Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy
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...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2017-09-01
|
| Series: | Molecular Therapy: Methods & Clinical Development |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050117300700 |
| _version_ | 1811277534709415936 |
|---|---|
| author | Yuka Igarashi Toru Uchiyama Tomoko Minegishi Sirirat Takahashi Nobuyuki Watanabe Toshinao Kawai Masafumi Yamada Tadashi Ariga Masafumi Onodera |
| author_facet | Yuka Igarashi Toru Uchiyama Tomoko Minegishi Sirirat Takahashi Nobuyuki Watanabe Toshinao Kawai Masafumi Yamada Tadashi Ariga Masafumi Onodera |
| author_sort | Yuka Igarashi |
| collection | DOAJ |
| description | 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+ cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT. |
| first_indexed | 2024-04-13T00:18:26Z |
| format | Article |
| id | doaj.art-ed70ec7c4d1c4466aa7a2d0b95a82b56 |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-04-13T00:18:26Z |
| publishDate | 2017-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-ed70ec7c4d1c4466aa7a2d0b95a82b562022-12-22T03:10:52ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012017-09-016C81610.1016/j.omtm.2017.05.005Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene TherapyYuka Igarashi0Toru Uchiyama1Tomoko Minegishi2Sirirat Takahashi3Nobuyuki Watanabe4Toshinao Kawai5Masafumi Yamada6Tadashi Ariga7Masafumi Onodera8Department of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, JapanDepartment of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, JapanDepartment of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, JapanDepartment of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, JapanDepartment of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, JapanDepartment of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, JapanDepartment of Pediatrics, Hokkaido University Graduate School of Medicine, Hokkaido 060-8638, JapanDepartment of Pediatrics, Hokkaido University Graduate School of Medicine, Hokkaido 060-8638, JapanDepartment of Human Genetics, National Center for Child Health and Development, Tokyo 157-8535, JapanClinical 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+ cells exhibited vector integration in bone marrow, indicating that the transgene-positive cells in PB might have differentiated from a small population of stem cells or lineage-restricted precursor cells. sc-ddPCR is a simplified and powerful tool for the detailed assessment of transgene-positive cell distribution in patients treated with SCGT.http://www.sciencedirect.com/science/article/pii/S2329050117300700gene therapyvector integrationsingle celldigital droplet PCRADA-SCID |
| spellingShingle | Yuka Igarashi Toru Uchiyama Tomoko Minegishi Sirirat Takahashi Nobuyuki Watanabe Toshinao Kawai Masafumi Yamada Tadashi Ariga Masafumi Onodera Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy Molecular Therapy: Methods & Clinical Development gene therapy vector integration single cell digital droplet PCR ADA-SCID |
| title | Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy |
| title_full | Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy |
| title_fullStr | Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy |
| title_full_unstemmed | Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy |
| title_short | Single Cell-Based Vector Tracing in Patients with ADA-SCID Treated with Stem Cell Gene Therapy |
| title_sort | single cell based vector tracing in patients with ada scid treated with stem cell gene therapy |
| topic | gene therapy vector integration single cell digital droplet PCR ADA-SCID |
| url | http://www.sciencedirect.com/science/article/pii/S2329050117300700 |
| work_keys_str_mv | AT yukaigarashi singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT toruuchiyama singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT tomokominegishi singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT sirirattakahashi singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT nobuyukiwatanabe singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT toshinaokawai singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT masafumiyamada singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT tadashiariga singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy AT masafumionodera singlecellbasedvectortracinginpatientswithadascidtreatedwithstemcellgenetherapy |