University of Heidelberg

AAV - Background

  • Adeno-associated viruses (AAV) are small, non-pathogenic, single-stranded DNA viruses that belong to the Parvoviridae family and that exist in over 100 naturally occurring variants or so-called serotypes. The wildtype virus is naturally replication-defective and can propagate only in the presence of a helpervirus, which is typically the name-giving Adenovirus or Herpes simplex virus 1.
  • In addition to its intriguing unique biology, AAV is highly interesting from a biomedical standpoint since it can readily be engineered as a recombinant vector for human gene transfer/therapy. As such, it provides a number of attractive features over all other viral vector systems under development, including its inherent apathogenicity, its small genome (comprising only two genes of ~4.7 kilobases) which facilitates molecular engineering, as well as its low chromosomal integration frequency and ensuing minimal risk of genotoxicity.
  • Perhaps most remarkable is AAV’s amenability to pseudotyping, i.e., the cross-packaging of a given vector genome into any capsid of choice via co-transfection of human 293T cells with the recombinant vector and the selected capsid-encoding plasmid. Since the AAV capsid is the major determinant of the viral tropism (i.e. the specificity for certain target cells), pseudotyping a vector genome is a simple yet enormously powerful strategy to re-target recombinant AAVs to cell types of choice in vitro or in vivo.
  • Further potentiating the power and promise of AAV pseudotyping is the option to create synthetic AAV capsids that are derived from naturally occurring AAV serotypes via directed molecular evolution. In this respect, the most versatile and powerful method is DNA family shuffling technology which was first introduced to the AAV field in a study published 2008 by Grimm and colleagues, and which has subsequently been used by a large number of groups.
  • The hallmark of this method is controlled fragmentation of specific AAV variants (selected based on features that are interesting for the application), followed by re-assembly into chimeras via 2 PCRs. Cloning of the resulting pool of typically >1e6 different sequences into a replication-competent AAV plasmid context allows the production of a highly diverse library of viral particles which can be selected on target cells for candidates of interest. See a video of the entire procedure (Kienle & Senis et al., JoVE, 2012).
  • To date, we have expanded this method to a collection of 12 AAV serotypes from which we have created >20 distinct libraries, several of which we are screening in various collaborations to select novel synthetic capsids that can potently infect e.g. pancreatic β-cells, dorsal root ganglia, iPS cells or human T-cells. Moreover, we have engineered a unique battery of 108 further AAV capsid mutants in which we have inserted 9 different re-targeting peptides into our 12 natural serotypes, as an alternative powerful strategy to expand the AAV tropism. Our ultimate goal is to use the most effective and specific AAV variant on each cell type for therapeutic delivery of anti-viral (HIV, HBV, HCV) RNAi sequences that we optimize in parallel, or as novel tools for iPS cell reprogramming and purging.  
Contact: E-Mail (Last update: 19/12/2013)