RD2-MolPack-Chim3,a Packaging Cell Line for Stable Production of Lentiviral Vectors for Anti-HIV Gene Therapy

Abstract

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Baboon envelope pseudotyped LVs outperform VSV-G-LVs for gene transfer into early-cytokine-stimulated and resting HSCs

“…[64][65][66][67][68] Importantly, 2 stable cell lines for high-titer RDTR-LV production were engineered. 30,69 This points toward BaEVTR-gp as an important candidate for establishing a stable LV packaging cell line because its long-term expression is not cytotoxic to 293T cells ( Figure 1D), and these vectors allow high-level HSC transduction. Of note, we confirmed that these BaEV-LVs also allow highlevel gene transfer in human T and B cells (A.G-G. and E.V., unpublished data, June 2013).…”

“…23,25 An RD114 mutant glycoprotein (RDTR) allows efficient pseudotyping of LVs, which confers efficient transduction of hCD34 1 HSCs. [24][25][26][27][28][29][30] RD114 belongs to the betaretroviruses, also including the baboon endogenous retrovirus (BaEV). Both of these viruses use the neutral amino acid (aa) transporter 2 (ASCT-2), present on hCD34…”

Construction of stable packaging cell lines for clinical lentiviral vector production

“…Compared to currently available PCLs, WinPac cells can support the production of SIN LV at superior titers compared to the other constitutive LV PCL reported to date27. In contrast to inducible PCLs proposed for clinical LV production, like the GPRG cell line16, continuous production using WinPac cells is easier to scale up and avoids the rapid decline in titers following induction.…”

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

“…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 .…”