Construction of stable packaging cell lines for clinical lentiviral vector production

Abstract

Lentiviral vectors are useful experimental tools for stable gene delivery and have been used to treat human inherited genetic disorders and hematologic malignancies with promising results. Because some of the lentiviral vector components are cytotoxic, transient plasmid transfection has been used to produce the large batches needed for clinical trials. However, this method is costly, poorly reproducible and hard to scale up. Here we describe a general method for construction of stable packaging cell lines that continuously produce lentiviral vectors. This uses Cre recombinase-mediated cassette exchange to insert a codon-optimised HIV-1 Gag-Pol expression construct in a continuously expressed locus in 293FT cells. Subsequently Rev, envelope and vector genome expression cassettes are serially transfected. Vector titers in excess of 106 transducing units/ml can be harvested from the final producer clones, which can be increased to 108 TU/ml by concentration. This method will be of use to all basic and clinical investigators who wish to produce large batches of lentiviral vectors.

Concepts for the Production of Viruses and Viral Vectors in Cell Cultures

“…Non-replicating viral vectors for gene therapy are typically expressed in packaging cell lines containing only the essential structural virus genes [13]. This results in a virus construct that contains therapeutic RNA and regulatory elements to activate gene expression.…”

Lentivector Producer Cell Lines with Stably Expressed Vesiculovirus Envelopes

“…We therefore transfected WinPac cells, 10 which stably express HIV gag-pol and rev , with a COCV.G expression vector (Figure S2A) and isolated single-cell clones by selection in phleomycin. Functional clones were identified by transient transfection of a lentivector construct containing the gfp gene; data from 13 of these clones are shown in Figure S2B.…”

“…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 …”

Open access? Widening access to chimeric antigen receptor (CAR) therapy for ALL

“…This would suggest that, for a given CAR design and targeted tumour antigen, these transduction platforms may not be equivalent in terms of the quality of CAR T cell product generated 56,57 . Whilst much research effort is focussed on generating lentiviral producer cell lines for clinical purposes 58,59 , at present, viral yields generally remain lower than those generated by transient transfection.…”