Lentiviral Transduction of SARS-CoV-2 Delta Spike

Lentiviral vectors for expression of SARS-CoV-2 Spike (Delta variant B.1.617.2, NCBI accession # OX014251.1) were designed and synthesized from Genscript together with the two packaging plasmids (pMD2.G and psPAX2). Full length sequence for Spike was used. Additionally, the delta Spike sequence was modified for codon optimization and insertion of 2P mutation for stabilization of the sequence [14 (link)]. To facilitate the trafficking of Spike to the exosomes, the proteins were linked to N-terminal of the exosome specific tetraspanin CD9 by a synthetic transmembrane domain and a secretion signal peptide: this allowed the correct membrane localization of Spike. Lentiviral particles for transduction were generated by transfecting 293T cells with pMG.2 (Genescript), psPAX2 (Genescript) and STX-S_pLenti (Genscript) expressing Spike at a ratio of 5:5:1 using Lipofectamine 3000 according to the manufacturer’s instruction. Spike lentiviral particles were collected at 72 hours post transfection and used to transduce 293F parental cells to generate STX-S respectively.

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Cacciottolo M., Li Y., Nice J.B., LeClaire M.J., Twaddle R., Mora C.L., Adachi S.Y., Young M., Angeles J., Elliott K, & Sun M. (2023). Nanograms of SARS-CoV-2 spike protein delivered by exosomes induce potent neutralization of both delta and omicron variants. PLOS ONE, 18(8), e0290046.

Publication 2023

pMD2.G psPAX2 STX-S_pLenti

293t cells Cells Codon Exosome Insertion mutation Lipofectamine Membrane Parental Plasmids Proteins Sars cov 2 Secretion Signal peptide Tetraspanin Transfection Vectors

Corresponding Organization : Capricor Therapeutics (United States)

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Variable analysis

independent variables

Expression of SARS-CoV-2 Spike (Delta variant B.1.617.2, NCBI accession # OX014251.1)

dependent variables

Generation of lentiviral particles for transduction

control variables

Packaging plasmids pMD2.G and psPAX2
Codon optimization and 2P mutation for stabilization of the Spike sequence
Linking Spike to the exosome specific tetraspanin CD9
Transfection of 293T cells with pMG.2, psPAX2, and STX-S_pLenti at a ratio of 5:5:1 using Lipofectamine 3000

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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