Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency.

Abstract

A number of stable producer cell lines for high-titer Mo-MuLV vectors have been constructed. Development has previously centered on increasing end-point titers by producing maximal levels of Mo-MuLV Gag/Pol, envelope glycoproteins, and retroviral RNA genomes. We describe the production yields and transduction efficiency characteristics of two Mo-MuLV packaging cell lines, FLYA13 and TEFLYA. Although they both produce 4070A-pseudotyped retroviral vectors reproducibly at >1 x 10(6) LFU ml(-1), the transduction efficiency of unconcentrated and concentrated virus from FLYA13 lines is poor compared with vector preparations from TEFLYA lines. A powerful inhibitor of retroviral transduction is secreted by FLYA13 packaging cells. We show that the inhibitory factor does not affect transduction of target cells by RD114-pseudotyped vectors. This suggests that the inhibitory factor functions at the level of envelope-receptor interactions. Phosphate starvation of target cells shows a two-fold increase in Pit2 receptor mRNA and causes some improvement in FLYA13 virus transduction efficiency. Western blots show that FLYA13 viral samples contain an eight-fold higher ratio of 4070A envelope to p30gag than that of virus produced by TEFLYA producer cell lines. This study correlates overexpression of 4070A envelope glycoprotein in retroviral preparations with a reduction of transduction efficiency at high multiplicities of infection. We suggest that TEFLYA packaging cells express preferable levels of 4070A compared with FLYA13, which not only enables high-titer stocks to be generated, but also facilitates a high efficiency of transduction of target cells.

• Authors: J H Slingsby, D Baban, J Sutton, M Esapa, T Price, S M Kingsman, A J Kingsman, A Slade
• Journal: Human gene therapy (Hum Gene Ther) Vol. 11 Issue 10 Pg. 1439-51 (Jul 01 2000) ISSN: 1043-0342 [Print] United States
• PMID: 10910141 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Antibodies, Monoclonal
• Culture Media
• Phosphates
• RNA, Messenger
• Receptors, Virus
• leukemia virus receptor, gibbon ape

Topics

• Animals
• Antibodies, Monoclonal
• Blotting, Western
• Cell Line
• Culture Media
• Dose-Response Relationship, Drug
• Gene Transfer Techniques
• Genes, env (genetics)
• Genes, gag (genetics)
• Genes, pol (genetics)
• Humans
• Moloney murine leukemia virus (genetics)
• Phosphates (pharmacology)
• RNA, Messenger (metabolism)
• Rats
• Receptors, Virus (metabolism)
• Retroviridae (genetics)
• Transduction, Genetic
• Tumor Cells, Cultured