Kinetics of lentiviral vector transduction in human CD34(+) cells.
Unlike cell lines, human hematopoietic stem cells (HSCs) are less efficiently transduced with HIV-1 vectors, potentially limiting this approach. To investigate which step (internalization, reverse transcription, nuclear transport, and integration) limits lentiviral transduction, we evaluated the kinetics of lentiviral transduction in human CD34(+) cells. We transduced HeLa and CD34(+) cells with self-inactivating HIV-1 vector at low and tenfold higher multiplicity of infection (MOI) and evaluated vector amounts at various time points based on the rationale that if a given step was not limiting, tenfold greater vector amounts would be obtained at the tenfold higher MOI. We observed slower internalization (>60 min), a peak in reverse transcription at 24 hours, and completion of integration at 3 days in CD34(+) cells. In HeLa cells, there were approximately tenfold greater amounts at high MOI at all time points. When compared with HeLa cells, CD34(+) cells exhibited larger differences in vector amounts between high and low MOIs at 2-6 hours and a smaller difference at 12 hours to 10 days, revealing a limitation in human CD34(+) cell transduction around 12 hours, which corresponds to reverse transcription. In serial measurements of reverse transcription at 24 hours, vector amounts did not decrease once detected among CD34(+) cells. When using an HSC expansion medium, we observed less limitation for starting reverse transcription and more efficient transduction among CD34(+) cells in vitro and in xenografted mice. These data suggest that it is the initiation of reverse transcription that limits lentiviral transduction of human CD34(+) cells. Our findings provide an avenue for optimizing human CD34(+) cell transduction.
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD. Electronic address: uchidan@nhlbi.nih.gov.
Naoya
N
Uchida
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD.
Rashidah
R
Green
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD.
Josiah
J
Ballantine
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD.
Luke P
LP
Skala
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD.
Matthew M
MM
Hsieh
Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institute (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD.
John F
JF
Tisdale
D000818:Animals; D018952:Antigens, CD34 / Q000276:immunology*; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014161:Transduction, Genetic
D018952:Antigens, CD34