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.

Figure 1

SIN LVs used in this study. (a) Schemes of the SIN-RD114-TR LVs. CMV, cytomegalovirus promoter; ΔU3, deleted U3; IN, intron; BGI, rabbit β-globin intron; RRE, Rev Responsive Element; A, polyA sequence; IRES, internal ribosome entry site; SD, splice donor; SA, splice acceptor; Ψ, packaging signal; WPRE, woodchuck hepatitis post-transcriptional regulatory element; cPPT, central polypurine tract. (b) Scheme of the SIN-GFP TV. AAV-ITR, adeno-associated virus-inverted terminal repeat; SV40-P, Simian Virus 40 promoter; zeoR, zeocin resistance gene.

Figure 2

Characterization of the SIN-RD114-TR vectors. (a) Western blot analysis of cellular extracts (45 μg/sample) obtained from HEK-293T cells 72 hours after either mock-transfection (lane 2) or transfection with the indicated plasmids (plasmid DNA transfected, 2 μg/1 × 10⁶ cells) (lanes 1 and 3). (b) Northern blot analysis of total RNA (7.5 μg/sample) extracted from HEK-293T 48 hours after transfection of the indicated plasmids (lanes 1 and 3). The size of the expected full length and internal cassette transcripts corresponds to 6.3- and 3.9-kb, respectively. The membrane was hybridized with a 550-bp RD114-TR-specific probe. (c) Western blot analysis of cellular extracts (35 μg/sample) obtained from HEK-293T cells 48 hours after transfection of the indicated plasmids (plasmid DNA transfected, 2 μg/1 × 10⁶ cells). The membranes were hybridized with the anti-RD114-TR-specific Ab recognizing the precursor (PR, 75-kDa) and the transmembrane (TM, 18-kDa) subunit of the RD114-TR envelope and, after stripping, with the anti-actin Ab. (d) Western blot analysis of cellular extracts (35 μg/sample) obtained from PK-7 cells 72 hours after transduction with the indicated VSV-G-pseudotyped SIN-RD114-TR LVs. The membranes were hybridized as described for panel c. The asterisk (*) indicates nonspecific band.

Figure 3

Integrity of the vector genes in the packaging, producer and target cells. Southern blot analysis of genomic DNA (10 μg/sample) extracted from the indicated cells and hybridized with the specific probes: (a) 661-bp CMV probe; (b) 550-bp RD114-TR probe; (c and d) 339-bp GFP probe. In panel d, the CEM A3.01 and PB T lymphocytes were either mock-transduced (lane 7) or transduced at MOI = 25.The genomic DNA was extracted 10 days after transduction.

Figure 4

Transduction efficiency of activated peripheral blood (PB) T lymphocytes with either RD3-MolPack24 or VSV-G-pseudotyped SIN-GFP-zeo LVs. (a) Peripheral blood mononuclear cells (PBMC) were preactivated with CD3/CD28 Dynabeads for 48 hours and cultured in the presence of IL-7 and IL-15. T lymphocytes were then transduced with the RD3-MolPack24 LVs at the indicated MOI and 6 and 14 days after transduction, GFP expression was evaluated by FACS analysis to calculate transduction efficiency (MOI 25 day 6 p.t., range 89.2–94.5%, n = 3, MOI 25 day 14 p.t., range 83.5–96.3%, n = 4; MOI 3 day 6 p.t., range 82.4–96.9%, n = 3, MOI 3 day 14 p.t., range 84.8–97.8%, n = 4; MOI 1.5 day 6 p.t., range 63.4–90.9%, n = 3, MOI 1.5 day 14 p.t., range 65–92.5%, n = 4). (b) Quantification of the SIN-GFP-zeo vector copy number (VCN) in T lymphocytes of panel a by quantitative polymerase chain reaction (q-PCR) using specific primer-probe sets recognizing the packaging (Ψ) signal of the integrated vector and the genomic telomerase gene as control (MOI 25 day 6 p.t., range 6.2–8.5, n = 3, MOI 25 day 14 p.t., range 3.9–7.9, n = 4; MOI 3 day 6 p.t., range 4.6–11.9, n = 3, MOI 3 day 14 p.t., range 3.7–12.7, n = 3; MOI 1.5 day 6 p.t., range 2.7–7.3, n = 3, MOI 1.5 day 14 p.t., range 2.1–6.5, n = 3). (c) PBMC were preactivated as in (a) and then transduced with 36 ng of p24Gag equivalents of either RD3-MolPack24 LVs or VSV-G-pseudotyped LVs carrying the SIN-GFP-zeo TV. Six and 14 days after transduction, the GFP expression was evaluated by FACS analysis (RD114-TR day 6 p.t., range 66.7–90.9%, n = 3, RD114-TR day 14 p.t. range 84.8–89.7%, n = 2; VSV-G day 6 p.t. range 92.4–98.8%, n = 3; VSV-G day 14 p.t., range 91.9–96.4%, n = 2). (d) Quantification of the SIN-GFP-zeo VCN in T lymphocytes of panel (c) as described in (b) (RD114-TR day 6 p.t., range 2.6–5.2%, n = 3, RD114-TR day 14 p.t. range 3.7–5.3%, n = 2; VSV-G day 6 p.t. range 15.3–31.8%, n = 3; VSV-G day 14 p.t., range 7.7–15.8%, n = 2). Results are mean ± standard error of the mean of n = 3 independent experiments, unless otherwise indicated. *P ≤ 0.05; **P ≤ 0.01.

Figure 5

Transduction efficiency of activated PB T lymphocyte subsets with either RD3-MolPack24 or VSV-G-pseudotyped SIN-GFP-zeo LVs. (a) PBMC were preactivated with CD3/CD28 Dynabeads for 48 hours and cultured in the presence of IL-7 and IL-15. T lymphocytes were then transduced with 36-ng p24Gag equivalent of RD3-MolPack24 LVs and 6 days after transduction, GFP expression was evaluated in CD3⁺/CD8⁺ and CD3⁺/CD8⁻ T-cell subsets (a), and in T-cell memory subpopulations of CD3⁺/CD8⁺ and CD3⁺/CD8⁻ T-cell subsets (c) and (d) by FACS analysis. (b) Relative frequency of CD3⁺/CD8⁺ and CD3⁺/CD8⁻ T cell subsets in LV- and mock-transduced cells. (e and f) Relative frequency of T central memory (T_CM), T-stem-cell memory (T_SCM), T effector memory (T_EM) and T effector memory RA⁺ (T_EMRA) in either CD3⁺/CD8⁺ (e) or CD3⁺/CD8⁻ Tcell subsets (f) of LV- and mock-transduced cells. Results are mean ± standard error of the mean of n = 4 independent experiments (day 6 p.t.). *P ≤ 0.05; **P ≤ 0.01.