A Rev-Independentgag/polEliminates Detectable psi-gag Recombination in Lentiviral Vectors

Abstract

Lentiviral vectors (LVs) are being developed for clinical use in humans for applications including gene therapy and immunotherapy. A safety concern for use of LVs in humans is the generation of replication-competent lentivirus (RCL), which may arise due to recombination between the split genomes of third-generation LVs. Although no RCL has been detected to date, design optimizations that minimize recombination events between split genome vectors would provide an added safety benefit that may further reduce the risk of RCL formation. Here we describe design elements introduced to the gag/pol plasmid with the intention of eliminating psi-gag recombination between the vector genome and gag/pol. These design changes, consisting of codon optimization of the gag/pol sequence and the deletion of the Rev-responsive element, abrogate the requirement for Rev in expression of Gag protein, thus the resulting gag/pol construct being Rev independent (RI gag/pol). We show that generating vector using the RI gag/pol construct has no effect on particle production or transduction titers. The RI and wild-type gag/pol vectors function equivalently as antigen-specific immunotherapy, potently inducing antigen-specific CD8 T cells that protect against challenge with vaccinia virus. Most importantly, the designed RI gag/pol eliminated detectable psi-gag recombination. Interestingly, we detected recombination between the vector genome and gag/pol from regions without sequence homology. Our findings imply that although unpredictable recombination events may still occur, the RI gag/pol design is sufficient to prevent psi-gag recombination.

Production of lentiviral vectors

“…Since the TV contains a portion of the gag sequence codon optimization of the gag-pol sequence renders the so-called ψ-gag recombination (for instance, reported by Sastry et al 31 ) impossible. 32 An additional advantage of codon optimization is about 100-fold reduced frequency of potential recombination events. 30 …”

Virological and Preclinical Characterization of a Dendritic Cell Targeting, Integration-deficient Lentiviral Vector for Cancer Immunotherapy

“…However, the fact that not all transduced lymph node–resident DCs expressed SIGNR1 may indicate that either receptor downregulation occurs, or other unknown receptors can be utilized for binding and entry. Pseudotyping of ID-VP02 with the envelope of vesicular stomatitis virus which has broad cellular tropism also resulted in strong immunogenicity, 11 however, because the vector described here was designed to be specific for human DC, we have not performed head-to-head comparative efficacy studies in mice. Published results in mice suggest that direct expression of antigens in DC may be up to 100,000-fold more potent compared with cross-presentation with respect to activation of CD8 cells.…”

“…The design and production of ID-VP02 was described previously. 6 , 11 ). Briefly, ID-VP02 is produced through transient transfection of 293T cells with 5 plasmids: the transfer vector (ID-VP02 genome), a modified gagpol transcript (RI -gagpol ), accessory protein Rev from HIV-1, accessory protein Vpx from SIVmac, and the E1001 envelope glycoprotein variant of Sindbis virus.…”

“… 13 A codon optimized gagpol plasmid allows for production that is devoid of the HIV-Rev response element (RRE), minimizing the chance of psi-gag recombination and thereby reducing the likelihood for formation of Replication Competent Lentivirus during vector production. 11 , 14 , 15 Vpx from SIVmac is included as an accessory protein to overcome SAMHD1-mediated restriction in human DCs by promoting its degradation. 8 , 9 The genome contains an antigen cassette downstream of the human Ubiquitin-C promoter that has been modified to have its natural intron deleted (ΔUbiC).…”

Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

“…The established psi–gag qPCR method for RCL detection was also incompatible with our assay format because sequences in the VP02 packaging construct have been codon-optimized to substantially reduce psi–gag recombination. 17 , 25 Therefore it is likely that any RCL, should it arise, would occur via alternate, unpredictable recombination events for which it would be difficult to design qPCR primers. Furthermore, these and other RCL sequences that could be targeted for qPCR amplification ( e.g.…”