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.
Highlights
Human immunodeficiency virus strain 1 (HIV-1) is the most well characterized of the lentiviruses, a family of viruses that reverse-transcribe and stably integrate their genetic information into the host genome
Rational Design of a U5-AUG Mutant Structures of the HIV-1 leader determined previously by in-gel selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE)[24] were used to guide the design of U5-AUG helix-strengthening mutations aimed at enhancing dimerization of the RNA.[14,15,26,27]
The terminal Gag processing step, p25 to p24 cleavage, is most sensitive to detect processing defects[38] but did not differ between the WT and U5s (Figure 2F). These results suggest that 50 UTR mutations in the transfer vector RNA (vRNA) do not affect Gag provided in trans; any effect on the functionality of the mutant transfer vector was not caused by Gag expression, processing, or budding
Summary
Human immunodeficiency virus strain 1 (HIV-1) is the most well characterized of the lentiviruses, a family of viruses that reverse-transcribe and stably integrate their genetic information into the host genome. When used as lentiviral vectors (LVs), they can achieve gene delivery and sustained gene expression.[1] Vector RNA packaging predominantly depends on the 50 RNA leader sequence of the virus, which is the most conserved region of the HIV genome;[2] it contains cis-acting elements that regulate a variety of viral processes, including transcriptional activation, reverse transcription, splicing, and translation as well as genome dimerization and packaging[3,4] (Figure 1A) This 50 leader RNA is present in its entirety in the HIV-1based gene therapy vector system. Mutations that strengthen U5-AUG base-pairing result in elevated levels of dimerization,[15,16] suggesting that this structural element promotes dimerization and, packaging
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