Integration-deficient lentiviral vectors: a slow coming of age

Abstract

Lentiviral vectors are very efficient at transducing dividing and quiescent cells, which makes them highly useful tools for genetic analysis and gene therapy. Traditionally this efficiency was considered dependent on provirus integration in the host cell genome; however, recent results have challenged this view. So called integration-deficient lentiviral vectors (IDLVs) can be produced through the use of integrase mutations that specifically prevent proviral integration, resulting in the generation of increased levels of circular vector episomes in transduced cells. These lentiviral episomes lack replication signals and are gradually lost by dilution in dividing cells, but are stable in quiescent cells. Compared to integrating lentivectors, IDLVs have a greatly reduced risk of causing insertional mutagenesis and a lower risk of generating replication-competent recombinants (RCRs). IDLVs can mediate transient gene expression in proliferating cells, stable expression in nondividing cells in vitro and in vivo, specific immune responses, RNA interference, homologous recombination (gene repair, knock-in, and knock-out), site-specific recombination, and transposition. IDLVs can be converted into replicating episomes, suggesting that if a clinically applicable system can be developed they would also become highly appropriate for stable transduction of proliferating tissues in therapeutic applications.

Figure 1

Generation of episomes from lentiviral vectors. The product of vector reverse transcription is a linear double-stranded DNA (dsDNA) with LTRs at both ends (deleted in the U3 region in self-inactivating vectors, here denoted as dLTR). This DNA is imported into the nucleus as part of the viral preintegration complex. (a) Conventional lentiviral vectors, harboring a functional IN, can be integrated into the host genome as proviruses. However, the linear DNA can also be circularized in several possible ways: nonhomologous end-joining produces 2-LTR circles, while intramolecular homologous recombination between the LTRs in linear DNA or 2-LTR episomes, or ligation of nicks (shown as arrow-bead structures) in intermediate products of reverse transcription, lead to 1-LTR circles. (b) When proviral integration is blocked through class I IN mutations, increased amounts of vector episomes are produced. att, IN attachment sites at the ends of viral DNA. LTR, long terminal repeat.

Figure 2

Stability of eGFP expression in cells transduced with integrating vectors and IDLVs. Proliferating HeLa cells were transduced at MOI (a) 0.05, (b) 0.5, or (c) 5 (based on titering by flow cytometry for GFP expression) with integrating vector or IDLV (vector #11 on Table 1). The percentage of eGFP-expressing cells was estimated at the indicated times by flow cytometry. eGFP, enhanced green fluorescent protein; IDLV, integration-deficient lentiviral vector; MOI, multiplicity of infection.

Figure 3

Transgene expression levels and effect of internal promoter with IDLVs. Proliferating HeLa cells were transduced at low vector doses (<0.1 eGFP vector unit/cell) with integration-proficient vectors or IDLVs encoding eGFP, and eGFP fluorescence analyzed by flow cytometry 3 days post-transduction. (a) eGFP expression levels in mock-transduced cells or cells transduced with integrating or integration-deficient lentivector carrying a CMV-eGFP-WPRE expression cassette, shown schematically above the dot-plots (vector #11 on Table 1). (b) eGFP expression levels in mock-transduced cells or cells transduced with integrating or integration-deficient lentivector carrying an SFFV-eGFP-WPRE expression cassette, shown schematically above the dot-plots (vector #3 on Table 1). CMV, cytomegalovirus; eGFP, enhanced green fluorescent protein; IDLV, integration-deficient lentiviral vector; SFFV, spleen focus-forming virus; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element.