Transduction of Human Hematopoietic Cells and Cell Lines Using a Retroviral Vector Containing a Modified Murine CD4 Reporter Gene

To investigate conditions for improving transduction efficiencies of human hematopoietic stem or progenitor cells using retroviral vectors, we constructed a retroviral vector containing a modified murine CD4 cDNA reporter gene with a truncated cytoplasmic domain to prevent signaling. The advantages of using this truncated murine CD4 reporter gene include: (i) CD4 is well characterized with well-known cell signaling pathways, (ii) truncation of the cytoplasmic domain of CD4 has been demonstrated to abrogate signaling, (iii) the truncated murine CD4 is easily detectable on the cell surface with no cross-reaction to human CD4, (iv) a variety of monoclonal antibodies directed against the murine CD4 molecule are available commercially, and (v) expression of a truncated CD4 molecule in a transgenic mouse in vivo does not interfere with hematopoiesis. We cloned the truncated murine CD4 reporter gene into the retroviral vector LXSN, packaged this vector using PG13 retrovirus packaging cells, and transduced hematopoietic cell lines representing erythroid, myeloid, megakaryocyte, and lymphoid lineages using vector-containing medium harvested from the murine CD4 producer line. After seven daily exposures to vector-containing medium, all cell lines expressed murine CD4 on the cell surface, and 5–7% of human CD34⁺ cells expressed murine CD4 on the cell surface after 3 days of exposure to murine CD4 vector-containing medium. Colony-forming cell assays assessing progenitor cells demonstrated the presence of transduced cells in the CD34⁺ population. These results demonstrate the utility of using a modified murine CD4 gene in a retroviral vector to allow optimization of in vitro transduction conditions of human hematopoietic cells and to facilitate identification of the lineages that have been transduced using different growth factors, prior to clinical trials using retroviral vectors.

Overview summary

Current clinical protocols using retroviral vectors have been hampered by low transduction efficiencies. To investigate the conditions required to improve transduction efficiency, a retroviral vector was constructed containing a modified murine CD4 cDNA as a reporter gene. Vector-containing medium harvested from the murine CD4 producer line was used to transduce human hematopoietic cell lines, representing various cell lineages, and human CD34⁺ cells. Murine CD4 was present on the surface of transduced cells, demonstrating the utility of using a modified murine CD4 gene in a retroviral vector to allow optimization of in vitro transduction conditions of human hematopoietic cells prior to transduction of hematopoietic cells in clinical trials using retroviral vectors.