Interlaboratory Assessment of Candidate Reference Materials for Lentiviral Vector Copy Number and Integration Site Measurements

Published

April 21, 2025

Author(s)

Hua-Jun He, Zhiyong He, Steven Lund, Barbara Paugh, Jennifer McDaniel, Justin Zook, Sierra Miller, Samantha Maragh, Simona Patange, Mahir Mohiuddin, Alessandro Tona, John Elliott, Kenneth Cole, Sheng Lin-Gibson

Abstract

Reference materials are essential for accurately measuring integrated lentiviral vector (LV) copy number (VCN) and integration sites for the safety and efficacy of lentivirus-based gene therapy. We conducted an interlaboratory study on NIST candidate reference materials for VCN measurements. The candidate reference materials comprise of five human genomic DNA samples or fixed cells from clonal Jurkat cell lines with defined VCNs ranging from 0 to 4. All 12 participating laboratories were able to identify the VCN in the five blinded samples using either quantitative real-time PCR (qPCR), digital PCR (dPCR) or next generation sequencing (NGS) assays. At all participating sites, no integrated LV was identified in the VCN0 sample. As expected, qPCR results showed higher variation compared to dPCR results in samples containing LV targets. dPCR results showed good concordance with the expected values for both VCN and vector copy concentration measurements. After alignment of NGS bioinformatic pipelines, all three NGS results reported identical VCNs and integration sites. The fixed clonal VCN cells were also used to evaluate an emerging imaging-based technology called molecular combing. This interlaboratory assessment supported the utility and suitability of the NIST VCN candidate reference materials in the analytical parameters of different VCN measurement methods to ensure quality assurance.

Citation

Molecular Therapy Methods & Clinical Development

Volume

33

Issue

2

Pub Type

Journals

Download Paper

https://doi.org/10.1016/j.omtm.2025.101472

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Keywords

Lentiviral vector (LV), vector copy number (VCN), integration site analysis, NGS, reference material, gene therapy

Citation

He, H. , He, Z. , Lund, S. , Paugh, B. , McDaniel, J. , Zook, J. , Miller, S. , Maragh, S. , Patange, S. , Mohiuddin, M. , Tona, A. , Elliott, J. , Cole, K. and Lin-Gibson, S. (2025), Interlaboratory Assessment of Candidate Reference Materials for Lentiviral Vector Copy Number and Integration Site Measurements, Molecular Therapy Methods & Clinical Development, [online], https://doi.org/10.1016/j.omtm.2025.101472, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=958707 (Accessed October 7, 2025)