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Igarashi Y, Uchiyama T, Minegishi T, et al. Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy. Mol Ther Methods Clin Dev. 2017;6:8-16doi: 10.1016/j.omtm.2017.05.005.
Igarashi, Y., Uchiyama, T., Minegishi, T., Takahashi, S., Watanabe, N., Kawai, T., Yamada, M., Ariga, T., & Onodera, M. (2017). Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy. Molecular therapy. Methods & clinical development, 68-16. https://doi.org/10.1016/j.omtm.2017.05.005
Igarashi, Yuka, et al. "Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy." Molecular therapy. Methods & clinical development vol. 6 (2017): 8-16. doi: https://doi.org/10.1016/j.omtm.2017.05.005
Igarashi Y, Uchiyama T, Minegishi T, Takahashi S, Watanabe N, Kawai T, Yamada M, Ariga T, Onodera M. Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy. Mol Ther Methods Clin Dev. 2017 May 25;6:8-16. doi: 10.1016/j.omtm.2017.05.005. eCollection 2017 Sep 15. PMID: 28626778; PMCID: PMC5466583.
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Mol Ther Methods Clin Dev. 2017 May 25;6:8-16. doi: 10.1016/j.omtm.2017.05.005. eCollection 2017 Sep 15.

Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy.

Molecular therapy. Methods & clinical development

Yuka Igarashi, Toru Uchiyama, Tomoko Minegishi, Sirirat Takahashi, Nobuyuki Watanabe, Toshinao Kawai, Masafumi Yamada, Tadashi Ariga, Masafumi Onodera

Affiliations

  1. Department of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, Japan.
  2. Department of Pediatrics, Hokkaido University Graduate School of Medicine, Hokkaido 060-8638, Japan.

PMID: 28626778 PMCID: PMC5466583 DOI: 10.1016/j.omtm.2017.05.005

Abstract

Clinical improvement in stem cell gene therapy (SCGT) for primary immunodeficiencies depends on the engraftment levels of genetically corrected cells, and tracing the transgene in each hematopoietic lineage is therefore extremely important in evaluating the efficacy of SCGT. We established a single cell-based droplet digital PCR (sc-ddPCR) method consisting of the encapsulation of a single cell into each droplet, followed by emulsion PCR with primers and probes specific for the transgene. A fluorescent signal in a droplet indicates the presence of a single cell carrying the target gene in its genome, and this system can clearly determine the ratio of transgene-positive cells in the entire population at the genomic level. Using sc-ddPCR, we analyzed the engraftment of vector-transduced cells in two patients with severe combined immunodeficiency (SCID) who were treated with SCGT. Sufficient engraftment of the transduced cells was limited to the T cell lineage in peripheral blood (PB), and a small percentage of CD34

Keywords: ADA-SCID; digital droplet PCR; gene therapy; single cell; vector integration

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