VP22 flips the switch on cell death
Authors: Matthew D Weitzman
Journal: Molecular Therapy (2003)

Delivery of therapeutic agents in a safe and efficient manner remains a challenge for molecular therapeutic approaches. There are a plethora of strategies—both viral and non-viral types—by which to penetrate the cell, and there are advocates of both types. The viral and non-viral camps each boast of obvious advantages to their approach, and both readily point to the limitations of the other. What is clear is that the latter camp will undoubtedly benefit from lessons that viruses have taught us about cell entry, molecular trafficking, and gene regulation. A new particle for delivery and regulated activation of a therapeutic cargo provides an interesting example of how viral lessons can be harnessed into non-viral approaches and is described by Brewis et al. in this issue of Molecular Therapy [1Brewis, N. D., Phelan, A., Normand, N., Choolun, E., and O'Hare, P. (2003). Particle assembly incorporating a VP22-BH3 fusion protein, facilitating intracellular delivery, regulated release and apoptosis. Mol. Ther. this issueGoogle Scholar]. The particles are assembled from fusion proteins based on the VP22 polypeptide of herpes simplex virus (HSV). VP22 forms part of the viral tegument, a region of the HSV virion that lies between the capsid and the envelope. The VP22 protein possesses the unusual ability to translocate between mammalian cells [2Elliott G. O'Hare P. Intercellular trafficking and protein delivery by a herpesvirus structural protein.Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar]. It appears largely cytoplasmic in cells in which it is initially expressed, but can then move to adjacent cells where it accumulates mainly in the nucleus. The exact mechanism of this transport and its precise role in the viral lifecycle remain unclear. It may prepare neighboring cells for oncoming viral infection, and it has been suggested to mediate the transfer of viral RNA from cell to cell [3Sciortino M.T. Taddeo B. Poon A.P.W. Mastino A. Roizman B. Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection.Proc. Natl. Acad. Sci. USA. 2002; 99: 8318-8323Crossref PubMed Scopus (100) Google Scholar]. Heterologous proteins fused to VP22 can hitch a ride for intracellular transport [2Elliott G. O'Hare P. Intercellular trafficking and protein delivery by a herpesvirus structural protein.Cell. 1997; 88: 223-233Abstract Full Text Full Text PDF PubMed Scopus (908) Google Scholar], providing a powerful system to amplify the effect of a therapeutic gene product. Proteins that have been delivered in this fashion include reporter proteins such as GFP, functional proteins such as the p53 tumor suppressor, enzymes such as Flp recombinase and T7 RNA polymerase, and the pro-drug thymidine kinase of HSV. The group of Peter O'Hare, who originally uncovered the remarkable intercellular trafficking of VP22, has recently expanded its transport repertoire. In addition to carrying a protein fused to VP22, they show that it can ferry other molecular cargoes into the cell. They found that addition of a soluble fraction of purified VP22 to oligonucleotides induces the spontaneous assembly of particles, for which they coined the term “vectosomes” [4Normand N. van Leeuwen H. O'Hare P. Particle formation by a conserved domain of the herpes simplex virus protein VP22 facilitating protein and nucleic acid delivery.J. Biol. Chem. 2001; 276: 15042-15050Crossref PubMed Scopus (61) Google Scholar]. These particles are regular spheres with diameters ranging from 50 nm to 2 μm and are stable in serum. They do not show any toxicity but can enter many cell types and reside stably in the cytoplasm. A surprising observation was that these particles could be activated by light to release the oligonucleotides. In the absence of light, the fluorescein-labeled oligos were observed as intensely luminous dots in the cytoplasm of cells that have taken up the particles. Upon light activation the particles are disrupted and the oligonucleotides are redistributed throughout the nucleus and cytoplasm. This is nicely captured by time-lapse microscopy in an impressive accompanying video. The mechanism of activation by illumination remains unclear, but appears to be dependent on the presence of the fluorophore attached to the oligonucleotide. In the new report, Brewis et al. fused a pro-apoptotic BH3 domain to the VP22 polypeptide and assembled vectosomes that enter cells without evidence of toxicity [1Brewis, N. D., Phelan, A., Normand, N., Choolun, E., and O'Hare, P. (2003). Particle assembly incorporating a VP22-BH3 fusion protein, facilitating intracellular delivery, regulated release and apoptosis. Mol. Ther. this issueGoogle Scholar]. Upon light activation the particles were rapidly disrupted, resulting in cell death by apoptosis. Importantly, there was no apoptotic effect from particles assembled with a similar fusion protein containing a mutation in the BH3 peptide, although the cells had taken up these particles as well. This synchronized, regulated release demonstrates that fusion proteins incorporated into vectosomes retain functional activity. As a final step towards application of such as a system, the BH3-VP22 vectosomes were tested in vivo for their ability to inhibit tumor progression in a mouse model. Cells were pre-loaded in vitro with the vectosomes prior to subcutaneous implantation and then activated by illumination. A modest but significant effect was seen only with BH3 vectosomes coupled with light activation. Obviously, there are many aspects that remain to be characterized for this new delivery system. The size constraints for the fusion peptide and the nucleic acid portion need to be systematically determined. Future work should also unravel the requirements for assembly of the spherical particles and the pathways of cellular uptake. More important questions are the stability in vivo and the potential immunogenicity of vectosome particles. The synchronized and regulated release of a death signal from the inert VP22-based particles may be useful in studies of the mechanism and temporal activation of apoptotic events. Although apoptosis is the end for the cells in this particular study, clearly it is just the beginning for future derivations of this delivery system. There are many therapeutic applications that might be envisaged, depending on the components incorporated into either the fusion protein or the oligonucleotide. The dual nature of the particles provides a flexible system that could be used to provide two regulated punches to the cell. The O'Hare group has previously used antisense oligonucleotides against the c-raf kinase to give rise to light-induced inhibition of cell proliferation [4Normand N. van Leeuwen H. O'Hare P. Particle formation by a conserved domain of the herpes simplex virus protein VP22 facilitating protein and nucleic acid delivery.J. Biol. Chem. 2001; 276: 15042-15050Crossref PubMed Scopus (61) Google Scholar]. In addition to antisense, one could deliver ribozymes to direct RNA cleavage, small interfering RNAs to knock-down protein levels, and RNA/DNA hybrids or triple helix-forming oligonucleotides for homologous recombination and gene targeting. The future for vectosomes looks bright, illuminated by a number of fortuitous observations from basic studies of a viral protein. Innovative exploitations of viral mechanisms such as this one will continue to propel the development of both viral and non-viral vectors for gene therapy.