Multi Approaches to Produce Super Infectious rAAV Particles at Super High Titer:
Not like adenovirus, it is relatively harder to achieve high yield of rAAV with regular rAAV production procedures. By adopting the following multi strategies, we have managed to produce super infectious (~30 times better infectious capability than conventional rAAV) rAAV particles at super high titer (up to 1E+15 GC).
– AAV·HT™ Packaging Cell: We have screened different HEK293 cell types and developed a highly productive strain——AAV·HT™ packaging cell which per our validation data produces ~10 times more rAAV particles than regular HEK293.
– Modify rAAV cis Vector: Customer has an option to choose rAAV cis vector with a truncated WPRE cassette downstream of transgene. Inclusion of WPRE cassette in rAAV cis vector allows to produce ~8 times more rAAV particles than rAAV cis vector without WPRE.
– Modify rAAV Capsid: rAAV capsid mutations at several specific sites significantly (~30 folds better in vitro & in vivo) enhanced rAAV transduction efficiency. Customers have an option to choose packaging rAAV with mutant capsids to produce more infectious rAAV vector.
– Package Double-stranded AAV (dsAAV): Double-stranded AAV (dsAAV) also known as self-complementary AAV (scAAV) is confirmed to deliver up to 50 times better transduction efficiency in both vitro and vivo. Custom service is available to generate and package gene of interests to a scAAV vector*.
– Advanced Double CsCl Ultra-centrifugation Protocol: The main feature of the advanced protocol is treatment with a special formulated detergent after 3xfreeze/thaw cycle and pre-precipitation step before loading to ultra-centrifugation. The advanced protocol produces super purified (clinical trial grade) and super infectious rAAV vector at high yield. See Figure 8 for details. The differences with conventional protocol are being framed in red.
Figure 7. Multi Approaches to Produce Super Infectious rAAV Particles at Super High Titer
Outline and comparison of advanced protocol and conventional protocol. The differences in advanced protocol are being framed in red.
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