886. An Adenovirus Vector with a Chimeric Fiber Derived from Ovine Adenovirus for Novel Tropism Discovery

Abstract

Gene therapy vectors based on human adenovirus (Ad) serotypes 2 and 5 continue to show increasing promise as gene therapy delivery vehicles, particularly in the context of cancer gene therapy. However, many clinically important tissues are refractory to Ad5 infection, including numerous cancer tissue types, due to reduced levels of the coxsackie and adenovirus receptor (CAR). Thus, development of novel Ad vectors demonstrating CAR-independent tropism may lead directly to therapeutic gain. Ovine adenovirus type 7, isolate 287 (OAdV7-287), has recently been described as the prototype of the new genus Atadenovirus, which contains several animal Ads. OAd287 encodes a fiber molecule containing 25 repeating units, and has a unique C-terminal knob domain of 121 amino acids. OAdV7-287 abortively infects several human, rodent and rabbit cell lines in vitro, and efficiently transduces human cells in vitro. Unlike Ad5, however, systemically administered OAd-based vectors do not predominately localize to the liver in rodents, but are distributed more evenly among the major organs. Based on these observations, we hypothesized that replacement of the Ad5 fiber with the non-human OAdV7-287 fiber would result in an Ad5 vector with a novel, CAR-independent, tropism in vitro and liver |[ldquo]|detargeting|[rdquo]| in vivo. We constructed an E1-deleted, luciferase transgene Ad5 vector encoding a chimeric fiber from ovine adenovirus 7-287 that includes the Ad5 tail region (Ad5/OAdV7), utilizing fiber-switching technology established in our laboratory. Viral particles were harvested from 911 cells, purified by standard cesium chloride gradient centrifugation and yielded titers in excess of 2.7|[times]|10e12 vp/mL. Characterization of Ad5/OAdV7 vector infectivity in CAR-deficient CHO Chinese hamster ovary cells, low-CAR MCF7 human breast cancer cells, T24 human bladder cancer cells and PC-3 human prostate cancer cells showed 10-to-30 fold greater luciferase reporter gene activities versus the isogenic AdLuc1 control vector. Furthermore, competitive inhibitory knob blocking assays in high-CAR cells using Ad5 recombinant knob protein demonstrated these increases to be independent of CAR receptor binding. Herein, we have demonstrated the construction, rescue, purification, and initial infectivity characterization of a novel CAR-independent, infectivity enhanced chimeric fiber Ad vector. These data strongly suggest that our genetic xeno-fiber paradigm alters Ad vector tropism, a key gene therapy vector design principle.