Advantages of HR system over Tn7 system in term of product safety
We designed qPCR assays to amplify specifically product-related DNA impurities, i.e. gentamycin and kanamycin gene sequences, in the newly produced rAAV batches. In the Tn7-derived rAAV batches, we detected >109 copies of gentamycin out of 1012 viral genomes while this value is remarkably lower <5,0 x 107 copies in HR-derived batches (Fig. 5a ). The kanamycin resistance gene sequence was not detectable whatever the system used to produce recombinant baculoviruses (data not shown). LOQ = 7.0 x 106copies/ml.
Finally, the identity and purity of rAAV vectors derived from HR and Tn7 systems were determined by western blot analysis and SDS-PAGE silver staining (Fig. 5b ). The profile of the proteins detected by silver stained SDS-PAGE was comparable in all cases, but the capsid signal was more intense in rAAV stocks produced with the HR system when loading the same amount of vg per well, and the same was true for western blot analysis. It correlates with previously observed increased of total capsids in HR condition as measured by ELISA (Fig. 4b ). Altogether, these observations suggested that HR system definitively reduces the risk of bacterial sequences encapsidation into the final rAAV biotherapeutic and, thus, it increases the safety profile of the product as recommended by regulatory agencies, taking into account that the delta chitinase-cathespin version may lead to more empty rAAV particles at harvest.