Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells.
Efficient gene transfer to hematopoietic stem cells by Moloney murine leukemia virus-derived retroviral vectors benefits from ex vivo culture and cytokine support. Both also increase the risks of apoptosis and differentiation among cells targeted for transduction. In an effort to maximize the retention of stem cell properties in target cells, we developed a transduction protocol with a focus on minimizing graft manipulation, cytokine stimulation, and ex vivo exposure duration. Based on their wide host range and ability to transduce quiescent cells, human immunodeficiency virus (HIV)-derived lentivirus vectors are ideally suited for this purpose. Our present studies in a murine model show that whole bone marrow cells are readily transduced after a 1-hour vector exposure in the presence of stem cell factor and CH296 fibronectin fragment. Using this rapid transduction protocol, we achieved long-term, multilineage reconstitution of murine recipients with up to 25% GFP-expressing cells in primary and secondary recipients. Our results demonstrate the unique ability of HIV-derived vectors to transduce hematopoietic stem cells in the absence of enrichment, under minimal cytokine stimulation, and following brief exposures.
Kurre
P
P
Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA. kurrepe@ohsu.edu
Anandakumar
P
P
Kiem
H-P
HP
D000818:Animals; D001853:Bone Marrow / Q000821:virology*; D002452:Cell Count; D002478:Cells, Cultured; D003470:Culture Media; D005353:Fibronectins / Q000494:pharmacology; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology*; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D019337:Hematologic Neoplasms / Q000473:pathology / Q000628:therapy; D006412:Hematopoietic Stem Cells / Q000821:virology*; D051379:Mice; D010446:Peptide Fragments / Q000494:pharmacology; D019089:Stem Cell Factor / Q000494:pharmacology; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods*
D003470:Culture Media; D005353:Fibronectins; D010446:Peptide Fragments; D019089:Stem Cell Factor; D049452:Green Fluorescent Proteins