Gene therapy of benign gynecological diseases

Abstract

Gene therapy is the introduction of genetic material into patient's cells to achieve therapeutic benefit. Advances in molecular biology techniques and better understanding of disease pathogenesis have validated the use of a variety of genes as potential molecular targets for gene therapy based approaches. Gene therapy strategies include: mutation compensation of dysregulated genes; replacement of defective tumor-suppressor genes; inactivation of oncogenes; introduction of suicide genes; immunogenic therapy and antiangiogenesis based approaches. Preclinical studies of gene therapy for various gynecological disorders have not only shown to be feasible, but also showed promising results in diseases such as uterine leiomyomas and endometriosis. In recent years, significant improvement in gene transfer technology has led to the development of targetable vectors, which have fewer side-effects without compromising their efficacy. This review provides an update on developing gene therapy approaches to treat common gynecological diseases such as uterine leiomyoma and endometriosis.

Fig. 1

Schematic diagram depicts a) Adenovirus virion b) Adenovirus infection pathway [16].

Fig. 2

Structure of Adenovirus DNA [22].

Fig. 3

Schematic diagram of the wild type and mutant estrogen receptors [117].

Fig. 4

HSV1TK/GCV By standard effect: A; normal cells with low GIJ protein level B; tumor cells with high GIJ protein level [90].

Fig. 5

Adenovirus-infected human leiomyoma cells. LM-15 cells were optimally transfected with Ad-LacZ at a high multiplicity of infection (MOI) of 10 PFU/cell. At a low MOI of 5 PFU/cell, about 50% of cells were successfully transfected [104].

Fig. 6

Direct intratumor injection of adenovirus with dominant negative estrogen receptor (ER) inhibited tumor growth. Direct intratumor injection of the corresponding agent was performed on day 16 after cell implantation. (A) Ad-ER-DN treatment caused immediate overall arrest of tumor growth. The difference among treatment and control groups was highly significant (P=0.007). Results are mean±SD (n=10) of two independent experiments. (B) Tumors treated with Ad-ER-DN demonstrated inhibition of tumor progression [104].

Fig. 7

Effect of Ad-DNER on transcriptional activity of endogenous ER of rat uterine leiomyoma cells (ELT3 cell line). The ER transcriptional activity was assayed using Ad-ERE-luc, and the relative light units were normalized to the cell number. Results are plotted as mean±SE of three independent experiments. P<0.01 was considered a significant difference from control (a) or from Ad-DNER 1 PFU/cell (b) [105].

Fig. 8

Ad-DNER injection into Eker rat uterine leiomyoma significantly shrinks total leiomyoma volume (compared to both vehicle control and Ad-LacZ treated animals). Tumor volume was calculated as a percentage of its corresponding day zero (pretreatment) volume and presented as M±SE of at least three animals at each time point. a,b indicate significant difference from vehicle control and Ad-LacZ treated animals, respectively, at P<0.05 [106].

Fig. 9

Ad-DNER treatment of fibroid lesions in Eker rats induces modulation of several estrogen regulated genes controlling both (A) intrinsic (p53, BAX, Bcl2 and PARP) and (B) extrinsic (DAXX and fas L) apoptotic pathways. Data represent uterine leiomyoma tissues collected at 8, 15 and 30 days post Ad-DNER treatment compared to either vehicle control or Ad-lacZ treated group: (A) Protein levels assessed by western blot; (B) DAXX and FAS L mRNA expression; (C) demonstrate increased Caspase 3 activity; (D) increase in the percentage of positive TUNNEL cells in the Ad-DNER treated lesions. The letters a or b indicate significant difference from vehicle control or Ad-lac Z treated lesions respectively at P<0.05 using two tailed student T test in (C) caspase 3 experiment and Tukey test as post-ANOVA test in (B) RT-PCR and (D) TUNNEL assay experiments [106].

Fig. 10

Safety studies of the Ad-DNER gene therapy approach after a single direct intra-fibroid treatment of Eker rat. (A) Assessment of adenovirus particles dissemination into Eker rat body organs by PCR amplification of the specific Ad5 E4 region detected by 714pb DNA band on 1% agarose gel. The band was detected mainly in tumor tissues while faint bands were detected also in uterus (lane 11) and liver (Lane 12) respectively, in 40% and 30% of the treated animals. (B) Although Ad-DNER particles were detected in the liver of 40% of Eker rats injected with adenovirus, Ad-DNER did not produce any significant change in liver function tests (AST, ALT and total bilirubin) evaluated 30 days post adenovirus injection, compared to vehicle control animals [106].

Fig. 11

Adenovirus-mediated delivery of herpes simplex virus 1 thymidine kinase gene (Ad-HSV1TK) (3×10¹⁰ PFU/cm³ of tumor) by a single direct injection into Eker rat uterine leiomyoma followed by a subcutaneous injection of GCV 50 mg/kg/day for 10 days significantly shrinks Eker rat uterine leiomyoma volume when compared with both Ad-LacZ/GCV and vehicle-treated animal groups. Uterine leiomyoma tumor volume was measured by both MRI scanning and caliper measurement. Tumor volume was calculated as a percentage from day 0. Each time point was represented by mean± SE of 3 animals. ^a,bIndicate a significant difference from control and Ad-LacZ/GCV-treated groups, respectively, at P<0.05 using the Tukey test as a post-ANOVA test [107].

Fig. 12

Evaluation of modified adenovirus vectors in human leiomyam cells. (A) fiber-modified adenoviruses; Ad5-SLPI-luc, Ad5-heparanase-luc, and Ad5-MSLN-CRAD-luc and (B) transcriptional targeted adenoviruses;Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc showed higher luciferase expression levels in both human primary (PLM) and immortalized leiomyoma (huLM) cells at 10 PFU/cell when compared with Ad5-luc at the same dose level, while survivin showed uterine leiomyoma cells off-profile. Luciferase activities (RLU) were normalized to the protein content and expressed as mean±standard error of the mean (SEM) of 4 separate experiments and plotted as percentage of Ad5-luc activity. a; Indicates significant differences compared with corresponding Ad5-luc at 10 PFU/cell (P<0.05) [108].

Fig. 13

Evaluation of modified adenovirus transduction efficiency in normal human liver cell line (THLE3) at 10 PFU/cell. Luciferase activities (RLU) were normalized to the protein content, and expressed as mean±standard error of the mean (SEM) of 4 separate experiments, and plotted as percentage of corresponding Ad5-luc activity. a,c; indicate significant difference from Ad5 (P<.001 and P<.01, respectively) [108].

Fig. 14

Luciferase transactivation in endometriosis cells mediated by transductionally targeted adenoviruses at MOI of 10 PFU/cell.

Fig. 15

Luciferase transactivation in endometriosis cells mediated by transcriptionally targeted adenoviruses at MOI of 10 PFU/cell.

Fig. 16

Luciferase transactivation in liver tissues mediated by transductionally and transcriptionally targeted adenoviruses at MOI of 10 PFU/cell.