RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope

Abstract

To date, gene therapy with transiently derived lentivectors has been very successful to cure rare infant genetic diseases. However, transient manufacturing is unfeasible to treat adult malignancies because large vector lots are required. By contrast, stable manufacturing is the best option for high-incidence diseases since it reduces the production cost, which is the major current limitation to scale up the transient methods. We have previously developed the proprietary RD2-MolPack technology for the stable production of second-generation lentivectors, based on the RD114-TR envelope. Of note, opposite to vesicular stomatitis virus glycoprotein (VSV-G) envelope, RD114-TR does not need inducible expression thanks to lack of toxicity. Here, we present the construction of RD2- and RD3-MolPack cells for the production of self-inactivating lentivectors expressing green fluorescent protein (GFP) as a proof-of-concept of the feasibility and safety of this technology before its later therapeutic exploitation. We report that human T lymphocytes transduced with self-inactivating lentivectors derived from RD3-MolPack cells or with self-inactivating VSV-G pseudotyped lentivectors derived from transient transfection show identical T-cell memory differentiation phenotype and comparable transduction efficiency in all T-cell subsets. RD-MolPack technology represents, therefore, a straightforward tool to simplify and standardize lentivector manufacturing to engineer T-cells for frontline immunotherapy applications.

Codon Optimization Leads to Functional Impairment of RD114-TR Envelope Glycoprotein

“…We initially analyzed the expression of the RD114-TRWT envelope in RD3-MolPack-GFP producer cells and in their derived LVs 23 to confirm previous studies describing proper processing and trafficking to the plasma membrane of the wild-type (WT) envelope 19 . RD3-MolPack-GFP cells contain 12 copies of the integrated self inactivating (SIN)-RD114-TRWT-IN-rev-responsive element (RRE) transfer vector (TV) (Figure 1A, scheme 2), and the originated RD114-TRWT pseudo-typed LVs are proficient in cell transduction, as reported previously 23 .…”

“…Upon recognition and engagement of functional subunits to specific receptors, fusion between viral and cell membranes mediates the entry of the vector into target cells. RD114-TR-pseudotyped retroviral vectors are suitable for both ex vivo and in vivo gene therapy applications because they can be concentrated by centrifugation and are resistant to human serum complement inactivation 20, 21, 22, 23…”

“…To improve and simplify the expression of the RD114-TR envelope during development of the RD-MolPack packaging technology for stable and constitutive manufacturing of LVs,21, 23 we codon-optimized the entire RD114-TR open reading frame (ORF). This idea stemmed from our previous observation that RD114-TR expression is achieved only when the β-globin intron (BGI) is inserted between the promoter and the RD114-TR cDNA of the expression cassette of many different expression plasmids tested 23 .…”

“…This idea stemmed from our previous observation that RD114-TR expression is achieved only when the β-globin intron (BGI) is inserted between the promoter and the RD114-TR cDNA of the expression cassette of many different expression plasmids tested 23 . To explain this constraint, we hypothesized that BGI may attenuate the negative effect of interfering sequences existing in the RD114-TR cDNA.…”

Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes

“…However, these have not yet been employed for clinical LV production. Another approach has used a non-toxic gammaretroviral envelope from the feline endogenous virus RD114 with a modified cytoplasmic tail to allow LV incorporation 8, 9, 10, 11. Although a producer cell clone for clinical use has been developed, this has not progressed to a clinical trial 12 …”