Recent advances in lentiviral vector development and applications

Abstract

Lentiviral vectors (LVs) have emerged as potent and versatile vectors for ex vivo or in vivo gene transfer into dividing and nondividing cells. Robust phenotypic correction of diseases in mouse models has been achieved paving the way toward the first clinical trials. LVs can deliver genes ex vivo into bona fide stem cells, particularly hematopoietic stem cells, allowing for stable transgene expression upon hematopoietic reconstitution. They are also useful to generate induced pluripotent stem cells. LVs can be pseudotyped with distinct viral envelopes that influence vector tropism and transduction efficiency. Targetable LVs can be generated by incorporating specific ligands or antibodies into the vector envelope. Immune responses toward the transgene products and transduced cells can be repressed using microRNA-regulated vectors. Though there are safety concerns regarding insertional mutagenesis, their integration profile seems more favorable than that of gamma-retroviral vectors (gamma-RVs). Moreover, it is possible to minimize this risk by modifying the vector design or by employing integration-deficient LVs. In conjunction with zinc-finger nuclease technology, LVs allow for site-specific gene correction or addition in predefined chromosomal loci. These recent advances underscore the improved safety and efficacy of LVs with important implications for clinical trials.

Figure 1

Lentiviral vector production by trans-complementation. Packaging cells are transfected with the lentiviral vector plasmid and three helper (i.e., packaging) constructs encoding Gag, Pol, Rev, and VSV-G. Only the vector contains the packaging sequence ψ, whereas the packaging constructs are devoid of ψ. The LV is flanked by the 5′ and 3′ LTR sequences that have promoter/enhancer activity and are essential for the correct expression of the full-length vector transcript. The LTRs also play important role in reverse transcription and integration of the vector into the target cell genome. Assembled vector particles are harvested from the supernatant and, if required, subjected to further purification and concentration. Constructs encoding different envelope genes allow for the production of distinct LV pseudotypes that exhibit different tropisms. The self-inactivating (SIN) LTR sequences that contain a partial deletion (δ), Woodchuck post-transcriptional regulatory element (WPRE), central polypurine tract (cPPT), and Rev responsive element (RRE) are indicated. LV, lentiviral vector.

Figure 2

LV targeting into specific cell types. The LV Env protein is amenable to engineering allowing the display of cell type–specific ligands or single chain antibody fragment (scFv). These ligands or scFv can then bind onto cell surface receptors that are specifically expressed on the desired target cells. Some of the ligand-receptor interaction may activate the target cells and consequently enhance transduction. Some Env proteins like the amphotropic MLV Env can be engineered to display the stem cell factor (SCF) as ligand for the SCF cellular receptor (i.e., c-kit) allowing enhanced transduction of CD34⁺ HSC. Alternatively, thrombopoietin can be incorporated along with SCF into the same LV particle. These “early-acting” cytokines may enhance transduction by activating the target cells. A mutated version of the RD114 envelope is used as fusogen. An alternative retargeting paradigm is based on the display of scFv on the measles hemaggluttinin envelope H protein, whereas the native tropism of this measles Env was ablated. The measles envelope F protein acts as fusogen. LV, lentiviral vector.

Figure 3

LV chromosomal targeting. (a) A pair of integration-defective LVs vectors (IDLVs) each expressing ZFN are co-transduced with an IDLV providing the exogenous donor DNA template. (b) Gene correction is catalyzed by ZFN-mediated double-stranded DNA break (DSB) and homologous recombination repair using an exogenous DNA as the template. The zinc finger domains provide specificity, whereas Fok1 catalyzes the DSB. The exogenous DNA carries the corrected version (red) of the gene of interest. LV, lentiviral vector.

Figure 4

micro-RNA regulated LV. Ectopic transgene expression in APC results in the induction of an antigen-specific T-cell dependent immune response that consequently eliminates the gene-engineered APC and hepatocytes that express the transgene product (i.e., GFP). To avoid ectopic transgene expression in APC, miR target sequences can be incorporated in the LV that are designed to be complementary to a miR that is expressed in APC (i.e., miR142-3p) but not in hepatocytes. The specific interaction of the APC-specific miR with its cognate miR target sequence embedded within the mRNA encoded by the vector results in its degradation via the cellular miR processing machinery. This prevents inadvertent transgene expression in APC but not in hepatocytes. Consequently, there is no induction of an antigen-specific T-cell response, and the LV-transduced APC and hepatocytes are able to persist resulting in long-term expression of the gene of interest. APC, antigen-presenting cell; cPPT, central polypurine tract; GFP, green fluorescent protein; LV, lentiviral vector; WPRE, Woodchuck post-transcriptional regulatory element.