129. Infectivity Enhancement of an Adenovirus Vector Via Genetic Incorporation of the Reovirus Spike Protein Sigma 1

Abstract

During the last decade, human adenoviruses (Ad) have been exploited as vectors for gene transfer and gene therapy of various monogenic and polygenic diseases, with limited success. Early gene therapy efforts revealed that the clinical benefit of these gene delivery modalities was irrevocably linked to specific localization of the therapeutic agent. Many clinically relevant tissues are refractory to Ad5 infection, including several cancer tissue types, due to negligible CAR levels. Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Based on a clear understanding of native Ad cell recognition, development of genetically targeted vectors has rationally focused on the fiber knob domain, the primary determinant of Ad tropism. Reovirus serotype type 3 (T3) utilizes a fiber-like attachment protein, called sigma 1, to infect human cells. The sigma 1 molecule is comprised of an N-terminal tail region of alpha helix and eight beta-repeats, and a C-terminal globular head domain. Sigma 1 contains two receptor-binding domains (RBDs): one within the fiber tail that binds sialic acid (SA), and the other in the globular head that binds to junction adhesion molecule (JAM). The head RBD plays a key role in determining the tropism of T3 reoviruses, which have CAR-independent tropism. Based on these observations, we hypothesized that replacement of the Ad5 fiber with reovirus attachment protein sigma 1, a fiber-like molecule, would result in an Ad5 vector with a novel, CAR-independent tropism. We constructed a dual-fiber Ad5 vector, designated Ad5-sigma 1, encoding a luciferase reporter gene and two fibers: the first fiber is a chimeric fiber comprised of the tail domain of Ad5 fiber fused to the reovirus sigma 1 and the second fiber is the wild-type Ad5 fiber. Virus particles were rescued and purified and characterization of the dual-fiber Ad5-sigma 1 virions confirmed the presence of both fibers. Functionally, Ad5-sigma 1 was found to be able to utilize both receptors, CAR and JAM, for virus attachment to cells. In CAR positive cells, Ad5-sigma 1 gene transfer was reduced 80% by competition with recombinant Ad5 knob. Gene transfer was also inhibited by antibodies directed against JAM and sigma 1 in the L929 murine fibroblast cell line, known to express JAM and SA but not CAR. Ad5-sigma 1 provided 223-fold greater gene transfer versus the isogenic control vector in the L929 cells. In addition, Ad5-sigma 1 gene transfer was increased 6-fold in SA-positive, low-CAR chinese hamster ovary cells versus the isogenic control Ad5 vector. Furthermore, Ad5-sigma 1 provided 3.1- to 10.7- fold greater gene transfer versus control in a panel of ovarian cancer cell lines, known for relatively low CAR expression. We developed an infectivity enhanced Ad5 vector to address the key issue of limited Ad5 vector efficiency. The expanded tropism of the Ad5-sigma1 vector represents a crucial attribute for an Ad-based gene therapy vector specifically for ovarian cancer, and may represent an agent with future clinical utility.