Simple and efficient new adeno-associated virus (AAV) qPCR titration method

SEOANE ROCHA, CAMILA; PALMA, MARÍA BELÉN; LOMBARDI, ANTONELLA; AMÍN, GUADALUPE; IRIBARNE, AILEN; MARTINEZ, KEVIN; INDA, ANA MARÍA; MIRIUKA, SANTIAGO G.; MORO, LUCÍA N.; GARCÍA, MARCELA N.

Mar del Plata

Congreso; Reunión Conjunta SAIC, SAI&FAIC, SAFIS 2022; 2022

Adeno-associated virus (AAV) are small nonenveloped viruses with a linear, single stranded DNA of approximately 4.7kb. AAV has gained growing interest as a human gene therapy vector for decades. This is due to its non-pathogenicity in humans, persists episomal, its high transduction efficacy, low inflammatory response, and broad host cell tropism. Currently, methods for purification of AAV can be expensive because they require equipment of ultrahigh speed gradient centrifugation and in the absence of this it is possible to obtain cell detritus together with the viral fraction. The strategy to titrate AAV particles is the quantification by qPCR using primers that target ITRs sequences of the viral genome, but these cell debris mentioned above could interfere with the titration. The aim is to propose a new AAV qPCR titration method including an additional step of transduction with the obtained viral fraction. We carried out Qpcr titration of AAV2-GFPturbo produced from pAAV-CASI-Cas9C-2AturboGFP (#80942, AddGene), directly from the viral fraction or by the proposed new method. For this, HEK 293T cells are transduced with 1, 5 and 10uL of the viral fraction obtained, incubated for 6h and then the viral genome is extracted to quantify only the infective viral particles. Finally, qPCR was performed. The results show that we obtained a value 1,06x10ˆ9Vg/ml with the new titration method, compare with 2,19x10ˆ11Vg/ml obtained with the conventional titration method. Therefore, it can be said that the proposed method was efficient since we obtained a value similar to those published (1x10ˆ9Vg/ml). In summary, we have proposed a new, accurate, reliable AAV titration method. Furthermore, it´s a simple method which can be carried out in laboratories that do not have access to the necessary equipment to purify completely the viral fraction. In addition, as the additional step is not specific to the viral serotype, this method could be applied to the rest of the AAV serotypes.