a reduction in cyclin E levels, thus promoting exit from the cell cycle (Tanner et al. 1998). These observations led to the hypothesis that the overexpression of p27Kip1 in the arteries after vascular injury may attenuate the proliferative response, which was subsequently demonstrated in balloonedinjured porcine arteries (Tanner et al. 2000). Likewise, other studies showed that the overexpression of any of the TCM Vol. 13, No. 3, 2003
cyclin-dependent kinase inhibitor (p16Ink4, p21Cip1, or p57Kip2) in various models of restenosis also led to a reduction in neointimal hyperplasia (reviewed in Nabel 2002). In addition, the overexpression of a fusion protein containing active portions of both p27Kip1 and p16Ink4 significantly reduced neointima formation in the porcine coronary arteries after balloon injury (Tsui et al. 2001). Many of the signal transduction pathways that are activated by mitogenic stimuli result in the activation of E2Fresponsive genes, such as cyclin E and cyclin A, that are essential for cell-cycle progression. These genes are negatively regulated by members of the Rb family, which, when hyperphosphorylated by cyclin D–cdk4/6 and cyclin E–cdk2 complexes, results in the release of the transcription factor E2F and activation of transcription (reviewed in Nabel 2002). Thus, several strategies have sought to reduce VSMC proliferation by inhibiting the transcriptional activity of the E2F transcription factor. Early work in the vascular gene therapy field showed that the overexpression of a nonphosphorylatable, constitutively active form of Rb lead to a reduction in neointima formation in rat and porcine models of restenosis (Chang et al. 1995). More recently, the overexpression of a related family member Rb2 (p130) also has been shown to inhibit VSMC proliferation and neointima formation by sequestering E2F (Claudio et al. 1999). In addition, another study (Wills et al. 2001) created a potent transcriptional repressor of E2F-dependent transcription by fusing the C-terminal fragment of Rb (p56) to the DNA and DP1-binding domains of E2F, which was shown to be effective in limiting neointima formation. In an alternative strategy, the delivery of decoy oligonucleotides that contain multiple E2F binding sites was effective in inhibiting neointima formation in animal studies and improved the patency of conduits used in coronary artery bypass graft (Mann et al. 1999). Likewise, recent studies (Ahn et al. 2002, Kume et al. 2002) demonstrated the benefit of using decoy oligonucleotides in inhibiting the activity of other transcription factors involved in cellular proliferation, such as AP-1. In contrast, the use of antisense technology has allowed for the inhibition of genes that promote cell division and thus has also been found to be effective TCM Vol. 13, No. 3, 2003
in reducing neointimal hyperplasia in various models of arterial injury. In addition, a recent study (Morishita et al. 1993a) has looked at the combined effect of inhibiting multiple cell-cycle genes such as proliferating-cell nuclear antigen and cdc2. Whereas these approaches were shown to be effective in inhibiting neointima formation, their efficacy may be improved with the advent of RNAi. This conserved process involves the silencing of specific genes by double-stranded RNA (Fire et al. 1998). Recently, the use of short doublestranded RNA oligonucleotides or small interfering RNA (siRNA) to specifically “knock-down” genes of interest was shown to be an effective tool in mammalian cells in vitro (Hannon 2002, Paddison et al. 2002). Furthermore, the generation of constructs that utilize a truncated RNA polymerase II promoter or an RNA polymerase III promoter to overexpress short-hairpin RNA species makes the prospect of combining these constructs with recombinant adenoviral technology realistic (Paddison et al. 2002, Xia et al. 2002). Recently, viral-mediated delivery of siRNA was shown to specifically reduce expression of targeted genes in various cells, both in vitro and in vivo (Xia et al. 2002). The merging of this RNAi with current modes of gene transfer may allow for the “knock-down” of any particular gene in vivo. This may be particularly useful in cases in which current clinical methods of gene expression inhibition have been ineffective. Thus, the choice of candidate genes for modification has become broader, and therefore might lead to further developments in the understanding and treatment of vascular diseases.
References Ahn JD, Morishita R, Kaneda Y, et al.: 2002. Inhibitory effects of novel AP-1 decoy oligodeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neointimal formation in vivo. Circ Res 90:1325–1332. Akyürek LM, Nabel EG: 2002. Gene therapy of thrombotic and hemorrhagic disorders. In Loscalzo J, Schafer AI, eds. Thrombosis and Hemorrhage, 3rd ed. Philadelphia, Lippincott, Williams & Wilkins, pp 1096–1108. Akyürek LM, Nallamshetty S, Aoki K, et al.: 2001. Coexpression of guanylate kinase with thymidine kinase enhances prodrug cell killing in vitro and suppresses vascular smooth muscle cell proliferation in vivo. Mol Ther 3:779–786.
Akyürek LM, Yang ZY, Aoki K, et al.: 2000. SM22 promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo. Mol Med 6:983–991. Amado RG, Chen ISY: 1999. Lentiviral vectors—the promise of the gene therapy within reach? Science 285:674–676. Chang MW, Barr E, Lu MM, Barton K, et al.: 1995. Adenovirus-mediated over-expression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest 96:2260–2268. Claudio PP, Fratta L, Farina F, et al.: 1999. Adenoviral RB2/p130 gene transfer inhibits smooth muscle cell proliferation and prevents restenosis after angioplasty. Circ Res 85:1032–1039 Clowes WA: 1997. Vascular gene transfer using smooth cells. Ann NY Acad Sci 811:293–297. Fire A, Xu S, Montgomery MK, et al.: 1998. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. Gao X, Huang L: 1995. Cationic liposomemediated gene transfer. Gene Ther 2:710–722. Hannon GJ: 2002. RNA interference. Nature 418:244–251. Isner JM, Walsh K, Symes J, et al.: 1996. Arterial gene transfer for therapeutic angiogenesis in patients with peripheral artery disease. Hum Gene Ther 7:959–988. Kaneda Y, Nakajima T, Nishikawa T, et al.: 2002. Hemagglutinating virus of Japan (HVJ) envelope vector as a versatile gene delivery system. Mol Ther 6:219–226. Kasahara N, Dozy AM, Kan YW: 1994. Tissue-specific targeting of retroviral vectors through ligand-receptor interactions. Science 266:1373–1376. Kim I-H, Jockowicz, Piedra PA, et al.: 2001. Lifetime correction of genetic deficiency in mice with a single injection of helperdependent adenoviral vector. Proc Natl Acad Sci USA 89:13,282–13,287. Kim S, Lin H, Barr E, et al.: 1997. Transcriptional targeting of replication-defective adenovirus transgene expression to smooth muscle cells in vivo. J Clin Invest 100:1006–1014. Klugherz BD, Jones PL, Cui X, et al.: 2000. Gene delivery from a DNA controlledrelease stent in porcine coronary arteries. Nat Biotechnol 18:1181–1184. Klugherz BD, Song C, DeFelice S, et al.: 2002. Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Hum Gene Ther 10:443-454. Kume M, Komori K, Matsumoto T, et al.: 2002. Administration of a decoy against the activator protein-1 binding site suppresses neointimal thickening in rabbit balloon-injured arteries. Circulation 105: 1226–1232.
105
Leclerc G, Gal D, Takeshita S, et al.: 1992. Percutaneous arterial gene transfer in a rabbit model. Efficiency in normal and balloon-dilated atherosclerotic arteries. J Clin Invest 90:936–944. Mann MJ, Whittemore AD, Donaldson MC, et al.: 1999. Ex vivo gene therapy of human vascular bypass grafts with E2F decoy: the PREVENT single-centre, randomised, controlled trial. Lancet 354:1493–1498. Morishita R, Gibbons GH, Ellison KE, et al.: 1993a. Single intraluminal delivery of antisense cdc2 kinase and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of neointimal hyperplasia. Proc Natl Acad Sci USA 90:8474–8478. Morishita R, Gibbons GH, Kaneda Y, et al.: 1993b. Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells. Hypertension 21:894– 899. Nabel EG: 1995. Gene therapy for cardiovascular disease. Circulation 91:541–548. Nabel EG: 2002. CDKs and CKIs: Molecular targets for tissue remodeling. Nat Rev Drug Discov 1:587–598. Nabel EG, Plautz G, Boyce FM, et al.: 1989. Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science 244:1342–1344. Nicklin SA, Reynolds PN, Brosnan MJ, et al.: 2001. Analysis of cell-specific promoters for viral gene therapy targeted at the vascular endothelium. Hypertension 2001:38:65–70. Paddison PJ, Caudy AA, Bernstein E, et al.: 2002. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16:948–958. Panetta CJ, Miyauchi K, Berry D, et al.: 2002. A tissue-engineered stent for cell-based vascular gene transfer. Hum Gene Ther 13:433–441. Plautz G, Nabel EG, Nabel GJ: 1991. Introduction of vascular smooth muscle cells expressing recombinant genes in vivo. Circulation 83:578–583. Ross R: 1993. The pathogenesis of athersclerosis: a perspective for the 1990s. Nature 362:801–809. Taniyama Y, Tachibana K, Hiraoka K, et al.: 2002. Development of safe and efficient novel nonviral gene transfer using ultrasound: enhancement of transfection efficiency of naked plasmid DNA in skeletal muscle. Gene Ther 9:372–380. Tanner FC, Boehm M, Akyürek LM, et al.: 2000. Differential effects of the cyclindependent kinase inhibitors p27(Kip1), p21(Cip1), and p16(Ink4) on vascular smooth muscle cell proliferation. Circulation 101: 2022–2025. Tanner FC, Yang ZY, Duckers E, et al.: 1998. Expression of cyclin-dependent kinase inhibitors in vascular disease. Circ Res 82: 396–403.
106
Tirlapur UK, Konig K: 2002. Targeted transfection by femtosecond laser. Nature 418: 290.
and neointima formation. Gene Ther 8: 1847–1854.
Tsui LV, Camrud A, Mondesire J, et al.: 2001. p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasi and coronary artery occlusion. Circ Res 89:323–328.
Xia H, Mao Q, Paulson HL, et al.: 2002. siRNA-mediated gene silencing in vitro and in vivo. Nature Biotech 20:1006–1010.
Wills KN, Mano T, Avanzini JB, et al.: 2001. Tissue-specific expression of an anti-proliferative hybrid transgene from the human smooth muscle alpha-actin promoter suppresses smooth muscle cell proliferation
Yang X, Atalar E, Li D, et al: 2001. Magnetic resonance imaging permits in vivo monitoring of catheter-based vascular gene delivery. Circulation 104:1588–1590. PII S1050-1738(02)00255-4
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Genetic Manipulation of Primate Embryonic and Hematopoietic Stem Cells with Simian Lentivirus Vectors Yutaka Hanazono,* Takayuki Asano, Yasuji Ueda, and Keiya Ozawa
During the past several years, many articles have described how human embryonic stem (ES) cells and adult hematopoietic stem cells (HSCs) can differentiate into cardiac muscle, blood vessels, and various other types of cells. The articles raised the expectation that these stem cells may become useful for the treatment of a variety of diseases, including cardiovascular diseases. Genetic manipulation of ES cells and HSCs would be important for such future applications of the cells. Until now, retroviral vectors have been used primarily for stable expression of transgenes in murine ES cells and HSCs. Because murine models may not predict reliably the biology of ES cells and HSCs in humans, we have utilized primate ES cells and HSCs as targets of gene transfer. We have shown that primate ES cells and HSCs can be transduced efficiently with lentiviral vectors derived from the simian immunodeficiency virus, and that the high transgene expression persists without transcriptional silencing. This highly efficient gene transfer method allows for safe and faithful gene delivery to primate ES cells and HSCs to test potential research and therapeutic applications. (Trends Cardiovasc Med 2003;13:106–110) © 2003, Elsevier Science Inc.
Yutaka Hanazono, Takayuki Asano, and Keiya Ozawa are at the Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, Tochigi, Japan. Yasuji Ueda is at the DNAVEC Research Inc., Ibaraki, Japan. * Address correspondence to: Yutaka Hanazono, MD, PhD, Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi, Kawachi, Tochigi 329-0498, Japan. Tel.: (81) 285-58-7402; fax: (81) 285-448675; e-mail: [email protected]. © 2003, Elsevier Science Inc. All rights reserved. 1050-1738/03/$-see front matter
• Stem Cell Therapy and Animal Models With the establishment of embryonic stem (ES) and embryonic germ (EG) cell lines (Reubinoff et al. 2000, Shamblott et al. 1998, Thomson et al. 1998), great attention has been given to the therapeutic potential of these cells from both a biologic and medical perspective. This potential is based on their remarkable ability to differentiate into most of the specialized cells of the three EG layers and their ability TCM Vol. 13, No. 3, 2003