TY - JOUR
T1 - Droplet digital polymerase chain reaction-based quantitation of therapeutic lentiviral vector copies in transduced hematopoietic stem cells
AU - Phuphanitcharoenkun, Suphanun
AU - Bhukhai, Kanit
AU - Phanthong, Phetcharat
AU - Prasongtanakij, Somsak
AU - Linn, Aung Khine
AU - Sutjarit, Nareerat
AU - Anurathapan, Usanarat
AU - Leboulch, Philippe
AU - Payen, Emmanuel
AU - Hongeng, Suradej
AU - Borwornpinyo, Suparerk
N1 - Publisher Copyright:
© 2024
PY - 2024/6
Y1 - 2024/6
N2 - Background aims: Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). Methods: After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10−3 ng and 5 × 10−6 ng of target copy per reaction. Results: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Conclusions: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.
AB - Background aims: Gene therapy using lentiviral vectors (LVs) that harbor a functional β-globin gene provides a curative treatment for hemoglobinopathies including beta-thalassemia and sickle cell disease. Accurate quantification of the vector copy number (VCN) and/or the proportion of transduced cells is critical to evaluate the efficacy of transduction and stability of the transgene during treatment. Moreover, commonly used techniques for LV quantification, including real-time quantitative polymerase chain reaction (PCR) or fluorescence-activated cell sorting, require either a standard curve or expression of a reporter protein for the detection of transduced cells. In the present study, we describe a digital droplet PCR (ddPCR) technique to measure the lentiviral VCN in transduced hematopoietic stem and progenitor cells (HSPCs). Methods: After HSPCs were transduced with an LV encoding the therapeutic β-globin (βA-T87Q) gene, the integrated lentiviral sequence in the host genome was amplified with primers that targeted a sequence within the vector and the human RPP30 gene. The dynamic range of ddPCR was between 5 × 10−3 ng and 5 × 10−6 ng of target copy per reaction. Results: We found that the ddPCR-based approach was able to estimate VCN with high sensitivity and a low standard deviation. Furthermore, ddPCR-mediated quantitation of lentiviral copy numbers in differentiated erythroblasts correlated with the level of βA-T87Q protein detected by reverse-phase high-performance liquid chromatography. Conclusions: Taken together, the ddPCR technique has the potential to precisely detect LV copy numbers in the host genome, which can be used for VCN estimation, calculation of infectious titer and multiplicity of infection for HSPC transduction in a clinical setting.
KW - beta-thalassemia
KW - droplet digital PCR
KW - gene therapy
KW - lentiviral vector
KW - vector copy number
UR - http://www.scopus.com/inward/record.url?scp=85189084073&partnerID=8YFLogxK
U2 - 10.1016/j.jcyt.2024.02.018
DO - 10.1016/j.jcyt.2024.02.018
M3 - Article
AN - SCOPUS:85189084073
SN - 1465-3249
VL - 26
SP - 586
EP - 591
JO - Cytotherapy
JF - Cytotherapy
IS - 6
ER -