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.