High-Level rAAV Vector Production by rAdV-Mediated Amplification of Small Amounts of Input Vector.

Abstract

The successful application of recombinant adeno-associated virus (rAAV) vectors for long-term transgene expression in clinical studies requires scalable production methods with genetically stable components. Due to their simple production scheme and the high viral titers achievable, first generation recombinant adenoviruses (rAdV) have long been taken into consideration as suitable tools for simultaneously providing both the helper functions and the AAV rep and cap genes for rAAV packaging. So far, however, such rAdV-rep/cap vectors have been difficult to generate and often turned out to be genetically unstable. Through ablation of cis and trans inhibitory function in the AAV-2 genome we have succeeded in establishing separate and stable rAdVs for high-level AAV serotype 2 Rep and Cap expression. These allowed rAAV-2 production at high burst sizes by simple coinfection protocols after providing the AAV-ITR flanked transgene vector genome either as rAAV-2 particles at low input concentrations or in form of an additional rAdV. With characteristics such as the ease of producing the required components, the straightforward adaption to other transgenes and the possible extension to further serotypes or capsid variants, especially the rAdV-mediated rAAV amplification system presents a very promising candidate for up-scaling to clinical grade vector preparations.