Lentiviral vectors pseudotyped with the baculovirus envelope protein GP64 transduce primary cultures of human airway epithelia (HAE) at their apical surface. Our goal in this study was to harness a directed evolution approach to develop a novel envelope glycoprotein with increased transduction properties for HAE. Using error-prone PCR, a library of GP64 mutants was generated and used to prepare a diverse pool of lentiviral virions pseudotyped with GP64 variants. The library was serially passaged on HAE and three GP64 mutations were recovered. Single-, double- and the triple-combination mutant envelope glycoproteins were compared with wild-type GP64 for their ability to transduce HAE. Our results suggest that lentiviral vectors pseudotyped with evolved GP64 transduced HAE with greater efficiency than wild-type GP64. This effect was not observed in primary cultures of porcine airway epithelial cells, suggesting that the directed evolution protocol was species specific. In summary, our studies indicate that serial passage of a GP64 mutant library yielded specific variants with improved HAE cell tropism, yielding tools with the potential to improve the success of gene therapy for airway diseases.
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We thank Kimberly Bekas and Mayumi Oakland for their technical assistance. Lokesh Gakhar of the Protein Crystallography Core Facility assisted with the protein modeling. The In Vitro Models and Cell Culture Core provided the primary cell cultures. The Viral Vector Core produced and titered the viral vector. This work was supported by the National Institutes of Health: P01 HL-51670, P01 HL-091842 and R01 HL-105821, and the Center for Gene Therapy of Cystic Fibrosis: P30 DK-54759.
PBM is a founder of and holds equity in Talee Bio. DVS is a co-inventor and has a patent related to directed evolution of lentiviral vectors. The other authors declare no conflict of interest.
Supplementary Information accompanies this paper on Gene Therapy website
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