Factors that influence VSV‐G pseudotyping and transduction efficiency of lentiviral vectors—in vitro and in vivo implications

Abstract

Pseudotyping viral vectors with vesicular stomatitis virus glycoprotein (VSV-G) enables the transduction of an extensive range of cell types from different species. We have discovered two important parameters of the VSV-G-pseudotyping phenomenon that relate directly to the transduction potential of lentiviral vectors: (1) the glycosylation status of VSV-G, and (2) the quantity of glycoprotein associated with virions. We measured production-cell and virion-associated quantities of two isoform variants of VSV-G, which differ in their glycosylation status, VSV-G1 and VSV-G2, and assessed the impact of this difference on the efficiency of mammalian cell transduction by lentiviral vectors. The glycosylation of VSV-G at N336 allowed greater maximal expression of VSV-G in HEK293T cells, thus facilitating vector pseudotyping. The transduction of primate cell lines was substantially affected (up to 50-fold) by the degree of VSV-G1 or VSV-G2 incorporation, whereas other cell lines, such as D17 (canine), were less sensitive to virion-associated VSV-G1/2 quantities. These data indicate that the minimum required concentration of virion-associated VSV-G differs substantially between cell species/types. The implications of these data with regard to VSV-G-pseudotyped vector production, titration, and use in host-cell restriction studies, are discussed.

Analysis of factor VIII mediated suppression of lentiviral vector titres

“…Furthermore, the VSV-G envelope used was more severely affected than any other envelope tested including a more commonly used VSV-G isoform differing by single amino-acid residue (threonine rather than alanine at position 336) known to be involved in glycosylation. 21 These factors resulted in a catastrophic drop in titre, whereas for other vector system/envelope combinations a less obvious decrease would only be unmasked by thorough characterization of vector and may explain why this phenomenon has thus far gone unnoticed. The precise mechanism of the inhibitory effect remains to be elucidated: FVIII has been shown to colocalize with VSV-G throughout its secretion 22 and we speculate that high-level expression of FVIII may overwhelm the secretory machinery blocking normal trafficking of the envelope glycoprotein.…”

“…The following quantities of plasmid were used per 10 cm dish: 4 mg genome plasmid, 2 mg EIAV synthetic Gag/Pol (pESYNGP) 28 and 0.08 mg VSV-G envelope plasmid (pRV67 29 unless otherwise stated). Concentrated vectors were prepared using 440 mg genome plasmid, 220 mg EIAV synthetic Gag/Pol (pESYNGP) 28 and 8.8 mg VSV-G (phG[VSVG2]) 21 envelope per 10-layer cell factory. Vector containing marker genes was titred by serial dilution on D17 cells as previously described; 27 transduction with vectors encoding EGFP was assessed by flow cytometry and those encoding lacZ by X-gal staining and visual scoring of colonies.…”

Development of inducible EIAV-based lentiviral vector packaging and producer cell lines

“…Furthermore, high expression of cellular VSV-G has been shown to directly correlate with efficient pseudotyping of vector particles, which is an important parameter in improving the transduction efficiency of certain target cells. 36 …”

“…The minimal EIAV genome plasmids pONY8.4GCZ 37 and pONYK1-ORT 36 are SIN vectors that contain the lacZ gene and a tricistronic cassette (encoding three enzymes required for dopamine synthesis), respectively, under the control of an internal hCMVp. The VSV-G envelope plasmid, pHG, has been described earlier.…”

“…The VSV-G envelope plasmid, pHG, has been described earlier. 36 pESGPK contains codon optimised EIAV Gag/Pol and is a derivative of pESYNGP, 38 in which the ampicillin resistance gene has been replaced by kanamycin.…”