Hum. Reprod. Advance Access originally published online on January 8, 2008
Human Reproduction 2008 23(3):514-524; doi:10.1093/humrep/dem410
Toward gene therapy of uterine fibroids: targeting modified adenovirus to human leiomyoma cells
1 Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX 77555-0587, USA 2 Faculty of Pharmacy, Department of Pharmacology and Toxicology, Al-Azhar University, Cairo, Egypt 3 Gene Therapy Center, University of Alabama at Birmingham, AL 35924, USA 4 Department of Obstetrics and Gynecology, Center for Women's Health Research, Meharry Medical College, 1005 Dr D.B. Todd Jr Blvd., Nashville, TN 37208, USA
5Correspondence address. Tel: +1-615-963-3148; Fax: +1-615-963-3125; E-mail: ahendy{at}mmc.edu
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Abstract |
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BACKGROUND: To circumvent the paucity of the primary adenovirus (Ad5) receptor and the non-specific Ad5 tropism in the context of uterine leiomyoma cells, Ad5 modification strategies would be beneficial.
METHODS: We screened several modified adenoviruses to identify the most efficient and selective virus toward human leiomyoma cells to be used as candidate for delivering therapeutic genes. We propagated: wild-type Ad5-luc, fiber-modified viruses: ad5 RGD-luc, Ad5-Sigma–luc, Ad5/3-luc and Ad5-CAV2-luc, as well as transcriptional targeted viruses: ad5 survivin-luc, Ad5-heparanase-luc, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc, on 293 cells and purified them by double CsCL density centrifugation. Then we transfected primary cultures of human leiomyoma cells derived from fibroids of four different patients, telomerase-immortalized human leiomyoma cell line (huLM), telomerase-immortalized normal human myometrial cell line (HM9) and immortalized normal human liver cells (THLE3) with the viruses at 5, 10 and 50 plaque-forming units (PFU)/cell. After 48 h, luciferase activities were measured and normalized to the total cellular protein content.
RESULTS: Ad5-RGD-luc and Ad5-CAV2-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc at 5, 10 and 50 pfu/cell showed significantly higher expression levels of luciferase activity in both primary and immortalized human leiomyoma cells when compared with Ad5-Luc. Additionally, these modified viruses demonstrated selectivity toward leiomyoma cells, compared with myometrial cells and exhibited lower liver cell transduction, compared with Ad5-luc, at the same dose levels.
CONCLUSIONS: Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc are promising delivery vehicles in the context of leiomyoma gene therapy.
Key words: uterine leiomyoma/gene therapy/adenovirus targeting strategies
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Introduction |
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Uterine leiomyomas are the most common gynecological tumors and affect more than 25% of reproductive-age women (Buttram et al., 1981
; Stewart, 2001). One study revealed prevalence as high as 77% (Cramer and Patel, 1990). Symptomatic fibroids are associated with a host of clinical problems including menorrhagia, pelvic pressure, pelvic pain, spontaneous abortions and infertility (Farhi et al., 1995; Eldar-Geva et al., 1998; Hart et al., 2001; Surrey et al., 2001). Leiomyoma-associated manifestations are responsible for about one-third of all hospital admissions for gynecological services and 
370 000 of all hysterectomies performed annually in the United States alone (Cramer and Patel, 1990; Lepine et al., 1997). Although different treatment modalities for uterine leiomyomas are currently available, including classical surgical options (hysterectomy and myomectomy) (Cramer and Patel, 1990; Lepine et al., 1997; Vilos, 2000), non-invasive options such as myolysis and uterine artery embolization (Vilos et al., 1998; Liu, 2000; Payne and Haney, 2003), as well as medicinal options such as GnRH agonists and the progesterone antagonist, RU 486(mifepristone) (Buttram and Reiter, 1981; Andreyko et al., 1987; Kettel et al., 1994; Eisinger et al., 2003), unfortunately none of these are optimal and they each imply their own limitations. In general, the surgical approach, the mainstay for leiomyoma treatment (Lefebvre et al., 2003), is costly, especially considering the long post-operative recovery time and in 15–38% of cases (VeKaut, 1993), it carries a risk of major complications such as post-operative hemorrhage, fever, injury to adjacent organs and possible extensive pelvic adhesions and, hence, the preclusion of future fertility (Vercellini et al., 1998; Al Hendy and Salama, 2006). Therefore, the development of novel therapeutic strategies for uterine leiomyoma would be greatly welcomed, especially those that are localized, conservative and do not interfere with hormonal levels.
Gene therapy is a promising approach; it involves delivery of genetic materials to target cells to achieve therapeutic benefits, such as interfering with certain harmful gene functions, restoring lost function or initiating a new function (Al Hendy and Salama, 2006). Although there are many ways to deliver therapeutic genes to target tumors, the adenoviral (Ad) vector is the most commonly used vehicle in gene therapy clinical trials (Hallenbeck and Stevenson, 2000; Nettelbeck et al., 2000). The use of Ad vectors in gene therapy is advantageous because of many positive attributes including the following: (i) Ad has the ability to provide efficient in vivo gene transfer to both dividing and non-dividing cells (Bangari and Mittal, 2006); (ii) Ad has exhibited higher in vivo stability; (iii) Ad stocks can be prepared to ultra-pure high concentrations (1013 particles/ml), which allow the delivery of large amounts of viral particles in finite volumes (Kozarsky and Wilson, 1993); (iv) Ad is non-oncogenic and stays episomic, which makes it relatively safe (Rein et al., 2006); (v) Ad can accommodate larger transgenes of upto 7.5 kb (Vorburger and Hunt, 2002); (vi) Ad supports high levels of gene targeting to the nucleus, which results in significant gene expression (Hallenbeck and Stevenson, 2000). However, the gene delivery efficiency of human serotype 5 recombinant adenoviruses (Ad5) in tumor gene therapy clinical trials to date has been limited, mainly due to the development of Ad-neutralizing antibodies within the human population limiting the gene transfer to target cells (Worgall et al., 1997), short life span of gene expression due to local immune response against Ad5 (Yang et al., 1995), the inability of Ad5-based vectors to transduce important therapeutic target cell types (Stone et al., 2005), the paucity of the primary coxsackie/Ad receptor (CAR) in many human tumors, as well as virus dissemination to normal tissues (Yang et al., 1995; Bergelson et al., 1997; Tomko and Philipson, 1997). To circumvent some of these drawbacks, several virus modifications have been performed to augment the infectivity and/or increase the selectivity of Ad toward target tumor cells (Connelly, 1999; Barker et al., 2003; Everts and Curiel, 2004; Breidenbach et al., 2006).
To achieve the levels of enhanced transduction efficiency required in the context of uterine leiomyoma, it may be necessary to route the Ad5 vectors via CAR-independent pathways. One such modification is the insertion of short peptide (21 amino acid) composed of arginine, glysine and aspartate (RGD) to the H1 loop of the wild Ad5 fiber knob domain to reroute Ad5 binding to the cellular integrin (Dmitriev et al., 1998; Cripe et al., 2001). Another strategy is to employ knob switching by creating chimeric fibers possessing the knob domains of alternate human Ad serotypes beside the Ad5 fiber ex, Ad5/3 and Ad-sigma, which are created by substituting 1 Ad5 fiber with adenovirus serotype 3 (Ad3) or reovirus fiber, respectively. Functionally, Ad5-sigma utilizes CAR, sialic acid and the junctional adhesion molecule 1 (JAM1) for cell entry (Mercier et al., 2004), whereas Ad5/3 is redirected to bind the putative Ad3 receptors (CD80, CD86 or CD46) (Kanerva et al., 2002). Recently, more radical modifications based on xenotype knob switching with non-human adenovirus have been exploited, e.g. Ad5-CAV2 in which the Ad5 fiber knob is switched to that of the canine Ad serotype 2 (Soudais et al., 2000; Glasgow et al., 2004).
To achieve a higher level of tumor cell selectivity, transcriptional targeted viruses have been developed. In transcriptional targeting, the expression of the gene of interest is placed under control of a tumor-specific promoter (TSP) that maintains a tumor-on status, a normal tissue-off status and/or liver-off status. Another improvement is the introduction of the conditionally replicating adenovirus (CRAD) concept. In these vectors, the viral genome and subsequent replication is modified in such a way that it will only be complemented by the tumor cell (Alemany et al., 2000; Heise and Kirn, 2000; Hermiston, 2000), e.g. Ad5-MSLN-CRAD. The Ad5 vectors used in this study have been listed and described in Table I.
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Uterine leiomyoma is an attractive target for gene therapy because of several inherent biologic features. The disease is localized and well circumscribed in the uterus with fibrous capsules that could conceivably simplify targeting the viral load to the tumor (Al Hendy and Salama, 2006
). We have recently applied several gene therapy strategies for uterine leiomyoma; these include the use of an adenovirus-delivered dominant-negative estrogen receptor (Ad DNER) mutant (ER1-536) under a cytomegalovirus (CMV) promoter (Ad5-DNER) (Al-Hendy et al., 2004) or an Ad-herpes simplex thymidine kinas/genciclovir (HSV-TK/GCV) (Al-Hendy et al., 2000; Salama et al., 2007). We had considerable success with these approaches in the leiomyoma nude mice model in vitro and recently in the spontaneous Eker rat model (Hassan et al., 2007). To optimize our approach toward leiomyoma gene therapy, as well as improve the safety profile of this novel treatment, we have presented the utility of several modified Ad vectors in the human leiomyoma cell line. Our goal was to identify the best Ad vector to enable the targeting of therapeutic genes to human leiomyoma cells with minimal effect on normal myometrial cells, as well as normal liver cells.
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Material and methods |
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Recombinant adenovirus
Large-scale production of adenovirus vectors was performed as we have described previously (Al-Hendy et al., 2000
) with a typical batch yield of 2 x 1010 plaque-forming units (PFU)/ml. Ad vectors used in this study are listed in Table I.
Cell cultures
For experimental models, we used primary cultures of human leiomyoma cells (PLM 148, PLM 155, PLM 158 and PLM 144) derived from fibroid tumors removed during hysterectomies of four different patients. Human leiomyoma tissues were collected according to the policies of the Institutional Review Board of the University of Texas Medical Branch, Galveston, TX, USA, and used to establish PLM cells, as described previously (Rauk et al., 1995; Al-Hendy et al., 2004). The immortalized human uterine leiomyoma cell line (huLM), which expresses both estrogen receptors and progesterone receptors, was a kind gift from Dr Darlene Dixon (National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA). The cells were cultured and maintained as described previously (Carney et al., 2002). To represent normal cells (controls), we used a telomerase-immortalized human myometrial cell line (HM9); this cell line was cultured and maintained as described previously (Carney et al., 2002; Al-Hendy et al., 2004). We also used the THLE3 cell line, an immortalized liver cell line derived from adult human normal liver cells. The THLE3 cell line was purchased from American Type Culture Collection (Manassas, VA, USA) and was maintained in bronchial epithelial growth cell medium (BEGM Bullet Kit, CC3170, Clonetics Corporation, Walkersville, MD, USA). This medium includes 500 ml of basal medium and separate frozen additives; we discarded the gentamycin/amphotericin and epinephrine and added an extra 5 ng/ml of epidermal growth factor (EGF), 70 ng/ml of phosphoethanolamine, and 10% fetal bovine serum (FBS). The cells were maintained at 37ºC and 5% CO2/air.
Transfection with adenovirus vectors and luciferase reporter assays
The transfection with Ad vectors was conducted as we have described previously (Al-Hendy et al., 2004; Salama et al., 2007). Briefly, various cell lines (PLM, huLM, HM9 and THLE3) were cultured in 12-well plates (105/well). The cells were transfected with 5, 10 or 50 pfu/cell of various Ad vectors using transfection medium containing 1% antibiotic and 2% FBS for 4 h with continuous gentle shaking. The media was then replaced with fresh media, and incubation was continued for 48 h. Luciferase transactivation was determined using luciferase enzyme assay systems, according to the manufacturer's protocol (Promega, Madison, WI, USA). The luciferase activity was normalized against total protein content, as measured with a BCA Kit (Pierce Biotech, Rockford, IL, USA) (Reynolds et al., 1999). The experiments were repeated four times using different patient-derived PLM cells to ensure generalizability of results.
Statistical analysis
The results of the luciferase transactivation were expressed as mean ± standard error of the mean (SEM) of four different experiments. Statistical analysis was determined using two-tailed Student's t-test to compare groups. A P-value <0.05 was considered significant.
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Fiber-modified virus showed higher luciferase transactivation compared with wild-type adenovirus in human leiomyoma cells
We evaluated the gene delivery efficiency of fiber-modified adenoviruses (infectivity-enhanced Ad5 vectors) at three dose levels (5, 10 and 50 pfu/cell) by assessing the mediated luciferase activity of the modified virus panels in 2-cell models of human uterine leiomyoma: the immortalized leiomyoma cell line (huLM) and the primary leiomyoma cell line (PLM) prepared from four different patients. As shown in Fig. 1, Ad5-RGD-luc, Ad5-sigma-luc, Ad5/3-luc and Ad5-CAV2 -luc supported higher luciferase transactivation than wild-type Ad5-Luc in both huLM and PLM cells. Ad-RGD-luc at 10 pfu/cell had 700% and 450% of Ad5-luc (wild type) activity in huLM and PLM cells, respectively (P < 0.001) (Fig. 1A). Ad-sigma-luc at 10 pfu/cell showed 330% (P < 0.05) and 240% (P < 0.001) when compared with Ad5-luc in huLM and PLM cells, respectively (Fig. 1A). Ad5/3-luc at 10 pfu/cell showed 424% and 254% of Ad5-luc activity in huLM and PLM cells, respectively, in all of its results (P < 0.001) (Fig. 1A), whereas Ad5 CAV2-luc at 10 pfu/cell exhibited 800% and 680% of Ad5-luc activity in huLM and PLM cells, respectively (P < 0.001) (Fig. 1A). The same trend was obtained at both 5 pfu/cell and 50 pfu/cell for all fiber-modified viruses included in this study (data not shown). As shown in Fig. 1B, the modified adenoviruses (Ad5-RGD-luc, Ad5/3-luc and Ad5 CAV2-luc) at the low dose (5 pfu/cell) still demonstrated higher transduction efficiency (225%, 173 P < 0.001; 148% and 250%, P < 0.001, respectively) when compared with Ad5-luc at 10 pfu/cell, a dose we have previously shown to mediate optimal transfection by Ad5-luc (Al-Hendy et al., 2004
). Collectively, Ad5-CAV2-luc had the highest transduction level, followed by Ad5-RGD-luc and Ad5/3-luc in various huLM cells (Fig. 1).
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Targeting of fiber-modified adenovirus to leiomyoma versus normal myometrium
We evaluated the fiber-modified viruses to determine if they are selective toward leiomyoma compared with normal myometrium cells. We screened the transduction efficiency of these fiber-modified viruses in immortalized normal human myometrium cells (HM9) versus huLM cells. Interestingly, Ad5-CAV2-luc showed the best selective profile toward uterine leiomyoma versus HM9 cells. Luciferase activity occurred at a greater level in huLM cells (489%) than the corresponding luciferase activity in HM9 cells at 10 pfu/cell (P < 0.001) (Fig. 2). Ad5-RGD-luc was second in showing selectivity toward leiomyoma cells, as it expressed 357% of HM9 luciferase activity (P < 0.001) (Fig. 2), whereas Ad5-sigma-luc and Ad5/3-luc showed 205% (P < .001) and 180% of their corresponding luciferase activity in HM9 cells, respectively, at 10 pfu/cells (Fig. 2). These results were comparable with Ad5-luc, which showed luciferase activity in uterine leiomyoma cells of 130% of its corresponding HM9-luc value at 10 pfu/cell. The same order of selectivity was shown at 5 and 50 pfu/cell (data not shown). In conclusion, Ad5-CAV2-luc and Ad5-RGD-luc are highly selective toward huLM cells compared with HM9 cells.
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Evaluation of the activity of four TSPs in human leiomyoma cells compared with adenovirus wild type
Three TSPs showed significantly higher luciferase activity in both PLM and huLM cells when compared with Ad5-luc (Fig. 3). Ad5-SLPI-luc showed the highest luciferase activity, as it had 1400% and 1100% of Ad5-luc activity at 10 pfu/cell in huLM and PLM cells, respectively (P < 0.001) (Fig. 3). Ad5-MSLN-CRAD-luc was the second modified virus from this panel that expressed higher lucifirase activity, exhibiting 870% and 730% of corresponding wild Ad5 luc activity in the same cell order at 10 pfu/cell (P < 0.001) (Fig. 3A).
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As shown in Fig. 3A, the heparanase promoter was third in showing higher luciferase activity, as it expressed 307% (P < 0.001) and 257% (P < 0.001) at 10 pfu/cell of Ad-5 luc activity in huLM and PLM cells, respectively. The survivin promoter exhibited the lowest activity (18.2% and 1.7%, P < 0.001) of Ad5-luc activity (10 pfu/cell) in huLM and PLM cells, which suggests that the survivin promoter achieved a uterine tumor-off profile. This panel of modified viruses exhibited the same trend of luciferase activity in huLM and PLM cells at both 5 and 50 pfu/cell (data not shown).
Figure 3B demonstrates a very impressive result: ad5-SLPI-luc, Ad5-MSLN-CRAD-luc and Ad5-heparanase-luc at the lowest dose (5 pfu/cell) still exhibit significantly higher luciferase activity (800%, P < 0.001; 380%, P < 0.001; 150% P < 0.05, respectively) when compared with Ad5-luc at 10 pfu/cell, which is the dose that we have previously demonstrated to mediate optimal transduction efficiency by Ad-5 in huLM cells (Al-Hendy et al., 2004).
Transcriptional targeting of TSPs to uterine leiomyoma cells versus normal myometrium cells
We tested the transductional efficiency of the transcriptional targeted viruses in huLM cells versus HM9 cells to evaluate their selectivity toward leiomyoma compared with normal myometrium. As shown in Fig. 4, Ad-MSLN-CRAD-luc, Ad-SLPI-luc, Ad-heparanase-luc and Ad5-survivin-luc showed a higher selectivity profile toward human uterine huLM cells, as they expressed the following luc activities: 838% (P < 0.001), 473% (P < 0.001), 180% (P < 0.01) and 199% (P < 0.05), respectively, of their luc activity in HM9 at 10 pfu/cell compared with Ad5luc, which expressed 130% of its luc activity in HM9 (Fig. 4). The same selectivity profile was exhibited by these viruses at 5 and 50 pfu/cell (data not shown). Conclusively, among the transcriptional targeted virus panel, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc were the most selective toward huLM cells compared with HM9 cells.
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Evaluation of modified viruses in normal human liver cells
Because Ad5-luc has a higher liver tropism and causes hepatoxicity, which is considered to be a major obstacle in adenovirus gene therapy and virotherapy (Huard et al., 1995
; Hemminki and Alvarez, 2002), we evaluated potential liver infectivity caused by the two panels of viruses (fiber-modified panel: ad5-RGD-luc, Ad5-sigma-luc, Ad5/3-luc and Ad5-CAV2-luc; and transcriptional targeted panel: ad5-SLPI-luc, Ad5-heparanase-luc, Ad5-survivin-luc and Ad5-MSLN-CRAD-luc) in immortalized normal human liver cells (THLE3) by utilizing the luciferase reporter gene activity. In this regard, fiber-modified Ad5-sigma-luc and Ad5/3-luc from the first panel showed higher liver transduction as they mediated luciferase activity of 228% (P < 0.001) and 435% (P < 0.001), respectively, at 10 pfu/cell when compared with Ad5-luc at the same dose (Fig. 5A). Conversely, Ad5-RGD-luc showed a non-significant decrease in luciferase activity in THLE3 cells compared with Ad5 wild type at 10 pfu/cell, as it exhibited 93% of Ad5-luc activity at the same dose level; Ad5-CAV2-luc expressed the lowest luciferase activity in this virus panel, showing 60% of Ad5-luc value at 10 pfu/cell (P < 0.01) (Fig. 5A). As shown in Fig. 5B, the transcriptional targeted viruses (Ad5-SLPI-luc, Ad5-heparanase-luc, Ad-survivin-luc and Ad5-MSLN-CRAD-luc) exhibited a general trend of lower liver cell transduction compared with Ad5-luc at 10 pfu/cell (70%, P < 0.01; 43%, P < 0.001; 4.3%, P < 0.001 and 65% P < 0.01, respectively, of Ad5-luc activity). The same trend was expressed by all viruses included in this study at both 5 and 50 pfu/cell (data not shown).
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Interestingly, when compared with Ad5-luc at 10 pfu/cell, all transcriptional targeting viruses at 50 pfu/cell (Ad-survivin-luc, Ad5-heparanase-luc and Ad5-MSLN-CRAD-luc) showed lower liver transduction than Ad5-luc (Fig. 5B). The transcriptional targeted viruses at 50 pfu/cell had 16.4% (P < 0.001), 70% and 92%, respectively, of luc activity mediated by Ad5- luc at 10 pfu/cell; however, Ad5-SLPI-luc at 50 pfu/cell exhibited a non-significant increase in luc activity compared with Ad5 at 10 pfu/cell (110%). Three of the fiber-modified viruses had significantly higher liver cell transduction efficiency at 50 pfu/cell (highest dose) when compared with Ad5 at 10 pfu/cell. Interestingly, at 50 pfu/cell, Ad5-CAV2-luc did not have significantly higher luc activity (135% of Ad5-luc at 10 pfu/cell) (Fig. 5B).
In conclusion, transcriptional targeted viruses showed lower liver infectivity, whereas fiber-modified viruses had a higher liver transduction infectivity. The exceptions were Ad5-CAV2- luc, which expressed a lower liver transfection level, and Ad5-RGD-luc, which was similar to Ad5 wild type (Fig. 5A and B).
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The development of novel therapeutic strategies would be a welcome addition for uterine leiomyoma, especially if it is conservative, relatively safe and implies a localized method of treatment that would ablate the uterine fibroid without interfering with ovulation, local blood supply to uterus and systemic sex hormonal milieus and, hence, fertility, which are major side effects of current available treatment options for uterine leiomyomas (Andreyko et al., 1987
; VeKaut, 1993; Kettel et al., 1994; Vercellini et al., 1998; Vilos et al., 1998; Vilos, 2000; Eisinger et al., 2003; Payne and Haney, 2003; Al Hendy and Salama, 2006).
In this regard, adenovirus-mediated gene therapy of leiomyoma is a potentially promising approach. We have recently demonstrated this potential by applying Ad5-DNER and Ad5-HSV-TK/GCV gene therapy to the treatment of uterine leiomyoma in mice (Al-Hendy et al., 2004; Salama et al., 2007). These modalities severely inhibited cell proliferation and resulted in a marked increase of apoptotic cells, as well as regression of in vivo lesions in nude mice (Al-Hendy et al., 2004) and Eker rats (Hassan et al., 2007). These strategies, however, had some limitations, e.g. the non-specific tissue tropism of native Ad5. It is well-established that Ad5-luc is CAR-dependent for cell entry (Kanerva et al., 2002). Conversely, CAR is scarce in tumor tissues (Bergelson et al., 1997; Tomko and Philipson, 1997) or, at best, is relatively lacking in target cells in comparison with normal cells (Mueck et al., 2004). We have recently tested the distribution of Ad5-luc when injected intratumorally in the Eker rat model of uterine leiomyoma. The Ad5-luc particles were disseminated to myometrium and liver in 40% and 30% of animals, respectively (Hassan et al., 2007). To overcome these problems, adenovirus modification strategies need to be explored.
In this study, we planned to optimize the Ad5-mediated gene delivery to uterine leiomyoma by screening two panels of modified adenovirus, including the fiber-modified panel (Ad5-RGD-luc, Ad5/3-luc, Ad5-sigma- luc and Ad5-CAV2-luc) and the TSP-containing panel (Ad5-SLPI-luc, Ad5-survivin-luc, Ad5 heparanase-luc and Ad5-MSLN-CRAD-luc), in both PLM and immortalized huLM cells, as well as in HM9 cells and THLE3 cells as representatives of normal tissue (controls). In these sets of experiments, we compared the modified adenoviruses to wild type Ad5 to identify the best adenoviral vector that would enable targeting therapeutic genes to huLM cells with minimal effect on HM9 cells or distant organs such as the liver (Huard et al., 1995). To confirm our results, we chose to screen our two panels of modified Ad5 in both PLM and huLM cells to avoid the potential variability of CAR in primary cultures (Bauerschmitz et al., 2002), as well as possible deviations of immortalized cells from their native ancestor cell characters (Le et al., 2006). Of note, although the primary cell cultures were prepared from four different patients, the behaviors of the modified viruses were fairly similar in all samples (data presented as mean ± SEM from the four patients) and gave a similar trend of efficiency that was observed in huLM cells (Figs 1 and 3). Interestingly, the modified Ad5 vectors showed higher luciferase activity in the immortalized human leiomyoma cell line versus primary leiomyoma cells which might conceivably reflect variability in both primary CAR and modified Ad specific receptors which has been observed before in other cell line models (Stoff-Khalili et al., 2005).
The two adenovirus panels used in this study exploit two main strategies: ad5-transduction targeting and Ad5-transcriptional targeting. Transductional targeting aims at deletion of the broad tropism of Ad5 toward normal epithelial cells and/or enhances virus infectivity of CARdeficient tumor cells (Noureddini and Curiel, 2005). In this study, we explored the possibility of achieving higher transduction levels in the context of human leiomyoma using certain transductionally enhanced Ad5, including Ad5-RGD-luc, Ad5-CAV2-luc, Ad5-sigma-luc and Ad5/3-luc. We showed that Ad-RGD-luc and Ad-CAV2-luc support significantly higher reporter gene activity in leiomyoma cells than unmodified Ad-5-luc at all tested multiplicities of infection (MOIs) (5, 10 and 50 pfu/cell; 10 pfu/cell is represented in Fig. 1).
Ad5-CAV2-luc is an infectivity-enhanced adenovirus that shows expanded tropism created by switching the xeno knob of the original Ad5 knob with that of canine adenovirus serotype 2 (Glasgow et al., 2004). The receptor for this particular virus has not yet been identified but is thought to be different from the receptors for CAR and integrins (Soudais et al., 2000; Noureddini and Curiel, 2005), and it displays a distinct tropism not exhibited by human Ad5 (Soudais et al., 2000). Ad5-CAV2-luc was reported to enhance gene delivery to various tumor cell types such as primary ovarian cells (Soudais et al., 2000; Glasgow et al., 2004). Our data agree with these reports and demonstrate that Ad5-CAV2-luc consistently exhibited the highest gene transfer of all fiber-modified viruses used in this study in both PM and huLM cells at all dose levels (Fig. 1). Our data, however, are different from data obtained by Stoff-Khalili et al. (2005) in breast cancer cells, as they reported that Ad5-CAV2-luc mediated only a modest increase. These results are probably due to the paucity of Ad5-CAV2-luc receptors in breast cancer cells (Stoff-Khalili et al., 2005).
Ad-RGD-luc is an expanded tropism adenovirus that contains a targeting peptide (Arg-GlyAsp) attached to the HI loop of its fiber to redirect its entry through integrin receptors on the cell surface (Dmitriev et al., 1998; Reynolds et al., 1999). Previous reports have shown significantly increased transductional efficiency mediated by this virus when compared with unmodified Ad-5-luc in integrin expressing ovarian cancers (Kanerva et al., 2002), squamous cell carcinomas of the head and neck (Dehari et al., 2003) and CAR-negative cell lines (Wu et al., 2002). Our data are in agreement with these reports, as Ad5-RGD-luc showed higher transduction efficiency in uterine leiomyoma cells compared with Ad5-luc viruses. Leiomyoma tissues express various types of integrins (Taylor et al., 1996). The results were also not unexpected, considering both the smooth muscle nature of uterine leiomyoma (Chegini et al., 2002) and a study by Curiel (1999), who demonstrated that the RGD-containing adenovirus consistently exhibits enhanced gene delivery to endothelial and smooth muscle cells in vitro.
The Ad5-transcriptional targeting strategy also represents a powerful molecular tool to achieve leiomyoma cell-specific expression of transgenes encoded with viral vectors. We evaluated four TSPs for their activity in huLM cells: survivin, secretory leukoprotease inhibitor (SLPI), heparanase and mesothelin (MSLN) promoters. The activity of these four promoters has not been evaluated previously in huLM cells. Their TSP activity, however, has been reported to be highly expressed in many tumor types (Chang and Pastan, 1996; McElvaney et al., 1997; Barker et al., 2003; Zhu et al., 2004); therefore, they were considered potential candidates for the development of leiomyoma-specific transcriptional targeting of adenovirus mediated gene expression. According to our results, Ad-SLPI-luc and Ad-MSLN-CRAD-luc showed the highest reporter gene activities in uterine leiomyoma cells at MOIs of 5, 10 and 50 pfu/cell when compared with Ad5-luc (10 pfu/cell is represented in Fig. 3).
SLPI is a potent inhibitor of serine proteases such as elastase and cathepsin G from neutrophiles, trypsine and chemotrypsin from pancreatic cancer and chymase/tryptase from mast cells (Thompson and Rabinovitch, 1996; McElvaney et al., 1997). SLPI plays a role in the protection of mucosal surfaces by helping to prevent injury associated with inflammation (Eisenberg et al., 1990). The SLPI promoter is regulated by the estrogen in the cycling and pregnant rat endometrium (Chen et al., 2004). It is also highly expressed in some estrogen-dependent conditions such as breast cancer (Garver et al., 1994; Barker et al., 2003) and endometriosis (Suzumori et al., 1999). The SLPI protein is detected in higher amounts in cervical mucus secretion (Wallner and Fritz, 1974; Casslen et al., 1986) and in pregnant uteri of different species such as the pig (Farmer et al., 1990), horse and cow (Zhang et al., 2002), rhesus monkey and human endometrium during the menstrual cycle (King et al., 2000; Okulicz and Ace, 2003). SLPI also inhibits the production and activity of matrix metalloproteinases (MMPs) in monocytes and macrophages (Zhang et al., 1997). Furthermore, SLPI knockout mice showed enhanced elastase activity and increased activation of MMPs, which then leads to reduced matrix deposition and impaired cutaneous wound healing (Ashcroft et al., 2000). Collectively, these reports are consistent with a role of SLPI in the regulation of proteolytic cascades and inflammatory events. To our knowledge, this is the first report that suggests enhanced SLPI-promoter activity in huLM cells. On the basis of the reported functions of SLPI, we speculate that SLPI expression is increased in leiomyoma tissue, since leiomyoma is an estrogen-dependent neoplasm (Chen et al., 2005) with lower levels of MMP activity (Dou et al., 1997) and an abundant accumulation of extracellular matrix and collagen deposition (Kawaguchi et al., 1985; Stewart et al., 1994). Our results support this concept, as Ad-SLPI-luc showed significantly higher luciferase activity in leiomyoma cells (Fig. 3); this suggests that SLPI can be used as a uterine leiomyoma TSP for adenovirus gene therapy targeting.
Ad-MSLN-CRAD is a type II promoter-inducible conditionally replicative adenovirus (CRAd), in which mesothelin (MSLN), TSP, replace endogenous viral promoters to control early translated genes (E1A) of the virus (Haviv and Curiel, 2003). This restricts viral replication to target tissues, actively expressing the transcription factors that stimulate this TSP and lead to virus replication, oncolysis and subsequent release of the virus progeny. Normal tissue with little or no TSP activity is spared due to lack of replication (Rocconi et al., 2007). This replication cycle is important because it allows dramatic local amplification of the input dose, and, in theory, a CRAd could replicate until all tumor cells are lysed (Haviv and Curiel, 2003). MSLN, one of the most attractive candidates in tumor gene therapy and virotherapy, is overexpressed in different tumor types such as ovarian cancer, mesotheliomas and different squamous cell cancers of the cervix, vulva, lungs and esophagus (Chang and Pastan, 1994, 1996). MSLN is not present in normal tissues, except in mesothelial cells, and is not shed into the bloodstream in significant amounts (Breidenbach et al., 2005). This limited tissue distribution of the MSLN gene could represent a useful TSP candidate for targeting the adenovirus to uterine leiomyomas. Our results indicate that the MSLN promoter is transcriptionally active in uterine leiomyoma cells (PM and huLM). The MSLN promoter exhibited higher luciferase activities compared with the CMV promoter of unmodified Ad5 at all tested doses (Fig. 3).
We targeted adenoviruses that showed high activity in immortalized human leiomyoma cells (huLM) to evaluate the modified adenovirus specificity in leiomyoma gene therapy, and we also evaluated them against normal immortalized human myometrial cells (HM9). Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc achieved significantly higher activity in huLM than in HM9 cells (Figs 2 and 4) when compared with Ad5-luc at 5, 10 and 50 pfu/cell. This indicates the significant tissue selectivity of these viruses toward the neoplastic leiomyoma cell versus the normal myometrial cells.
The adenovirus has a propensity to localize to the liver (Huard et al., 1995; Sullivan et al., 1997), and leakage of low Ad5 titer into bloodstream results in detectable gene expression in the liver (Hiltunen et al., 2000). Furthermore, the liver sequesters the majority of systemically administered adenovirus particles via hepatic macrophage (Kupffer cell) uptake (Alemany et al., 2000; Tao et al., 2001). This is compounded by the fact that hepatocytes express higher amounts of CAR and integrin (Einfeld et al., 2001). This localization to the liver can potentially lead to adenovirus-mediated inflammation and liver toxicity (Lieber et al., 1997; Hemminki and Alvarez, 2002). We decided to evaluate the ability of various modified Ad5 at different dose levels (5, 10 and 50 pfu/cell) to transduce the normal human THLE3 liver cell line. Our results demonstrated that Ad5-CAV2 from the fiber-modified panel and all of the transcriptional targeted viruses supported lower liver luciferase transactivation compared with Ad5-luc (10 pfu/cell represented in Fig. 5A). This is in agreement with previous reports that used either human liver tissue slices (Rein et al., 2004; Stoff-Khalili et al., 2005; Breidenbach et al., 2006) or animal models (Lu et al., 2005). Implementation of targeted Ad5 gene therapy in uterine leiomyoma cells provides great potential to achieve higher gene transfer at lower Ad5 doses. It also affords a wider safety range, as the targeted adenoviruses can be utilized at higher dose levels to maximize therapeutic impact with lower liver side effects compared with Ad5. Additionally, serotype switching, such as Ad5-CAV2-luc, could circumvent any pre-existing neutralizing Ad5 antibodies (Wu et al., 2002).
In conclusion, Ad5-CAV2-luc, Ad5-RGD-luc, Ad-SLPI-luc and Ad5-MSLN-CRAD-luc showed higher reporter gene activity in huLM cells, and lower activity in both HM9 and THLE3 cells compared with unmodified Ad5-luc. These modified adenoviruses are promising vectors for targeted adenovirus-mediated gene therapy in uterine leiomyoma. Such modified (improved) adenoviral vectors will need to be tested in Eker rat, an experimental animal model for uterine leiomyoma (Everitt et al., 1995) before contemplation of a human clinical trial.
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TopAbstractIntroductionMaterial and methodsResultsDiscussionFundingAcknowledgementsReferences |
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NIH/NICHD 1 R01 HD04622801 to A.A.; DOD W81XWH-05-1-0035 and NIH R01CA083821 to D.C.
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TopAbstractIntroductionMaterial and methodsResultsDiscussionFundingAcknowledgementsReferences |
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We would like to thank Dr Zeng Zhu (Gene Therapy Center, University of Alabama at Birmingham, AL, USA) for his comprehensive review of this manuscript and his valuable comments for data presentation.
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Al Hendy A, Salama S. Gene therapy and uterine leiomyoma: a review. Hum Reprod Update (2006) 16:385–400.
Al-Hendy A, Lee EJ, Wang HQ, Copland A. Gene therapy of uterine leiomyomas: adenovirus-mediated expression of dominant negative estrogen receptor inhibits tumor growth in nude mice. Am J Obstet Gynecol (2004) 191:1621–1631.[CrossRef][Web of Science][Medline]
Al-Hendy A, Magliocco AM, Al Tweigeri T, Braileanu G, Crellin N, Li H, Strong T, Curiel D, Chedrese PJ. Ovarian cancer gene therapy: repeated treatment with thymidine kinase in an adenovirus vector and ganciclovir improves survival in a novel immunocompetent murine model. Am J Obstet Gynecol (2000) 182:553–559.[CrossRef][Web of Science][Medline]
Alemany R, Balague C, Curiel DT. Replicative adenoviruses for cancer therapy. Nat Biotechnol (2000) 18:723–727.[CrossRef][Web of Science][Medline]
Andreyko JL, Marshall LA, Dumesic DA, Jaffe RB. Therapeutic uses of gonadotropin-releasing hormone analogs. Obstet Gynecol Surv (1987) 42:1–21.[Medline]
Ashcroft GS, Lei K, Jin W, Longenecker G, Kulkarni AB, Greenwell-Wild T, Hale-Donze H, McGrady G, Song XY, Wahl SM. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing. Nat Med (2000) 6:1147–1153.[CrossRef][Web of Science][Medline]
Bangari DS, Mittal SK. Current strategies and future directions for eluding adenoviral vector immunity. Curr Gene Ther (2006) 6:215–226.[CrossRef][Web of Science][Medline]
Barker SD, Dmitriev IP, Nettelbeck DM. Combined transcriptional and transductional targeting improves the specificity and efficacy of adenoviral gene delivery to ovarian carcinoma. Gene Ther (2003) 10:1198–1204.[CrossRef][Web of Science][Medline]
Bauerschmitz GJ, Barker SD, Hemminki A. Adenoviral gene therapy for cancer: from vectors to targeted and replication competent agents. Int J Oncol (2002) 21:1161–1174.[Web of Science][Medline]
Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW. Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science (1997) 275:1320–1323.
Breidenbach M, Rein DT, Everts M, Glasgow JN, Wang M, Passineau MJ, Alvarez RD, Korokhov N, Curiel D. Mesothelin-mediated targeting of adenoviral vectors for ovarian cancer gene therapy. Gene Ther (2005) 12:187–193.[CrossRef][Web of Science][Medline]
Breidenbach M, Rein DT, Schondorf T, Khan KN, Herrmann I, Schmidt T, Reynolds PN, Vlodavsky I, Haviv YS, Curiel DT. A new targeting approach for breast cancer gene therapy using the heparanase promoter. Cancer Lett (2006) 240:114–122.[Web of Science][Medline]
Bruner-Tran KL, Zhang Z, Eisenberg E, Winnekar RC, Osteen KG. Down-regulation of endometrial matrix metalloproteinase-3 and -7 expression in vitro and therapeutic regression of experimental endometriosis in vivo by a novel nonsteroidal progesterone receptor agonist, tanaproget. J Clin Endocrinol Metab (2006) 91:1554–1560.
Buttram VC, Reiter RC. Uterine leiomyomata: etiology, symptomatology, and management. Fertil Steril (1981) 36:433–445.[Web of Science][Medline]
Carney SA, Tahara H, Swartz CD, Risinger JI, He H, Moore AB, Haseman JK, Barrett JC, Dixon D. Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics. Lab Invest (2002) 82:719–728.[Web of Science][Medline]
Casslen B, Andersson A, Nilsson IM, Astedt B. Hormonal regulation of the release of plasminogen activators and of a specific activator inhibitor from endometrial tissue in culture. Proc Soc Exp Biol Med (1986) 182:419–424.[CrossRef][Medline]
Chang K, Pastan I. Molecular cloning and expression of a cDNA encoding a protein detected by the K1 antibody from an ovarian carcinoma (OVCAR-3) cell line. Int J Cancer (1994) 57:90–97.[Web of Science][Medline]
Chang K, Pastan I. Molecular cloning of mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers. Proc Natl Acad Sci USA (1996) 93:136–140.
Chegini N, Ma C, Tang XM, Williams RS. Effects of GnRH analogues, ‘add-back’ steroid therapy, antiestrogen and antiprogestins on leiomyoma and myometrial smooth muscle cell growth and transforming growth factor-beta expression. Mol Hum Reprod (2002) 8:1071–1078.
Chen D, Xu X, Cheon YP, Bagchi MK, Bagchi IC. Estrogen induces expression of secretory leukocyte protease inhibitor in rat uterus. Biol Reprod (2004) 71:508–514.
Chen W, Yoshida S, Ohara N, Matsuo H, Morizane M, Maruo T. Gonadotropin-releasing hormone antagonist cetrorelix down-regulates proliferating cell nuclear antigen and epidermal growth factor expression and up-regulates apoptosis in association with enhanced poly(adenosine 5'-diphosphate-ribose) polymerase expression in cultured human leiomyoma cells. J Clin Endocrinol Metab (2005) 90:884–892.
Connelly S. Adenoviral vectors for liver-directed gene therapy. Curr Opin Mol Ther (1999) 1:565–572.[Web of Science][Medline]
Cramer SF, Patel A. The frequency of uterine leiomyomas. Am J Clin Pathol (1990) 94:435–438.[Web of Science][Medline]
Cripe TP, Dunphy EJ, Holub AD, Saini A, Vasi NH, Mahller YY, Collin MH, Snyde JD, Krasnykh V, Curiel DT, et al. Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells. Cancer Res (2001) 61:2953–2960.
Curiel DT. Strategies to adapt adenoviral vectors for targeted delivery. Ann NY Acad Sci (1999) 886:158–171.[CrossRef][Web of Science][Medline]
Dehari H, Ito Y, Nakamura T, Kobune M, Sasaki K, Yonekura N, Kohama G, Hamada H. Enhanced antitumor effect of RGD fiber-modified adenovirus for gene therapy of oral cancer. Cancer Gene Ther (2003) 10:75–85.[CrossRef][Web of Science][Medline]
Dmitriev I, Krasnykh V, Miller CR, Wang M, Kashentseva E, Mikheeva G, Belousova N, Curiel DT. An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism. J Virol (1998) 72:9706–9713.
Dou Q, Tarnuzzer RW, Williams RS, Schultz GS, Chegini N. Differential expression of matrix metalloproteinases and their tissue inhibitors in leiomyomata: a mechanism for gonadotrophin releasing hormone agonist-induced tumour regression. Mol Hum Reprod (1997) 3:1005–1014.
Einfeld DA, Schroeder R, Roelvink PW. Reducing the native tropism of adenovirus vectors requires removal of both CAR and integrin interactions. J Virol (2001) 75:11284–11291.
Eisenberg SP, Hale KK, Heimdal P, Thompson RC. Location of the protease-inhibitory region of secretory leukocyte protease inhibitor. J Biol Chem (1990) 265:7976–7981.
Eisinger SH, Meldrum S, Fiscella K, Le Roux HD, Guzick DS. Low-dose mifepristone for uterine leiomyomata. Obstet Gynecol (2003) 101:243–250.[CrossRef][Web of Science][Medline]
Eldar-Geva T, Meagher S, Healy DL, MacLachlan V, Breheny S, Wood C. Effect of intramural, subserosal, and submucosal uterine fibroids on the outcome of assisted reproductive technology treatment. Fertil Steril (1998) 70:687–691.[CrossRef][Web of Science][Medline]
Everitt JI, Wolf DC, Howe SR, Goldsworthy TL, Walker C. Rodent model of reproductive tract leiomyomata. Clinical and pathological features. Am J Pathol (1995) 146:1556–1567.[Abstract]
Everts M, Curiel DT. Transductional targeting of adenoviral cancer gene therapy. Curr Gene Ther (2004) 4:337–346.[Web of Science][Medline]
Farhi J, Ashkenazi J, Feldberg D, Dicker D, Orvieto R, Ben-Rafael Z. Effect of uterine leiomyomata on the results of in-vitro fertilization treatment. Hum Reprod (1995) 10:2576–2578.
Farmer SJ, Fliss AE, Simmen RC. Complementary DNA cloning and regulation of expression of the messenger RNA encoding a pregnancy-associated porcine uterine protein related to human antileukoproteinase. Mol Endocrinol (1990) 4:1095–1104.
Garver RI, Goldsmith KT, Rodu B, Hu PC, Sorcher EJ, Curiel DT. Strategy for achieving selective killing of carcinomas. Gene Ther (1994) 1:46–50.[Web of Science][Medline]
Glasgow JN, Bauerschmitz GJ, Curiel DT, Hemminki A. Transductional and transcriptional targeting of adenovirus for clinical applications. Curr Gene Ther (2004) 4:1–14.[CrossRef][Web of Science][Medline]
Hallenbeck PL, Stevenson SC. Targetable gene delivery vectors. Adv Exp Med Biol (2000) 465:37–46.[Web of Science][Medline]
Hart R, Khalaf Y, Yeong CT, Seed P, Taylor A, Braude P. A prospective controlled study of the effect of intramural uterine fibroids on the outcome of assisted conception. Hum Reprod (2001) 16:2411–2417.
Hassan MH, Salama S, Zhang D, Al-Hendy A. Towards fibroids gene therapy: adenovirus mediated delivery of dominant negative estrogen receptor shrinks uterine leiomyoma tumors in Eker rats. Reprod Sci (2007) 14:80A.
Haviv YS, Curiel DT. Engineering regulatory elements for conditionally-replicative adeno-viruses. Curr Gene Ther (2003) 3:357–385.[CrossRef][Medline]
Heise C, Kirn DH. Replication-selective adenoviruses as oncolytic agents. J Clin Invest (2000) 105:847–851.[Web of Science][Medline]
Hemminki A, Alvarez RD. Adenoviruses in oncology: a viable option? BioDrugs (2002) 16:77–87.[CrossRef][Web of Science][Medline]
Hermiston T. Gene delivery from replication-selective viruses: arming guided missiles in the war against cancer. J Clin Invest (2000) 105:1169–1172.[Web of Science][Medline]
Hiltunen MO, Turunen MP, Turunen AM, Rissanen TT, Laitinen M, Kosma VM, Yla-Herttuala S. Biodistribution of adenoviral vector to nontarget tissues after local in vivo gene transfer to arterial wall using intravascular and periadventitial gene delivery methods. FASEB J (2000) 14:2230–2236.
Huard J, Lochmuller H, Acsadi G, Jani A, Massie B, Karpati G. The route of administration is a major determinant of the transduction efficiency of rat tissues by adenoviral recombinants. Gene Ther (1995) 2:107–115.[Web of Science][Medline]
Kanerva A, Wang M, Bauerschmitz GJ, Lam JT, Desmond RA, Bhoola SM, Barnes MN, Alvarez RD, Siegal GP, Curiel DT, et al. Gene transfer to ovarian cancer versus normal tissues with fiber-modified adenoviruses. Mol Ther (2002) 5:695–704.[CrossRef][Web of Science][Medline]
Kawaguchi K, Fujii S, Konishi I, Okamura H, Mori T. Ultrastructural study of cultured smooth muscle cells from uterine leiomyoma and myometrium under the influence of sex steroids. Gynecol Oncol (1985) 21:32–41.[CrossRef][Web of Science][Medline]
Kettel LM, Murphy AA, Morales AJ, Yen SC. Clinical efficacy of the antiprogesterone RU486 in the treatment of endometriosis and uterine fibroids. Hum Reprod (1994) 9:116–120.
King AE, Critchley HO, Kelly RW. Presence of secretory leukocyte protease inhibitor 598 in human endometrium and first trimester decidua suggests an antibacterial protective role. Mol Hum Reprod (2000) 6:191–196.
Kozarsky KF, Wilson JM. Gene therapy: adenovirus vectors. Curr Opin Genet Dev (1993) 3:499–503.[CrossRef][Medline]
Krasnykh VN, Mikheeva GV, Douglas JT, Curiel DT. Generation of recombinant adenovirusvectors with modified fibers for altering viral tropism. J Virol (1996) 70:6839–6846.
Le LP, Rivera AA, Glasgow JN, Ternovoi VV, Wu H, Wang M, Smith BF, Siegal GP, Curiel DT. Infectivity enhancement for adenoviral transduction of canine osteosarcoma cells. Gene Ther (2006) 13:89–399.
Lefebvre G, Vilos G, Allaire C, Jeffrey J, Arneja J, Birch C, Fortier M, Wagner MS. The management of uterine leiomyomas. J Obstet Gynaecol Can (2003) 25:396–418.[Medline]
Lepine LA, Hillis SD, Marchbanks PA, Koonin LM, Morrow B, Kieke BA, Wilcox LS. Hysterectomy surveillance—United States, 1980–1993. MMWR CDC Surveill Summ (1997) 46:1–15.[Medline]
Lieber A, He CY, Meuse L, Schowalter D, Kirillova I, Winther B, Kay MA. The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion of recombinant adenovirus vectors. J Virol (1997) 71:8798–8807.[Abstract]
Liu WM. Laparoscopic bipolar coagulation of uterine vessels to treat symptomatic leiomyomas. J Am Assoc Gynecol Laparosc (2000) 7:125–129.[CrossRef][Web of Science][Medline]
Lu B, Makhija SK, Nettelbeck DM, Rivera AA, Wang M, Komarova S, Zhou F, Yamamoto M, Haisma HJ, Alvarez RD, et al. Evaluation of tumor-specific promoter activities in melanoma. Gene Ther (2005) 12:330–338.[CrossRef][Web of Science][Medline]
McElvaney NG, Stoller JK, Buist AS, Prakash UB, Brantly ML, Schluchter MD, Crystal RD. Baseline characteristics of enrollees in the National Heart, Lung and Blood Institute Registry of alpha 1-antitrypsin deficiency. Alpha 1-Antitrypsin Deficiency Registry Study Group. Chest (1997) 111:394–403.[CrossRef][Web of Science][Medline]
Mercier GT, Campbell JA, Chappell JD, Stehle T, Dermody TS, Barry MA. A chimeric adenovirus vector encoding reovirus attachment protein sigma1 targets cells expressing junctional adhesion molecule 1. Proc Natl Acad Sci USA (2004) 101:6188–6193.
Mueck AO, Seeger H, Huober J. Chemotherapy of breast cancer-additive anticancerogenic effects by 2-methoxyestradiol? Life Sci (2004) 75:1205–1210.[CrossRef][Web of Science][Medline]
Nettelbeck DM, Jerome V, Muller R. Gene therapy: designer promoters for tumour targeting. Trends Genet (2000) 16:174–181.[CrossRef][Web of Science][Medline]
Noureddini SC, Curiel DT. Genetic targeting strategies for adenovirus. Mol Pharm (2005) 2:341–347.[CrossRef][Medline]
Okulicz WC, Ace CI. Temporal regulation of gene expression during the expected window of receptivity in the rhesus monkey endometrium. Biol Reprod (2003) 69:1593–1599.
Payne JF, Haney AF. Serious complications of uterine artery embolization for conservative treatment of fibroids. Fertil Steril (2003) 79:128–131.[CrossRef][Web of Science][Medline]
Rauk PN, Surti U, Roberts JM, Michalopoulos G. Mitogenic effect of basic fibroblast growth factor and estradiol on cultured human myometrial and leiomyoma cells. Am J Obstet Gynecol (1995) 173:571–577.[CrossRef][Web of Science][Medline]
Rein DT, Breidenbach M, Curiel DT. Current developments in adenovirus-based cancer gene therapy. Future Oncol (2006) 2:137–143.[CrossRef][Medline]
Rein DT, Breidenbach M, Nettelbeck DM, Kawakami Y, Siegal GP, Huh WK, Wang M, Hemminki A, Bauerschmitz GJ, Yamamoto M, et al. Evaluation of tissue-specific promoters in carcinomas of the cervix uteri. J Gene Med (2004) 6:1281–1289.[CrossRef][Web of Science][Medline]
Reynolds P, Dmitriev I, Curiel D. Insertion of an RGD motif into the HI loop of adenovirus fiber protein alters the distribution of transgene expression of the systemically administered vector. Gene Ther (1999) 6:1336–1339.[CrossRef][Web of Science][Medline]
Rocconi RP, Zhu ZB, Stoff-Khalili M, Rivera AA, Lu B, Wang M, Alvarez RD, Curiel DT, Makhija SK. Treatment of ovarian cancer with a novel dual targeted conditionally replicative adenovirus (CRAd). Gynecol Oncol (2007) 105:113–121.[CrossRef][Web of Science][Medline]
Salama S, Kamel M, Christman G, Wang H, Al-Hendy A. Gene therapy of uterine fibroids: adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir treatment inhibits growth of human and rat leiomyoma cells. Gynecol Obstet Invest (2007) 651:61–70.
Schagen FH, Wensveen FM, Carette JE, Dermody TS, Gerritsen WR, van Beusechem VW. Genetic targeting of adenovirus vectors using a reovirus sigma1-based attachment protein. Mol Ther (2006) 13:997–1005.[CrossRef][Web of Science][Medline]
Soudais C, Boutin S, Hong SS, Chillon M, Danos O, Bergelson JM, Boulanger P, Kremer EJ. Canine adenovirus type 2 attachment and internalization: coxsackievirus adenovirus receptor, alternative receptors, and an RGD-independent pathway. J Virol (2000) 74:10639–10649.
Stewart EA. Uterine fibroids. Lancet (2001) 357:293–298.[CrossRef][Web of Science][Medline]
Stewart EA, Friedman AJ, Peck K, Nowak RA. Relative overexpression of collagen type I and collagen type III messenger ribonucleic acids by uterine leiomyomas during the proliferative phase of the menstrual cycle. J Clin Endocrinol Metab (1994) 79:900–906.[Abstract]
Stoff-Khalili MA, Stoff A, Rivera AA, Mathis JM, Everts M, Wang M, Kawakami Y, Waehler R, Mathews QL, Yamamoto M, et al. Gene transfer to carcinoma of the breast with fiber-modified adenoviral vectors in a tissue slice model system. Cancer Biol Ther (2005) 4:1203–1210.[Web of Science][Medline]
Stone D, Ni S, Li ZY, Gaggar A, DiPaolo N, Feng Q, Sandig V, Lieber A. Development and assessment of human adenovirus type 11 as a gene transfer vector. J Virol (2005) 79:5090–5104.
Sullivan DM, Jensen TG, Taichman LB, Csaky KG. Ornithine-delta aminotransferase expression and ornithine metabolism in cultured epidermal keratinocytes: toward metabolic sink therapy for gyrate atrophy. Gene Ther (1997) 4:1036–1044.[CrossRef][Web of Science][Medline]
Surrey ES, Lietz AK, Schoolcraft WB. Impact of intramural leiomyomata in patients with a normal endometrial cavity on in vitro fertilization-embryo transfer cycle outcome. Fertil Steril (2001) 75:405–410.[CrossRef][Web of Science][Medline]
Suzumori N, Sato M, Yoneda T, Ozaki Y, Takagi H, Suzmori K. Expression of secretory leukocyte protease inhibitor in women with endometriosis. Fertil Steril (1999) 72:857–867.[CrossRef][Web of Science][Medline]
Tao N, Gao GP, Parr M, Johnston J, Baradet T, Wilson JM, Barsoum J, Fawell SE. Sequestration of adenoviral vector by Kupffer cells leads to a nonlinear dose response of transduction in liver. Mol Ther (2001) 3:28–35.[CrossRef][Web of Science][Medline]
Taylor CV, Letarte M, Lye SJ. The expression of integrins and cadherins in normal human uterus and uterine leiomyomas. Am J Obstet Gynecol (1996) 175:411–419.[CrossRef][Web of Science][Medline]
Thompson K, Rabinovitch M. Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular-matrix bound basic fibroblast growth factor. J Cell Physiol (1996) 166:495–505.[CrossRef][Web of Science][Medline]
Tomko RP, Xu R, Philipson L. HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses. Proc Natl Acad Sci USA (1997) 94:3352–3356.
Tsuruta Y, Rein M, Nakayama M, Siegal GP, Pereboeva L, Breidenbach M, Curiel DT. Therapeutic efficacy of an oncolytic adenovirus monitored by in vivo imaging of an ovarian cancer model. Mol Ther (2006) 13:S116.
Van Houdt WJ, Haviv YS, Lu B, Wang M, Rivera AA, Ulasov IV, Lamfers ML, Rein D, Lesniak MS, Siegal GP, et al. The human survivin promoter: a novel transcriptional targeting strategy for treatment of glioma. J Neurosurg (2006) 104:583–592.[Web of Science][Medline]
Vercellini P, Maddalena S, De Giorgi O, Aimi G, Crosignani PG. Abdominal myomectomy for infertility: a comprehensive review. Hum Reprod (1998) 13:873–879.
VeKaut BS. Changing trends in treatment of leiomyomata uteri [abstract]. Curr Opin Obstet Gynecol (1993) 5:301.[Web of Science][Medline]
Vilos GA. Treatment options for uterine fibroids [abstract]. Can J Diagn (2000) 17:55–59.
Vilos GA, Daly LJ, Tse BM. Pregnancy outcome after laparoscopic electromyolysis. J Am Assoc Gynecol Laparosc (1998) 5:289–292.[CrossRef][Web of Science][Medline]
Vorburger SA, Hunt KK. Adenoviral gene therapy. Oncologist (2002) 7:46–59.
Wallner O, Fritz H. Characterization of an acid-stable proteinase inhibitor in human cervical mucus. Hoppe Seylers Z Physiol Chem (1974) 355:709–715.[Web of Science][Medline]
Worgall S, Wolff G, Falck-Pedersen E, Crystal RG. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum Gene Ther (1997) 8:37–44.[Web of Science][Medline]
Wu H, Seki T, Dmitriev I, Uil T, Kashentseva E, Han T, Curiel DT. Double modification of adenovirus fiber with RGD and polylysine motifs improves coxsackievirus-adenovirus receptor-independent gene transfer efficiency. Hum Gene Ther (2002) 13:1647–1653.[CrossRef][Web of Science][Medline]
Yang Y, Li Q, Ertl HC, Ertl HC, Wilson JM. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol (1995) 69:2004–2015.[Abstract]
Zhang D, Simmen RC, Michel FJ, Zhao G, Vale-Cruz D, Simmen FA. Secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes. J Biol Chem (2002) 277:29999–30009.
Zhang Y, DeWitt DL, McNeely TB, Wahl SM, Wahl LM. Secretory leukocyte protease inhibitor suppresses the production of monocyte prostaglandin H synthase-2, prostaglandin E2, and matrix metalloproteinases. J Clin Invest (1997) 99:894–900.[Web of Science][Medline]
Zhu ZB, Makhija SK, Lu B, Wang M, Kaliberova L, Liu B, Rivera AA, Nettelbeck DM, Mahareshti PJ, Leath CA, et al. Transcriptional targeting of tumors with a novel tumor-specific survivin promoter. Cancer Gene Ther (2004) 11:256–262.[CrossRef][Web of Science][Medline]
Submitted on June 15, 2007; resubmitted on October 16, 2007; accepted on December 5, 2007.
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