In Vivo Potency Assay for Adeno-Associated Virus-Based Gene Therapy Vectors Using AAVrh.10 as an Example.

Publication Type	Academic Article
Authors	De B, Chen A, Salami C, Van de Graaf B, Rosenberg J, Pagovich O, Sondhi D, Crystal R, Kaminsky S
Journal	Hum Gene Ther Methods
Volume	29
Issue	3
Pagination	146-155
Date Published	06/08/2018
ISSN	1946-6544
Keywords	Dependovirus, Genetic Therapy, Genetic Vectors
Abstract	The development of a drug product requires rigorous methods of characterization and quality control to assure drug potency. Gene therapy products, a relatively new strategy for drug design with very few licensed examples, represent a unique challenge for the measure of potency. Unlike traditional drugs, potency for a gene therapeutic is a tally of the measures of multiple steps, including infectivity, transcription, translation, protein modifications, proper localization of the protein product, and protein function. This is particularly challenging for products based on the adeno-associated virus (AAV) platform, which has poor in vitro infectivity, limiting the sensitivity and thus the usefulness of cell-based assays. A rigorous in vivo assay has been established that separately evaluates infection, transcription, and resulting protein levels with specifications for each based on real time polymerase chain reaction (DNA and RNA) and standard protein assays. For an acceptance criterion, an administered vector must have vector DNA, transgene mRNA, and transgene expressed protein each concurrently meet individual specifications or the production lot fails. Using the AAVrh.10 serotype as a model vector and three different transgenes as examples, the assay is based on intravenous administration of the vector to male mice. At 2 weeks, the harvested liver is homogenized and assessed for vector genome levels (to assess for vector delivery), mRNA (to assess vector infectivity and transcription), and protein in the liver or serum (to assess protein expression). For all AAV vectors, the assay is robust and reproducible: vector DNA (linearity 102-109 copies, coefficient of variation) intra-assay <0.8%, inter-assay <0.5%; mRNA intra-assay <3.3%, inter-assay <3.4%. The reproducibility of the assay for transgene expressed protein is product specific. This in vivo potency assay is a strategy for characterization and a quantitative lot release test, providing a path forward to meet regulatory drug requirements for any AAV gene therapy vectors.
DOI	10.1089/hgtb.2017.246
PubMed ID	29706115