Retargeting vesicular stomatitis virus glycoprotein pseudotyped lentiviral vectors with enhanced stability by in situ synthesized polymer shell

Abstract

The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus "targeting nanovirus." The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes.

FIG. 1.

Schematic illustration of the synthesis and delivery of vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral nanocapsules: (I) Surface modification of the lysine group of the envelope protein by NAS; (II) in situ polymerization of monomer (acrylamide), crosslinker (GMA), and targeting agent (acrylamide tailored-cRGD) at the surface of the modified virus; and (III) targeting delivery of the nanovirus to cells. GMA, glycerol dimethacrylate; cRGD, cyclic arginine-glycine-aspartic acid. Color images available online at www.liebertpub.com/hgtb

FIG. 2.

Shielding of virus infectivity by polymer shell. 1×10⁵ Hela cells were transduced with equal amounts of VSV-G, cRGD-nVSV-G, and nVSV-G (p24=10 ng). Enhanced green fluorescent protein (EGFP) expression was monitored by flow cytometry 3 days post transduction. Color images available online at www.liebertpub.com/hgtb

FIG. 3.

Targeted transduction of Hela cells by the nanovirus is cRGD dependent. 1×10⁵ Hela cells were transduced with equal amount of VSV-G, cRGD-nVSV-G, and cRAD-nVSV-G (p24=10 ng) using the best ratio of N-acryloxysuccinimide (NAS) and monomer obtained from Figure S1. EGFP expression was monitored by flow cytometry 3 days post transduction. Color images available online at www.liebertpub.com/hgtb

FIG. 4.

Entry kinetics of the targeting nanovirus by β-lactamase assay. Both VSV-G pseudotypes and targeting nanovirus incorporating the BlaM-Vpr fusion protein (p24=100 ng) were used to transduce 1×10⁵ Hela cells for 5, 15, 30, 45, 60, 90, and 120 min. The percentage of cells with β-lactamase activity was measured by flow cytometry (*p<0.05). Color images available online at www.liebertpub.com/hgtb

FIG. 5.

Fusion and reverse transcription of the targeting nanovirus. (A) 1×10⁵ Hela cells were treated or not treated with vacuolar-type H+-ATPase inhibitor bafilomycin-A for 30 min then transduced with VSV-G or cRGD-nVSV-G (p24=10 ng) with or without bafilomycin-A for 4 hr. (B) Transduction by the nanovirus is inhibited by reverse transcriptase AZT. 1×10⁵ Hela cells were treated or not treated with AZT for 30 min then transduced with VSV-G or cRGD-nVSV-G (p24=10 ng) for 4 hr with or without AZT. Color images available online at www.liebertpub.com/hgtb