Superior lentiviral vectors designed for BSL-0 environment abolish vector mobilization

Abstract

Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single-LTR to support production of high vector titers. Here, we demonstrate that configuring the internal vector expression cassette in opposite orientation to the LTRs abolishes mobilization of SIN vectors. This additional SIN mechanism is in part premised on induction of host PKR response to double-stranded RNAs comprised of mRNAs transcribed from cryptic transcription initiation sites around 3’SIN-LTR’s and the vector internal promoter. As anticipated, PKR response following transfection of opposite orientation vectors, negatively affects their titers. Importantly, shRNA-mediated knockdown of PKR rendered titers of SIN HIV-1 vectors comprising opposite orientation expression cassettes comparable to titers of conventional SIN vectors. High titer vectors carrying an expression cassette in opposite orientation to the LTRs efficiently delivered and maintained high levels of transgene expression in mouse livers. This study establishes opposite orientation expression cassettes as an additional PKR-dependent SIN mechanism that abolishes vector mobilization from integrated and episomal SIN lentiviral vectors.

β-Globin Lentiviral Vectors Have Reduced Titers due to Incomplete Vector RNA Genomes and Lowered Virion Production

“…PKR is an interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase that protects cells against viral infections ( Pindel and Sadler, 2011 ). Vectors with an internal expression cassette in the reverse orientation were shown by Kafri and colleagues to trigger PKR response to inhibit protein translation ( Hu et al., 2018 ; Poling et al., 2017 ). To test whether PKR was activated by Lenti/βAS3-FB, we knocked out PKR in 293T cells via CRISPR-Cas9, generated an isogenic PKR−/− cell clone, and confirmed the allelic disruption by TIDE sequencing and protein expression by western blot ( Figures 6 D, S5 A, and S5B).…”

“…Previous studies have showed that knocking out certain cellular factors improved the production of LVs ( Hu et al., 2018 ; Park et al., 2018 ). In this paper, we showed that knocking out PKR in the packaging cells restored p24 production in the reverse-oriented vectors, suggesting that PKR was activated to inhibit P24 production in reverse-oriented LVs.…”

Safety and efficiency modifications of SIV-based integrase-defective lentiviral vectors for immunization

“…Previous work has shown that U3 deletions leading to self-inactivation of LV significantly reduced vector mobilization from integrated templates. 11 , 25 Conversely, circular episomal forms of HIV-based SIN-LV supported mobilization of vector templates and subsequent production of high vector titers. 25 , 26 …”

Small interfering RNA target for long noncoding RNA PCGEM1 increases the sensitivity of LNCaP cells to baicalein

“…The lentiviral vector is a gene therapy vector modified based on HIV‐1, which belongs to retrovirus and is widely applied in studies of in vitro living cell transfection and gene therapy (Benskey & Manfredsson, 2016; Hu et al, 2018). HIV‐1 lentiviral vector particles carrying target genes not only retain efficient infection and integration but also avoid damage to host cells in which viruses replicate.…”