Evaluation of Cell Penetrating Peptide/Adenovirus Particles for Transduction of Car-negative Cells

Abstract

Adenovirus (Ad) is a promising gene therapy vector and is used currently in more than 23% of clinical gene therapy trials. The viral vector, however, has drawbacks such as immunogenicity, promiscuous tropism, and the inability to infect certain types of cells. The focus of this work was to develop an improved vector through electrostatic formation of a complex between negatively charged adenovirus and positively charged cell-penetrating peptides (CPPs), including Tat, Penetratin, polyarginine and Pep1. The resulting complexes were demonstrated to be capable of transducing cells that lack the coxsackie-adenovirus receptor (CAR) and are otherwise difficult to infect with native Ad. The transduction efficiency of the complexes was optimized by varying the multiplicity of infection, complex formation time and ratio of CPPs to Ad. The complexes improved the transduction efficiency on CAR-negative cells by more than 100-fold compared to unmodified Ad. Physicochemical characterization, including measurements of the size and zeta-potential of the complex, was performed to determine the suitability of the complex for in vivo gene delivery studies and investigate correlations between physicochemical properties and gene delivery efficiency. The size of CPP/Ad complex is initially less than 300 nm, but stability studies performed in the presence of serum indicate that the complex aggregates with serum after an extended time. The results of the present study indicate electrostatic modification of Ad with CPPs provides a relevant platform for developing effective Ad-based gene therapy vectors.