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Hybrid HIV/MSCV LTR Enhances Transgene Expression of Lentiviral Vectors in Human CD34⁺ Hematopoietic Cells

^a Department of Pathology and Laboratory Medicine and
^b Department of Medicine, University of Pennsylvania, Philadelphia, USA;
^c Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

Key Words. Lentiviral vector • MSCV-based vector • Human CD34⁺ cells • Hybrid LTR

John Kim Choi, M.D., Ph.D., University of Pennsylvania, 413A SCL, 422 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. Telephone: 215-573-6527; Fax: 215-573-6523; e-mail: jkchoi{at}mail.med.upenn.edu;  Alan M. Gewirtz, M.D., University of Pennsylvania School of Medicine, Department of Pathology and Internal Medicine, Rm 713 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104, USA. Telephone: 215-898-4499; Fax: 215-573-2078; e-mail: gewirtz{at}mail.med.upenn.edu

HIV-based lentiviral vectors can transduce nondividing cells, an important advantage over murine leukemia virus (MLV)-based vectors when transducing slowly dividing hematopoietic stem cells. However, we find that in human CD34⁺ hematopoietic cells, the HIV-based vectors with an internal cytomegalovirus (CMV) promoter express transgenes 100- to 1,000-fold less than the MLV-based retroviral vector murine stem cell virus (MSCV). To increase the expression of the integrated lentivirus, we replaced CMV promoter with that of the Rous sarcoma virus or MSCV and obtained a modest augmentation in expression. A more dramatic effect was seen when the CMV enhancer/promoter was removed and the HIV long-terminal repeat (LTR) was replaced by a novel HIV/MSCV hybrid LTR. This vector retains the ability to transduce nondividing cells but now expresses its transgene (enhanced green fluorescent protein) 10- to 100-fold greater than the original HIV-based vector. When compared under identical conditions, the HIV vector with the hybrid LTR transduced a higher percentage of CD34⁺ cells than the MSCV-based retroviral vector (19.4% versus 2.4%). The number of transduced cells and level of transgene expression remain constant over 5-8 weeks as determined by long-term culture-initiating cells, fluoresence-activated cell sorting, and nonobese diabetic/severe combined immunodeficiency repopulation assay.

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