Efficient Pseudotyping of Different Retroviral Vectors Using a Novel, Codon-Optimized Gene for Chimeric GALV Envelope

Abstract

The Gibbon Ape Leukemia Virus envelope protein (GALV-Env) mediates efficient transduction of human cells, particularly primary B and T lymphocytes, and is therefore of great interest in gene therapy. Using internal domains from murine leukemia viruses (MLV), chimeric GALV-Env proteins such as GALV-C4070A were derived, which allow pseudotyping of lentiviral vectors. In order to improve expression efficiency and vector titers, we developed a codon-optimized (co) variant of GALV-C4070A (coGALV-Env). We found that coGALV-Env mediated efficient pseudotyping not only of γ-retroviral and lentiviral vectors, but also α-retroviral vectors. The obtained titers on HEK293T cells were equal to those with the classical GALV-Env, whereas the required plasmid amounts for transient vector production were significantly lower, namely, 20 ng coGALV-Env plasmid per 106 293T producer cells. Importantly, coGALV-Env-pseudotyped γ- and α-retroviral, as well as lentiviral vectors, mediated efficient transduction of primary human T cells. We propose that the novel chimeric coGALV-Env gene will be very useful for the efficient production of high-titer vector preparations, e.g., to equip human T cells with novel specificities using transgenic TCRs or CARs. The considerably lower amount of plasmid needed might also result in a significant cost advantage for good manufacturing practice (GMP) vector production based on transient transfection.

The Dual-Pseudotyped Lentiviral Vector with VSV-G and Sendai Virus HN Enhances Infection Efficiency through the Synergistic Effect of the Envelope Proteins

“…There is also a technical concern that the addition of SeV-HN plasmid DNA increases the total amount of DNA for LV production. Codon optimization of the viral envelope protein significantly reduces plasmid consumption [ 44 ]. Therefore, we attempted to minimize the SeV-HN plasmid vector and constructed HN with the Kozak sequence (kHN) and human codon-optimized HN with the Kozak sequence (khcHN) to increase the levels of SeV-HN envelope protein in the LV particles ( Figure 3 A).…”

“…The careful balance of the two envelope proteins in viral particles to provide maximum effectiveness is crucial for dual-pseudotyped viral vectors. Moreover, despite the codon-optimization approach allowing for the synthesis of a large amount of protein using only a small amount of plasmid during the transient transfection, it has been less frequently used for viral envelope proteins [ 44 ]. We achieved technical optimization and showed that 0.1 unit of pCAG-khcHN (human codon-optimized SeV-HN with the Kozak sequence) plasmid vector per 1.0 unit of pCMV-VSV-G plasmid vector is adequate to maintain the balance of VSV-G and SeV-HN in the viral particles for V/HN-LV production.…”

CD45-Directed CAR-T Cells with CD45 Knockout Efficiently Kill Myeloid Leukemia and Lymphoma Cells In Vitro Even after Extended Culture

“…Retroviral vector particles pseudotyped with the Gibbon-ape leukemia virus (GALV) envelope were produced by transiently transfecting HEK293T cells. To do so, we used the calcium phosphate method and titrated the obtained vector containing supernatants on HEK293T, both steps as described previously [21]. Titers of concentrated (by centrifugation) supernatants were in the range of 1-60 ×10 6 transducing units per mL.…”

“…Cancers 2024, 16, x FOR PEER REVIEW 4 of 18 calcium phosphate method and titrated the obtained vector containing supernatants on HEK293T, both steps as described previously [21]. Titers of concentrated (by centrifugation) supernatants were in the range of 1-60 ×10 6 transducing units per mL.…”