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Published On September 22, 2010

CLINICAL RESEARCH

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Zinc fingers could pull gene therapy back from the brink—but only if more researchers can get their hands on the remarkable proteins.

TO UNDERSTAND THE GROWING EXCITEMENT ABOUT ZINC FINGER NUCLEASESa medical innovation with an odd name and a controversial history—think about how AIDS wrecks the human immune system. In its normal progression, the human immunodeficiency virus, HIV, uses the protein produced by a particular gene to gain entry into immune system T cells. When a sufficient number of T cells are destroyed, often after several years, the immune system can no longer function properly, leaving the body at the mercy of every passing infection.

However, for a fortunate few people who happen to have a mutation in that gene, HIV poses little or no threat, because inactivation of the protein prevents the AIDS virus from entering T cells. If scientists could replicate the effect of that natural mutation, it might confer immunity to HIV. That has long been the hope of gene therapy—that a particular gene could be somehow altered or replaced to produce a desired therapeutic effect. There has never been an effective way to cause a particular mutation in a particular gene, however, and the usual method of dumping corrected versions of human genes into the body has seldom worked as planned.

Now there’s a new approach involving an engineered protein known as a zinc finger nuclease, or ZFN. In studies with the University of Pennsylvania and the University of Southern California, scientists from a California company, Sangamo BioSciences, have “knocked out” the HIV-implicated gene in human T cells and hematopoietic stem cells, thus blocking the AIDS virus’s access to T cells. Sangamo has moved on to a Phase I clinical trial with HIV-positive patients, whose cells were changed with zinc fingers and then transfused back into their blood. The hope is that the patients now have immune cells invulnerable to HIV. In January the University of Pennsylvania researcher in charge of the study reported results from one patient whose body appears to have tolerated the mutated T cells, which were still active 20 weeks after being introduced. If larger trials show similar results, HIV may become easier to treat.

During the past few years, zinc fingers have been popping up everywhere in biomedical research, with applications not only in gene therapy but also in creating knockout rats and manipulating embryonic stem cells. “This technology is transformative,” says Dan Voytas, a researcher at the University of Minnesota. “We are opening the door to a new era in genome modification.”

But there’s a catch. Until recently, the most established way to make zinc fingers was Sangamo’s patented method, and only Sangamo and its close collaborators could use it. Now the company has licensed its technology for research and other applications to Sigma-Aldrich, a large biosciences firm that makes chemicals and biochemical products and kits, and Sigma will create customized zinc finger nucleases for scientists who can afford a price tag of approximately $30,000. (Discounts do exist for academic researchers and those buying multiple nucleases, says Sigma, adding that the price will come down as volume increases, and if a scientist orders a ZFN the company has already made, the cost drops to about $10,000.) Others can turn to the Zinc Finger Consortium, a group of academic researchers that has developed its own way to make the proteins. Though the effectiveness of these proteins hasn’t been compared directly with Sangamo’s, a handful of laboratories have successfully created them, and many more have expressed interest in doing so. The proteins cost much less than Sangamo’s, but while Sangamo’s ZFNs come ready to use, the consortium’s protocol requires a dedicated effort (and significant staff time) of several months to get up and running. All of this raises a question: Business is essential for developing new technologies, but is the business of making zinc fingers actually getting in the way of the science of curing disease?

OUR BODY IS MADE UP OF THOUSANDS OF PROTEIN MOLECULES that form muscle, skin and organs, among other things, with genes in our DNA holding the recipes for these proteins. To create a protein from a recipe, the gene’s code first must be copied (onto a molecule called messenger RNA, or mRNA), then transferred to a cell’s ribosome, in which amino acids are pieced together to form the protein based on the DNA/mRNA code. Of the molecules that guide this process, one of the most important is called a transcription factor. Transcription factors attach to DNA and dictate how much mRNA—and, by extension, how much protein—a gene produces. A protein itself, a transcription factor is a long chain of amino acids that bend into helixes and, like the letters in a neon sign, wind into flat sheets, building upon one another to form a sort of three-dimensional protein knot. A zinc finger is one component of the transcription factor—a helix plus a sheet—the part that binds to DNA.

Zinc fingers were discovered in 1985, when Sir Aaron Klug and his colleagues at the MRC Laboratory of Molecular Biology in Cambridge, England, were studying theXenopus frog and noticed that a particular bit of protein kept showing up in one of the frog’s transcription factors. It turned out that this was the transcription factor that actually bound to the DNA. The scientists also discovered that one ion of zinc was lodged within this section, holding the fragment into a tight structure, like a knuckle stabilizing a finger clutching the DNA. Thus the name—zinc fingers. And though they’re not the only type of structure that binds to DNA, zinc fingers do it in a uniquely useful way that lends itself to relatively easy manipulation.

Each zinc finger fits a very specific target—a set of three DNA nucleotides—and linking together several zinc fingers into a DNA-binding “hand” that recognizes 6, 9, 12 or even 18 nucleotides enables scientists to match the hand to a gene’s particular sequence of nucleotides. A landmark 1991 paper by Carl Pabo and Nikola Pavletich at Johns Hopkins, in the journal Science, reported the first structure of zinc fingers bound to DNA and proposed using them to design customized DNA-binding proteins. Subsequent work by Pabo, Klug and others showed that each repeat chemically recognized and was drawn to certain nucleotide letters in the DNA alphabet (A, C, G or T). By the mid-1990s, these researchers had proved that they could, in fact, target their favorite genes with zinc fingers.

That’s around the time when businessman Edward Lan­phier began to take a keen interest in zinc fingers. Pabo’s work convinced Lanphier of the technology’s potential as a research and clinical tool, and he began licensing intellectual property from several major research institutions that had been applying for patents on zinc fingers. In 1995, Lanphier consolidated this intellectual property and founded Sangamo BioSciences, whose researchers went on to obtain an extensive patent portfolio of their own.

SCIENTISTS REALIZED THAT WHILE IT WAS INTERESTING to be able to use zinc fingers to target particular genes, what really mattered was what could be done once the target was engaged—such as knocking it out. By knocking out specific genes—that is, turning off their ability to produce proteins—scientists can determine the genes’ functions and gauge the impact of particular genetic mutations. Before zinc fingers, mice had been the only mammals whose genes could be knocked out quite specifically, and then only through an inefficient process called gene targeting, which requires screening tens of thousands of cells to find a mutation. (It is not yet known why the technology works efficiently only in mice.)

In 1996 researchers at Johns Hopkins University fused zinc fingers to a scissorslike nuclease enzyme to create a zinc finger nuclease. Subsequently, in the early 2000s, a group at the University of Utah used ZFNs to bind to targeted genes in fruit flies and other organisms and then cut them up. The cell’s natural repair mechanism would try to fix the break and often cause mutations in the gene, creating permanent changes in the genome. That new method represented a big advance in several areas of research that had never had such a straightforward way to knock out targeted genes. In 2008 two academic labs, including one working with Sangamo, reported knocking out targeted zebrafish genes for the first time.

Then, just last year, academic researchers working with Sangamo and Sigma knocked out genes in rats. Sangamo licensed the technology to Sigma, which now has the exclusive right to develop and sell knockout rats and has already created rat models of Parkinson’s disease, Alzheimer’s disease and other neurological disorders. The Scientist magazine named the knockout rat one of the top five innovations of 2009.

Yet as essential to research as knockouts are, it’s the possibility of using ZFNs for gene therapy that could have the biggest impact. Gene therapy has been around for years, but what had seemed a relatively simple concept has proved devilishly difficult to execute. The notion is that when you know a defective gene causes a disease, such as cystic fibrosis or sickle cell anemia, you could inject a healthy copy of the gene into a patient with the disease. Ideally the patient would resume making the normal protein that the implicated gene should have produced, thus curing the disease. But the reality is more complex.

To work, a gene needs to be carried into a cell, usually by an inactivated virus, and then inserted somehow into a patient’s DNA. But no one knows exactly where the gene will end up, and though most of the time the location will be innocuous, it also can be deadly. In a famous case in 2002, researchers tried to cure children with severe combined immunodeficiencies by introducing the healthy gene associated with the condition into bone marrow stem cells (bone marrow is where immune cells are produced) taken from their bodies. Although the method worked in most patients, in four children the retrovirus carrying the gene inserted itself near a cancer-promoting gene, turning it on, and the patients developed leukemia.

Using zinc fingers may avoid some pitfalls of traditional gene therapy. The new method has the potential to actually fix a defective gene, through a natural cell process called homology-directed repair. If a gene is damaged during cell division, homology-directed repair fixes it by referring to a sister copy of that gene’s chromosome. When a zinc finger nuclease zeroes in on a defective gene and cuts it, researchers can then trick the cell into using a healthy gene that they added instead of the defective one as a template. That way, scientists needn’t worry about where a new copy of the gene is going to insert itself. “This gives you the possibility of actually going in and restoring the defective gene to normal,” says J. Keith Joung, a pathologist at Massachusetts General Hospital who co-founded the Zinc Finger Consortium with Voytas.

UNTIL RECENTLY THAT POSSIBILITY WAS VERY HARD TO REALIZE, because good zinc finger nucleases were very hard to make. With all the patents it has acquired, Sangamo has had a virtual monopoly on effective ZFNs, and now, with Sigma marketing Sangamo’s technology, researchers can order customized ZFNs targeting particular genes. It takes about eight weeks for the company to produce a ZFN, and it’s not cheap—about $30,000 for a custom pair (ZFNs work only in pairs). The reasons for the high cost, says Sigma, are staffing (several scientists are needed to create each customized zinc finger) and the costs to license all of the patents owned by others—typically universities and their researchers. For those whose budget can’t accommodate that kind of outlay, there have been other protocols for making zinc fingers, but they have been difficult and time-consuming.

So in late 2005, Joung, Voytas and other members of the Zinc Finger Consortium began developing an alternative protocol for producing zinc finger nucleases and, after three years of testing, unveiled OPEN (for Oligomerized Pool ENgineering). It uses a library of zinc fingers, with each batch or pool containing multiple zinc fingers that attach to a particular DNA nucleotide sequence—GAA, CCG, AGC and so on. Taking advantage of the techniques of molecular biology, researchers can stitch together three zinc fingers from various pools to bind to the unique sequence of the gene they’re targeting. They can then screen the resulting batch for the hand of zinc fingers that binds best to their gene.

By testing the whole hand rather than individual fingers, OPEN takes into account the subtle interactions that occur between fingers, thus producing a more effective research tool than those created by earlier methods other than Sangamo’s. The OPEN protocol is published online, and Joung distributes pools of zinc fingers to any academic investigator who fills out the appropriate paperwork and pays a nominal processing fee.

So far, more than 150 academic laboratories have requested the zinc finger pools, more than 70 have completed the paperwork and received the materials, and at least 6 have made their own ZFNs. What’s more, researchers using OPEN-generated ZFNs have created mutations in specific genes in human embryonic stem cells and in induced pluripotent stem cells (adult cells that have been manipulated into a state similar to that of embryonic cells). Because stem cells of either kind can potentially become any cell in the body, correcting a gene at the single-cell stage could be a permanent fix for every subsequent cell descended from that stem cell. Researchers might one day make iPS cells from a person with a disease, use ZFNs to correct the problem genes in those cells, grow them into healthy tissue outside the body, then insert that tissue back into the person.

But here’s the rub: A laboratory using OPEN could take as long as nine months to ramp up, but once it has, it can make multiple ZFNs simultaneously in eight weeks or fewer. “It’s a long, exacting protocol,” says Liz Wolffe, Sangamo’s director of corporate communications. “It’s a significant investment of time and energy, and it’s not as cheap as the OPEN people say, because you also have to buy all the reagents.” (The cost for obtaining all of the OPEN pools and reagents needed, which can be used to make hundreds of ZFNs, is approximately $5,000.)

THAT'S THE CRUX OF THE DEBATE ABOUT HOW ZINC FINGERS are produced and distributed: How much control should Sangamo and Sigma exert? “Sangamo did a great job putting all those patents together, and its mission is to realize some financial gain on that investment,” says Voytas. “I have no problem with that. But they are exercising a lot of control over what you can do with the reagents.” Scientists who purchase zinc fingers from Sigma must sign a license that imposes certain restrictions. To prevent scientists from selling or even sharing ZFNs, there’s a limit to how many animals can be produced. Nor can a scientist share anything made using the zinc fingers outside her organization.

“All that kind of goes against what we do in academia,” says Voytas. “We need to have flexibility in how we use these reagents. Journals require that the reagents be made available, and funding authorities require us to disseminate our reagents.” According to the grants policy of the National Institutes of Health, the biggest U.S. funding source, “[grant] recipients are expected to avoid signing agreements that unduly limit the freedom of investigators to collaborate and publish, or that automatically grant co-authorship or copyright to the provider of an invention used primarily as a research tool.... NIH expects recipients to determine the appropriate means of effecting prompt and effective access to research tools…to further advance scientific research and discovery.… Investigators are expected to submit unique biological information to the appropriate data banks; otherwise, they are not truly accessible to the scientific community.”

Sigma, maintaining that its licenses meet industry standards and are consistent with NIH guidelines, points to other technologies with similar licenses, and says that it does allow for the deposit of certain materials in data banks. And without such a license, scientists could just remake and give away the ZFNs, and no one would have to buy anything from Sigma or Sangamo, which expended a lot of time and money to produce the ZFNs. To bolster its case, Sigma cites another passage in the NIH policy: “Reasonable restrictions on the dissemination of research tools are sometimes necessary to protect legitimate proprietary interests and to preserve incentives for commercial development.” In addition, if a researcher makes a discovery using Sangamo’s ZFNs and wants to commercialize it, he or she can buy a less restrictive commercial license.

Might Sangamo’s monopoly on ZFN patents actually help this emerging field? In “Proprietary Science, Open Science and the Role of Patent Disclosure: The Case of Zinc-Finger Proteins,” a paper published in Nature Biotechnology in February 2009, the authors argue that it would be quite costly to develop and use zinc fingers if it meant negotiating licensing terms with multiple universities and companies holding patents on the technology. As it is, anyone wanting to commercialize the proteins only has to deal primarily with one company. Moreover, the paper suggests that the current situation, in which OPEN coexists with Sangamo/Sigma, may actually benefit research involving zinc fingers: “The presence of roughly comparable proprietary [Sangamo] and open-science [OPEN] alternatives could produce a productive tension resembling the competition between the public and private human genome sequencing endeavors.”

But at the OPEN end of things, some dispute that contention. Last spring, at the end of a long day of presentations at a Zinc Finger Consortium conference in a Boston hotel, several participants—both those who are part of the Consortium and those who aren’t—grumbled that their work was being stonewalled, and they blamed Sangamo and Sigma. The companies’ bid to control not just the reagents the scientists were buying but also anything the scientists created using those reagents was going too far, they said. To these scientists, it seemed like a matter of intellectual property versus intellectual freedom, and they clearly felt the companies were on the wrong side of the argument.

 

DOSSIER

1. “Proprietary Science, Open Science and the Role of Patent Disclosure: The Case of Zinc-Finger Proteins,” by Subhashini Chandrasekharan et al.,Nature Biotechnology, Feb. 9, 2009. The authors explore the pros and cons of a single company controlling the intellectual property for a potentially game-changing technology.

2. “The Discovery of Zinc Fingers and Their Applications in Gene Regulation and Genome Manipulation,” by Aaron Klug, Annual Review of Biochemistry, July 2010. Klug provides a detailed account of how he discovered zinc fingers and notes their potential applications.

3. “Oligomerized Pool Engineering (OPEN): An ‘Open-Source’ Protocol for Making Customized Zinc-Finger Arrays,” by Morgan Maeder et al.,Nature Protocols, Sept. 17, 2009. A how-to manual for this weeks-long protocol used to make one’s own set of zinc fingers.

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