LONDON - A viral protein that is naturally transported into neighboring cells may be able to overcome one of the main hurdles of gene therapy: delivering the gene or its product into enough cells to bring about a therapeutic effect.

Researchers in the U.K. have shown that the VP22 protein of herpes simplex virus type 1 (HSV-1) can ferry other functional proteins into neighboring cells. They were able to bring about significantly raised levels of apoptosis (programmed cell death) in tumor cells by linking VP22 to p53, the protein which is lacking or mutated in many human cancers.

Anne Phelan, postdoctoral research scientist at the Marie Curie Research Institute, in Oxted, U.K., told BioWorld International, “If our in vitro data are mirrored in vivo, we hope to see a major improvement in delivery of therapeutic agents to human cancers with the help of this system.“

Phelan, together with Gill Elliott and Peter O'Hare, also of the Marie Curie Research Institute, investigated whether the HSV-1 protein VP22 could transport functional p53 into cells.

In their paper in the May edition of Nature Biotechnology, titled “Intercellular delivery of functional p53 by the herpesvirus protein VP22,“ they reported that it can.

Getting Genes Into Cells Remains Difficult

The team already knew that VP22 could transport large proteins from cells in which it was made into surrounding cells. Elliott and O'Hare reported early last year that if cells were transfected with expression vectors containing the DNA for VP22 and for green fluorescent protein (GFP) - a natural protein which fluoresces on exposure to ultraviolet light - then the VP22-GFP fusion protein made in these cells finds its way into neighboring cells.

Phelan said, “The obvious next step was to find out whether we could transport a therapeutic protein, with the aim of achieving gene therapy. The problem with gene therapy at present is that conventional delivery systems such as viral vectors and liposome vectors allow you to get the gene into only quite a small percentage of cells - and that is not always enough to give a very good therapeutic effect.“

VP22 held out the promise that if conventional methods could deliver the desired gene to one in 10 cells, then linking the gene to that for VP22 might allow delivery of the desired gene product to possibly all cells.

O'Hare and his colleagues decided to test whether VP22 could deliver p53. This tumor suppressor protein is the most commonly mutated protein in human cancers: more than 50 percent of cancers have mutations in this gene. Furthermore, various studies have shown that, with many such tumors, delivering non-mutated wild-type p53 can halt growth of the tumor and induce apoptosis.

The team set about making expression vectors that contained the gene for VP22 linked to the gene for p53. When these were transfected into mammalian cells, a proportion of the cells made fusion proteins consisting of VP22 and p53. Tests showed these fusion proteins entered the nuclei of surrounding cells in the culture.

To find out whether the transported p53 was functional, Phelan next transfected cells from a p53-negative osteosarcoma cell line with the VP22-p53 expression vector. It already had been shown that if p53 is reintroduced into these cells, their growth is suppressed and apoptosis induced. The team found that VP22-p53 and p53-VP22 both caused a statistically significant loss in cell viability, compared with controls.

In Nature Biotechnology, they wrote: “[This is] consistent with a more widespread cytotoxic effect in both primary expressing cells and cells that have obtained the fusion protein by cell to cell spread.“

A further experiment examined the extent to which the VP22-p53 fusion proteins could induce apoptosis in the p53-negative osteosarcoma cell line. Phelan said, “It was important to show that the p53-VP22 fusion proteins were functional in inducing apoptosis not only in the primary expressing cells but also in the secondary recipient cells, because there is no point in delivering a protein to lots of other cells if it is not functional when it gets there.“

This assay, she added, enabled the team to confirm that the fusion protein was functional in both primary expressing and recipient cells.

The group is now examining which gene delivery system it should employ in order to evaluate what effect coupling VP22 to therapeutic genes might have in whole animals. Several systems for delivering the gene for p53 into tumor cells already have been devised, with reasonable success, they wrote. These include retroviral and adenoviral vectors, as well as liposome delivery systems.

Research May Improve Gene Transfer Efficiency

Commenting on the paper in an analysis article in the same issue of Nature Biotechnology, Tania Fernandez and Hagan Bayley, postdoctoral associate and professor, respectively, in the department of medical biochemistry and genetics at the Texas A&M Health Science Center, in College Station, Texas, said the approach offers great promise for gene therapy and as a general means of introducing proteins into cells.

In their article, titled “Ferrying proteins to the other side,“ Fernandez and Bayley reiterated that one major problem with gene therapy is the poor efficiency of infection with viruses which cannot replicate or are otherwise crippled. They added, “The results from O'Hare's team suggest their approach could provide a possible solution to this difficulty by introducing a marvelous new type of bystander effect.“

Phelan, Elliott and O'Hare also noted VP22 may be helpful in delivering other types of protein to cells, including transcription factors, enzymes and antigens. It may also boost the effectiveness of other currently experimental cancer treatments, such as those in which prodrugs are activated by locally produced enzymes.

For example, Phelan said, one strategy relies on introducing the gene for the enzyme thymidine kinase into cancer cells. When expressed, this enzyme phosphorylates the drug ganciclovir, turning it into a cytotoxic agent.

“The cytotoxic version then passes into surrounding cells, and this is known as the bystander effect,“ Phelan explained. “But if thymidine kinase was coupled to VP22, then you would have the bystander effect coupled with transport of this fusion protein, and this would mean that the therapy is delivered much more efficiently.“ *

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