HGP is 10: the gene therapy challenge

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.’