Login to Your Account

Zinc Finger Proteins Can Be Used as Bootstraps, Lassos

By Anette Breindl
Science Editor

In the biotech world, zinc finger nucleases got their start as gene correction tool. But their ability to precisely target and cut specific DNA sequences could be used for other purposes as well, and two recent papers reported methodological advances that could ultimately expand the uses of the technology.

In one of those papers, which appeared in the July 1, 2012, advance online edition of Nature Methods, a team from the University of California at Los Angeles described delivering zinc finger nucleases as proteins, rather than in a viral vector or as nonviral DNA or RNA.

Senior author Carlos Barbas III told BioWorld Today that he and his team first tried to deliver zinc finger proteins directly to cells by using so-called protein transduction domains, which are short basic proteins of viral origin, connected to the protein that is to be delivered into the cell.

Those attempts did not work, or at least not well enough to be of practical use. But during the course of the work, Barbas and his team realized that the zinc finger proteins themselves are basic overall, and might be able to essentially bootstrap themselves into cells.

In their experiments, Barbas and his colleagues delivered purified zinc finger proteins to an experimental cell line that contained a fluorescence gene that is not expressed due to a frameshift mutation. When zinc finger proteins penetrate such a cell, they can restore expression of the fluorescence gene by inserting or deleting base pairs that counteract the frameshift effect. The team showed that they could ultimately restore EGFP expression to more than 10 percent of the cells by direct delivery of the zinc finger protein.

Barbas and his team next turned their attention to the CD4 cell surface protein CCR5, which is a co-receptor for HIV entry. Cells lacking the receptor are immune to HIV infection, and "Berlin patient" Timothy Ray Brown appears to have been cured of his HIV infection through a bone marrow transplant from a donor with a mutation in the CCR5 gene. (See BioWorld Insight, May 23, 2011.)

Through repeated applications of protein, they were able to disrupt CCR5 expression in roughly 8 percent of cells.

Levels of zinc finger proteins in transduced cells remained low, which is in line with the overall experience that "protein transduction is very inefficient," Barbas said, In this case, however, that low efficiency is no great issue, because "you need just enough protein to affect cleavage of the DNA."

In fact, it may be an advantage. Barbas and his team looked at how site-specific the zinc finger proteins were, and found that they had fewer off-target effects than zinc finger nuclease genes that are delivered via viral vectors.

"Futuristically, it might allow for an injectable therapy for a variety of genetic diseases." Huntington's disease, for example, stems from a triplet expansion that is in theory straightforward to remove with the right zinc finger protein.

"Right now, what we envision is treating stem cells ex vivo" in an autologous transplantation approach, for example, to create helper T cells that lack the CCR5 receptor and thus are resistant to HIV.

Philip Gregory, too, named "an ex vivo cell-based approach" as the most likely first application of the results that Barbas and his team described in Nature Methods.

Gregory is vice president of research and chief scientific officer at Sangamo Biosciences Inc., which is developing zinc finger nucleases for several indications including HIV and hemophilia. He said that there is "great interest" in developing zinc finger nucleases beyond the gene correction application where they got their start.

He noted that the Sangamo team, too, has seen a reduction in off-target effects by tweaking their delivery mechanisms. But direct delivery of proteins, he said, is also a developmental path "that should be further explored."

"It's an early result," he said. "That's why it's a Nature Methods paper and not a Nature Medicine paper yet. But the fact that it works in research is always the first step."

Gregory is the co-author of a recent Cell paper that showed yet another way to use zinc finger proteins. Scientists from the University of Pennsylvania used zinc finger proteins to bring together a distant DNA enhancer and the beta-globin gene in a so-called chromatin loop, and initiate transcription of the latter in cells where it is normally inactive.

The existence of chromatin loops during transcription had long been known. But it was, Gregory said, "a chicken-and-egg question" whether such loops helped initiate transcription, or formed because transcription was occurring. The results published in Cell showed that artificially inducing such a loop "is sufficient to activate gene expression," Gregory said. Such gene expression, he clarified, does not reach levels it would when the gene is activated naturally. "But the fact that it turns on at all was sort of stunning."

For now, the work published by Gregory and his co-authors in Cell, like the study in Nature Methods, describes basic research.

But Gregory said there will be increased interest in trying to use zinc finger proteins to activate or increase the transcription of genes for therapeutic purposes. And because fetal hemoglobin can substitute for adult hemoglobin in diseases like sickle-cell anemia, "beta-globinopathies are a great target" for such efforts.