(560f) Modification of Fiberless Adenovirus With a Polymer/Peptide Conjugate for Enhanced Transduction and Reduced Immune Response

Recombinant adenovirus vectors are relatively safe and have been used widely as gene delivery vectors in gene therapy trials. Adenovirus capsid and fiber proteins, however, mediate an inflammatory response and initiate clearance of the virus in patients with preexisting immunity. In addition, adenovirus has a tropism toward cells that have abundant coxsackie-adenovirus receptor (CAR) and the inability to efficiently infect cells that lack this receptor. The adenovirus fiber protein plays a role in both of these drawbacks. Clearance of the vector, or immune inactivation, is the result of a reaction of neutralizing antibodies against epitopes on the surface of the virus (e.g. hexon and fiber proteins), and the virus tropism is based on the interaction of the fiber proteins with CAR.  Adenovirus vectors could be significantly improved if the fiber protein could be replaced with an alternate material that would function in place of the protein while reducing the associated drawbacks.

In our study we removed the fiber protein and developed a vector composed of fiberless adenovirus and a cell-penetrating peptide/polyethylene glycol conjugate (CPP-PEG) with the aim of diminishing the drawback associated with the virus. The CPP (Tat and Penetratin) aided in transporting the virus into cells in a CAR-independent manner while PEG acted to reduce the innate and adaptive immune responses to the virus particle.  The type of CPP, PEG molecular weight, and degree of PEGylation were varied to maximize the transduction efficiencies and minimize the immune response. The resulting vector showed 30-fold enhancement in gene transduction on cells lacking the CAR receptor while retaining 75 % of its transduction efficiency on cells with CAR. Modifying the vector with CPP-PEG lowered the level of proinflammatory cytokine IL-6 produced by mouse macrophage cells by 80 % compared to unmodified adenovirus. Furthermore, the vector was able to avoid immune inactivation by neutralizing antibodies. The results of this study demonstrated that the function of the fiber protein was effectively replaced with a CPP-PEG conjugate, which in turn reduced the tendency of the adenovirus to be neutralized by inactivating antibodies and introduced the ability to efficiently transform cells with and without the native receptor.