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Sangamo Study Shows Its ZFP Technology Can Rapidly Generate Knockout Cell Lines

Sangamo BioSciences has published data recently showning that its zinc finger DNA-binding protein nucleases, called ZFNs, can rapidly generate knockout cell lines in CHO cells.
The findings, published in the March 21 issue of the Proceedings of the National Academy of Sciences, suggest that ZFN-containing cell lines are important tools in pharmaceutical research and therapeutic product development, a Sangamo official told this week.
“We introduced the zinc finger nucleases into the cell on a plasmid DNA; they are then expressed by the cell, and the nucleases then bind to the DNA as specified by the DNA binding domain that we can engineer at Sangamo,” said Philip Gregory, vice president for research at Sangamo.
Sangamo’s core technology enables researchers to design and engineer zinc finger protein DNA-binding domains that will recognize and bind to a specific genomic sequence.
“What happens when the protein binds to that sequence is determined by the functional domain we have attached to the ZFP,” said Gregory. In the case of the nuclease, Sangamo scientists have attached a nuclease domain that allows the ZFP to cut DNA precisely at the sequence bound by the ZFP end.
Once the DNA break is made, the cell has extremely robust pathways for the repair of that double strand break. “We can usurp those pathways to drive to different genetic outcomes,” Gregory said.
“If we simply allow the cell to repair the break, it does so, but it does so in an error-prone fashion,” Gregory said. “That creates mutations in the DNA at the site of the break, and if we have targeted that cut to the exon of the gene, it will destroy that exon and, of course, destroy the gene product.
“If we also provide a donor DNA molecule through the process called gene targeting, that process uses homologous recombination, or HR, to target the new gene into a locus specified by the new gene and encoded on the donor DNA,” said Gregory.
The reason why that is used only in mouse embryonic stem cells is that the efficiencies of the HR process are incredibly low in other cell types, particularly in transformed or somatic cell types of mammalian origin, such as CHO cells.
“However, it has been known for about 20 years that in the context of double-stranded breaks, an accelerated, more efficient version of HR called homology-directed repair takes place,” Gregory said. He mentioned that homology-directed repair is approximately 1,000 times more efficient than homologous recombination.
“By putting the double-strand break in the genome precisely where the ZFP cuts, we stimulate homology-directed repair to accelerate the gene targeting process,” said Gregory.
Sangamo’s zinc finger protein technology represents a new way to target the genome of a living cell, David Smoller, president of research biotech at Sigma-Aldrich, which exclusively licensed the technology from Sangamo for use in the laboratory research reagent area.
“We are a small biotech company, and we are essentially unable to deal with the demand, so we sought a partner who could help us deliver ZFP reagents to the research community,” said Gregory.

“By putting the double-strand break in the genome precisely where the ZFP cuts, we stimulate homology-directed repair to accelerate the gene targeting process.”

According to Smoller, Sigma-Aldrich’s goal “is to generate a set of products and get the technology to market,” Smoller told CBA News this week. “We currently have a custom cell line engineering service that is mainly directed to pharma in their efforts to make cells that produce proteins.”
Smoller said that Sigma-Aldrich and Sangamo continue to refine the ZFP technology so that researchers “can speed up the time required to develop a cell line, and the numbers, so that we can have knockouts or knockins for a lot of different genes.” He said that the aim is to market these cell lines either as off-the-shelf kits or as custom services.
As zinc finger protein technology becomes more robust, its use in standard laboratory research has become much more interesting to basic researchers, both in academia and biopharma, for use in drug discovery and high-throughput screening, among other applications, said Gregory.
“We started originally looking at this zinc finger transcription factor technology as a therapeutic, and most recently have been looking at the nuclease technology, both as a therapeutic and a research reagent,” said Gregory. The agreement with Sigma “will move us aggressively into the field of research reagents.”  
The financial details of the partnership between Sangamo and Sigma-Aldrich were not disclosed.
In Zinc
Sangamo was founded in 1995, and the company was “early in licensing all of the ZFP IP that existed,” Gregory said, adding that the company has spent the last 12 years developing zinc finger DNA-binding proteins as a way of targeting particular events in the genome of different types of cells.
Gregory explained that Sangamo’s ZFP transcription factor technologies permit researchers to turn gene expression on or off. “The ‘off’ version is kind of like RNAi, but it was done at the DNA level,” he said.
Genes can also be activated, which is the principle on which Sangamo’s lead clinical molecule, which has just completed accrual for a phase 2 clinical trial, is based, said Gregory. The molecule, currently known as SB-509, is an activator of VEGF and is being tested in patients with diabetic neuropathy.   
“We have also used our technology [to generate] high-throughput screening systems,” said Gregory, adding that the company has published several papers demonstrating that ZFP transcription factors can be used to generate cell lines that express a particular drug target that they normally would not.
“The idea is that we can produce isogenic cell lines that differentially express the target gene of interest,” Gregory explained. He said that the work presented in those papers was done in collaboration with Wyeth and Johnson and Johnson. The PNAS paper was done in collaboration with Pfizer.
“The other side of this technology is the new stuff that we have been working on, which is using ZFP DNA-binding domains, but linking them to a nuclease domain to create, if you like, a designer pair of scissors that cuts the genome at a precise point,” said Gregory. The technology can be used to knock out genes, insert genes, or insert very precise information, “such that if we cut within the exon of the gene, for example, we can correct a natural mutation. Or from a tools perspective, [we can] insert a mutation that is found in the natural population to see what it would do in a disease model.”
The PNAS paper is the first time researchers have used a ZFN to delete a gene in a mammalian cell, said Gregory. “We have done this in the context of a CHO cell, in this example, to create a cell that now requires thymidine and hypoxanthine to be added to the culture medium for growth,” he said.
That allows the Sangamo researchers to put the gene that the cells need into a plasmid that can be inserted into these cells as a way of selecting for the presence of the plasmid, and therefore “the presence of the antibody expression cassette or the protein expression cassette or what ever the client wants to put into that cell line,” said Gregory.
“Where I think we are going to go next is to the knockout of multiple sets of genes in the same host cell line,” Gregory said. He pointed out that this has not been possible through the use of HR, because of the need to use selected markers.

“It is really going to open up for researchers the opportunity to create complex knockouts of different genes for intricate pathway analysis, and to create novel host lines that are very powerful from a screening perspective.”   

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