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

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+ 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.

Direct Assessment of Single-Cell DNA Using Crudely Purified Live Cells: A Proof of Concept for Noninvasive Prenatal Definitive Diagnosis

“…sc-ddPCR was performed according to a previously described method (Figure 1). 20 For a reference signal, a hexachloro-6-carboxyfluoresceinelabeled RPP30 probe and primer set was used, which was the same as that used in the original method, 20 and for target, 1 mL of TaqMan Copy Number Assay (catalog number Hs01026408_cn; Thermo Fisher Scientific, Waltham, MA) was used, which contained a 6-carboxyfluoresceinelabeled SRY probe and primer set. One unit per well of the KAPA2G Robust HotStart DNA polymerase (KAPA Biosystems, Wobum, MA) in the modified sc-ddPCR system, whereas we added 4 units per well of the KAPA2G Robust HotStart DNA polymerase in the original sc-ddPCR system.…”

“…One unit per well of the KAPA2G Robust HotStart DNA polymerase (KAPA Biosystems, Wobum, MA) in the modified sc-ddPCR system, whereas we added 4 units per well of the KAPA2G Robust HotStart DNA polymerase in the original sc-ddPCR system. 20 To generate PCR mix containing additional DNA polymerase and the TaqMan primers and probe sufficient for 20 mL reactions in each well, the volume of the cell suspension was adjusted using 1Â PBS. For each maternal blood sample, we used 93 wells to analyze all of the cells after performing MACS.…”

“…To minimize the effect of detergents on the cells and TaqMan probe in each droplet, we generated droplets for 24 wells at a time using the QX200 Droplet Generator (Bio-Rad, Hercules, CA) just after mixing all of the reagents with the cell suspension. 20 After the generation of droplets, we performed PCR and subsequently analyzed the signal of each droplet using the QX200 Droplet Reader (Bio-Rad). The number of generating droplets were analyzed with the t-test in statistical software package R version 3.5.2 (https://www.r-project.org).…”

“…19 We previously reported on a single-cellebased droplet digital (sc-dd) PCR system that analyzes single-cell DNA without whole-genome amplification, cell fixation, or cell staining. 20 With this sc-ddPCR system, numerous cells are simultaneously separated and analyzed by encapsulation in a droplet that contains digital PCR reagents. In this previous study, we successfully analyzed single-cell genomic DNA from cultured cells and nucleated blood cells that were highly purified using fluorescence-activated cell sorting.…”

“…In this previous study, we successfully analyzed single-cell genomic DNA from cultured cells and nucleated blood cells that were highly purified using fluorescence-activated cell sorting. 20 However, sc-ddPCR has not been applied to cruder cell suspensions because the impurities therein decrease the reaction efficiency of multiplex PCR or increase the fragility of each droplet.…”