Microbiology
With their knack for insinuating their own genes into a cell's DNA, retroviruses seem the perfect vehicle for getting therapeutic genes into sick cells. But harnessing these nimble microbes has not been easy. Finding a way to steer them to needy cells--and only those cells--has been one of the major challenges facing gene therapy. A pair of HMS researchers may have found a solution to this critical problem. They have created a novel molecular device that enables genetically modified retroviruses not only to seek out but enter specific cells in culture.
Whereas previous attempts have focused on modifying the retrovirus to give it better aim, the new method leaves the virus vehicle untouched. Instead, it links the virus and target cells by means of a molecular bridge consisting of two proteins--epidermal growth factor (EGF) and the avian leukosis virus (ALV) receptor. These, in turn, bind proteins found on specific human cells and viruses, respectively. The specificity of the binding--between EGF and its human cell surface receptor and between the ALV receptor and the viral protein--is what allows the virus to hook up with particular cell targets.
| John Young and Sophie Snitkovsky (not shown) have discovered a way to help viruses deliver therapeutic genes to specific cell targets and only those targets. |
When applied to cultured cells, the protein hybrid allowed the virus not only to bind but also enter its designated cell targets, the researchers report in the June 9 Proceedings of the National Academy of Sciences. This is the first time this kind of gene therapy delivery system has been shown to enable successful targeting and infecting of selected cells in vitro.
"The novel thing here is that this device works efficiently. It allows for the first time the possibility that we can use this approach to target any cell type we want," says John Young, associate professor of microbiology and molecular genetics at HMS. He developed the method in collaboration with graduate student Sophie Snitkovsky. If it is successful in vivo, the method could be used in gene therapy protocols to deliver particular genes to selected groups of cells, including tumors and diseased tissues such as the defective lungs of cystic fibrosis patients.
Until recently, many gene therapy trials have employed ex vivo strategies--genetically modifying target cells outside the body and reimplanting them in the body--to achieve this goal. But this is an invasive and generally impractical procedure with variable results. Efforts to design in vivo delivery systems have focused on modifying the virus, for example, by building into the envelope a structure that directs the virus to a specific cell type. But the tinkering appears to interfere with the ability of the virus to enter the cells.
"We wanted to find an approach where we could preserve as much as possible the normal virus-envelope receptor interaction," says Young. Supported in part by an HMS Funds for Discovery Award, he and Snitkovsky created the EGF-ALV receptor protein hybrid and applied it to a series of cell types, including one with the normal EGF receptor and one without. Only those cells with the EGF receptor bound the molecular device.
Normally, viruses get their genes into cells by attaching
to a compatible receptor found on certain cells (left). Cells lacking this
viral receptor but having others, such as the EGF receptor, are immune to
infection (center). To infect these cells, the researchers took a viral
receptor and fused it to the EGF ligand, giving the virus and cell each
something to bind to. The resulting protein bridge allowed the virus not
only to attach but also enter its cell target.
To see if the virus, once linked, could actually enter cells, the researchers introduced a gene conferring resistance to the antibiotic neomycin into the viruses. They added the gene-carrying viruses to the cells, and then exposed the cells to neomycin. To their surprise, the researchers saw cell colonies--demonstrating that cells had been infected by the viruses carrying the resistance gene. In fact, a much higher percentage of the cells were infected than expected. "It was really quite remarkable. I think jumping up and down is the way you'd describe our reaction," says Young.
The success may be due in part to the simplicity of the ALV system. Unlike HIV, which requires multiple protein receptors to enter a cell, ALV seems to use a single protein receptor. While the ALV receptor thus appears to be uniquely suited to the protein bridge approach, EGF is only one of many possible protein partners. Young and Snitkovsky speculate the method could be used on a variety of cell surface markers, including those found on cancer cells. They are currently exploring other possibilities in conjunction with graduate student Adrienne Boerger and other colleagues at HMS. They are also working on ways to deliver the protein hybrid in vivo.
"The way I look upon this experiment is that it has opened the door to the possibility of targeting by this mechanism," says Young. "It solves that problem in tissue culture. Translating that to the in vivo situation is the focus of our current work."
--Misia Landau
Focus 7/17/98