Bringing molecular biology to bear on adhesion prevention: Postsurgical adhesion reduction using intraperitoneal inoculation of hyaluronic acid-inducing adenoviral vector in a murine model

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 × 10⁷ viral particles in 1 ml of fluid on day -1. Sham injection was performed on group 4 SF mice. On day O, 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 = 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 = 0.0004, 0.039, and 0.0001, respectively). Significance persisted despite correction for multiple comparisons (p = 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 = 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.