Efficient marking of murine long-term repopulating stem cells targeting unseparated marrow cells at low lentiviral vector particle concentration.
HIV-1-derived lentivirus vectors offer unique biological properties for gene delivery to hematopoietic stem cells and, when used at high multiplicities of infection (m.o.i.), permit efficient gene transfer after minimal target cell stimulation. However, such a strategy has been shown to promote multicopy proviral integration, potentially increasing the risk of insertional mutagenesis. To minimize cell manipulation, we targeted unseparated marrow and demonstrated that transduction at an m.o.i. of 1 resulted in up to 12% vector-modified peripheral blood leukocytes and successful repopulation of secondary recipients with vector-marked cells. Real-time PCR showed on average 1.8 proviral integrants per GFP-marked cell. By comparison, a cohort of animals transplanted with cells transduced at m.o.i. of 10 under otherwise unchanged conditions showed up to 45% marking with an average of 7 copies per GFP-expressing cell. Both m.o.i. groups demonstrated sustained proviral expression with stable GFP fluorescence intensity. In summary, we have identified conditions for lentiviral gene transfer involving minimal ex vivo target cell manipulation and have shown that the m.o.i. is a critical determinant of proviral copy number in lentivirus-transduced murine long-term repopulating cells. Thus, gene transfer efficiencies may be limited when single-copy integration is desired and additional strategies such as in vivo selection may be required to improve the frequency of gene-modified cells.
Kurre
Peter
P
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Anandakumar
Ponni
P
Harkey
Michael A
MA
Thomasson
Bobbie
B
Kiem
Hans-Peter
HP
D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry; D003114:Colony-Forming Units Assay; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006098:Granulocytes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000032:analysis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D016254:Mutagenesis, Insertional / Q000235:genetics; D013601:T-Lymphocytes / Q000737:chemistry; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics; D014771:Virion / Q000235:genetics; D016662:Virus Integration
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins