Interlaboratory assessment of candidate reference materials for lentiviral vector copy number and integration site measurements

While lentiviral vectors have played a critical role in the emergence of gene-modified cell therapies, safety concerns remain regarding potential insertional mutagenesis. Regulatory authorities strongly recommend risk assessment and management of vector copy numbers (VCNs), integration profiles, and integration sites in the lentivirus-based cell and gene therapy products. However, accurately measuring these parameters remains a significant challenge due to the lack of standardized methodologies and VCN reference materials (RMs). Toward this challenge, we conducted an interlaboratory study on NIST candidate RMs for VCN measurements. The candidate RMs comprise five human genomic DNA samples or fixed cells from clonal Jurkat cell lines with defined VCNs ranging from 0 to 4. All 12 study participants were able to identify the VCN in the five blinded samples using quantitative PCR (qPCR), digital PCR (dPCR), or next generation sequencing (NGS) assays. Consensus value of VCN and integration sites in these candidate RMs were achieved. The fixed clonal VCN cells were also used to evaluate an emerging imaging-based technology called molecular combing. This interlaboratory assessment demonstrated the utility, commutability, and suitability of the NIST VCN candidate RMs for quality assurance and improved confidence in VCN, integration profile, and integration site measurements.

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Journal
Molecular Therapy — Methods & Clinical Development
Published
2025-04-21
DOI
https://doi.org/10.1016/j.omtm.2025.101472
Primary Topic
Mathematics, Computing, and Information Processing
Type
article
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article

Interlaboratory assessment of candidate reference materials for lentiviral vector copy number and integration site measurements

Boro Dropulić, Mohamed Khalid Mohiuddin, Paul M. Welch, Barbara S. Paugh et al.
Molecular Therapy — Methods & Clinical Development
Mathematics, Computing, and Information Processing
article

Interlaboratory assessment of candidate reference materials for lentiviral vector copy number and integration site measurements

Boro Dropulić, Mohamed Khalid Mohiuddin, Paul M. Welch, Barbara S. Paugh, Yu Qiu, Rimas J. Orentas, Sierra Miller, Gabrielle S. Dijksteel, Laure Turner, Carole A. Foy, Monica Raimo, Harma Feitsma, J. Kandell, Daniëlle Steenmans, Michael Sheldon, Jennifer McDaniel, Chieh-Yuan Li, Sheng Lin‐Gibson, Hua‐Jun He, Zhiyong He, Justin M. Zook, Alessandro Tona, Ellen Stelloo, Shareef Nahas, Jan Spanholtz, Samantha Maragh, Alexandra S. Whale, Sana Ahmed‐Seghir, Uma Lakshmipathy, Simona Patange, Oxana Slessareva, Tu Feng, Yongjun Fan, Yan Ding, Kenneth D. Cole, David C. Corney, Benjamin Schroeder, Steven P. Lund, Katie Dungca, Rostyslav Makarenko, Engin Altunlu, Shai Senderovich, Dilek Yener
article en

Abstract

While lentiviral vectors have played a critical role in the emergence of gene-modified cell therapies, safety concerns remain regarding potential insertional mutagenesis. Regulatory authorities strongly recommend risk assessment and management of vector copy numbers (VCNs), integration profiles, and integration sites in the lentivirus-based cell and gene therapy products. However, accurately measuring these parameters remains a significant challenge due to the lack of standardized methodologies and VCN reference materials (RMs). Toward this challenge, we conducted an interlaboratory study on NIST candidate RMs for VCN measurements. The candidate RMs comprise five human genomic DNA samples or fixed cells from clonal Jurkat cell lines with defined VCNs ranging from 0 to 4. All 12 study participants were able to identify the VCN in the five blinded samples using quantitative PCR (qPCR), digital PCR (dPCR), or next generation sequencing (NGS) assays. Consensus value of VCN and integration sites in these candidate RMs were achieved. The fixed clonal VCN cells were also used to evaluate an emerging imaging-based technology called molecular combing. This interlaboratory assessment demonstrated the utility, commutability, and suitability of the NIST VCN candidate RMs for quality assurance and improved confidence in VCN, integration profile, and integration site measurements.

Molecular Therapy — Methods & Clinical DevelopmentVol. 33(2)
National Institute of Standards and Technology (US), National Physical Laboratory (GB), Age UK (GB), Ghent University (BE), Mission Bio (United States) (US), Environmental and Occupational Health Sciences Institute (US), Cergentis (Netherlands) (NL), Université Bourgogne Franche-Comté (FR), Lentigen Technology (United States) (US), Thermo Fisher Scientific (United States) (US), Eagle Mount (US), Genomic Vision (France) (FR), Green Cross International (US)
Openalex Percentile: Top 91%
Mathematics, Computing, and Information Processing
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