A Novel Method for Quantitation of AAV Genome Integrity Using Duplex Digital PCR

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2023 Dec 14

PMID 38096204

Authors

Lauren Tereshko

Xiaohui Zhao

Jake Gagnon

Tinchi Lin

Trevor Ewald

Yu Wang

Marina Feschenko

Cullen Mason

Affiliations

Soon will be listed here.

Abstract

Recombinant adeno-associated virus (rAAV) vectors have become a reliable strategy for delivering gene therapies. As rAAV capsid content is known to be heterogeneous, methods for rAAV characterization are critical for assessing the efficacy and safety of drug products. Multiplex digital PCR (dPCR) has emerged as a popular molecular approach for characterizing capsid content due to its high level of throughput, accuracy, and replicability. Despite growing popularity, tools to accurately analyze multiplexed data are scarce. Here, we introduce a novel statistical model to estimate genome integrity from duplex dPCR assays. This work demonstrates that use of a Poisson-multinomial mixture distribution significantly improves the accuracy and quantifiable range of duplex dPCR assays over currently available models.

Citing Articles

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PMID: 39670179 PMC: 11634990. DOI: 10.1016/j.omtm.2024.101381.

References

Dube S, Qin J, Ramakrishnan R . Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One. 2008; 3(8):e2876. PMC: 2483940. DOI: 10.1371/journal.pone.0002876. View

Regan J, Kamitaki N, Legler T, Cooper S, Klitgord N, Karlin-Neumann G . A rapid molecular approach for chromosomal phasing. PLoS One. 2015; 10(3):e0118270. PMC: 4349636. DOI: 10.1371/journal.pone.0118270. View

Whale A, Huggett J, Tzonev S . Fundamentals of multiplexing with digital PCR. Biomol Detect Quantif. 2016; 10:15-23. PMC: 5154634. DOI: 10.1016/j.bdq.2016.05.002. View

Radukic M, Brandt D, Haak M, Muller K, Kalinowski J . Nanopore sequencing of native adeno-associated virus (AAV) single-stranded DNA using a transposase-based rapid protocol. NAR Genom Bioinform. 2021; 2(4):lqaa074. PMC: 7671332. DOI: 10.1093/nargab/lqaa074. View

Marguet E, Forterre P . Protection of DNA by salts against thermodegradation at temperatures typical for hyperthermophiles. Extremophiles. 1998; 2(2):115-22. DOI: 10.1007/s007920050050. View

Morley A . Digital PCR: A brief history. Biomol Detect Quantif. 2016; 1(1):1-2. PMC: 5129430. DOI: 10.1016/j.bdq.2014.06.001. View

Tai P, Xie J, Fong K, Seetin M, Heiner C, Su Q . Adeno-associated Virus Genome Population Sequencing Achieves Full Vector Genome Resolution and Reveals Human-Vector Chimeras. Mol Ther Methods Clin Dev. 2018; 9:130-141. PMC: 5948225. DOI: 10.1016/j.omtm.2018.02.002. View

Greer S, ZAMENHOF S . Studies on depurination of DNA by heat. J Mol Biol. 1962; 4:123-41. DOI: 10.1016/s0022-2836(62)80046-1. View

Furuta-Hanawa B, Yamaguchi T, Uchida E . Two-Dimensional Droplet Digital PCR as a Tool for Titration and Integrity Evaluation of Recombinant Adeno-Associated Viral Vectors. Hum Gene Ther Methods. 2019; 30(4):127-136. PMC: 6707039. DOI: 10.1089/hgtb.2019.031. View

10.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N . The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16):2078-9. PMC: 2723002. DOI: 10.1093/bioinformatics/btp352. View

11.

Hindson B, Ness K, Masquelier D, Belgrader P, Heredia N, Makarewicz A . High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem. 2011; 83(22):8604-10. PMC: 3216358. DOI: 10.1021/ac202028g. View

12.

Pinheiro L, Coleman V, Hindson C, Herrmann J, Hindson B, Bhat S . Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 2011; 84(2):1003-11. PMC: 3260738. DOI: 10.1021/ac202578x. View

13.

EIGNER J, Boedtker H, Michaels G . The thermal degradation of nucleic acids. Biochim Biophys Acta. 1961; 51:165-8. DOI: 10.1016/0006-3002(61)91028-9. View

14.

Prantner A, Maar D . Genome concentration, characterization, and integrity analysis of recombinant adeno-associated viral vectors using droplet digital PCR. PLoS One. 2023; 18(1):e0280242. PMC: 9876284. DOI: 10.1371/journal.pone.0280242. View

15.

Marguet E, Forterre P . DNA stability at temperatures typical for hyperthermophiles. Nucleic Acids Res. 1994; 22(9):1681-6. PMC: 308049. DOI: 10.1093/nar/22.9.1681. View

16.

Shmidt A, Egorova T . PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations. Pharmaceuticals (Basel). 2022; 15(1). PMC: 8779925. DOI: 10.3390/ph15010023. View

17.

Peak M, Robb F, Peak J . Extreme resistance to thermally induced DNA backbone breaks in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol. 1995; 177(21):6316-8. PMC: 177479. DOI: 10.1128/jb.177.21.6316-6318.1995. View

18.

Lindahl T, Nyberg B . Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972; 11(19):3610-8. DOI: 10.1021/bi00769a018. View

19.

Amarasinghe S, Su S, Dong X, Zappia L, Ritchie M, Gouil Q . Opportunities and challenges in long-read sequencing data analysis. Genome Biol. 2020; 21(1):30. PMC: 7006217. DOI: 10.1186/s13059-020-1935-5. View

20.

Basu A . Digital Assays Part I: Partitioning Statistics and Digital PCR. SLAS Technol. 2017; 22(4):369-386. DOI: 10.1177/2472630317705680. View