Mol Ther Methods Clin Dev. 2021 May 14
Pete Clarner, Shukkwan K. Lau, Twinkle Chowdhury, Edward Guilmette, Patrick Trapa, Shih-Ching Lo, and Shen Shen
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AAV vector packaging AAV-RNAi vectors were produced at PackGene Biotech (Worcester, MA, USA). Titers of AAV vectors were quantified by ddPCR using transgene-specific primer/probe sets. Request Quote

Research Field: method to determine the expression and potency of AAV vectors

Keywords: one-step RT-ddPCR, assay development, AAV gene therapy, vector expression, potency assay

AAV Serotype: AAV9 and AAVrh10

Dose: 1.5 X 1014 vg/NHP

Routes of Administration: 1.5 x10^14 vg of each AAV-CAGGs-mCherry was administered per animal in 2.5 mL injected into cerebrospinal fluid of cynomolgus monkeys via a bolus ICM injection.

Targeted organ: brain

Animal or cell line strain: adult cynomolgus monkeys


Robust assays to quantify adeno-associated virus (AAV) vector expression and potency are essential for gene therapy development. These assays inform the efficacy, safety, and pharmacodynamic profiles of AAV development candidates. Additionally, for gene downregulation strategies such as RNAi, knockdown of endogenous genes reflects the mechanism of action of such development candidates. Therefore, a method to quantify target mRNA repression is necessary for measuring vector potency both in vitro and in vivo. Here, we report the development of a one-step reverse-transcription droplet digital PCR (RT-ddPCR) method to analyze expression of AAV vectors and the potency of AAV-RNAi vectors. This one-step RT-ddPCR method simplifies the workflow, allows for duplexing reactions, and enables absolute quantification of transcripts without standard materials. With a gene augmentation vector, we demonstrate the application of RT-ddPCR in quantifying vector expression in vitro and in non-human primate (NHP) samples. This novel method is demonstrated to be precise and linear within the range of 0.05–25 ng of RNA input. Using an AAV-RNAi vector, we further demonstrate the utility of this RT-ddPCR method in quantifying potency. Orthogonal potency assays, including ELISA and functional readout, correlate well with RT-ddPCR results. Therefore, one-step RT-ddPCR can be implemented in the analytical and pharmacological characterization of AAV vectors.

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