Gene Transfer Approaches for Gynecological Diseases pot

Gene Transfer Approaches for Gynecological Diseases Mari Raki, 1,2 Daniel T. Rein, 3 and Anna Kanerva 1,2,4 Akseli Hemminki 1,2, * 1 Cancer Gene Therapy Group, Rational Drug Design Program, University Helsinki, 00014 Helsinki, Finland 2 Department of Oncology and 4 Department of Obstetrics and Gynecology, Helsinki University Central Hospital, 00029 Helsinki, Finland 3 Department of Obstetrics and Gynecology, University of Du ¨ sseldorf Medical Center, 40001 Du ¨ sseldorf, Germany *To whom correspondence and reprint requests should be addressed at P.O. Box 63, University of Helsinki, 00014 Helsinki, Finland. Fax: +358 9 1912 5465. E-mail: akseli.hemminki@helsinki.fi. Available online 2 May 2006 Gene transfer presents a potentially useful approach for the treatment of diseases refractory to conventional therapies. Various preclinical and clinical strategies have been explored for treatment of gynecological diseases. Given the direst need for novel treatments, much of the work has been performed with gynecological cancers and ovarian cancer in particular. Although the safety of many approaches has been demonstrated in early phase clinical trials, efficacy has been mostly limited so far. Major challenges include improving gene transfer vectors for enhanced and selective delivery and achieving effective penetration and spread within advanced and complex tumor masses. This review will focus on current and developmental gene transfer applications for gynecological diseases. Key Words: gene transfer, gene therapy, ovarian cancer, cervical cancer, gynecological disease Contents Introduction 154 Gene Therapy for Ovarian Cancer 154 Targeting Vectors to Ovarian Cancer Cells 155 Replacement of an Altered Tumor Suppressor Gene 155 Inhibition of Growth Factor Receptors 157 Molecular Chemotherapy 157 Antiangiogenic Gene Therapy 157 Virotherapy 158 Gene Therapy for Other Gynecological Cancers 159 Gene Therapy for Other Gynecological Disorders 159 Future Directions 161 Acknowledgments 161 References 161 INTRODUCTION An increasing understanding of the molecular mecha- nisms that cause human disease has rationalized gene transfer as an approach for the treatment of diseases resistant to more conventional therapies. Gene therapy aims at transfer of genes for correction of either genetic or somatic disease phenotypes or for expression of molecules within or near target cells for therapeutic effect. Vehicles for gene transfer include both nonviral and viral vectors, such as adenovirus, retrovirus, adeno- associated virus (AAV), and herpes simplex virus (HSV). Nonviral gene transfer is most co mmonly based on plasmid DNA, particle bombardment, or cationic lip- osomes. Viral gene delivery has already been optimized by evolution and is therefore generally more effective, while nonviral approaches are pharmacologically more attractive. GENE THERAPY FOR OVARIAN CANCER Ovarian cancer is the leading cause of death from gyne- cological malignancies in developed countries [1].Mostcases are diagnosed at an advanced state, and long-term survival of patients with metastatic disease is rare. Although chemo- REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 154 Copyright C The American Society of Gene Therapy 1525-0016/$30.00 therapy approaches featuring taxanes and platinums, when given following optimal cytoreductive surgery, can increase the survival of patients, treat ment of metastatic disease eventually results in drug resistance and disseminated disease cannot be cured. Therefore, novel trea tment approaches are n eeded. Gene therapy, even at its current rather adolescent stage, is an attractive modality for ovarian cancer a s t his c ancer frequently p resents w ith metastases confined to the peritoneal cavity, creating a rationale for locoregional delivery. Targeting Vectors to Ovarian Cancer Cells The majority of gene therapy approaches for ovarian cancer (Table 1) are based on adeno virus serotype 5 (Ad5), which binds to the coxsackie–adenovirus receptor (CAR). A number of approaches have been tested in phase I clinical trials with impressive safety data. Moreover, successful gene transfer has been demonstrated in most cases in which it has been analyzed. In contrast, only rare examples of efficacy have been published. This is partly influenced by trial design (phase I trials usually have safety as the main endpoint), but nevertheless the lack of res- ponse implies that there is a discrepancy between preclin- ical and clinical efficacy. Onepossiblereasonisthattheremightbea tendency for researchers to use models that allow effective transduction, and therefore variable CAR expression has been recognized only upon analysis of clinical substrates. Another reason might be the greater complexity of advanced solid tumor masses in compar- ison to relatively rapidly growing xenografts. By extension, this implies that it is crucial to perform extensive sampling and biopsies in phase I trials to acquire material for correlative studies. Obviously, this is hampered by compliance and cost issues and the fact that traditionally phase I trials have mostly looked at safety. Heretofore, all published studies have been per- formed with CAR-binding viruses. Unfortunately, con- current studies have suggested that expression of CAR is frequently dysregulated in many types of advanced cancers, including ovarian cancer [2]. Various strategies have been evaluated to modify adenovirus tropism to circumvent CAR deficiency, for increased transduction of tumor cells and reduced normal tissue tropism. Transductional targeting can be achieved by utilizing bispecific molecules that block the interaction with CAR and redirect the virus to a novel receptor. Several ligands, including basic fibroblast growth factor [3], anti-TAG-72 [4], and anti-CD40 [5], have been physically linked to an Ad5-fiber-binding moiety for enhanced transduction. Another strategy involves genetic modifications of the viral capsid. Enhanced infectivity of ovarian cancer cells has been demonstrated by incorporating an integ- rin-binding RGD-4C motif in the HI loop of the fiber knob [6]. Fiber pseudotyping has also been evaluated. Substitution of the knob do main of Ad5 wi th the corresponding domain of serotype 3 (Ad3) allows bind- ing and entry through the Ad3 receptor, which is expressed to a high degree on ovarian cancer cells [7,8]. High tolerability of adenoviruses in cancer trials has allowed administration of large doses. In most trials, the maximum tolerated dose has not been reached and the maximum affordable dose has become limiting instead. Nevertheless, some trials have reported abdominal pain or liver enzyme elevations [9,10], suggesting that trans- duction of normal tissue has the potential for toxicity. Also, while very safe in comparison to, e.g., chemothe- rapy, it is now well known that adenoviruses can cause eve n fatal immune reactions [11].Therefore,ithas become attractive to restrict expression of viral genes or transgenes to tumor cells by using tumor-specific pro- moters (TSPs) in a strategy called transcriptional target- ing. Several TSPs have been evaluated for ovarian cancer specificity, including L-plastin [12], midkine [13], cyclo- oxygenase-2 (cox-2) [13], ovar ian-specific promoter-1 [14], secretory leukoprotease inhibitor promoter (SLPI) [15,16], and mesothelin [17]. Although transcriptional targeting can reduce toxicity associated with transgene expression in nontarget tissues, it does not reduce immunological recognition of virus particles and infected cells. An immune response toward infected tumor cells can be useful for eradication of metastases and protection against relapse. In contrast, an acute immune reaction or clearance of infected nontarget cells can be harmful. Specific transductional targeting of viruses to target cells is a useful way to retain the potentially beneficial aspects of a vector-targeted immune response while reducing immunological toxic- ity. Other approaches for reducing immune responses toward adenovirus are discussed in the last section. Replacement of an Altered Tumor Suppressor Gene Mutation of the p53 tumor suppressor gene is one of the most frequent genetic changes in cancer and it has been found in nearly 60% of advanced ovarian cancers [18]. Preclinical studies have demonstrated that adenovirus- mediated delivery of wild-type p53 inhibits growth of ovarian cancer cells both in vitro and in vivo [19,20] (Fig. 1A). p53 gene transfer to ovarian cancer cells using catio- nic nonviral vector has also been reported [21]. Adp53 was evaluated in a phase I/II trial and the treatment was well tolerated [9,22,23]. Gene transfer and biological activity were also demonstrated [24]. These findings led to a randomized phase II/III trial in which Adp53 was given intraperitoneally in combi- nation with chemotherapy. Although complete results have unfortunately not been published, the first interim analysis suggested a lack of therapeutic effect but increased toxicity and the study was closed [25].In parallel with most trials wit h this approach, trans- REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 155 Copyright C The American Society of Gene Therapy TABLE 1: Overview of gene therapy approaches for gynecological cancers Construct Vehicle Approach Study Main result Reference Ovarian cancer Adp53 Adenovirus Delivery of p53 Phase I/II/III clinical trial Phase I/II: No dose-limiting toxicity Phase III: No advantage, toxicity seen [9,23,25] LXSN-BRCA1sv Retrovirus Delivery of BRCA-I Phase I/II clinical trial Phase I: Safe, tumor reduction in 25% Phase II: No responses [26,27] Ad21 Adenovirus Inhibition of erbB2 Phase I clinical trial No dose-limiting toxicity, no responses [33] DCC-E1A Cationic liposome Inhibition of erbB2 Phase I clinical trial Safe, no responses [35] AdHSV-TK Adenovirus HSV-TK suicide gene therapy Phase I clinical trial No dose-limiting toxicity, no responses [39] ADV-RSV-tk Adenovirus HSV-TK suicide gene therapy Phase I clinical trial No dose-limiting toxicity [10] ONYX-015 Adenovirus (CRAd type I) Virotherapy Phase I clinical trial Dose-limiting toxicity in one patient, no responses [60] Sense and antisense OPCML Plasmid Delivery of OPCML In vitro and in vivo Inhibition of ovarian cancer cell growth [101] Ad-mda-7 Adenovirus Delivery of mda-7/IL-24 In vitro Inhibition of ovarian cancer cell growth, induction of apoptosis, targeting to CD40 or EGFR [102,103] EGFR-DNR Retrovirus Delivery of truncated EGFR (erbB1) In vivo Inhibition of cancer cell growth, enhanced sensitivity to cisplatin [104] HSV-T3 HSV type 1 HSV-TK suicide gene therapy In vitro Gene transfer to ovarian cancer cells [105] rAAV-P125Aendo AAV Antiangiogenic gene therapy In vivo Inhibition of ovarian cancer cell growth, inhibition of angiogenesis [48] Ad5-D24RGD Adenovirus (CRAd type I) Virotherapy In vitro and in vivo Killing of ovarian cancer cells, infectivity enhancement [52–54] Ad5/3-D24 Adenovirus (CRAd type I) Virotherapy In vitro and in vivo Killing of ovarian cancer cells, infectivity enhancement [61] Dearing reovirus serotype 3 Reovirus Virotherapy In vitro and in vivo Inhibition of ovarian cancer cell growth [106] MV-CEA Measles virus Virotherapy In vitro and in vivo Killing of ovarian cancer cells, expression of soluble marker peptide [107] MV-CEA MV-Moraten MuV-JL Measles and mumps viruses Virotherapy In vitro and in vivo Intercellular fusion of ovarian cancer cells, cell death [108] Cervical cancer Ad-p73 Adenovirus Delivery of p73 In vitro Growth inhibition of E6-positive cells [69] AAV-TK AAV HSV-TK suicide gene therapy In vitro Cell killing of HPV-positive cells [66] Ad5-D24RGD Adenovirus (CRAd type I) Virotherapy In vitro and in vivo Inhibition of cervical cancer cell growth [67] Ad-MN/Ca9-E1a Adenovirus (CRAd type II) Virotherapy In vitro and in vivo Inhibition of cervical cancer cell growth [109] Tissue-specific promoters Adenovirus Transcriptional targeting In vitro High activity of MK and VEGF promoters in cervical cancer cell lines and primaries [110] Endometrial carcinoma Adp21, Adp53 Adenovirus Delivery of p21 or p53 In vitro Inhibition of endometrial cancer cell growth, induction of apoptosis [71] SFG-F/S-IRES-tk Retrovirus HSV-TK suicide gene therapy In vitro Inhibition of endometrial cancer cell growth [111] pNF nB-TK Plasmid HSV-TK suicide gene therapy In vitro and in vivo Inhibition of endometrial cancer cell growth [72] REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 156 Copyright C The American Society of Gene Therapy duction of advanced and bulky tumor masses may not have been sufficient for significant therapeutic effect, while transduction of normal tissues may have been the reason for side effects. Retrovirus has also been clinically studied for ovarian cancer therapy, utilizing transfer of BRCA1 [26,27].A phase I study using intraperitoneal delivery showed partial response in 25% of patients, and the majority had stable disease. However, a subsequent phase II study showed no responses and vector stability was poor. Other viral and nonviral approaches are listed in Table 1. Inhibition of Growth Factor Receptors Growth factor receptors such as erbB1–erbB4 of the epidermal growth factor receptor family can be targeted for replacement or inactivation. Deshane et al. con- structed a gene that encodes an intracellular single- chain antibody (intrabody) against erbB2/HER-2/neu [28]. This receptor is highly expressed in 10–15% of ovarian cancers with correlation with poor prognosis [29]. Adenovirus (Ad21)-mediated transfer of the intra- body to ovarian tumors resulted in induction of apoptosis and cytotoxicity in vitro and enhanced efficacy and survival in animal models of ovarian cancer [30–32]. The strategy was subsequently evaluated in a phase I trial [33]. Intraperitoneal treatment was well tolerated without dose-limiting toxicity, and gene transfer was demonstrated but no responses were detected. Adenoviral E1A has been shown to downregulate erbB2 expression with concomitant growth inhibition [34]. Hortobagyi et al. evaluat ed cationic liposome- mediated E1A transfer in a phase I trial with breast and ovarian cancer patients [35]. Expression of E1A and downregulation of erbB2 expression were demonstrated in peritoneal samples. Fo llowing dose escalation, abdominal pain eventually identified the maximum tolerated dose, but stable disease was detected in only 17% of patients, a rather low figure perhaps reflecting the effectiveness of plasmid-based transduction in the context of advanced disease. A similar strategy was used in another phase I trial [36]. Molecular Chemotherapy Molecular chemotherapy (a.k.a. suicide gene therapy) is a strategy based on delivery of genes encoding a prodrug- activating enzyme (Fig. 1B). The most popular approach in the context of ovarian cancer has been herpes simplex virus thymidine kinase (HSV-TK), which converts the prodrug ganciclovir (GCV) into a toxic metabolite. The HSV-TK/GCV system is associated with a bbystander effect,Q i.e., killing of uninfected neighboring cells. Based on promising preclinical results [37,38] Alvarez et al. utilized intraperitoneal delivery of a replication-deficient adenovirus (AdHSV-TK) followed by intravenous GCV [39]. No dose-limiting side effects were seen and 38% of patients had stable disease for the duration of the study. Transgene expression could be detected from ascites samples of patients. Another phase I study combined intrape ritoneal AdHSV-TK with intravenous acyclovir and topo tecan [10]. Again, no dose-limiting adverse effects were seen, and the most common side effect was myelosuppression most likely related to chemo- therapy. The median survival of these patients was 18.5 months [40]. As an example of bench-to-bedside-and- back translational work, when clinical specimens revealed variable expression of CAR, the efficacy of the HSV-TK/GCV approach was subsequently enhanced in vitro and in vivo by incorporating an integrin-binding RGD-4C motif into the adenoviral fiber [41,42], and a trial is forthcoming. Antiangiogenic Gene Therapy Antiangiogenic gene transfer inhibits formation of neo- vasculature required for tumor growth and may also act by collapsing immature tumor-associated vascular struc- tures (Fig. 1C). Ovarian cancer cells have been shown to express proangiogenic growth factors such as vascular endothelial growth factor (VEGF) [43]. Effects of VEGF are mediated through the endothelium-specific VEGF recep- tors such as Flt-1 [44]. Soluble FMS-like tyrosine kinase receptor 1 (sFlt-1) is a splice variant of Flt-1 and binds to VEGF, inhibiting its angiogenic actions and may also prevent dimerization of wild-type Flt-1. Mahasreshti et al. evaluated the effect of adenovirus-mediated sFlt-1 transfer against ovarian carcinoma [45,46]. Intraperitoneal deliv- ery of an integrin-targeted virus encoding sFlt-1 inhibited ovarian tumor growth and increased the survival of mice. However, intravenous delivery of the same construct resulted in hepatotoxicity. Inhibition of angiogenesis was demonstrated after intraperitone al injection of an AAV expres sing sFlt-1 [47]. Also other antiangiogenic genes such as mutant Construct Vehicle Approach Study Main result Reference Teratocarcinoma Ad5-flt-1luc Adenovirus Transcriptional targeting via flt-1 promoter In vitro High transgene expression in teratocarcinoma cells [112] TABLE 1 (continued) REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 157 Copyright C The American Society of Gene Therapy endostatin have been packaged into AAV for in vivo efficacy [48]. Lentiviruses have not been widely used for ovarian cancer therapy, but transfer of interferon-a has been evaluated in a murine model [49]. Antitumor effects were associated with a decrease in the formation of hemorrhagic ascites and a reductioninmicrovessel density. Virotherapy Utilization of the oncolytic potential of viruses for killing of tumor cells predates the concept of gene therapy by more than half a decade [50]. Nevertheless, due to safety concerns, most modern gene therapy approaches have been based on viruses that are unable to replicate in infected cells. However, the main result from a generation of clinical trials with these agents is that the utility of replication-deficient viruses may be limited when faced with advanced and bulky disease. Thus, intratumoral diffusion of nanosize carriers such as viruses may be a limiting step. While tumor targeting and infectivity enhancement have improved transduc- tion rates of replication-deficient viruses preclinically, to our knowledge no trials have been initiated yet, although a number are in preparation (Table 1). A specific obstacle with regard to analysis of oncolytic viruses on clinical specimens is the limited viability of the latter in vitro. This can be partly overcome by maintaining clinical samples as multicellular tumor clusters or spheroids [51]. This technology has been applied to analysis of transductionally targeted oncolytic adenoviruses [52–55], but correlation to clinical respon- siveness is not yet available. FIG. 1. Gene therapy approaches. (A) Replacement of a mutated tumor suppressor gene. Delivery and expression of a wild-type gene results in apoptosis and cancer cell death. (B) Molecular chemotherapy. Delivery and expression of a suicide gene results in conversion of a nontoxic prodrug into a cytotoxic metabolite. (C) Antiangiogenic gene therapy. Deliv- ery of a soluble VEGF receptor results in sequestra- tion of VEGF and subsequent inhibition of neovascularization. (D) Virotherapy. Viral infection of cancer cells results in replication, oncolysis, and release of virions to surrounding cells. REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 158 Copyright C The American Society of Gene Therapy To improve tumor penetration, various naturally occurring, inherently tumor-selective or engineered oncolytic viruses have been utilized, including adeno- virus, HSV, Newcastle disease virus, vaccinia, reovirus, measles virus, and vesicular stomatitis virus [56]. Conditionally replicating adenoviruses (CRAds) are the most widely studied members of this group (Fig. 1D), and more than 500 cancer patients have been treated with CRAds [2,57]. In type I CRAds, tumor-specific replication is achieved by engineering deletions in genes critical for efficient viral replication in normal but not in tumor cells [58]. The most widely studied CRAd, ONYX-015 (dl1520), carries deletions in E1B, exhibits reduced binding of p53, and replicates selectively in tumor cells [59]. ONYX- 015 has been evaluated in a phase I ovarian cancer trial [60]. Treatment resulted in grade 3 abdominal pain and diarrhea in one patient but the maximum tolerated dose was not reached, and the bmaximum affordable doseQ was 10 11 viral particles. However, there were no clinical or radiological responses in any patients. In addition to ONYX-015, many type I CRAds have been evaluated preclinically. Integrin-targeted Ad5- D24RGD and serotype 3 receptor-targeted Ad5/3-D24 contain a 24-bp deletion in the retinoblastoma (Rb) binding site of E1A. Therefore, these viruses replicate selectively in cancer cells deficient in the Rb/p16 pathway. Recent studies have demonstrated that both agents deliver a powerful antitumor effect to ovarian cancer cells in vitro, to clinical ovarian cancers, and in orthotopic models of ovarian cancer, and both viruses are now proceeding toward clinical testing [52,53,61]. Type II CRAds are designed to achieve replicative specificity based on heterologous promoters placed into the adenovirus genome to control the expression of the early genes such as E1A, which is essential for viral replication. The utility of these agents is subservient to the identification of promoters that induce the appro- priate inductivity vs specificity profile [62]. Promoters that have shown utility for ovarian cancer include IAI.3B, cox-2, and SLPI [55,63,64]. GENE THERAPY FOR OTHER GYNECOLOGICAL CANCERS While ovarian cancer is the most problematic gynecolog- ical cancer in developed societies, cervical cancer remains the leading cause of mortality worldwide [1]. Unfortu- nately, neither improvements in surgery nor radiotherapy has significantly decreased mortality [65], and patients with advanced, recurrent, or metastatic disease still have a poor chance of being cured. The pathogenesis of cervical cancer follows a natural history characterized by human papillomavirus (HPV) infection, a long latency period, and progression in a fraction of patients through dysplasia and carcinoma in situ to invasive cancer and metastatic disease. Only a few viral strains are specifically responsible for cervical neoplasms, of which HPV16 accounts for more than one-half of reported cases. Carson et al. demonstrated a novel gene-based strategy to prevent virus replication in HPV-infected cells through the conditional expression of the HSV-TK gene [66]. Delivery of HSV-TK with AAV followed by GCV treatment resulted in efficient cell killing of HPV-positive cells. CRAds represent another promising treatment alter- native. In a recent study, Ad5-D24RGD demonstrated effective oncolysis in cervical cancer cells [67]. Moreover, therapeutic efficacy could be demonstrated in a mouse model of cervical cancer with both intratumoral and intravenous application. Importantly, no toxicity was seen with human peripheral blood mononuclear cells. Another interesting approach, which takes advantage of similarities between gene products of DNA viruses, is complementation of adenovirus mutants by HPV genes [68]. An alternative approach to inhibiting the growth of cervical cancer cells is based on the observation that tumor suppressor p53 functions are downregulated in most cervical cancer cells. The product of HPV oncogene E6 binds to and inactivates p53 by promoting its degradation. p73 is similar to p53 in structure and function but not degraded by the HPV E6 gene product. Das et al. demonstrated growth inhibition of E6-positive cell lines in vitro following infection with Ad-p73 [69]. Endometrial carcinoma is the most common neoplasm of the female reproductive tract and it accounts for nearly one-half of all gynecologic malignancies. Although usu- ally curable with surgery, sometimes aggressive tumors such as uterine papillary serous carcinomas (UPSC) are seen. Immunohistochemical studies suggest that p53 is aberrant in 50–90% of UPSC tumors in comparison to 10– 30% in typical endometrioid adenocarcinomas [70].Ina recent study, adenoviral delivery of p53 or p21 resulted in growth suppression and induction of apoptosis in a UPSC cell line [71]. Another interesting gene therapy approach for endo- metrial cancer is based on the obs ervation that the gonadotropin-releasing hormone receptor (GnRH-R) is expressed by the majority of ovarian and endometrial cancers. GnRH-R is a promising tumor-specific target due to limited normal tissue expression. Grundker et al. demonstrated the efficacy of HSV-TK/GCV controlled by GnRH-R-specific elements in intraperitoneal and subcutaneous mouse models of endometrial and ovarian cancer [72]. GENE THERAPY FOR OTHER GYNECOLOGICAL DISORDERS Leiomyomas are benign, proliferating, estrogen-depend- ent uterine tumors, which become clinically relevant only when they enlarge enough to elicit symptoms such REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 159 Copyright C The American Society of Gene Therapy as abnormal bleeding [73].Further,theycancause infertility and miscarriages. Current treatment is usually hysterectomy or myomectomy. However, the disease is localized to the uterus, which makes it an ideal target for local gene therapy via ultrasound-guided injections, laparoscopy, or hysteroscopy (Table 2). A plasmid-based strategy with HSV-TK/GCV was assessed in vitro both in human clinical samples and in a rat leiomyoma cell line. A bystander effect was demonstrated, and interestingly, it was increased with estradiol treatment [74]. In a murine leiomyoma xenograft model adenovirus-mediated expression of a dominant negative estrogen receptor inhibited subcutaneous tumor growth and cell prolifer- ation, while increased apoptosis was found [75]. Endometriosis, the growth of ectopic endo metrial tissue, is an estrogen-dependent disease that causes pain and infertility. Moreover, there is an association between untreated endometriosis and development of ovarian cancer. Typically, it is treated with surgical removal of the lesions and medical therapy aiming at a hypoestro- genic state [73]. An important feature of active endome- triosisispronouncedvascularization,andtherefore antiangiogenic gene therapy has been evaluated [76].In a murine model, intraperitoneal delivery of an adenovi- rus encoding the angiogenesis inhibitor angiostatin caused a decrease in the number, size, and density of blood vessels. More importantly, established endome- triosis was eradicated in all treated mice within 18 days [76]. Fortin et al. evaluated the efficacy of HSV-TK/GCV for treatment of endometriosis. Human endometrial fragments were inf ected ex vivo with an adenovirus containing HSV-TK and injected subcutaneously into nude mice. GCV treatment induced a significant regres- sion in endometrial implants [77]. Placental disorders and dysfunction cause significant fetal and maternal mo rbidity, including fetal growth retardation, preeclampsia or eclampsia, and mortality. Initially, there is defective development of the early placenta and its maternal blood supply. The clinical syndrome arises from subsequent generalized maternal endothelial dysfunction [73]. Pathologically, a hypoxic and dysfunctional placenta releases factors such as sFlt-1, which binds VEGF and p lacental g rowth factor [78]. Increased understanding of these mechanisms facilitates development of gene therapeutic strategies for treatment of preeclampsia and prolonging the pregnancy. Senut et al. delivered gene-modified placental cells to the rodent placenta in vivo and demonstrated that gene products were secreted throughout gestation without deleterious effects [79]. Plasmid DNA and adenoviruses have been guided with angiography to uterine arteries in rabbits for transfection of trophoblast cells. Transfection efficiency was as high as 34% with adenovirus, while plasmid complexes led to much lower rates [80]. Insulin-like growth factors (IGFs) I and II are critical in fetal growth because of their role in placental development and function, and reduced levels have been reported in intra- uterine growth retardation. Adenoviruses encoding IGF-I or IGF-II were utilized for in vitro gene transfer to fresh, human primary placental fibroblasts. IGFs exerted both autocrine and paracrine effects on cell proliferation, migration, and survival [81]. Molecular defects have been implicated in embryo implantation disorder, making it a possible target for gene therapy. Homeobox (HOX) genes are transcription factors necessary for embryonic development. Unlike in most adult tissues, HOXA10 and HOXA11 expression persists in the endometrium, and they are essential for endometrial development and receptivity in response to sex steroids. Interestingly, it has been shown that mice with disruption of the HOXA10 gene are infertile because of implantation failure [82]. More importantly, defects in endometrial HOX gene expression in infertile women have been demonstrated [82]. Thus, augmenting HOX gene expression with gene therapy to improve implanta- tion becomes attractive and has already been achieved TABLE 2: Gene therapy approaches for noncancer gynecological diseases Construct Vehicle Rationale Study Reference Leiomyomas pNGVL1-tk Plasmid HSV-TK suicide gene therapy In vitro [74] Ad-ER-DN Adenovirus Inhibition of estrogen receptor In vitro and in vivo [75] Endometriosis AdAngiostatin Adenovirus Antiangiogenic gene therapy In vitro and in vivo [76] AdTK Adenovirus HSV-TK suicide gene therapy In vitro and in vivo [77] Placental disorders Ad-LacZ, LacZ plasmid Adenovirus, Liposome/plasmid Angiographically guided utero-placental transfer of marker gene In vivo [80] Ad-IGF-I, Ad-IGF-II Adenovirus Delivery of IGF-1 or IGF-II In vitro [81] Embryo implantation disorder HOXA10 cDNA Liposome/plasmid Delivery of HOXA10 In vitro and in vivo [83] REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 160 Copyright C The American Society of Gene Therapy with intrauterine administration of HOXA10 plasmid/ liposome complex to mice [83]. In general, nonmalignant gynecological diseases are less severe and more treatable than gynecological cancers. Therefore, clinical translation of gene therapy strategies probably requires even more stringent safety information. Moreover, given the immunogenic nature of adenovi- ruses, other vectors such as lentiviruses and AAV may be more attractive for this group of diseases. FUTURE DIRECTIONS Recent evidence su ggests that relatively conventional gene therapy approaches, when applied following max- imal cytoreduction, can increase the survival of cancer patients [84]. Nevertheless, only a few pioneering studies have managed to harness fully the power of correlative analysis in phase I trials and these studies have implied that traditional delivery systems usually result in insuffi- cient gene transfer when faced with advanced tumor masses [85]. To improve the quality and quantity of correlative data in early phase trials, it is important to increase our capacity for detection of the persistence and magnitude of virus replication. Because obtaining serial biopsies is difficult due to safety, cost, and compliance issues, noninvasive strategies are most attractive. Some promising approaches include functional imaging of transgenes, incorporation of secretable marker proteins, and detection of fluorescent proteins incorporated into virus capsids [42,86,87]. Several strategies are currently being explored to improve transduction of target cells and effective pene- tration of solid tumors. For example, gene transfer by viral vectors can be enhanced by using modified agents that are retargeted to receptors highly expressed on target cells [88]. Nonetheless, viral spread in the tumor can be limited by physical barriers such as stromal cells and matrix and necrotic, hypoxic, or hyperbaric regions. For overcoming these obstacles, selectively oncolytic viruses may be useful and targeting oncolytic viruses to tumor cells is a logical sequel [52,61 ].Forfurther potentiation, replication-competent v iruses can be armed with therapeutic transgenes such as cytokines, suicide genes, and fusogenic, proteolytic, or antiangio- genic moieties [89]. A powerful approach for increasing efficacy is utiliza- tion of gene transfer in combination with conventional anticancer therapies in a multimodal antitumor approach [90], which has recently been validated in randomized trials [57,91,92]. Gene therapy differs from traditional modalities with regard to mechanism and side effects, providing a possibility for additive or synergistic inter- actions [93,94]. The aforementioned intratumoral complexities hinder also conventional antitumor approaches such as chemo- therapy, and it is known that effective treatments usually require multiple rounds of administration; solid tumors can usually be reduced only layer by layer. Thus, clinical gene transfer might benefit from readministration of virus, whose efficacy may be inhibited by neutralizing antibodies (NAb). Strategies for facilitating re-treatment include alternating related viruses with different capsids (sero-switch) [95], cotreatment with immunosuppressive drugs for temporary abrogation of NAb induction [96],or physical removal of NAbs by using immunopheresis or an adenovirus capsid protein column [97]. Most importantly, it remains crucial to translate preclinical advances quickly into clinical trials, because only in patients can we find out which approaches work and which do not. Comprehensive correlative analysis of specimens obtained in these trials allows the translational process to cycle rapidly back to the lab for development of next generation agents. It may be that the biggest obstacle cancer gene therapy faces is the continually increasing difficulty in rapidly setting up phase I trials in an ever- tightening regulatory environment. Other challenges include improving gene delivery and potency to levels compatible with clinical responses. Also, given the recent success of monoclonal antibodies and small molecular inhibitors as effective and relatively nontoxic antitumor agents, gene therapy needs to deliver emphatic clinical results to attract resources compatible with transforma- tion of a promising approach to a clinically successful strategy. Fortunately, recent watershed clinical trials [57,84,92,98–100] have demonstrated that the theoretical considerations behind gene delivery for therapeutic effect are sound, and the technology remains a viable and potent approach for treatment of diseases resistant to available modalities. A CKNOWLEDGMENTS This work was supported by HUCH Research Funds (EV O), the Academy of Finland, the Emil Aaltonen Foundation, the Finnish Cancer Society, the University of Helsinki, the Sigrid Juselius Foundation, the Sohlberg Foundation, the Biocentrum Helsinki, the Instrumentarium Research Fund, the Finnish Oncology Association, the Research and Science Foundation Farmos, and Regional Funds of the Finnish Cultural Foundation. RECEIVED FOR PUBLICATION AUGUST 31, 2005; REVISED DECEMBER 13, 2005; ACCEPTED FEBRUARY 6, 2006. REFERENCES 1. Parkin, D. M., Bray, F., Ferlay, J., and Pisani, P. (2005). Global cancer statistics, 2002. CA Cancer J. Clin. 55: 74 – 108. 2. Kanerva, A., and Hemminki, A. (2005). 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REVIEW ARTICLE doi:10.1016/j.ymthe.2006.02.019 MOLECULAR THERAPY Vol. 14, No. 2, August 2006 163 Copyright C The American Society of Gene Therapy . developmental gene transfer applications for gynecological diseases. Key Words: gene transfer, gene therapy, ovarian cancer, cervical cancer, gynecological. rationalized gene transfer as an approach for the treatment of diseases resistant to more conventional therapies. Gene therapy aims at transfer of genes for correction