In the Oct. 20 issue of Nature, researchers at the Wellcome Trust Sanger Institute and Sangamo BioSciences, reported some dramatic results in the field of stem cell therapeutics. 
Using fibroblasts from human patients with alpha1-antitrypsin deficiency, a recessive metabolic disorder of the liver stemming from a single point mutation in the A1AT gene, the team produced patient-specific induced pluripotent stem (iPS) cells, corrected both chromosomal copies of the point mutation, and differentiated them back into “hepatocyte-like cells.” When those cells were then transplanted into mice, they colonized the mouse liver for at least five weeks, producing measurable A1AT protein.
It was perhaps the best demonstration yet — albeit only in an animal model — that stem cell biology sits potentially on the cusp of a completely new kind of clinical therapy, one in which a patient’s own cells are rebooted into an embryonic-like state, genetically repaired, differentiated back into a desired cell type, and returned to the body.
“This is the first demonstration, to our knowledge, of the generation of mutation-corrected patient-specific iPSCs, which could realize the therapeutic promise of human iPSCs,” the authors wrote.
There’s a long road ahead before this kind of technology is ready for the clinic, of course. Still, it’s a key step. After all, stem cell genome editing isn’t new — researchers have been editing the genomes of mouse embryonic stem cells for years using homologous recombination. That’s how they make mouse knockouts. But other mammalian cells, including humans ones, don’t perform homologous recombination as readily as do mESCs, says Keith Hansen, product manager in the emerging technology division at Sigma-Aldrich. As a result, genome editing in other cell types needs a little help.
In the case of the A1AT study, help came in the form of zinc-finger nucleases (ZFNs), a technology pioneered by Sangamo and commercialized for research use by Sigma-Aldrich. ZFNs are custom enzymes that induce a double-strand DNA break at a specified location in genomic DNA. In the absence of a “donor” template, the cell repairs these breaks via a relatively error-prone mechanism called “non-homologous end joining” (NHEJ).
If a homologous DNA donor is supplied, however, the cell will use it to repair the break instead via the far more reliable process of homologous recombination. In this case the researchers, led by the Wellcome Trust’s Allan Bradley, supplied a template that not only substituted the A1AT gene’s mutant A for a wild-type G, but also inserted a selectable marker flanked by recognition elements for the eukaryotic transposon, piggyBac. By subsequently introducing an additional piece of DNA encoding the piggyBac transposase enzyme, the authors were then able to excise their selectable marker without leaving any detectable genetic “scars” behind.
To be more precise, they left no unintended scars in the A1AT gene. They did have to introduce two point mutations in an upstream leucine codon in A1AT (forming a different leucine codon) to create the transposase’s recognition sequence.
But the process also produced a smattering of errors elsewhere as well. When Bradley and his team sequenced the exomes of both a corrected line and the parental fibroblasts they detected 29 mutations overall, 25 of which occurred during the iPS cell line derivation. Just four occurred during the genetic correction, “one during targeting and three during piggyBac excision,” the authors wrote. But none apparently impacted the cells’ pluripotency, differentiation potential, or ability to survive, populate, and function in a mouse liver in vivo. And that, says Ignacio Sancho-Martinez, a senior postdoc at the Salk Institute for Biological Studies, who has performed genome editing studies using virus-mediated homologous recombination, is the point.
In considering genome-editing technologies, functionality and efficiency should trump absolute precision, he says. “If you are in a clinical application you will need something that is cost-effective and functional,” Sancho-Martinez says. “You need to produce the cells, you need to correct the cells, and you might not care as much about how many mutations you introduce” so long as they are not detrimental — that is, that they don’t affect cell function or induce tumors.
ZFNS IN VIVO, AND IN THE CLINIC
A related study underscores the potential for ZFNs in the clinic. Katherine High of the University of Pennsylvania, with Holmes and others designed ZFNs to target the first intron of the human factor IX gene, which is implicated in hemophilia. But not in cells in a Petri dish. Instead, they used the ZFNs to insert a fully corrected partial cDNA containing factor IX exons 2 through 8 into cells in living mice. 
The strategy, says Philip Gregory, vice president for research and chief scientific officer at Sangamo, “renders everything downstream [of the insertion site] irrelevant, but retains an important feature of editing technology, which is that we can retain expression of the entire locus from its natural promoter.”
Constructing both the ZFN and donor template DNAs on adeno-associated viral vectors, they infected the mice via intraperitoneal injection. Ten weeks later, the researchers could detect corrected human factor IX alleles in the mouse liver at frequencies from 1% to 3%, as well as circulating factor IX protein the mouse plasma.
“The efficiencies are amazing,” says Gregory. For homologous recombination in vitro, single-digit frequencies “would be astonishing,” he explains. “To do this in the context of a living organ is why that paper was published in Nature.”
Others have adopted other strategies for ZFNs. Rudolf Jaenisch of the Whitehead Institute for Biomedical Research and the Massachusetts Institute of Technology showed they could be used with small single-stranded oligonucleotide templates (rather than double-stranded DNA), an advance that could simplify the creation of template DNAs.  Jaenisch’s team used the oligos and a GFP reporter to introduce mutations in hESCs for the study of Parkinson’s disease, without selectable markers.  The process was perhaps inefficient – just 5 of 720 tested GFP-positive clones were positive – but it works. (A selectable marker “makes life easier, but it’s unnecessary,” says Gregory.)
Johns Hopkins University School of Medicine researcher Linzhao Cheng used ZFNs to correct a gp91phox deficiency in iPS cells derived from patients with X-linked chronic granulomatous disease. Instead of correcting the mutant locus itself, Cheng’s team, led by research associate Jizhong Zou, used ZFNs to introduce an additional, functional copy in a genomic “safe harbor” locus called AAVS1. That single copy provided sufficient gp91phox activity to restore reactive oxygen species generation to iPS-derived neutrophils in vitro. 
Still others have applied ZFNs to adult stem cells. Christian Beausejour of the University of Montreal, and colleagues have used ZFNs in mesenchymal stem cells,  for instance, while an ongoing Phase 1 clinical trial uses ZFNs to target the CCR5 gene — an HIV-1 receptor — in CD4+ T cells.
OTHER WAYS TO EDIT A GENOME
Of course, there are other ways to edit a genome. One related technique involves transcription activator-like effector nucleases (TALENs), another class of engineered nucleases (commercialized by Cellectis) that also induces double-strand breaks.  ZFNs are assembled from peptide modules, each of which encodes a three-base segment of the enzyme’s recognition sequence. TALENs, in contrast, use one-base modules, theoretically providing greater flexibility.
In one recent study, Jaenisch and colleagues developed TALENs for five loci in ESC and iPSC clones, which they previously had targeted with ZFNs. The team observed comparable efficiencies between the two approaches. “As this approach couples a simple DNA recognition code with robust activity in human pluripotent stem cells, our data suggest that TALENs are a useful tool for investigator-specified targeting and genetic modification in human pluripotent cells,” the authors wrote.  In another study, a group of French researchers, with colleagues at Sangamo, demonstrated that injection of one-celled rat embryos with TALEN mRNAs produced modification in 51 of 88 pups, a quarter (13) of which were edited on both alleles. 
Yet still unresolved, notes Zou, are TALENs’ genomic specificity and genotoxicity, issues that must be addressed if TALENs are to be used clinically. “After almost 10 years of using ZFNs people [are starting] to pay more attention to specificity or genotoxicity,” he says. “The same principle should be used to investigate TALENs as well.”
Another genome editing strategy is homologous recombination itself, without the enabling nucleases. Juan Carlos Izpisua Belmonte of the Salk Institute for Biological Studies, for instance, used “helper-dependent adenoviral vector”-mediated homologous recombination to edit the lamin A gene in iPS cells from individuals with a form of progeria. 
According to Sancho-Martinez, the system’s dual positive and negative selection strategy makes a relatively inefficient molecular process fairly efficient in terms of correctly targeted clones (on the order of 70% to 90%, he says). But its best feature, he says, is in the size of the molecular “cargo” it can handle.
HDAdVs can deliver up to 37 kb, he says, thereby increasing the chances for “efficient targeting,” not to mention enabling the design of “generic” repair vectors — that is, vectors that can be applied to a range of different mutations. Some 400 mutations have been described in the lamin A gene to date, for instance, 250 or so in exons 2 through 12. Belmonte and his team designed a targeting vector that could fix any of these mutations at once; by comparison, Sancho-Martinez says, “If you would use the zinc-finger approach, you might need to design specific pairs for every single mutation if those are not present in close proximity.”
EDITING WRIT LARGE
Whether using nucleases or not, all the approaches outlined above have one thing in common: They can only repair or edit a single genomic locus at a time. For those interested in more extensive genomic rewrites, Harvard geneticist George Church has developed a pair of techniques dubbed MAGE (multiplex automated genome engineering) and CAGE (hierarchical conjugative assembly genome engineering).
MAGE is an approach in which a series of up to 10 loci can be modified simultaneously in bacteria in vivo; CAGE allows MAGE-modified genomes to be joined into a single unit. Using the two approaches, Church’s team has replaced all 314 TAG stop codons in the E. coli genome with TAA. 
Yet the approach, he says, is not limited to microorganisms. Using the two methods, researchers could create large genomic segments in bacterial artificial chromosomes and then transfer them to eukaryotic cells via homologous recombination and/or zinc finger/TAL recombinases and deaminases, technologies Church is developing to produce more accurate editing. “The nuclease makes double-strand breaks,” he explains, the typical result of which is NHEJ. “So it’s the race between repairing your double-strand break correctly or incorrectly,” he says, a race he says he now is side-stepping.
Now, as part of a large NHGRI-funded “Center of Excellence,” Church and his colleagues are editing iPS cell lines derived from his Personal Genome Project in a bid to study, en masse, the effects of genetic variation on phenotype.
For researchers intent on tweaking genomes in their own labs, such tools could help finally unlock the vast potential of stem cell science.
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