Gene Editing goes Live again

Gene editing is the new kid on the block in the fight against disease. This technology holds the potential to cure genetic diseases such as cystic fibrosis, Type I diabetes and Huntington’s. Although there are several gene editing technologies, the current favourite is CRISPR-Cas9. This was used in a Chinese patient for the first time in 2016 to treat lung cancer. One year on, and another gene editing solution gets its time in the spotlight. ZFN (or zinc finger nuclease) is currently being used in California to treat a patient with Hunter syndrome.  But how does this one work?

Unsurprisingly, not too differently. In fact, ZFNs precede their CRISPR gene editing counterparts in the laboratory by several years.

ZFNExperiments over 25 years ago identified that cuts made to the double stranded DNA stimulated the cell’s own repair mechanism (known as homologous recombination (HR)). Separate experiments at about the same time also identified a pair of bacterial scissors (Fok1) that could make those cuts.

Ubiquitous Zinc fingers occur naturally in all but bacterial cells. They grasp and hold on to several specific Dinky Amigos in the DNA strand. The zinc fingers can be engineered to target specific sequences. The Fok1 scissors are not fussy about which bits of DNA they cut. Fusing them to the zinc fingers means that they will cut at any place they are guided to. So a specific sequence of Dinky Amigos can be targeted and a break introduced across both strands of the DNA double helix at a mutation site. Once the break is complete, the HR repair mechanism then leaps into action, potentially increasing specificity for the target sequence by up to 10,000 fold and fusing the ends of the break back together again.

All is not entirely plain sailing, however. The cutting mechanism can get a bit carried away. If this happens,  too many areas of the DNA may be chopped and overwhelm the repair mechanism. To combat this, the zinc finger nucleases are used in pairs (dimers) which increase target specificity and thus the safety of the cell.

So to recap. Four zinc fingers (a pair of ZFNs with two fingers on each) are taught to grasp on to a specific DNA sequence – two on each side of the double helix. They break apart the Dinky Amigos’ line up on both strands at that specific place.  The cell HR then rushes in and beautifully fuses the Dinky Amigos back together again. All done and dusted!ZFN donor

But hang on – if the ends are fused together again, what was the point in the break in the first place? Moreover, in the dash to repair the break,  HR may be elbowed out of the way in favour of another mechanism – non homologous end joining (or NHEJ).  This is more likely to result in disruption at the break site  – potentially causing even more damage than the original mutation.

Enter a separate sequence of Dinky Amigos known as donor DNA. Not only does this act as a template which the cell mechanism can copy, it encourages the cell to favour the less damaging HR repair mechanism.  This introduces a repaired section of DNA into the strand. The disruptive Dinky Amigos are removed and replaced with well-behaved ones which restore correct function.

Billions of copies of the template strand and ZFN target scissors must be used to alter variant DNA in a patient. These repair kits are injected into the body in virus-like packages which will transport them into the cells. In this case, the microscopic ZFN scissors are destined for the hepatic (or liver) cells and designed to help manage the disease from the inside.

More than a decade after this system was first tested on human cells outside the body, the technology is ready to be trialled on cells inside a patient. The gene editing company Sangamo Therapeutics put out a call for patients to enter a clinical trial in the first half of the year and now one of those respondents will receive his therapy. Results on its efficacy should be available in just a few months. A positive result should add to the medical arsenal designed to combat diseases for which we currently have no cure.

 

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