Integration-specific In Vitro Evaluation of Lentivirally Transduced Rhesus CD34+ Cells Correlates With In Vivo Vector Copy Number

• Main Authors: Naoya Uchida, Molly E Evans, Matthew M Hsieh, Aylin C Bonifacino, Allen E Krouse, Mark E Metzger, Stephanie E Sellers, Cynthia E Dunbar, Robert E Donahue, John F Tisdale
• Format: Article
• Language: English
• Published: Elsevier 2013-01-01
• Series: Molecular Therapy: Nucleic Acids
• Subjects: CD34+ cells; hematopoietic stem cell transplantation; large animal model; lentiviral vector
• Online Access: http://www.sciencedirect.com/science/article/pii/S2162253116301810

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.