Rev-Independent Expression of Syntheticgag-polGenes of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus: Implications for the Safety of Lentiviral Vectors

Abstract

The safety of lentiviral vectors for clinical applications is still a major concern. The gag-pol expression plasmids and the lentiviral vectors used in previous studies contain homologous regions, which constitute a risk for recombination events. Synthetic gag-pol genes of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were therefore constructed, in which the codon usage was optimized for expression in human cells without altering the amino acid sequences. The synthetic gag-pol genes allowed efficient expression of these genes in the absence of Rev and the 5' untranslated leader region. Both the HIV-1 and the SIV synthetic gag-pol expression plasmids could mediate transduction of an SIV vector into nondividing human cells with titers of about 10(6) transducing units/ml. Similar titers were obtained with a four-plasmid vector-packaging system based on HIV-1. Using a biological assay, homologous recombination events between the synthetic gag-pol expression plasmids and an SIV vector were undetectable and in comparison with a previously used gag-pol expression plasmid at least approximately 100-fold less frequent. By eliminating regions of homology and sequences involved in packaging, synthetic gag-pol genes should improve the safety profile of lentiviral vectors.

Effects of CD2 locus control region sequences on gene expression by retroviral and lentiviral vectors

“…23 Vector-containing supernatants were generated by a 3-plasmid vector packaging system based on transient transfections of 293T cells with 6 g Hgp, a synthetic HIV gag-pol expression plasmid, 24 6 g ViG⌬BH or its derivatives, and 0.1 g VSV-G expression construct, as reported for Mo-MLV-based retroviral vectors. NIH-3T3 cells were used in preliminary experiments for titration of the vectors.…”

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors

“…Secondly, the use of EIAV as a positive control virus is inappropriate because the cis-acting sequences and regulatory proteins required for natural replication are missing and because the vectors are so divergent from their parental virus: the only lentiviral gene present in the EIAV packaging system is codon-optimized Gag-Pol, and the components are separated onto different plasmids; together these features are aimed at minimizing the potential for recombination. 2,[16][17][18][19][20] Thirdly, it is essential to assay for RCL on a human cell line, which is compatible with the fact that it is particularly human cell tropism that is of interest for clinical safety. This is due to the trend to use human cell production systems for manufacturing and as these are often not the natural host cells for the parental virus, there would potentially be selection for novel human cell tropic entities.…”

Protein transduction by pseudotyped lentivirus-like nanoparticles

“…20 The original codon-optimized HIV-1 gag-pol expression vector was described previously. 14 The Gag-GFP expression vector was described previously. 10 The pGFP-PH-gag-pol was constructed by inserting the NdeI-PshAI fragment from pEGFP-PLCd1 PH 21 into the corresponding sites of pPH-gag-pol.…”