The 4.5A cryo-EM Structure of AAV-DJ: An Engineered Gene Therapy Vector

Adeno-associated virus (AAV) is a leading candidate vector for gene therapy. Therapeutic gene delivery using AAV vectors is hindered by broad tissue tropism, low transduction efficiency, and a prevalent immunity to the natural virus. Recently, a capsid library created by random shuffling of several AAV serotypes was put under selective pressure in hepatocytes using pooled human polyclonal sera. A promising chimeric vector, AAV-DJ, emerged from this screen and has greatly enhanced liver specificity, while avoiding recognition by neutralizing antibodies. Our previous high-resolution crystal structures of several AAV serotypes have provided insights into the functions of AAV capsids, and serve as templates for the rational design of vectors with altered specificity. Crystallographic approaches, however, require large quantities of AAV and significant efforts to optimize crystal quality for high-resolution studies. In the current study, we overcome these limitations by usin g cryo-electron microscopy to determine the structure of the AAV-DJ capsid at 4.5A resolution. The structure reveals insights into the novel properties of the vector, provides a foundation for further characterization and development of next-generation vectors, and provides mechanistic insights into the process of directed evolution.