RESUMEN

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 1011 vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 1011 vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines.

Asunto(s)

Dependovirus , Vectores Genéticos , Humanos , Dependovirus/genética , Células HEK293 , Células HeLa , Transfección

RESUMEN

Objective To construct the small interfering RNA (siRNA) eukaryotic expression vector specific to human MGMT gene(pRNATin-H1.2/Neo MGMT siRNA) to observe its silencing effect on MGMT gene in vitro.Methods The pRNATin-H1.2/Neo MGMT siRNA expression vector was constructed by gene recombination,then transfected into the cultured HelaS3 cells.Inhibitory effect of siRNAs was detected by semi-quantitative RT-PCR.Results The pRNATin-H1.2/Neo MGMT siRNA expression vector was successfully constructed.Cells transfected with pRNATin-H1.2/Neo MGMT siRNA could obviously inhibit the expression level of MGMT gene.Conclusion The pRNATin-H1.2/Neo MGMT siRNA expression vector could inhibit the MGMT gene expression.