Scientists correct the typo behind a genetic liver disease

By Ed Yong | October 12, 2011 4:00 pm

Every year, thousands of people die because of typos in their genes. There’s a long list of debilitating or fatal genetic diseases that are caused by a single incorrect DNA letter among the three billion in our genome. It’s the equivalent of pulping an entire encyclopaedia on the basis of a single typo. But hope is at hand. We are fast approaching the point when we can proofread these errors out of our genes.

Kosuke Yusa  and Tamir Rashid have taken the latest step towards this goal, by developing a more efficient and less risky way of correcting genetic errors. They took cells from patients with a genetic liver disease, edited the gene responsible, and grew corrected liver cells that successfully treated mice with the same disease.

The disease in question is called alpha 1-antitrypsin deficiency (or alpha-1 for short). It affects 1 in every 2,000 Europeans and is caused by a single typo in the A1AT gene. The error stops people from making enough of the A1AT protein. The little they do make builds up in their liver, overloading it and leading to cirrhosis. At the moment, the only treatment is a liver transplant, and donors are hard to come by.

Yusa and Rashid fixed this problem in three steps: they took skin cells from alpha-1 patients with two bad copies of the A1AT gene; reprogrammed them into stem cells; fixed the error in both copies of the broken gene; and used the corrected stem cells to produce liver cells. This is the first time that anyone has corrected a faulty gene in stem cells derived from a human patient.

The first step relies on methods that were first developed in 2006. In August of that year, Shinya Yamanaka from Kyoto University found a way of reprogramming adult cells so they could create any type of tissue in the body. He could imbue adult cells, which are normally fixed in specific roles, with the limitless potential of stem cells. Since then, research on these “induced pluripotent stem cells” or iPSCs, has raced along at blistering speed. Scientists quickly realised that they could use iPSCs to create an unlimited supply of tissues and organs, tailored to a person’s own genome. They could even fix errors in the reprogrammed cells to create working tissues in people who were suffering from genetic disorders.

A few groups have successfully done this in mice. In 2007, Rudolf Jaenisch used the iPSCs to cure mice of a genetic disorder called sickle cell anaemia, caused by deformed blood cells. Jaenisch made iPSCs from skin cells in the rodents’ tails, corrected the faulty gene that was behind their disease, and injected the cells back into the mice. Just four weeks later, the mice started producing normal blood cells. In 2009, Yupo Ma used the same methods to treat a second genetic disease called haemophilia A, again in mice.

But there are two big problems with these methods. First, they’re not very efficient. If you start off with a large batch of iPSCs with the same faulty gene, you only ever correct a small proportion of them. To single them out, scientists rig the editing process so the corrected cells also gain a marker – say, a gene that makes them resistant to a particular antibiotic. That makes them easy to identify and isolate. Once this is done, the marker can be cut out. That leads to the second problem: the markers tend to leave small bits of DNA behind. In fixing one error, you could introduce several more. At worst, the DNA remnants could disrupt important genes and cause cancers.

Yusa and Rashid solved both of these problems. First, they designed a scissor-like protein called a ‘zinc finger nuclease’ to specifically cut the A1AT gene at its incorrect letter. By precisely targeting the typo, the team could efficiently replace it with the right letter.

Second, they created a marker that can be seamlessly removed. They relied on a jumping gene called piggyBac, which can cut itself out of its surrounding DNA and paste itself back into a different spot. It does so flawlessly; once it jumps out of a piece of DNA, you’d never be able to tell that it was once there. By loading their marker gene into piggyBac, Yusa and Rashid could yank it out once they had identified the corrected cells, without leaving any traces behind or disrupting any genes.

The duo used their corrected iPSCs to grow new liver cells, which produced normal working versions of the A1AT protein. They transplanted these cells into mice with the same faulty gene. Two weeks later, the cells had colonised the rodents’ livers, they were behaving normally, and they hadn’t produced any tumours.

That’s reassuring because even with their new technique, the results weren’t entirely faultless. Yusa and Rashid found that the liver cells had gained 29 new mutations since their days as skin cells. Most of these happened when they were reprogrammed back into stem cells. One was caused by the zinc finger nuclease, and three were due to piggyBac. This isn’t necessarily a problem; cells naturally acquire fresh mutations over time and most are of no consequence. Still, the mutations add a note of caution to an otherwise optimistic study.

The next step will obviously be to test the final transplant stage with human patients. The team want to take things slowly, testing the technique in degrees to make sure that it’s safe for patients. This will probably take several years but they are keen that the hype doesn’t outrun the pace of their work.

For people with alpha 1-antitrypsin deficiency, the technique could solve the shortage of liver transplants. You don’t need a donor when you can just grow a new liver from your own corrected stem cells. Even better, that liver would be genetically identical to the rest of your body, so you wouldn’t have to worry about rejection or immune problems.

The liver is a good place to start when trying to prove that this technique can work, because it regenerates very well naturally. Other organs might be trickier; new neurons, for example, would need to connect with the existing network. Even so, Yusa and Rashid’s study has implications for other genetic diseases. The same techniques could be used to correct other genetic faults – using the zinc finger nuclease to make things more efficient, and piggyBac to avoid disrupting the surrounding genes.

Reference: Yusa, Rashid, Marchand, Varela, Liu, Paschon, Miranda, Ordo, Hannan, Rouhani, Darche, Alexander, Marciniak, Fusaki, Hasegawa, Holmes, di Santo, Lomas, Bradley & Vallier. 2011. Targeted gene correction of a1-antitrypsin deficiency in induced pluripotent stem cells. Nature

Image by Sirsnapsalot

More on regenerative medicine:

CATEGORIZED UNDER: Medicine & health, Stem cells

Comments (8)

  1. Jeff

    This passage contains an error: “Yusa and Rashid found that the liver cells had gained 29 new mutations since their days as skin cells . . . One was caused by the zinc finger nuclease, and three were due to piggyBac.”

    The paper disagrees with the cause of the mutations that occurred during targeting and excision:

    “Twenty-four point mutations and one 1-bp deletion were detected in the primary
    iPSC line and four mutations arose during genetic correction: one
    during targeting and three during piggyBac excision. These mutations
    seemed to arise during culture as their genomic signatures were inconsistent
    with ZFN off-target sites or piggyBac integration sites
    (Supplementary Analysis).”

    [Good spot, Jeff. I’ve corrected the passage. – Ed]

  2. Eleanor

    ‘piggyBac’ – genius.

    Quick q.: if it affects 1 in 2000 of Europeans, they can’t all end up needing a liver transplant? Is it only if you end up with two faulty copies that it can become dangerous?

    Ta, excellent read as always.

  3. Jenn

    Eleanor… alpha 1 is fatal in many cases…. And in many cases it isn’t diagnosed properly… Do a google search for alpha 1 foundation… I’m on my phone so it isn’t easy to share links… In the usa it’s one of the leading causes of infant liver failure… It also causes copd in adults…it’s a fascinating disease…

  4. Just how frikkin’ clever is modern science? It’s easy to get blasé about our technological prowess, but this kind of stuff is really pretty mind-blowing.

  5. Eleanor, that 1 in 2000 figure is already for people with two faulty copies of the gene. A good deal more people than that are carriers for the mutation (depending on which country you’re in).

    But to answer your question, no they don’t all end up needing a liver transplant. Most people have COPD (like emphysema) as their most severe clinical symptom. The beauty of correcting the gene in liver cells is that this would most likely prevent the COPD as well.

  6. Vern

    This is great news. As an Alpha1 deficient male I have developed emphseyma. Thus far my liver is okay but if I was able to develop the correct transponders within my DNA, I would be able to maintain my lung function. In SK, prolastin injections treatments are not covered so it is to expensive. Currently on the list for a potential clinical trial so I will watch this story closely.

  7. Sue

    This is absolutely fascinating and very hopeful! I’m a lung affected “Alpha” who currently is on weekly IV therapy to replace the A1AT protein that I am deficient in. This augmentation therapy only helps prevents further damage to the lungs and will not repair the damage that has already taken place. Of course there are exceptions to the norm, but generally liver affected Alphas present with problems at birth or a very young age. Those who are lung affected generally do not present until approximately 35 years of age. Those who are not diagnosed (only 10% of the Alphas in the US are thought to be correctly diagnosed) have damage to the lung which slowly and silently occurs. By the time an Alpha starts noticing breathing problems and is properly diagnosed, they’ve already lost lung function that can never be regained, making transplant a very real possibility. Imagine the implications of identifying those with Alpha-1 at birth and correcting the mutation then!

  8. Jaime

    I really hope this becomes a reality for Alphas soon. I am a liver affected ZZ with cirrhosis, and I am not ready to accept having a transplant or die as my options in this life.


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