282151 Biophysical Characterization of Mutated Fiber Adenoviruses
Adenovirus is one the most widely used vectors in clinical trials for gene therapy. The majority of adenovirus internalizes into cells via highly specific interactions between the fiber protein on the virus and the Coxsackie-adenovirus receptor (CAR) on the surface of target cells. This broad natural tropism results in difficulty targeting adenovirus to specific diseased cells due to the ubiquitous presence of CAR receptors on various cells and tissues. Additionally, the fiber and capsid proteins on the surface of adenovirus are recognized by neutralizing antibodies (NAbs), which severely hamper the efficacy of adenovirus as a gene delivery and vaccine vector. The fiber protein consists of three domains that include the tail, shaft, and knob. The shaft domain consists of 22 repeating motif of approximately 15 residues and the pattern of hydrophobic residues can be aligned, and the knob domain is primarily responsible for interaction with the CAR receptor.
Our approach to overcoming issues of promiscuous tropism and immunogenicity is to remove the fiber protein from adenovirus, and replace its function with synthetic materials. The fiberless virus, however, is unstable and unable to assemble into correctly matured viral particles. To optimize the stability of the vector, while also reducing the natural tropism and immunogenicity, the knob protein was removed entirely and the length of the fiber protein was varied from 7-repeats, 14-repeats, and 21-repeats. The relative stability of these mutants was compared with the native adenovirus and fiberless adenovirus using circular dichroism, static and dynamic light scattering, intrinsic tryptophan fluorescence, and extrinsic fluorescence. The results indicate the minimum number of pseudo-repeats required for assembly of stable fiber-mutants adenoviral particles and are being used to identify the best candidate for future gene delivery studies.
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