Features

OxSciBlog has been building up to the 10th anniversary of the first draft human genome on Saturday with a series of articles looking at how different areas of research have been affected by knowing our DNA code.

We talk to Dr Deborah Gill of the Nuffield Department of Clinical Laboratory Sciences who, with Dr Steve Hyde, runs a group hoping to develop a gene therapy for cystic fibrosis.

Cystic fibrosis [CF] affects over 8,500 people in the UK and is the most common life-threatening single-gene disorder, says Deborah Gill. It occurs when two copies of a faulty gene are inherited together from a child’s parents. Around 1 in 25 people carry a faulty copy of the CF gene, and if two carriers have a child there’s a 1 in 4 chance of the child having cystic fibrosis.

The loss of this one gene affects many of the internal organs, particularly the lungs and gut where mucus begins to build up. Children tend to be diagnosed with the condition when they fail to thrive as normal and it becomes clear they are having difficulty absorbing food from the gut. Gradually, the patient’s lungs will become clogged with mucus and despite daily physiotherapy, the lungs become progressively more damaged.

‘There is no cure for cystic fibrosis,’ says Deborah Gill. ‘In addition to medication and nutritional supplements, the lung problems are managed using daily physiotherapy and antibiotics to both treat and avoid infections. Half of patients with cystic fibrosis currently live into their thirties.’

While those born today with cystic fibrosis are likely to live longer thanks to improvements in treatment, the number also hides a lot of teenage deaths from the condition, she explains.

Breathing it in
‘When the gene for cystic fibrosis was discovered in 1989, very quickly people began to think about gene therapy as a prospect,’ says Deborah. The aim was to replace the faulty or malfunctioning gene with a working copy wherever it is needed in the body. ‘Since then we’ve come up against every problem there is in gene therapy. It’s the delivery of the gene that’s the tricky bit.’

Although a functioning CF gene is absent throughout the body in cystic fibrosis patients, it is in the lungs where it is a real problem. ‘The lung is our focus for gene therapy as it is the cause of premature death in cystic fibrosis,’ explains Deborah. ‘The lung is essentially open to the atmosphere, so you should be able to simply breathe in a gene therapy. We want to capitalise on the existence of nebulisers for diseases like asthma and use this as a way of introducing a functioning version of the gene in an aerosol to patients.'

‘It’s a golden opportunity to get the gene in an inhaler and allow patients to just breathe it in,’ she says.

Gene therapies that have been investigated have largely fallen into two camps: those that have used types of viruses to get the genetic material into the cells where it is wanted and those that have used capsules made up of fat molecules or lipids.

Any gene therapy wanting to reach the cells in the lung lining will need repeated doses as these cells get renewed and replaced, but methods using current viruses are poor for continuing to get gene expression dose after dose.

Deborah Gill’s group uses lipid particles for their aerosol gene delivery approach. The lipids encase a circle of DNA called a plasmid. The plasmid includes a full version of the CF gene that isn’t functioning in cystic fibrosis patients. They use an aerosol droplet size that gets the gene where it is needed in the airways of the lungs.

Engineering for expression
While other groups have tinkered with the lipid capsules to try and get better results from gene therapy, Deborah Gill and colleagues have worked on engineering the plasmid DNA itself.

‘It’s possible to manipulate plasmid DNA in quite specific and sophisticated ways. It’s a method which is just beginning to be exploited,’ says Deborah. ‘We’ve been able to design the plasmids specifically to get long-term expression of the gene in the lungs.’

Expression describes the process where proteins are made from a gene. The CF gene delivered on the plasmid needs to be expressed continually if the gene therapy is to help relieve chronic lung symptoms in cystic fibrosis patients.

In the beginning, to control expression of the CF gene in their plasmid, the group used a standard DNA sequence, or promoter, taken from a virus. This is an approach that’s been used many times in gene therapy, and it works. You get large amounts of protein made from the CF gene – just not for very long. It turns out that the human body is more subtle than that.

The group found that if you use a general-purpose human promoter sequence in your plasmid instead, you get less CF protein made, but that production is sustained over much longer time periods. It fits much better with the body’s complex control systems that govern gene expression.

The group has also shown that the exact DNA sequence of the plasmid is important too, and needs to be carefully designed at all points. Human DNA tends to have a chemical group – a methyl group – added to the DNA everywhere a ‘C’ in the DNA code appears next to a ‘G’. An un-methylated CG group tends to be recognised as DNA from a virus or bacteria and destroyed. The plasmid DNA in gene therapy is un-methylated, but needs to hang around.

By carefully making sure their plasmid is free of all ‘CG’s in its code, the group has made sure they get expression of the CF gene for longer in the lungs and with less inflammation.

It’s through careful manipulations like this, that the group have fine-tuned their gene therapy approach for cystic fibrosis.

They have now begun clinical trials of their aerosol gene therapy in cystic fibrosis patients. These first studies in a handful of patients are designed to check for safety and to get the dose of the gene therapy right. They are not yet looking for CF gene expression to treat the condition, which will be the next step. But Deborah says she is ‘encouraged’ by the early results.

Prospects for gene therapy
The gene for cystic fibrosis was discovered over 20 years ago, yet we still don’t have a successful gene therapy.

‘It was massively overhyped at the time,’ Deborah admits. ‘As soon as the gene was discovered, many labs started working on a therapy for cystic fibrosis. There was a lot of repetition and this perhaps did the field a disservice. It seemed to many as if gene therapy didn’t work and the new biotechnology companies that had formed couldn’t make a return on people’s investments.’

‘But everyone was trying something new,’ she adds. ‘People hadn’t done gene therapy before. When this started in the early 1990s, no one had a way of making plasmid DNA suitable to give to patients. No methods had been published and there were no companies offering DNA sequences for sale. It was OK for cloning small pieces of DNA in the lab, but not for scaling up to make DNA like a pharmaceutical.’

That’s changed. ‘We are there now,’ she explains. ‘There are companies who can manufacture DNA on a large scale. It just takes a long time.’

In her view, the human genome project added a great deal to the tools available for her work in gene therapy. Although Deborah says she doesn’t get ‘excited’ by genomics – she says it appeals to those who like scale! – she believes the masses of information genomics has produced has ‘provided all the tools to make things really easy’.

‘It has revolutionised gene therapy on a support basis,’ she says. ‘There is great potential for exploiting that information and we haven’t scratched the surface yet.’

And while gene therapy has been slow to fulfil the initial hype and excitement, Deborah sees things changing rapidly in the field.

‘The discovery of micro RNAs – a delicate system where RNA molecules modulate gene expression – gives us a subtle and universal new way to manipulate protein expression,’ she adds. ‘The RNAs act like a rheostat for gene expression – continuously turning it up and down. This has become a really big new area for gene therapy in the last 2-3 years. We are nowhere near seeing just how this will affect the field yet.'

‘But the challenge of delivering the appropriate genetic material to the right part of the body remains the same for all. Still, we are getting a wider array of targets and tools to customise gene therapy for particular diseases.’

Deborah says, ‘I would like to see a world where we have a basket of different viral and plasmid approaches for gene delivery, where we know how to deliver to any organ such as the liver, or heart, and where we can stitch all these different elements together to treat a specific disease. Gradually this is all coming together.’

Streptococcus meets Saving Private Ryan

Humans are the casualties of a bacterial war for survival, new research shows.

The story comes from our friends at the Wellcome Trust who highlight work by Sam Brown of Oxford University's Department of Zoology published in Current Biology today.

Our bodies contain a wide range of bacteria which mostly do us no harm. But now and then a bacterium will evolve properties which are potentially deadly to its human host. The big question for evolutionary theory is, 'why?', when killing off its host could mean the bacterium is killing itself.

Sam explains: 'For many microbes, living in harmony with their host is the best option, so why do some suddenly turn nasty? Sometimes the answer is obvious – for example, the cold virus makes its host sneeze, helping it spread wider. But for other bacteria and viruses, which do not normally cause disease, the reason isn't at all clear.'

The team of Oxford and US scientists modelled in mice how the bacterium Streptococcus pneumoniae interacts with other bacteria, showing that competition for space can cause deadlier forms of bacteria to evolve.

S. pneumoniae usually exists in the nasal passage, where it sits quietly: as many as two in five people in some countries will carry the bug without being aware of it. When S. pneumoniae is forced to share space with Haemophilus influenzae, another common, ordinarily asymptomatic bacterium, the two battle for space.

But H. influenzae has an extra trick up its sleeve, calling on our immune system to help get rid of its competitor by recruiting white blood cells called neutrophils, which surround and attack the S. pneumoniae bacteria.

However, calling in an 'immune strike' on its own position has some unintended consequences.

For, in scenes of microscopic carnage reminiscent of the opening of Saving Private Ryan, whilst S. pneumoniae benefits, as H. influenzae are less able to withstand the immune attacks, when a sufficient amount of H. influenzae are present, the more virulent, armoured strain of S. pneumoniae out-competes its rivals: its thick sugar coating enables it to escape attack from the neutrophils, but, unfortunately for us, this coating also makes it more deadly when it enters the human blood stream.

'Creating a new armour is costly to S. pneumoniae in terms of the energy expended to make it, but it means the bacterium wins the battle with H. influenzae,' Sam tells us.

'However, it also means that if S. pneumoniae enters the blood stream, the immune system is unable to stop its rampant progress. Our bodies are unable to cope and the armoured bug could pay the ultimate price: death to its host and death to itself.'

Dr Sam Brown is a Wellcome Trust Research Career Development Fellow based at Oxford's Department of Zoology.

Thanks to Craig Brierley at Wellcome. 

There were 451 million clinical cases of Plasmodium falciparum malaria globally in 2007, according to new research by the Malaria Atlas Project [MAP].

The new study was led by Simon Hay of Oxford University's Department of Zoology and is published today in PLoS Medicine.

This estimate of how many people become ill because of the disease (which kills about 1m people a year) is almost double the previous one provided by the World Health Organization (it estimated 247m for 2007) and highlights the difficulties of tracking a global disease.

MAP, which is mainly funded by the Wellcome Trust, set out to tackle some of the uncertainties surrounding the number of cases of malaria gobally: estimates for the disease are made particularly difficult because in places where it is endemic diagnosis is often inaccurate and national reporting of cases incomplete.

The researchers used a recently published map of modern-day malaria risk and more advanced statistical techniques that better describe uncertainty. They found that there could have been between 349-552m clinical cases of P. falciparum worldwide in 2007 and came up with a combined estimate of 451m cases.

They also discovered that more than half of the estimated malaria burden and its associated uncertainty was contributed by India, Nigeria, the Democratic Republic of Congo, and Myanmar (Burma).

'The uncertainty in our knowledge of the true malaria burden in a mere four countries, confounds our ability to assess progress in relation to international development targets at the global level. It is clear that we urgently need an increased focus on reliably enumerating the clinical burden of malaria in these nations,' Simon told us.

'The divergence in our estimates and those of the World Health Organization is greatest in Asia and acute in India. We have sought to explore on a country by country basis how these differences arise, the relative uncertainty in the alternative burden estimation approaches and the potential insights that could be gained by hybridising the two.'

Oxford University's Bob Snow, who leads the MAP group in Kenya, said: 'Our estimates for P. falciparum malaria alone are almost twice those provided by the WHO, which include both P. falciparum and P. vivax malaria.'

Bob added: 'Getting the numbers right is fundamental to reporting on success or otherwise of increased donor funding. A valid question remains about whether agencies charged with the responsibility of supporting the delivery of malaria interventions should be the same ones expected to report progress.'

They're stars that never quite made the big time: mysterious cosmic objects known as 'brown dwarfs'.

Today the team behind the CoRoT space telescope report that they have found a rare example of a brown dwarf tightly orbiting its star.

I asked CoRoT team member Suzanne Aigrain of Oxford University's Department of Physics about 'brown dwarf deserts', the gap between giant planets and stars, and what would happen if our solar system had its very own brown dwarf...

OxSciBlog: What is a brown dwarf?
Suzanne Aigrain: A brown dwarf [BD] is a celestial object intermediate in mass between a planet and a star. It's helpful to recall the definition of a star: a star is a ball of gas held together by its own gravity and which radiates light produced by thermonuclear reactions in its core, mainly burning Hydrogen to produce Helium. A brown dwarf is an object very much like a star, but which is not massive enough to burn Hydrogen in its core.

As such, brown dwarfs are faint and radiate mainly in the infrared, slowly releasing the heat they accrued during their formation. On the other hand, according to the International Astronomical Union's definition, a planet is also held by its own gravity but it is a) in orbit around a star or brown dwarf and b) not massive enough to burn Deuterium (Deuterium is an element which burns even more easily than Hydrogen). Any object which has a mass below the Hydrogen limit but above the Deuterium limit is thus a brown dwarf. This is the case for CoRoT-15b.

The definitions I have given above leave a rather fuzzy area for the case of object which are below the Deuterium burning mass limit but are not in orbit around a star or brown dwarfs - these are sometimes called sub-brown dwarfs or free-floating planets.

OSB: What is the significance of CoRoT finding a BD? Are they rare?
SA: Brown dwarfs are not rare in themselves, on the contrary. It is difficult to detect and study them, because they are faint compared to stars, so we don't know as many of them as we know stars, but over the past 20 years, with the advent of better and better infrared detectors, we have been discovering many of them.

What is extremely rare, however, is to find one in a tight orbit around a star, as in the CoRoT-15 system. Until a few years ago, we knew of none at all, and this absence was called the 'brown dwarf desert'. Now we know of a handful, but CoRoT-15b has the shortest orbital period of any known brown dwarf. The very existence of CoRoT-15b in its tight orbit is interesting (see below), but the fact that it transits across the disk of its parent star makes it even more useful, because it enables us to measure its radius.

OSB: What can they tell us about how planets & stars evolve?
SA: Systems like CoRoT-15 are very important to understand star and planet formation as well as evolution. The majority of brown dwarfs are thought to be the result of the same process which forms stars. Stars form from giant clouds of gas and dust. Regions in these clouds which are marginally more dense than their surroundings attract more material onto themselves, and these over-densities grow and grow until thermonuclear fusion ignites in the core, and a star is born.

If a clump never grows large enough for that to happen - because the material within its gravitational influence runs out - you get a BD. So, from the formation point of view, there is nothing fundamentally different between a star and a brown dwarf, but whilst tight binary stars are quite common, tight binary systems involving a star and a brown dwarf are rare.

Why is CoRoT-15b different? Did it get kicked into its current orbit by a close encounter with another star? Could it have formed like a planet, which forms in the disk of material accreting onto a star, instead?

There are very few BDs in close binaries, and even fewer which transit their parent star. These are the only BDs whose radii we can measure, so they are very valuable. If you make a plot of radius versus mass for stars and planets, stars all more or less fall on a single line, which basically is the line you expect for a self-gravitating ball of Hydrogen. This line flattens out at low masses - from 0.1 solar masses to a Jupiter mass, the expected radius is about 1 Jupiter radius.

However, the measurements for exoplanets are scattered, with a range of radii observed for a given mass. This is because the radius of a planet is affected not just by its mass, but also by its composition (how much solid versus gaseous material it contains) and by the amount of light it receives from its parent star. CoRoT-15b fills an important gap in this diagram, between low-mass stars and planets. It's also extremely close to its star, so extremely hot, and hence a particularly strong test of just how much intense irradiation can affect the radius of an object of that mass.

OSB: How might our solar system be different if it contained a BD?
SA: We know that having a binary companion does not prevent planet formation, since we know of stars which have both one or more planets and a binary companion. If the Sun had a wide BD companion, the solar system would not necessarily be very different. We would definitely know about it, however: BDs are faint compared to stars, but a BD that close to us would not be missed.

On the other hand, if the BD was very close-in like CoRoT-15b, things would be very different. I'll consider two possibilities: If the BD formed in-situ, there would be no disk, or very little of it, around the Sun, for the planets of the solar system to form out of. There might have been a disk around the binary (we have seen such disks around other binaries) and it's conceivable that this disk might form planets. We currently know of no such circum-binary planets, but this is at least in part because it is harder to detect them.

But if the BD was captured (or kicked into a close orbit from a wider one) after the Sun had formed its planets, then that would most probably have a very dramatic impact, as the gravitational influence of the inbound BD would wreak havoc on the planets and most likely eject them from the solar system!

OSB: What do we hope further CoRoT finds could reveal about BDs?
SA: CoRoT already found another transiting BD, CoRoT-3b. It is less massive and less close in than CoRoT-15b, but the fact that CoRoT found two of these very rare systems shows that it is well-suited to detecting them. Along with the NASA mission Kepler, which is also searching for transiting planets, CoRoT can hope to discover several more systems like these in the next few years. They will tell us more about how these rare systems form, about what forms the difference between a massive planet and a BD, and about how BDs evolve when very close to their host star.

Dr Suzanne Aigrain is based at Oxford University's Department of Physics. 

As England's footballers struggle to deal with the effects of altitude on their bodies ahead of their first World Cup match in South Africa, they may wonder how people living day in day out at even higher levels get by at all.

A new study published this week in PNAS reveals that natural selection has enabled humans to better cope with living in Shangri La.

Shangri La here is not the mythical Eastern earthly paradise, but the very real province high up in the Tibetan plateau where the indigenous population lives at altitudes of 3,200m to above 4,000m – much greater than the 1500m Gerrard, Lampard and the rest will be playing at on Saturday.

The international team of researchers from the UK, Ireland, China and the US searched for evidence of selection at the genetic level among people living at these high altitudes where there is so little oxygen.

The study’s corresponding author, Professor Peter Robbins of Oxford’s Department of Physiology, Anatomy and Genetics, confirms that ‘Tibetans are better equipped for life at high altitude.’

People who move to live at high altitudes tend to be at risk of a chronic mountain sickness. The body makes too many red blood cells, giving excessive levels of haemoglobin, in an attempt to capture the little oxygen there is in the air. But native Tibetans manage to remain unaffected.

So the scientists compared the genetic profiles of groups of Tibetans living at these high altitudes with nearby populations in lowland China, and they found changes in a single gene were present at high frequency among the Tibetans.

The researchers showed that having a different version of the EPAS1 gene gives Tibetan populations an advantage: it keeps levels of haemoglobin low and so protects them against developing chronic mountain sickness. ‘This is where individuals make too many red cells and the blood becomes very sticky,’ says Peter Robbins. ‘[Having the EPAS1 variant] may also help with reproductive fitness at high altitude, but that is more speculative.’

This is good evidence that the lack of oxygen at these altitudes has exerted an evolutionary selection pressure over the 10,000 years or more people have been living in these areas.

‘There are probably not more than a handful of definitive examples of human evolution to their environment at the genetic level that have so far been described,’ says Peter Robbins. ‘There are genes for variation in skin colour, certain genes that confer protection against malaria (for malaria infested environments) and possibly one or two dietary adaptations (but these are a bit more speculative).’

There may also be some pay off from this work for future English footballers (or, depending on the result, fewer excuses):

EPAS1 – also known as HIF2a (hypoxia-inducible factor 2) – is a major gene involved in keeping oxygen levels stable, explains Peter Robbins. ‘As oxygen transport is the principal limiting factor to much endurance exercise, everything we can understand in relation to human variation here will probably help us to understand better the constraints on athletic performance.’

‘In addition, as low oxygen (hypoxia) is a feature of so much disease (eg lung disease, heart disease, vascular disease, cancer), understanding human variation in the response to low oxygen may help us to understand why some people seem so much better at surviving conditions of low oxygen than others.’