Mark Kay, Postdoctoral Faculty Sponsor
An optimized measles virus glycoprotein-pseudotyped lentiviral vector production system to promote efficient transduction of human primary B cells.
2022; 3 (1): 101228
Measles virus envelope pseudotyped LV (MV-LV) can achieve high B cell transduction rates (up to 50%), but suffers from low titers. To overcome current limitations, we developed an optimized MV-LV production protocol that achieved consistent B cell transduction efficiency up to 75%. We detail this protocol along with analytical assays to assess the results of MV-LV mediated B cell transduction, including flow cytometry for B cell phenotypic characterization and measurement of transduction efficiency, and ddPCR for VCN analysis.
View details for DOI 10.1016/j.xpro.2022.101228
View details for PubMedID 35284833
View details for PubMedCentralID PMC8914380
A novel role for gag as a cis-acting element regulating RNA structure, dimerization and packaging in HIV-1 lentiviral vectors
NUCLEIC ACIDS RESEARCH
2022; 50 (1): 430-448
Clinical usage of lentiviral vectors is now established and increasing but remains constrained by vector titer with RNA packaging being a limiting factor. Lentiviral vector RNA is packaged through specific recognition of the packaging signal on the RNA by the viral structural protein Gag. We investigated structurally informed modifications of the 5' leader and gag RNA sequences in which the extended packaging signal lies, to attempt to enhance the packaging process by facilitating vector RNA dimerization, a process closely linked to packaging. We used in-gel SHAPE to study the structures of these mutants in an attempt to derive structure-function correlations that could inform optimized vector RNA design. In-gel SHAPE of both dimeric and monomeric species of RNA revealed a previously unreported direct interaction between the U5 region of the HIV-1 leader and the downstream gag sequences. Our data suggest a structural equilibrium exists in the dimeric viral RNA between a metastable structure that includes a U5-gag interaction and a more stable structure with a U5-AUG duplex. Our data provide clarification for the previously unexplained requirement for the 5' region of gag in enhancing genomic RNA packaging and provide a basis for design of optimized HIV-1 based vectors.
View details for DOI 10.1093/nar/gkab1206
View details for Web of Science ID 000749588900034
View details for PubMedID 34928383
View details for PubMedCentralID PMC8754630
Development of a Novel Competitive qRT-PCR Assay to Measure Relative Lentiviral Packaging Efficiency
MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT
2020; 19: 307-319
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.
View details for DOI 10.1016/j.omtm.2020.09.010
View details for Web of Science ID 000598181600025
View details for PubMedID 33145367
View details for PubMedCentralID PMC7581820