Retroviral Gene Transfer Using Safe and Efficient Packaging Cell Lines

Abstract

One of the requirements for the use of retroviral vectors in human gene therapy is a packaging cell line which is incapable of producing replication-competent virus and which produces high titers of replication-deficient vector virus. Wild-type virus may be produced through recombinational events between the helper virus and a retroviral vector. We have constructed an ecotropic packaging cell line, GP + E-86, and an amphotropic packaging cell line, GP + envAm12, in which the viral gag and pol genes are on one plasmid and the viral env gene is on another plasmid. Both plasmids contain deletions of the packaging sequence and the 3' LTR. The fragmented helper virus genomes, when introduced into 3T3 cells, produce titers of retrovirus which are comparable to the titers produced from packaging cells containing the helper virus genome on a single plasmid. We have found no evidence for the generation of wild-type retrovirus using the GP + E-86 and GP + envAm12 packaging lines, either alone or in combination with the N2 retroviral vector. We also show that these packaging cell lines can be used to transfer the neoR gene of the N2 vector into mouse hematopoietic cells, followed by successful (48-52%), long-term (up to 200 days) transplantation into irradiated recipients. These results indicate that these packaging lines are safe and efficient for use in experiments designed for murine (using GP + E-86) and human (using GP + envAm12) gene therapy.