Development of a Novel Competitive qRT-PCR Assay to Measure Relative Lentiviral Packaging Efficiency

Abstract

Third-generation HIV-1-derived lentiviral vectors are successfully used as therapeutic agents in various clinical applications. To further promote their use, we attempted to enhance vector infectivity by targeting the dimerization and packaging properties of the RNA transfer vector based on the premise that these two processes are tightly linked. We rationally designed mutant vectors to favor the dimeric conformation, potentially enhancing genome packaging. Initial assessments using standard assays generated outputs of variable reproducibility, sometimes with conflicting results. Therefore, we developed a novel competitive qRT-PCR assay in a co-transfection setting to measure the relative packaging efficiencies of wild-type and mutant transfer vectors. Here we report the effect of the dimerization-stabilizing mutations on infectious and physical titers of lentiviral vectors together with their packaging efficiency, measured using our novel assay. Enhancing dimerization did not automatically lead to better vector RNA packaging, suggesting that, for vector functionality, sufficient flexibility of the RNA to adopt different conformations is more important than the dimerization capacity. Our novel competitive qPCR assay enables a more stringent analysis of RNA packaging efficiency, allowing a much more precise understanding of the links between RNA structure, packaging, and infectious titers that will be invaluable for future vector development.

A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors

“…A 2790 bp fragment from the WT pCCL-eGFP BglG and MS2 transfer vectors ( 38 ) containing the sequences between the CMV and hPGK promoters was amplified in a PCR reaction using the 5′-TACGGTAAACTGCCCACTTG-3′ and 5′-TAGGTCAGGGTGGTCACGAG-3′ primers in the AccuprimeTM Pfx Supermix (Thermo Fischer, #12344040). The 2790 bp PCR product was cloned into the pCR™Blunt II-TOPO ® vector (Invitrogen, #K280002) as per manufacturer′s instructions and was subsequently used to transform Stbl3 Escherichia coli competent cells (Invitrogen, #C737303).…”

“…Production of third generation lentiviral vectors was performed as previously described ( 38 ). Briefly 5 × 10 6 293T cells maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, #10-569-044) supplemented with 10% fetal bovine serum (GIBCO, #10082147) (cDMEM) were seeded in 10 cm 2 dishes in a final volume of 10 ml cDMEM.…”

“…Viral vector transduction efficiency was titrated as described previously ( 38 ). Briefly, 3 × 10 5 293T cells were seeded per well of a six-well plate in a final volume of 2 ml.…”

“…Intracellular RNA from transfected cells was extracted using the Qiagen RNeasy kit (#74004) as described by the manufacturer. Extracellular RNA extraction from lentiviral particles was performed as previously described ( 38 ).…”

More Than Just Gene Therapy Vectors: Lentiviral Vector Pseudotypes for Serological Investigation

“…Quantification of the LV titre can further be achieved by measuring the amount of viral genomes; as the viral genome is RNA, this can be performed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) [ 136 ]. More recently, a novel competitive qRT-PCR has been developed to improve the quantification of the viral genomic RNA packaged within the LV [ 137 ]. Finally, total particle content can be determined by nanoparticle tracking system analysis [ 138 , 139 ] and flow-based instruments [ 140 ].…”