Serotype 5 Adenovirus fiber (F7F41S) chimeric vectors incur packaging deficiencies when targeting peptides are inserted into Ad41 short fiber

摘要

Adenovirus is a well-established viral gene transfer model system that presents two major hurdles when being considered for cell specific targeting applications. First is the need to detarget the vector from inherent host binding mechanisms, and second is the need to establish a productive and stable method to retarget the vector to a desired cell receptor. In previous studies we had generated an adenovirus vector platform that lacks the normal targeting attributes derived from the fiber and penton capsid proteins. In the current study we characterized our detargeted Ad5-based vectors (Ad5.F7F41S and Ad5.F7F41SΔRGD) as platforms for novel retargeted viruses. The experimental strategy relied on incorporating small peptide ligands into several sites of the Ad 41 short fiber knob domain (AB, CD, HI, G and Cterm). Reengineering of Ad41 short fiber resulted either in a bypass to fiber 7 usage, or in a dominant negative packaging/production deficiency phenotype. Under specific growth conditions we could remedy some of the capsid deficiencies and generate high titer viruses. However when examined by Western blot analysis, the resulting viruses were still defective in capsid content. The tandem fiber F7F41S platform has revealed an unanticipated sensitivity of Adenovirus packaging to fiber 41 short structural modifications. These studies indicate fiber assembly into an intact virion or fiber influenced capsid stability as a bottleneck to efficient particle production. We also demonstrate that virus particles characterized as mature virions following CsCl banding can vary significantly in capsid protein content. Considering the complexity of virus entry into a target cell, modified “mature virions” may be compromised at the level of transduction not only through the intended modification, but also by virtue of secondary structural packaging conflicts.