PA3-17

To determine whether human retinal pigment epithelial (RPE) cells can be infected by retrovirus and modified by retrovirus-mediated gene transfer.

Recombination retroviral vector pLNCX-GFP (green fluorescent protein, GFP) was generated by inserting 780 bp GFP cDNA fragment into the MCS site of pLNCX. pLNCX-GFP was transfected into ecotropic packaging cell line PhiX-Eco and amphotropic packaging cell line PhiX-Ampho and PA317. Retroviral titer was tested by counting GFP expression of NIH3T3 cells. Then RPE cells were infected by using GFP retrovirus-containing supernatant.

GFP was expressed and retrovirus was produced upon pLNCX-GFP being transfected into packaging cell line. The GFP retrovirus was able to infect primary cultured human RPE cells and immortalized RPE cell line.

The retrovirus can introduce a foreign gene into RPE cells efficiently, thereby it can be used as an important tool to deliver gene into RPE for therapy of fundus diseases.

During the production of clinical-grade retroviral vector supernatant, we noted significant differences in the lactate production and glucose consumption of various producer cell lines submitted to the National Gene Vector Laboratory (NGVL). Since differences in growth characteristics could be important in determining the optimal culture conditions for maximizing titer, we studied the growth characteristics of three commonly used packaging cell lines: PA317, PG13 and GP+envAM12. A transformed phenotype, assessed by the ability to form colonies in semisolid media, was evident in all three packaging cell lines tested. In confluent cultures, the rates of glucose consumption and lactate production (per cell per hour) were similar for the three lines tested, but the growth rate and culture density varied. PA317 and PG13 continued to expand after reaching confluence, resulting in higher cell densities and subsequent rapid depletion of glucose within the 24-hr observation period. When the cell lines were evaluated for titer optimization, the slower growing packaging cell line GP+envAM12 generally provided the highest titer after 8 hr of culture in confluent roller bottles, while most vectors introduced into PA317 and PG13 cells yielded optimal titers after 24 hr of culture. We also found that the improved titers obtained by culturing cells at 32 degrees C previously reported for PA317 cells do not apply to other packaging cell lines. In particular, PG13 rapidly lost titer when grown at the lower temperature. Our findings suggest that optimization of titer requires careful consideration of the culture conditions, which should be individualized for the vector producer cell line.