Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors
| العنوان: | Development of a one-step RT-ddPCR method to determine the expression and potency of AAV vectors |
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| المؤلفون: | Edward Guilmette, Patrick Trapa, Shen Shen, Shih-Ching Lo, Pete Clarner, Shukkwan K. Lau, Twinkle Chowdhury |
| المصدر: | Molecular Therapy. Methods & Clinical Development Molecular Therapy: Methods & Clinical Development, Vol 23, Iss, Pp 68-77 (2021) |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | Gene knockdown, assay development, QH573-671, one-step RT-ddPCR, Genetic enhancement, RNA, Computational biology, Biology, QH426-470, potency assay, vector expression, RNA interference, Genetics, Molecular Medicine, Potency, Digital polymerase chain reaction, Original Article, Vector (molecular biology), Cytology, Molecular Biology, Gene, AAV gene therapy |
| الوصف: | 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. Graphical abstract Clarner et al. developed a one-step RT-ddPCR method to quantify both AAV vector expression and endogenous gene repression for AAV gene therapy vectors. This novel method was applied to analysis of cell-based assays, mouse studies, and NHP studies to demonstrate robust correlation with orthogonal methods. |
| تدمد: | 2329-0501 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::ad546c59ad543bedd861409815a15a77Test https://pubmed.ncbi.nlm.nih.gov/34589555Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....ad546c59ad543bedd861409815a15a77 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 23290501 |
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