A new retrovirus packaging cell for gene transfer constructed from amplified long terminal repeat-free chimeric proviral genes

Abstract

The retroviral gene transfer system is a powerful tool for somatic gene therapy. A retroviral stock with a high viral titer and lacking replication-competent virus (RCV) is desirable for this type of gene transfer. To fulfill these requirements, we made a new packaging cell line, designated ampli-GPE. To reduce the homology between proviral DNA in the packaging cell and retroviral vector, the gag-pol and env genes of Moloney murine leukemia virus were separated onto two different plasmids, pGP-KV and pENV-KV, respectively, in which the 5' long terminal repeat and the 3' long terminal repeat had been replaced by the mouse metallothionein I promoter or the human 13-globin gene containing the polyadenylation site as control units for the gag-pol and env genes. In addition, these plasmids contained 69%o of the bovine papillomavirus gene for gene amplification to obtain production of virus at a high titer. NIH 3T3 clones containing approximately 20 to 50 copies of the gag-pol and env genes were selected and designated ampli-GPE. When ampli-GPE was transfected with the N2 vector or pZipNeoSV(DHFR) derived from pZipNeoSV(X)1, we established clones producing titers of 5 x 106 and 1 x 106 CFU/ml, respectively. There was no sign of RCV generation in any virus-producing cells from ampli-GPE. However, virus-producing cells derived from 12 cells transfected with N2 did generate RCV. Thus, we showed that ampli-GPE, possessing the minimum complement of proviral genes, has potential for the development of a gene transfer system.

A Replication-Competent Retrovirus Arising from a Split-Function Packaging Cell Line Was Generated by Recombination Events between the Vector, One of the Packaging Constructs, and Endogenous Retroviral Sequences

“…A number of improved packaging cell lines which yield higher viral titers or pseudotyped particles for a variety of specific purposes have been constructed (5,14,20,25). All of these are based on the safety concept of split function to minimize the chances of producing replication-competent viruses.…”

Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors

“…These stable producer cell lines, however, have several major drawbacks: vector titers generated by them are generally too low to allow an efficient in vivo gene transfer, they may evolve to produce recombinant retroviral particles, and they are most suitable to provide vectors in vitro. Alternative improvements to overcome some of these limitations have been described previously, all of them based on the use of transient retroviral vector production systems (4,8,12,21,27,36,38,40). Although these systems differ in several respects, a common shared feature is that the retroviral packaging and vector components are introduced into cells by transfection procedures, thus limiting the efficiency of these new strategies.…”