Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors

Pete Clarner; Shukkwan K Lau; Twinkle Chowdhury; Edward Guilmette; Patrick Trapa; Shih-Ching Lo; Shen Shen

doi:10.1016/j.omtm.2021.05.003

Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors

Mol Ther Methods Clin Dev. 2021 May 14:23:68-77. doi: 10.1016/j.omtm.2021.05.003. eCollection 2021 Dec 10.

Authors

Pete Clarner¹, Shukkwan K Lau², Twinkle Chowdhury², Edward Guilmette¹, Patrick Trapa³, Shih-Ching Lo², Shen Shen¹

Affiliations

¹ Gene Therapy Accelerator Unit, Biotherapeutic and Medicinal Sciences, Biogen, 225 Binney Street, Cambridge, MA 02142, USA.
² Neuromuscular and Muscle Disorders Research Unit, Research, Biogen, 225 Binney Street, Cambridge, MA 02142, USA.
³ Drug Metabolism and Pharmacokinetics, Biotherapeutic and Medicinal Sciences, Biogen, 225 Binney Street, Cambridge, MA 02142, USA.

Abstract

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.

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