Protein delivery using engineered virus-like particles

Abstract

Over the years, researchers have developed several methods to deliver macromolecules into the cytosol and nucleus of living cells. However, there are limitations to all of these methods. The problems include (i) inefficient uptake, (ii) endosomal entrapment, (iii) delivery that is restricted to certain cell types, and (iv) damage to cells in the delivery process. Retroviral vectors are often used for gene delivery; however, integration of the genome of retroviral vector into the host genome can have serious consequences. Here we describe a safe alternative in which virus-like particles (VLPs), derived from an avian retrovirus, are used to deliver protein to cells. We show that these VLPs are a highly adaptable platform that can be used to deliver proteins either as part of Gag fusion proteins (intracellular delivery) or on the surface of VLPs. We generated VLPs that contain Gag-Cre recombinase, Gag-Fcy::Fur, and Gag-human caspase-8 as a proof-of-concept and demonstrated that the encapsidated proteins are active in recipient cells. In addition, we show that murine IFN-γ and human TNF-related apoptosis-inducing ligand can be displayed on the surface of VLPs, and that these modified VLPs can cause the appropriate response in cells, as evidenced by phosphorylation of STAT1 and induction of cell death, respectively.

Fig. 1.

Generation of functional VLPs and mode of action. (A) A schematic representation of the assembly of VLPs and the biological functions of the proteins delivered intracellularly. Cotransfection of HEK 293T cells with pGag-POI and pCMV-VSV-G plasmids results in the formation and subsequent release of VLPs into the culture supernatant. Four different VLPs containing GFP, Cre, Fcy:Fur, and caspase 8 fused to Gag are shown. (B) A schematic representation of receptor/ligand-mediated protein delivery. VLPs were made as described above by cotransfection of HEK 293T cells with plasmids containing Gag (with or without a protein fusion) and the NA/HA ligand. Biological function mediated by signal transduction from the ligand is also shown.

Fig. 2.

VLP-mediated GFP delivery into cells. The HEK293T cell line was transduced with VLPs consisting of (A) Gag-GFP pseudotyped with VSV-G envelope, (B) Gag-GFP without VSV-G envelope, (C) GFP and VSV-G envelope, and (D) nontransduced cells.

Fig. 3.

Cre recombinase delivery using a cell-penetrating peptide fusion (HIV TAT) and VLPs. (A) Activation of RFP expression following the action of Cre in a reporter cell line containing a stably integrated expression cassette (Lox1-GFP-Lox2-RFP). (B) A reporter 293T cell line containing a stably integrated expression cassette (Lox1-GFP-Lox2-RFP) was exposed to His-Cre, TAT-Cre, or VLP consisting of Gag-Cre pseudotyped with VSV-G. Expression of RFP shows that active Cre recombinase was able convert the genomic copy of the GFP/RFP expression cassette.

Fig. 4.

Activity and processing of Gag-Cre. Incorporation of a functional retroviral protease into VLPs caused the release of the Cre protein from the Gag-Cre fusion. (A) VLP without VSV-G envelope (control); (B) Gag-Cre VLPs pseudotyped with VSV-G envelope; and (C) Gag-Cre and Gag-protease VLPs pseudotyped with VSV-G.

Fig. 5.

Cytotoxic effects of 5FC in the presence of Gag-Fcy::Fur on the PC3 cell line. PC3 (a human prostate cancer line) cells were treated with complete culture medium (CCM) from untransfected cells; CCM from transfected cells with salmon sperm DNA (ssDNA) + VSV-G envelope; VLPs that contain Gag-GFP and VSV-G envelope or VLPs consisting of Gag-Fcy::Fur and VSV-G in the presence (+) or absence (−) of 60 μg/mL of 5FC.

Fig. 6.

Biological activity of VLPs containing split human caspase 8b. (A) The coding sequence of human capsase 8b was split into two separate components (p10 and p18) that were cloned in-frame into the Gag coding sequence. (B) Biological activity of split caspase 8b delivered via VLPs. Target cells (PC3) were either left untreated or incubated for 48 h with control VLPs consisting of Gag-(p10) + ssDNA (salmon sperm DNA), Gag-(p10) + VSV-G, Gag-(p18) + ssDNA, Gag-(p18) + VSV-G, Gag-(p10 + p18) + ssDNA, Gag-(p10) + VSV-G, and Gag-(p18) + VSV-G or with VLPs consisting of Gag-(p10 + p18) + VSV-G within the same particles. As shown, only the cells transduced with VLPs that contained both p10 and p18 and also contained VSV-G caused a significant reduction in cell viability.

Fig. 7.

TRAIL-mediated cell death. The PC3 cell line was treated with supernatants consisting of complete culture medium (N/T) or culture medium from a packaging cell line transfected with carrier ssDNA or culture medium supplemented with soluble human TRAIL or supernatants containing VLPs as Gag-GFP + VSV-G, Gag-GFP + NA-TRAIL. As shown, cell death is observed when cells were treated either with s-TRAIL or with VLPs consisting of Gag-GFP and NA-TRAIL. GFP was introduced into the VLPs to make it simple to monitor VLP formation.

Fig. 8.

Murine macrophage cells respond to VLPs that carry mouse IFN-γ. The mouse macrophage cell line was treated with VLPs containing HA–IFN-γ constructs. Cells were analyzed for the presence of phospho-STAT1 by flow cytometry. (A) Positive control (soluble IFN-γ). (B) VLPs containing HA–IFN-γ. (C) Gag-GFP + HA–IFN-γ + VSV-G. (D) Gag-GFP + VSV-G (negative control).