Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors
Brief intro:
- Author: Pete Clarner, Shukkwan K. Lau, Twinkle Chowdhury, Edward Guilmette, Patrick Trapa, Shih-Ching Lo, and Shen Shen
- Journal: Mol Ther Methods Clin Dev
- Doi: https://www.doi.org/10.1016/j.omtm.2021.05.003
- Publication Date: 2021 May 14
Products/Services used in the paper
Quotation shows PackGene: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.
Research Field:method to determine the expression and potency of AAV vectors
AAV Serotype:AAV9 and AAVrh10
Targeted organ:brain
Animal or cell line strain:adult cynomolgus monkeys
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.
About PackGene
PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.
