Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction

Human Gene Therapy · 6 authors, 1 centre

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TL;DR

A ddPCR-based assay was developed using 116-bp and 247-bp amplicons targeting the 18S rRNA gene to quantify residual host cell DNA in purified rAAV preparations produced in HEK293 cells.

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HEK293 cells were used as the host cell line, and two amplicon sizes (116 bp and 247 bp) targeting the 18S rRNA gene were evaluated. Using ddPCR without DNase treatment, host cell DNA was detected at 2.67–7.41 pg/10^9 vg for the 116-bp amplicon and 1.98–6.26 pg/10^9 vg for the 247-bp amplicon. DNase-resistant fractions ranged from 64.4–69.3% and 58.9–69.2% for the respective amplicons, indicating that DNA impurities are partially encapsidated.

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other

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research methods

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ORIGINAL PAPER

PMC10285681

Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction

Human Gene Therapy

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ABSTRACT

Abstract Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with the risk of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116- and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with copy numbers of three reference genes ( EIF5B , DCK , and HBB ). Results showed that 88.6–97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate residual host cell DNA as an impurity. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products. Keywords: recombinant adeno-associated virus, host cell DNA, 18S ribosomal RNA, droplet digital PCR, HEK293 cells

CITATION EXPORT

Kiyoko Higashiyama, Yuzhe Yuan, Noriko Hashiba et al.. Quantitation of Residual Host Cell DNA in Recombinant Adeno-Associated Virus Using Droplet Digital Polymerase Chain Reaction. Human Gene Therapy. (2023). https://doi.org/10.1089/hum.2023.006

AUTHORS

6 · 1 CENTRES

Kiyoko Higashiyama, Yuzhe Yuan, Noriko Hashiba, Kyoko Masumi-Koizumi, Keisuke Yusa, Kazuhisa Uchida

1Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.