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Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a

Abstract

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 min of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).

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Fig. 1: Schematic of CRAAVE assay workflow.
Fig. 2: Assay design and proof of concept.
Fig. 3: CRAAVE assay demonstration on Virovek AAV.
Fig. 4: Effect of assay temperature on detection kinetics.
Fig. 5: CRAAVE assay optimization on affinity purified AAV2.
Fig. 6: CRAAVE assay detection of different genomic constructs in 3 different affinity purified AAV serotypes.
Fig. 7: Michaelis-Menten kinetics of the CRAAVE assay.

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Data availability

Data presented in this study can be found in the article and supplementary file. Additional data are available from the corresponding author upon reasonable request.

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Acknowledgements

The authors would like to thank Merck KGaA and Millipore-Sigma for their support and supply of rAAV. The authors would also like to acknowledge the funding support from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL). We would also like to thank Christopher Cummings and Shriarjun Shastry from the Biomanufacturing, Training, and Education Center (BTEC) at NC State for their work in AAV production and purification to support this work.

Funding

We sincerely thank the funding support from the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) for this work.

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Contributions

ZH and QW conceived the project and designed the experiments. SM and AH provided input and discussion towards experimental design and manuscript revision. ZH, NL, NM, JK, LT, and DF performed the experiments. ALN, LO, HM, HG, and OR produced and purified the AAV. ZH and QW wrote the original manuscript, and all authors reviewed and edited the manuscript.

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Correspondence to Qingshan Wei.

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Hetzler, Z., Marinakos, S.M., Lott, N. et al. Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a. Gene Ther 31, 304–313 (2024). https://doi.org/10.1038/s41434-024-00449-x

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