title: Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein.
abstract: Lentiviral vectors (LVs) are a highly valuable tool for gene transfer currently exploited in basic, applied, and clinical studies. Their optimization is therefore very important for the field of vectorology and gene therapy. A key molecule for LV function is the envelope because it guides cell entry. The most commonly used in transiently produced LVs is the vesicular stomatitis virus glycoprotein (VSV-G) envelope, whose continuous expression is, however, toxic for stable LV producer cells. In contrast, the feline endogenous retroviral RD114-TR envelope is suitable for stable LV manufacturing, being well tolerated by producer cells under constitutive expression. We have previously reported successful, transient and stable production of LVs pseudotyped with RD114-TR for good transduction of T lymphocytes and CD34+cells. To further improve RD114-TR-pseudotyped LV cell entry by increasing envelope expression, we codon-optimized the RD114-TR open reading frame (ORF). Here we show that, despite the RD114-TRco precursor being produced at a higher level than the wild-type counterpart, it is unexpectedly not duly glycosylated, exported to the cytosol, and processed. Correct cleavage of the precursor in the functional surface and transmembrane subunits is prevented in vivo, and, consequently, the unprocessed precursor is incorporated into LVs, making them inactive.
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy; Great Ormond Street Institute of Child Health, University College London, London WC1N 1EH, UK.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Zucchelli
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Monika
authors.initials: M
authors.lastname: Pema
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Stornaiuolo
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Piovan
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Scavullo
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Erica
authors.initials: E
authors.lastname: Giuliani
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Sergio
authors.initials: S
authors.lastname: Bossi
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Corna
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Asperti
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Bordignon
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Gian-Paolo
authors.initials: GP
authors.lastname: Rizzardi
authors.affiliation: MolMed S.p.A., 20132 Milano, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Bovolenta
keywords: RD114-TR; RNA structure; codon optimization; envelope glycoprotein; gene therapy; gene vector delivery; gene vector engineering; lentiviral vector; packaging cell line