title: Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34(+) Cells Correlates With In Vivo Vector Copy Number.
abstract: Hematopoietic stem cell (HSC) gene therapy using integrating vectors has a potential leukemogenic risk due to insertional mutagenesis. To reduce this risk, a limitation of ≤2 average vector copy number (VCN) per cell is generally accepted. We developed an assay for VCN among transduced CD34(+) cells that reliably predicts in vivo VCN in 16 rhesus recipients of CD34(+) cells transduced with a green fluorescent protein (GFP) (or yellow fluorescent protein (YFP))-encoding lentiviral vector. Using GFP (or YFP)-specific probe/primers by real-time PCR, VCN among transduced CD34(+) cells had no correlation with VCN among granulocytes or lymphocytes in vivo assayed 6 months post-transplantation. This was a likely result of residual plasmids present in the vector preparation. We then designed self-inactivating long terminal repeat (SIN-LTR)-specific probe/primers, which detect only integrated provirus. Evaluation with SIN-LTR probe/primers resulted in a positive correlation of VCN among transduced CD34(+) cells with granulocytes and lymphocytes in vivo. The transduced CD34(+) cells had higher VCN (25.1 ± 5.6) as compared with granulocytes (2.8 ± 1) and lymphocytes (2.4 ± 0.7). In summary, an integrated provirus-specific real-time PCR system demonstrated nine- to tenfold higher VCN in transduced CD34(+) cells in vitro, as compared with VCN in vivo. Therefore, the restriction of ≤2 VCN before infusion might unnecessarily limit gene transfer efficacy.Molecular Therapy-Nucleic Acids (2013) 2, e122; doi:10.1038/mtna.2013.49; published online 17 September 2013.
authors.affiliation: Molecular and Clinical Hematology Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, Maryland, USA.
authors.forename: Naoya
authors.initials: N
authors.lastname: Uchida
authors.forename: Molly E
authors.initials: ME
authors.lastname: Evans
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Hsieh
authors.forename: Aylin C
authors.initials: AC
authors.lastname: Bonifacino
authors.forename: Allen E
authors.initials: AE
authors.lastname: Krouse
authors.forename: Mark E
authors.initials: ME
authors.lastname: Metzger
authors.forename: Stephanie E
authors.initials: SE
authors.lastname: Sellers
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
authors.forename: Robert E
authors.initials: RE
authors.lastname: Donahue
authors.forename: John F
authors.initials: JF
authors.lastname: Tisdale
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