High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells.
Transplantation of genetically corrected autologous hematopoietic stem cells is an attractive approach for the cure of sickle-cell disease and beta-thalassemia. Here, we infected human cord blood cells with a self-inactivating lentiviral vector encoding an anti-sickling betaA-T87Q-globin transgene and analyzed the transduced progeny produced over a 6-month period after transplantation of the infected cells directly into sublethally irradiated NOD/LtSz-scid/scid mice. Approximately half of the human erythroid and myeloid progenitors regenerated in the mice containing the transgene, and erythroid cells derived in vitro from these in vivo-regenerated cells produced high levels of betaA-T87Q-globin protein. Linker-mediated PCR analysis identified multiple transgene-positive clones in all mice analyzed with 2.1 +/- 0.1 integrated proviral copies per cell. Genomic sequencing of vector-containing fragments showed that 86% of the proviral inserts had occurred within genes, including several genes implicated in human leukemia. These findings indicate effective transduction of very primitive human cord blood cells with a candidate therapeutic lentiviral vector resulting in the long-term and robust, erythroid-specific production of therapeutically relevant levels of beta-globin protein. However, the frequency of proviral integration within genes that regulate hematopoiesis points to a need for additional safety modifications.
Imren
Suzan
S
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
Fabry
Mary E
ME
Westerman
Karen A
KA
Pawliuk
Robert
R
Tang
Patrick
P
Rosten
Patricia M
PM
Nagel
Ronald L
RL
Leboulch
Philippe
P
Eaves
Connie J
CJ
Humphries
R Keith
RK
D000818:Animals; D019070:Cell Lineage; D017690:Cell Transplantation; D002478:Cells, Cultured; D002877:Chromosomes, Human; D005312:Fetal Blood / Q000166:cytology*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D005914:Globins / Q000235:genetics / Q000378:metabolism*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D016086:Lentivirus / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D014161:Transduction, Genetic; D019076:Transgenes
D005914:Globins