Gene therapy targeting leiomyoma: adenovirus-mediated delivery of dominant-negative estrogen receptor gene shrinks uterine tumors in Eker rat model  Memy H. Hassan, M.Sc., Salama A. Salama, Ph.D., Dong Zhang, Ph.D., Hossam M.M. Arafa, Ph.D., Farid M.A. Hamada, Ph.D., Hala Fouad, M.D., Ph.D., Cheryl C. Walker, Ph.D., Ayman Al-Hendy, M.D., Ph.D.  Fertility and Sterility  Volume 93, Issue 1, Pages 239-250 (January 2010) DOI: 10.1016/j.fertnstert.2008.09.086 Copyright © 2010 Terms and Conditions

Figure 1 Adenovirus distribution in eker rat uterine leiomyoma tissue after a single direct injection of adenovirus-mediated delivery bacterial ß-galactosidase (Ad-LacZ) 3 × 1010 PFU/cm3 of tumor or vehicle only. Eker rat uterine leiomyoma tissue stained for X-gal 8 days after treatment. Presence of blue stain indicates delivery and expression of β-galactosidase gene. Fertility and Sterility 2010 93, 239-250DOI: (10.1016/j.fertnstert.2008.09.086) Copyright © 2010 Terms and Conditions

Figure 2 Adenovirus-mediated dominant-negative estrogen receptor gene (Ad-DNER) injection into Eker rat uterine leiomyoma significantly shrinks total leiomyoma volume (compared with 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,bIndicate significant difference from vehicle control and Ad-LacZ–treated animals, respectively, at P<.05. Other abbreviation as in Figure 1. Fertility and Sterility 2010 93, 239-250DOI: (10.1016/j.fertnstert.2008.09.086) Copyright © 2010 Terms and Conditions

Figure 3 Representative hematoxylin-eosin micrographs from vehicle control (a) and Ad-LacZ–treated (b) fibroid lesions collected from Eker rats at 30 days and Ad-DNER–treated fibroid lesions at 8 (c), 15 (d), and 30 days (e) after treatment. Original magnification ×40. Ad-DNER treatment induces histologic changes in Eker rat fibroid tissues, manifested by increase in apoptotic cells, decreased cellularity, distorted and spare nuclei with smudged chromatin pattern, and increased hyalinized intercellular matrix. Abbreviations as in Figures 1 and 2. Fertility and Sterility 2010 93, 239-250DOI: (10.1016/j.fertnstert.2008.09.086) Copyright © 2010 Terms and Conditions

Figure 4 Ad-DNER treatment of fibroid lesions in Eker rats induces modulation of several estrogen regulated genes controlling both (A) intrinsic (p53, Bax, Bcl-2, and PARP) and (B) extrinsic (Daxx and FasL) apoptotic pathways. Data represent uterine leiomyoma tissues collected at 8, 15, and 30 days after Ad-DNER treatment compared with either vehicle control or Ad-lacZ treated group: (A) protein levels assessed by Western blot; (B) Daxx and FasL mRNA expression; (C) increased caspase-3 activity; and (D) increase in the percentage of TUNEL-positive cells in the Ad-DNER–treated lesions. a,bSignificant difference from vehicle control or Ad-LacZ–treated lesions, respectively, at P<.05, using two-tailed Student t test in caspase-3 experiment (C) and Tukey test as post-ANOVA test in PCR (B) and TUNEL assay (D) experiments. ANOVA = analysis of variance; Daxx = death domain–associated protein; FasL = Fas ligand; PARP = poly (ADP-ribose) polymerase; PCR = polymerase chain reaction; TUNEL = terminal deoxynucleotide transferase–mediated dUTP nick-end labeling; other abbreviations as in Figures 1 and 2. Fertility and Sterility 2010 93, 239-250DOI: (10.1016/j.fertnstert.2008.09.086) Copyright © 2010 Terms and Conditions

Figure 5 Ad-DNER decreases the expression of proliferation- and extracellular matrix formation–related genes. Immunohistochemical analysis of proliferation-related gene expression of PCNA (A) and cyclin-D1 (B) in Eker rat uterine leiomyoma tissue sections collected from the animals treated with vehicle only (control) (a) or Ad-LacZ (b) collected 30 days, or Ad-DNER collected 8 (c), 15 (d), and 30 days (e) after treatment. (C) Expression of TGF-β3 gene at mRNA level detected by PCR amplification. a,bSignificant difference in Ad-DNER from vehicle control and Ad-LacZ–treated lesions at P<.05 using Tukey test as post-ANOVA test. PCNA = proliferating cell nuclear antigen; TGF = transforming growth factor; other abbreviations as in Figures 1, 2, and 4. Fertility and Sterility 2010 93, 239-250DOI: (10.1016/j.fertnstert.2008.09.086) Copyright © 2010 Terms and Conditions

Figure 6 Ad-DNER induces humoral immune response in Eker rat after direct intrauterine leiomyoma injection. Data are presented as mean and standard error of three animals' results per group. aSignificant difference from negative control at P<.05 using the Tukey test as a post-ANOVA test. Abbreviations as in Figures 1, 2, and 4. Fertility and Sterility 2010 93, 239-250DOI: (10.1016/j.fertnstert.2008.09.086) Copyright © 2010 Terms and Conditions

Figure 7 Safety studies of the Ad-DNER gene therapy approach after a single direct intrafibroid treatment of Eker rat. (A) Assessment of dissemination of adenovirus (Ad) particles into Eker rat body organs by PCR amplification of the specific Ad5 E4 region detected by the 714-pb DNA band on 1% agarose gel. The band was detected mainly in tumor tissues, and faint bands were detected also in uterus (lane 11) and liver (Lane 12) in 40% and 30% of the treated animals, respectively. (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 after Ad injection compared with vehicle control animals. ALT = alanine aminotransferase; AST = aspartate aminotransferase; other abbreviations as in Figures 1, 2, and 4. Fertility and Sterility 2010 93, 239-250DOI: (10.1016/j.fertnstert.2008.09.086) Copyright © 2010 Terms and Conditions