title: Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy.
abstract: Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer-probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.
authors.affiliation: 1 Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .|2 Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .
authors.forename: Huan-Ting
authors.initials: HT
authors.lastname: Lin
authors.affiliation: 2 Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .
authors.forename: Takashi
authors.initials: T
authors.lastname: Okumura
authors.affiliation: 3 Life Science Division, Bio-Rad Laboratories , Tokyo, Japan .
authors.forename: Yukinori
authors.initials: Y
authors.lastname: Yatsuda
authors.affiliation: 3 Life Science Division, Bio-Rad Laboratories , Tokyo, Japan .
authors.forename: Satoru
authors.initials: S
authors.lastname: Ito
authors.affiliation: 1 Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .|4 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine , Stanford, California.
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Nakauchi
authors.affiliation: 1 Division of Stem Cell Therapy, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .|2 Division of Stem Cell Processing/Stem Cell Bank, Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, University of Tokyo , Tokyo, Japan .
authors.forename: Makoto
authors.initials: M
authors.lastname: Otsu
mesh_terms: D056915:DNA Copy Number Variations / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics* / Q000302:isolation & purification; D006105:Granulomatous Disease, Chronic / Q000235:genetics* / Q000473:pathology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D060888:Real-Time Polymerase Chain Reaction; D033581:Stem Cell Transplantation; D016662:Virus Integration / Q000235:genetics
chemical_list: D017931:DNA Primers
keywords: chronic granulomatous disease; droplet digital PCR; induced pluripotent stem cells; integrating vectors; vector copy number