Rapid AAV-Neutralizing Antibody Determination with a Cell-Binding Assay

Abstract

Recombinant adeno-associated virus (rAAV) has been developed as a successful vector for both basic research and human gene therapy. However, neutralizing antibodies (NAbs) against AAV capsids can abolish AAV infectivity on target cells, reducing the transduction efficacy. Absence of AAV NAb has become a prerequisite qualification for patients enrolled in gene therapy trials. Nevertheless, accurate assessment of AAV NAb has remained a challenging task. Here we developed a rapid assay based on the observations that AAV NAb inhibits rAAV binding to the host cell surface and NAb titers are negatively related to the amount of AAV genomes binding to the target cells. By quantifying the AAV genome on the target cells in the presence of anti-sera, AAV NAb titers can be accurately determined. The titer determined by this assay correlates well with the classical transduction-based assays. A major advantage of this method is that it can be carried out with a 30-min binding assay without the lengthy wait for a transduction outcome. This assay is independent of transduction performance of AAV serotype in the target cells. Therefore, the AAV cell-binding assay for NAb determination offers an alternative method for in vivo NAb assay.

Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers?

“…16 In this regard, use of assays that do not rely on transduction performance, such as total antibody assays or the assay recently developed by Guo and colleagues, which relies on quantification of AAV binding to the target cells in vitro using a qPCR assay. 19 Further compounding the intrinsic complexity of each assay are the differences in the AAV investigational products themselves, in terms of infectivity titers and content of empty capsids, both of which influence transduction performance and thus may affect the NAb titer.…”

“…Other characteristics that impact NAb titers when evaluated using in vitro assays include the amount of serum used, the cell number on the plate and the reporter transgene . In this regard, use of assays that do not rely on transduction performance, such as total antibody assays or the assay recently developed by Guo and colleagues, which relies on quantification of AAV binding to the target cells in vitro using a qPCR assay …”

Development of New Strategies Using Extracellular Vesicles Loaded with Exogenous Nucleic Acid

“…However, since viral capsids are derived from wild-type AAV, AAV vectors can be impacted by preexisting adaptive immune responses, capsid neutralizing antibodies (Nabs), making the viral capsid the trigger of the immune response in several subjects undergoing AAV gene transfer [54]. An in vitro method was recently published to quantify AAV cell-binding inhibition by qPCR [55]; however, several hindrances associated with this assay have been reported [56]. Several strategies have been addressed to circumnavigate the capsid Nabs; the in vivo administration of a proteasome inhibitor (PI), bortezomib, has revealed a significantly reduced Nabs trigger against AAV vectors [57].…”

Identification of 11 candidate structured noncoding RNA motifs in humans by comparative genomics

“…Cell culture follows standard protocol (53). Brie y, cell lines HEK-293 (human embryonic kidneys) (purchased from Conservation Genetics CAS Kunming Cell Bank, Kunming, Yunnan, China) were passaged twice before collecting cells for RNA extraction.…”