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

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

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...

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Bibliographic Details
Published in:Gene therapy Vol. 31; no. 5-6; pp. 304 - 313
Main Authors: Hetzler, Zach, Marinakos, Stella M., Lott, Noah, Mohammad, Noor, Lass-Napiorkowska, Agnieszka, Kolbe, Jenna, Turrentine, Lauren, Fields, Delaney, Overton, Laurie, Marie, Helena, Hucknall, Angus, Rammo, Oliver, George, Henry, Wei, Qingshan
Format: Journal Article
Language:English
Published: London Nature Publishing Group UK 01-05-2024
Nature Publishing Group
Subjects:
631/1647/1513
631/337
631/61/201
9/10
Adenoviruses
Bioassays
Biomedical and Life Sciences
Biomedicine
Capsids
Cell Biology
CRISPR
CRISPR-Cas Systems
Dependovirus - genetics
Gene Expression
Gene Therapy
Genetic Vectors - genetics
Genome, Viral
Genomes
HEK293 Cells
Human Genetics
Humans
Nanotechnology
Parasites
Polymerase chain reaction
Serotypes
Transgenes
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Summary: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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ISSN:0969-7128
1476-5462
DOI:10.1038/s41434-024-00449-x