88. Structural Studies Into the Stability of Novel In Silico Reconstructed Ancestral AAV Vectors

Adeno-associated virus (AAV) vectors have emerged as therapy preferred clinical in vivo gene transfer system. Yet, their sensitivity to pre-existing immunity (PEI) and low carrying capacity remain major drawbacks that limit broad applications. We previously hypothesized and have shown that ancestral species of AAV, which we inferred using statistical and bio-informatic approaches, and generated synthetically, have reduced susceptibility to PEI. Here, our ambition was to study from a biophysical and structural perspective the evolutionary process of AAV using these novel reagents, and to develop correlates of those measures with virus and vector phenotypes.By the means of polyacrylamide gel electrophoresis, intrinsic fluorescence spectroscopy and differential scanning fluorescence, we derive the thermostability profiles of 6 contemporary AAV serotypes (AAV1, AAV2, AAV5, AAV8, AAV9, rh32.33) and 3 ancestral AAV serotypes, called Anc80, Anc81 and Anc82 and differing from AAV8 in VP1 protein sequence by 9.3%, 7.8% and 5.3%, respectively. A theoretical structure of Anc80L0065 VP3 monomer was derived by homology modeling and compared to the crystal structures of AAV2, AAV8 and rh32.33.Structural modeling indicates significant sequence and structure differences between ancestral and contemporary VP3 protein subunits in loops I, IV, VII and VIII, regions that were shown to be involved in antibody and cell receptor binding. Surprisingly, Anc80, Anc81 and Anc82 showed an increase in capsomer melting temperature of approximately 20°C in comparison to AAV8, while vector genome is accessible at lower temperatures. Evolutionary intermediates between Anc80 and AAV8 demonstrate unique melting properties that permit the identification of a limited set of capsid determinants to the thermodynamic stability of AAVs. Our reconstructed phylogeny of AAVs emerges as a relevant tool for sequence-function analysis of AAV vectors.