by Nessa Carey
2. Helena Devlin. ‘Scientists on brink of overcoming livestock diseases through gene editing’. The Guardian (17 March 2018).
3. Gao, Y., Wu, H., Wang, Y., Liu, X., Chen, L., Li, Q., Cui, C., Liu, X., Zhang, J., Zhang, Y. ‘Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects’. Genome Biol. (1 February 2017); 18(1): 13.
4. Zheng, Q., Lin, J., Huang, J., Zhang, H., Zhang, R., Zhang, X., Cao, C., Hambly, C., Qin, G., Yao, J., Song, R., Jia, Q., Wang, X., Li, Y., Zhang, N., Piao, Z., Ye, R., Speakman, J.R., Wang, H., Zhou, Q., Wang, Y., Jin, W., Zhao, J. ‘Reconstitution of UCP1 using CRISPR/Cas9 in the white adipose tissue of pigs decreases fat deposition and improves thermogenic capacity’. Proc. Natl. Acad. Sci. USA (7 November 2017); 114(45): E9474–E9482.
5. For a really useful review, see Lamas-Toranzo, I., Guerrero-Sánchez, J., Miralles-Bover, H., Alegre-Cid, G., Pericuesta, E., Bermejo-Álvarez, P. ‘CRISPR is knocking on barn door’. Reprod. Domest. Anim. (October 2017); 52, Suppl 4: 39–47.
6. Lv, Q., Yuan, L., Deng, J., Chen, M., Wang, Y., Zeng, J., Li, Z., Lai, L. ‘Efficient Generation of Myostatin Gene Mutated Rabbit by CRISPR/Cas9’. Sci. Rep. (26 April 2016); 6: 25029.
7. Crispo, M., Mulet, A.P., Tesson, L., Barrera, N., Cuadro, F., dos Santos-Neto, P.C., Nguyen, T.H., Crénéguy, A., Brusselle, L., Anegón, I., Menchaca, A. ‘Efficient Generation of Myostatin Knock-Out Sheep Using CRISPR/Cas9 Technology and Microinjection into Zygotes’. PLoS One (25 August 2015); 10(8): e0136690.
8. Wang, X., Yu, H., Lei, A., Zhou, J., Zeng, W., Zhu, H., Dong, Z., Niu, Y., Shi, B., Cai, B., Liu, J., Huang, S., Yan, H., Zhao, X., Zhou, G., He, X., Chen, X., Yang, Y., Jiang, Y., Shi, L., Tian, X., Wang, Y., Ma, B., Huang, X., Qu, L., Chen, Y. ‘Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system’. Sci. Rep. (10 September 2015); 5: 13878.
9. Marc Heller. ‘US agencies clash over who should regulate genetically engineered livestock’. E&E News (19 April 2018).
10. Lev, E. ‘Traditional healing with animals (zootherapy): medieval to present-day Levantine practice’. J. Ethnopharmacol (2003); 85: 107–118.
11. https://www.grandviewresearch.com/press-release/global-biologics-market
12. https://www.cjd.ed.ac.uk/sites/default/files/cjdq72.pdf
13. https://www.haea.org/HAEdisease.php
14. https://www.ruconest.com/about-ruconest/
15. Oishi, I., Yoshii, K., Miyahara, D., Tagami, T. ‘Efficient production of human interferon beta in the white of eggs from ovalbumin gene-targeted hens’. Sci. Rep. (5 July 2018); 8(1).
16. https://www.hra.nhs.uk/planning-and-improving-research/application-summaries/research-summaries/resource-use-associated-with-managing-lysosomal-acid-lipase-deficiency/
17. https://unos.org/data/
18. Yang, L., Güell, M., Niu, D., George, H., Lesha, E., Grishin, D., Aach, J., Shrock, E., Xu, W., Poci, J., Cortazio, R., Wilkinson, R.A., Fishman, J.A., Church, G. ‘Genome-wide inactivation of porcine endogenous retroviruses (PERVs)’. Science (27 November 2015); 350(6264): 1101–1104.
19. Niu, D., Wei, H.J., Lin, L., George, H., Wang, T., Lee, I.H., Zhao, H.Y., Wang, Y., Kan, Y., Shrock, E., Lesha, E., Wang, G., Luo, Y., Qing, Y., Jiao, D., Zhao, H., Zhou, X., Wang, S., Wei, H., Güell, M., Church, G.M., Yang, L. ‘Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas9’. Science (22 September 2017); 357(6357): 1303–1307.
20. http://www.frontlinegenomics.com/news/19625/pig-organs-future-transplants/
5
GENE EDITING OURSELVES
Humans are animals. This is not a value judgment, it’s just a biological fact. We already know that gene editing works in lots of animals from salmon to sheep, and from chickens to cattle. There is every reason to assume it will also work in humans. We already know that it’s successful in human cells in the lab. The next step is to find out if we can get the technique to work in living human beings.
Trying new techniques in humans usually follows a well-worn path, prescribed by technological, medical and ethical specialists. There are regulations to follow, permissions to obtain, monitoring processes to establish. You trial the process in cells, then in other animal species, then finally, after years of cautious, logical, sequential experiments and reviews, you and your team try the technology in an actual human being.
Or you can just call yourself a ‘biohacker’ and skip all that and experiment on yourself. Yes, really. Because the raw materials for gene editing are so cheap and easy to obtain, it’s alarmingly straightforward to generate the molecular reagents to try this at home. You literally can inject yourself with gene editing materials and absolutely no one can do anything about it.
Josiah Zayner is the first person we know who claims to have done this. Delightfully, he looks like everyone’s idea of a biohacker – runs a start-up tech business from a garage, wears unusual T-shirts, very much Not Working For The Man. Zayner has big ideas for increasing public access to gene editing. In his own words, ‘I want to live in a world where people get drunk and instead of giving themselves tattoos, they’re like, “I’m drunk, I’m going to CRISPR myself”.’*1
You might think it would be better to live in a world where drunk people are barred from tattoo parlours instead of having ever wider access to bad choices, but there’s always going to be a diversity of opinion.
Zayner certainly seems to have been willing to back up his own words. At a conference in October 2017 he injected himself in the arm with a gene editing preparation. This was designed to stimulate muscle growth. In fact, he was using gene editing to inhibit the myostatin gene, the same approach that’s already been successfully trialled for creating highly muscled sheep and rabbits.
In some ways Zayner was following in a long tradition of medical self-experimentation. Pierre Curie taped a packet of radium salts to his arm to demonstrate that radiation causes burns. Barry Marshall deliberately swallowed bacteria in order to test his hypothesis that stomach ulcers are caused by Helicobacter pylori infections (he was right, poor chap).
One feature that is quite noticeable from this history of medical self-experimentation is that the person involved frequently suffered harm as a result of the procedure. This is often one of the drivers for self-experimentation. The individual would probably never get ethical approval to perform the experiment on someone else, or their own ethical compass made it unacceptable to them to do so.
The data from animal studies suggest that gene editing is a safe procedure but this doesn’t mean there were no risks to Josiah Zayner when he performed his self-experimentation. The risks were less likely to be from the gene editing per se and more likely to arise from an immune response to the reagents, or an infection from a lack of sterility when they were prepared.
Happily, our human guinea pig didn’t suffer any adverse effects. But he didn’t get bigger muscles either. So, what does that tell us about the efficacy of gene editing in humans?
The answer is simple. It tells us absolutely nothing at all. We’ve no idea what was in the syringe that Zayner used to inject himself. There’s no reason to think it wasn’t gene editing reagents but we have no clue about the dose, whether they had been properly prepared, or a whole heap of other factors that could affect the likelihood of this experiment working. It was a great profile-raiser, but lifting weights rather than syringes remains the best way for the average human to increase their muscle mass right now.
Aiming for success
The only way we will be absolutely confident that gene editing works in humans, is safe, and can make a physiological difference to us, is if we run proper trials. These trials will require lots of high-quality manufacturing, oversight, monitoring, standardisation, long-term follow-up and enough subjects to generate statistically significant data and confidence in the outcomes. That’s going to be expensive, probably costing a minimum of tens of millions of dollars. Philanthropic donors are unlikely to put the money forward, mainly because there are le
ss risky and more immediate ways of improving human health and well-being if you have that kind of money to spend. Sewage systems, vaccination, mosquito nets and nutritional supplementation spring to mind. So that really only leaves the private sector. And the private sector will only make this investment if it believes it will ultimately generate profits. The most attractive way of doing this is to use gene editing to create new ways of treating serious illnesses.
Naturally if you are going to spend tens, or possibly hundreds, of millions of dollars trying to get a gene editing approach all the way to a registered product, you want to select a disease where you can be reasonably confident of success. There is a whole list of key factors. Can you be 100% certain that the patients you have diagnosed with the condition all have the same disease? This rules out disorders like schizophrenia where there are probably many different forms of the illness. Do you know exactly how the disease is caused in your patients? This rules out type 2 diabetes where it isn’t clear which is the key step in the development of the condition. Do you know what genetic change you need to create? This rules out multiple sclerosis, where we think multiple minor genetic variations interact with the environment to trigger the condition. Can you be sure that making the specific edit you have in mind will prevent or reverse pathology? This rules out Alzheimer’s disease. Drug trials targeting what we thought was the key pathway failed spectacularly recently,2 and the companies involved have probably lost billions of dollars as a consequence. Can you get the gene editing reagents to the tissues where they are most needed, in high enough levels? This probably excludes Parkinson’s disease, as the brain is quite a difficult tissue to access. Will the edited cells remain alive for long in the body and ideally also pass on their edited DNA to their daughter cells? This is important if you want to limit the number of times you need to give treatment. This may make it difficult to use this approach to target conditions such as muscle wasting in the elderly, where the muscles have used up all their regenerative capacity.
In fact, many of the most common and debilitating conditions aren’t likely to be good candidates for gene editing any time soon, because they are too challenging in one or more of these problem areas. When we think of the complexity of the issues, we might wonder if there are any conditions that do fit these criteria. And even if they do, will there be enough patients to make gene editing economically viable?
The answer is – almost astonishingly – yes to both these questions. Perhaps fittingly, given that gene editing developed from an arms race between bacteria and viruses, the diseases that will initially be tackled by this technology developed as part of an arms race between humans and a parasite.
Better blood
Red blood cells are vital in virtually all vertebrates. One of their major functions is to transport oxygen to where it’s needed and to carry carbon dioxide away from tissues before this gas reaches dangerous levels. The gas molecules bind to a pigment in red blood cells called haemoglobin, which gives the cells their colour. This pigment is made of four protein chains, of two different types, all associating together. In adult humans, two of the chains are called alpha and two are called beta. The red blood cells are absolutely stuffed full of haemoglobin.
In the genetic condition sickle cell disease, patients have mutations in the gene that codes for the beta chain of haemoglobin. The mutation is inherited from both the mother and the father, so the patients don’t have a normal gene for this protein. In patients with sickle cell disease, the haemoglobin protein folds up incorrectly, and distorts the whole shape of the red blood cell. This makes it harder for the red blood cells to travel through the smallest blood vessels, and they get stuck, leading to extreme pain. The red blood cells are also less efficient at carrying oxygen around the body, so the patient becomes breathless.
There is another set of conditions called thalassemias. In these disorders, the patients produce lower than normal amounts of either the alpha chain or the beta chain of haemoglobin. This makes the red blood cells more fragile and they don’t last very long. The patients develop anaemia (lack of red blood cells) and are breathless and tired. Just as in sickle cell disease, patients with thalassemias inherit mutations from both their parents.
Both conditions are surprisingly common. Astonishingly, about 1.1% of couples worldwide are at risk of having a child with a haemoglobin disorder.3 There are far higher numbers of people who have one mutant haemoglobin gene (carriers) than we would expect from normal genetic distribution. However, this is a localised effect, seen in some regions of the world and not in others. In the early 1950s a research group working in Kenya realised that a mutant haemoglobin gene was found much more frequently in areas where malaria was endemic than in areas where there was little risk of the disease. They went on to demonstrate that red blood cells from carriers of the haemoglobin mutation were much more resistant to infection with malaria than red blood cells with normal haemoglobin.4 This association was originally shown for the sickle cell mutation and was later shown to hold true for the thalassemia mutations, which also had a high carrier frequency in regions where malaria was common.
Although there are clear disadvantages if both of your copies of a haemoglobin gene are mutated – no one would want to have full-blown symptoms of sickle cell disease or thalassemia – these were outweighed genetically by the benefits of having one mutant copy. This advantage maintained the high levels of the carriers in the relevant geographical regions, as they won the arms race against the malaria parasite.
There are a number of features that make these haemoglobin disorders perfect first targets for therapies built around gene editing. They can be diagnosed with 100% certainty, and we can easily work out exactly which genetic change to target for a patient. In sufferers, both copies of the gene are mutated. In carriers, one copy is normal and one is mutated, and the carriers are healthy. So we know that in sufferers, converting one of their mutant copies to the normal one should be enough to restore them to a healthy condition, on par with the carriers. Although healthy red blood cells only last about 120 days in the body, we should still be able to treat with gene editing using only a small number of interventions. This is because we can extract stem cells from bone marrow, edit the DNA and then re-seed the bone marrow with the corrected cells. Once they re-establish in the bone marrow, the stem cells should continue to produce healthy red blood cells for decades.
There are also enough patients to make this economically worthwhile. Although the haemoglobin disorders evolved where malaria was rife – which is usually in poor regions – global migration means that the conditions are also fairly common in countries with well established healthcare infrastructure. About 100,000 Americans have full-blown sickle cell disease5 and the number in the European Union is in the region of 127,000.6 Crucially, there are no really effective therapies for these conditions.
The first approach being trialled is an intriguing one, driven by an unusual phenomenon that was observed in some patients. Clinicians have long known that there are some people who should be very ill with sickle cell disease or one of the thalassemias, but who seem remarkably healthy. Genetic analyses showed that the patients had inherited mutations from both their parents, and yet somehow they were fine.
Detailed genetic studies showed that these anomalous people were protected from the effects of the disease-causing mutation because they actually had another mutation as well. This may sound odd, as the word ‘mutation’ is often loaded with negative connotations, but it actually just refers to a change in DNA sequence. A mutation may have no effect, a negative one, or even a positive outcome for an individual.
Adults produce a form of haemoglobin known, unsurprisingly, as adult haemoglobin. But when a foetus is developing in the uterus it expresses a different form, called (with shocking originality) foetal haemoglobin. This is because oxygen levels in the uterine environment are different from those in the outside world. The foetus and adult produce different kinds of haemoglobin to ensure they are best suite
d to the environment. Foetal haemoglobin and adult haemoglobin are encoded by different genes.
After we are born, expression of the foetal haemoglobin genes gets dialled back and expression of the adult genes ramps up. After a few months, all the haemoglobin in the red blood cells is produced from the adult genes. But occasionally, there is a mutation in the control region for the foetal haemoglobin which means it isn’t switched off. Adults with this mutation keep producing foetal haemoglobin, but happily this doesn’t seem to do any harm.
The patients who should have had the symptoms of thalassemia or sickle cell disease, but who were fine, had all inherited the mutation in the control region of the foetal haemoglobin in addition to the disease-causing mutation in their adult haemoglobin gene. Their continued production of the foetal form of the protein protected them from the worst effects of the disease.
The gene editing company CRISPR Therapeutics has taken advantage of this clinical knowledge. Their strategy is to take bone marrow cells from patients with a haemoglobin disorder and edit the DNA in the lab, so that the resulting stem cells contain the protective mutation sometimes found so fortuitously in nature. They will then repopulate the patient’s bone marrow with these edited stem cells. The stem cells will produce red blood cells which express foetal haemoglobin, protecting the patient.
You might wonder why the company has chosen to take this approach, rather than correcting the disease-causing mutation in the adult haemoglobin gene. The reason is because their preferred strategy should work for any patient, no matter which mutation is causing their disease. This means they can create a standard gene editing protocol that works on all patients, rather than having to design reagents and procedures for each individual. This drives down costs and also makes the clinical trials easier to standardise and interpret.