Towards Gene Therapy of Postoperative Adhesions: Fiber and Transcriptional Modifications Enhance Adenovirus Targeting towards Human Adhesion Cells (original) (raw)
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Prevention of peritoneal adhesions: a promising role for gene therapy
World journal of gastroenterology : WJG, 2011
Adhesions are the most frequent complication of abdominopelvic surgery, yet the extent of the problem, and its serious consequences, has not been adequately recognized. Adhesions evolved as a life-saving mechanism to limit the spread of intraperitoneal inflammatory conditions. Three different pathophysiological mechanisms can independently trigger adhesion formation. Mesothelial cell injury and loss during operations, tissue hypoxia and inflammation each promotes adhesion formation separately, and potentiate the effect of each other. Studies have repeatedly demonstrated that interruption of a single pathway does not completely prevent adhesion formation. This review summarizes the pathogenesis of adhesion formation and the results of single gene therapy interventions. It explores the promising role of combinatorial gene therapy and vector modifications for the prevention of adhesion formation in order to stimulate new ideas and encourage rapid advancements in this field.
Journal of Gynecologic Surgery, 2006
Objective: Seprafilm, ® (Genzyme, Cambridge, MA) an absorbable adhesion barrier incorporating hyaluronic acid (HA), a high molecular mass glycosaminoglycan and important component of the extracellular matrix, has been shown to prevent adhesions in both experimental models and human subjects. Yet, the application of HA as a sheet at the time of surgery has several important logistic limitations. Recently, our laboratory has identified and cloned the genes encoding murine hyaluronic acid synthase 2 (mHAS2) and 3 (mHAS3) and engineered adenoviruses incorporating these genes, which, on intraperitoneal injection, significantly increases HA in peritoneal fluid. We hypothesized that intraperitoneal gene therapy with mHAS2 or mHAS3 via an adenoviral vector prior to a standardized cecal abrasion surgery would lead to a reduction in postoperative adhesion severity. Methods: Mice were assigned to one of four groups: (1) intraperitoneal inoculation with adenovirus encoding mHAS2; (2) mHAS3; (3) a control reporter adenovirus (RV) encoding GFP; or (4) intraoperative placement of a commercially available and murine-validated hyaluronic acid adhesion barrier (Seprafilm, ® SF). An a priori sample size calculation was performed. Mice in groups 1, 2, and 3 underwent injection of 2 ϫ 10 7 viral particles in 1 ml of fluid on day Ϫ1. Sham injection was performed on group 4 SF mice. On day 0, laparotomy was performed in random sequence by surgeon blinded to the experimental group. On day 7, adhesion scores (0-3) were assigned independently by two blinded investigators. Results: Mean adhesion scores (n ϭ 247) were 0.68 (mHAS2), 0.91 (mHAS3), 1.28 (RV), and 0.47 (SF). Pairwise comparisons using Wilcoxon rank-sum test revealed significant reduction in severity of adhesions between mHAS2, mHAS3, and SF compared to RV (p ϭ 0.0004, 0.039, and 0.0001, Original Articles respectively). Significance persisted despite correction for multiple comparisons (p ϭ 0.0002, Kruskal-Wallis). There was a direct relationship between intraperitoneal HA concentration and adhesion reduction. Only one death (RV) was secondary to adhesive disease; differential risk of death between groups was statistically significant (p ϭ 0.008) (highest in mHAS2 group). Conclusions: In a dose-response relationship, an intraperitoneal gene therapy approach to adhesion prevention in a murine model was successful, with adenoviruses most productive of HA resulting in the most significant reduction in adhesion scores compared to "empty" virus (RV). Although SF ® best reduced postoperative adhesions, the adenoviral gene delivery approach may prove to be more effective in clinical use when peritoneal injury is less localized or at laparoscopy where the application of SF is not possible. Further studies to elucidate the reason for the differential death rates (time bias may have played a role) and to validate results are in progress. (J GYNECOL SURG 22:7)
Surgery, 2009
Background. Tissue-plasminogen activator (tPA) demonstrated beneficial effects on peritoneal adhesion formation; however, its short half-life limits its continual fibrinolytic effect. Therefore, we delivered adenovirus encoding tPA to prevent adhesions. Methods. Rats were subjected to peritoneal injury and assigned to two protocols. In de novo adhesion protocol, adenovirus encoding human tPA gene (Ad-htPA) was instilled after peritoneal injury in group1 (n = 22), whereas group 2 received phosphate-buffered saline (PBS) (n = 24). In recurrent adhesion protocol, group 1 (n = 15) received the same Ad-htPA dose after adhesiolysis and group 2 (n = 13) received PBS. Adhesion severity was scored 1 week after ad-htPA instillation. Adhesions were analyzed for htPA mRNA by reverse transcription-polymerase chain reaction and levels of htPA, and fibrinolytic inhibitors PAI-1, TIMP-1, and TGF-b1 were measured using enzyme-linked immunosorbent assay. Results. htPA mRNA and protein were only expressed in adhesions from treated groups. A reduction in adhesion scores (P < .01) and in fibrinolytic inhibitors (P < .001) occurred in the treatment groups. Also, negative correlation was found (r = À.69, P < .01) between adhesion scores and htPA protein, but a positive correlation was found (r = .90, P < .01) between adhesion score and fibrinolytic inhibitors. No bleeding or wound complications were encountered. Conclusion. Administration of adenovector encoding htPA is safe and decreased de novo and recurrent peritoneal adhesions. (Surgery 2009;146:12-7.) From the
Toward gene therapy of uterine fibroids: targeting modified adenovirus to human leiomyoma cells
Human Reproduction, 2008
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), telomeraseimmortalized 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.
RNA Interference Targeting Hypoxia Inducible Factor 1α Reduces Post-Operative Adhesions in Rats
Journal of Surgical Research, 2007
To investigate the use of RNA interference mediated gene down-regulation targeting hypoxia inducible factor 1alpha (HIF-1alpha) and plasminogen activator inhibitor 1 (PAI-1) in an effort to prevent abdominal adhesion formation. Real time PCR and a PAI-1 protein activity assay were used in vitro to determine the efficacy of small interfering RNAs (siRNAs). For in vivo experiments, 57 white female rats were operated to generate ischemic and serosal injury to the uterine horns, and treated with saline, siRNA(Lamin A/C) (negative control), siRNA(HIF-1alpha), siRNA(PAI-1), or siRNA(HIF-1alpha) plus siRNA(PAI-1). The cationic polyer poly(ethylenimine) (PEI) was used as the delivery vehicle for all siRNAs delivered in vivo. Adhesions were analyzed by a blinded surgeon 8 days post-surgery. After in vitro transfection with siRNA, at least 69% gene down-regulation was obtained for all siRNAs tested. In vitro siRNA-mediated down-regulation of HIF-1alpha, PAI-1 or their simultaneous delivery resulted in a significant decrease of PAI-1 protein activity (at least P &amp;amp;amp;amp;amp;lt; 0.05). Administration of 4 nmol siRNA(HIF-1alpha)/PEI complexes after injury to the uterine horns achieved a statistical reduction of post-operative adhesion formation with a reduction by 52% (P &amp;amp;amp;amp;amp;lt; 0.05). Delivery of 4 nmol siRNA(PAI-1)/PEI complexes and the simultaneous delivery of 2 nmol siRNA(HIF-1alpha) plus 2 nmol siRNA(PAI-1), resulted in a reduction of abdominal adhesion by 36% and 42%, respectively, with the reduction being statistically significant when compared directly to the saline control (P &amp;amp;amp;amp;amp;lt; 0.01). These data show that administration of siRNA/PEI complexes within the peritoneal cavity can be used to prevent post-operative abdominopelvic adhesions.
Reproductive biology and endocrinology : RB&E, 2005
Injury to the peritoneum during surgery is followed by a healing process that frequently results in the attachment of adjacent organs by a fibrous mass, referred commonly as adhesions. Because injuries to the peritoneum during surgery are inevitable, it is imperative that we understand the mechanisms of adhesion formation to prevent its occurrence. This requires thorough understanding of the molecular sequence that results in the attachment of injured peritoneum and the development of fibrous tissue. Recent data show that fibroblasts from the injured peritoneum may play a critical role in the formation of adhesion tissues. Therefore, identifying changes in gene expression pattern in the peritoneal fibroblasts during the process may provide clues to the mechanisms by which adhesion develop. In this study, we compared expression patterns of larger number of genes in the fibroblasts isolated from adhesion and normal human peritoneum using gene filters. Contributions of TGF-beta1 and hy...
Adenovirus Serotype 5 Fiber Shaft Influences In Vivo Gene Transfer in Mice
Human Gene Therapy, 2003
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Journal of virology, 1997
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Gene Transfer to Ovarian Cancer Versus Normal Tissues with Fiber-Modified Adenoviruses
Molecular Therapy, 2002
Adenovirus serotype 5 (Ad5) displays unparalleled gene transfer efficacy to cells with high coxsackie-adenovirus receptor (CAR) expression. Unfortunately, cells isolated from clinical human cancers, both ovarian and other types, express highly variable and often low levels of CAR. Fortunately, native Ad5 tropism can be modified to circumvent CAR deficiency and to enhance infectivity. Ad5/3luc1 incorporates the serotype 3 fiber knob and binds to a receptor distinct from CAR, while the fiber of Ad5lucRGD is modified with an RGD-4C motif, allowing CAR-independent binding to integrins. We studied the liver tropism and blood clearance of these viruses after intravenous (i.v.) injection, and biodistribution after intraperitoneal (i.p.) injection to tumor-bearing mice. To estimate efficacy, we assessed gene transfer to purified human primary ovarian cancer cells, and in a mouse model of ovarian cancer. Ad5/3luc1 achieved improved gene transfer over Ad5lucRGD, and both infectivity-enhanced viruses were superior to the isogenic control with an unmodified Ad5 capsid. In the presence of malignant ascites, gene transfer was improved with both Ad5/3luc1 and Ad5lucRGD. Thus, retargeting to the Ad3 receptor enhances gene transfer to clinically relevant ovarian cancer substrates, while the mouse toxicity and biodistribution profile of both fiber-modified Ad vectors is comparable to Ad5.
Human Gene Therapy, 2004
Approaches to alter the native tropism of adenoviruses (Ads) are beneficial to increase their efficacy and safety profile. Liver tropism is important with regard to potential clinical toxicity in humans. Ad5/3 chimeras in which the Ad5 knob is substituted by the Ad3 knob, such as Ad5/3luc1, have been recently shown to increase infectivity of ovarian cancer cell lines and primary tumor cells, which express low levels of the coxsackie-adenovirus receptor (CAR), without increasing infectivity of liver cells. A novel strategy to address the problem of liver uptake and improve the tumor/liver ratio is genetic replacement of the Ad fiber shaft. Ad5.Ad3.SH.luc1 is an Ad5-based vector that contains the fiber shaft from Ad serotype 3 but the fiber knob from Ad serotype 5. To compare tumor/liver of Ad5.Ad3.SH.luc1 and Ad5/3luc1 in vivo, we created three different tumor and treatment models of ovarian cancer in mice, simulating intraperitoneal and intravenous administration of tumors. Ad5.Ad3.SH.luc1 displayed the lowest liver tropism of all viruses in all models tested. Intravenous administration of all viruses resulted in higher tumor transduction rates compared to intraperitoneal administration. Genetic shortening of the Ad5 fiber shaft significantly increases relative tumor/liver gene transfer. This could improve the effective tumor dose and reduce side effects, thereby increasing the bioavailability of therapeutic agents.