A complete testing approach to rAAV vectors

Recombinant adeno-associated virus (rAAV) vectors have emerged as powerful, diverse and precise gene therapy tools, offering promising solutions to previously unmeet genetic disorders. As modified versions of naturally occurring AAVs, they can be harnessed for their ability to target specific tissues and cells by engineering their tropism. AAV vector testing thus needs a comprehensive approach to best meet key technical parameters of different serotypes. AAV vector quality control testing should include various tests to assess for purity, identity, biosafety and potency of rAAVs while taking into account process development (production context, choice of construct, etc.).

With a growing number of GMP and non-GMP assays and methods for AAV vector testing, Clean Cells continues to show flexibility in development and routine application, having partnered with several organizations worldwide to develop serotype-specific assays (AAV2, AAV5, AAV6, AAV8, AAV9). Our access to established chimeric AAVs through a trusted partner, combined with our in-house capability to develop chimeric AAVs and our two detection platforms (HEK293 and HeLa), enables us to rapidly develop critical assays such as replication-competent AAV (rcAAV) testing for novel serotypes.

Comprehensive quality control approaches are key to AAV vector testing and characterization from different production backgrounds: assays should thus consider if the vectors have been produced with plasmids (typically on HEK293 cells) or through other methods (Sf/baculovirus system, stable producer cell line…). With a 200+ portfolio of qPCR assays developed and validated according to ICHQ2 guidelines, Clean Cells is at the forefront of detection and quantification of DNA sequences relating to AAV vector testing, in particular for residual DNA testing (host cell, plasmid, transgene, AdV5, etc.). Comprehensive and compendial testing packages in support of AAV testing have also been developed and validated to characterize substrates (cells, viruses) according to the worldwide regulatory requirements (EP, USP, JP, ICHQ5A). Our production platform provides essential support to sponsors in generating fully characterized cell banks (Sf, HEK293) and virus seeds (baculovirus) in a GMP setting.

Safety and potency of products should be established through robust and bespoke AAV vector testing methods, best tailored for each serotype. In particular, assays should show that the process yields qualitative vectors, able to properly transduce targeted cells while not generating replication competent AAVs. Through qualified ELISA, qPCR, dPCR and infectivity assay modalities, we can perform non-GMP and cGMP assays for AAV vector testing at different R&D and clinical stages with adequate validation levels, including:

As rAAV candidates progress toward clinical development, Clean Cells supports sponsors with bespoke testing approaches to assess transduction efficiency, transgene expression at the mRNA level by qPCR and at the protein level by ELISA, as well as the biological activity of the expressed protein. Drawing on our expertise in bioassay development and AAV characterization, we provide tailored support for robust and reliable potency assessment.

Novel tools have recently emerged allowing for increased flexibility in testing, which can be used to derisk processes and/or as complementary AAV vector testing methods intended for regulatory dossiers.

Next-Generation Sequencing is such a modality improving AAV vector QC and turnaround, allowing for quick determination of identity while having the potential to assist in biosafety by detecting contamination events. In addition to NGS, our testing platform welcomes rAAVs testing projects at every stage of development or clinical phase and systematically explores new techniques for continuous improvement of AAV vector testing and characterization.