To use an old rubric, Alnylam and Mirna are making a virtue from a necessity. Alnylam is targeting expression of a protein that is produced in the liver. Mirna is trying to develop treatments for liver cancer. Their molecules will be taken up by exactly the organ they want them to reach. The companies have adapted the structure or packaging of their molecules to try to ensure that once they are in the liver, they will survive long enough in the cells to do their job. SmallRNA approaches have been put forward for a number of other conditions, and the preliminary cellular and animal experiments often look good. But for a condition such as amyotrophic lateral sclerosis, where the nucleic acids will have to avoid the liver and be taken up by the brain,{397} it’s not clear yet how successful the industry will be in capitalising on this technology.
In Chapter 17, we saw how hopes of a promising new approach to treat Duchenne muscular dystrophy may be receding, after unexpectedly disappointing late-stage clinical trial failure. The methodology used in this approach was an example of a particular kind of junk DNA, known as antisense.
Antisense junk RNAs are probably a widespread feature of our genome, and it’s because of the double-stranded nature of DNA. We touched on this in Chapter 7, where the actual biological example we used was of Xist and its antisense counterpart, Tsix. We also used the analogy of the word DEER, which can be read backwards as the word REED. It just depends if the enzymes that make RNA copies of DNA reads one strand from left to right, or the opposite strand from right to left.
However, most words can’t be read in both directions. If we read the word BIOLOGY backwards, we get YGOLOIB, which doesn’t have a meaning. In the same way, messenger RNA from one direction in the genome may code for a protein, but the same region copied backwards simply codes for a junk RNA that cannot be translated into a protein. Sometimes this creates auto-regulatory loops in our cells, limiting expression of certain genes. An example of this is shown in Figure 19.2.
Figure 19.2 In some parts of the genome, both strands of DNA can be copied into RNA, in opposite directions. These are known as sense (creating RNAs that code for protein sequences) and antisense (which don’t code for protein sequences). The antisense RNA molecule can bind to the sense RNA molecule and affect its activity, in this example by inhibiting production of protein from the sense messenger RNA template.
Researchers have reported that about a third of protein-coding genes also produce junk RNA from the antisense strand. However, the antisense is usually produced at lower levels, often no more than 10 per cent.{398} Sometimes the antisense is just a short internal section of the gene. Other times the sense and antisense may start and end in different places so that they overlap but also have unique regions. Sometimes the machinery copying the sense DNA strand into sense RNA crashes into the machinery moving in the other direction to create the antisense RNA. Both sets of proteins fall off the DNA, and both RNA molecules are abandoned. There are even antisense strands for some long non-coding RNAs.
The effects of an antisense RNA binding to its sense RNA partner can vary. Figure 19.2 shows an example where this binding prevents the sense messenger RNA from being translated into protein. But there are other situations where the binding stabilises the messenger RNA, ultimately leading to higher protein expression.{399}
In the Duchenne muscular dystrophy trials that originally held such promise, the patients were treated with an antisense molecule that could recognise and bind to messenger RNA for dystrophin. The antisense molecule was chemically modified to prevent it from being broken down too quickly in the body. When the antisense molecule bound to the dystrophin messenger RNA, it prevented the splicing machinery from binding in the normal way. This altered the way the messenger RNA was spliced together, and got rid of the region that caused the most problems in mutant protein production.
The Duchenne muscular dystrophy trial ultimately failed but we shouldn’t take this as meaning the entire antisense field is tainted. In fact, it’s had its successes. In 1998 an antisense drug was licensed for use in immunocompromised patients who had developed a viral infection in the retina[66] that threatened their sight. The antisense molecule bound to a viral gene, and prevented the virus from reproducing.{400} It was an effective drug, which raises two questions. Why did this drug work so well? And given that it worked so well, why did the manufacturer stop selling it in 2004?
Both answers are quite straightforward. The drug worked well because it was injected straight into the eye. There was never a problem about it being scooped up by the liver, because it didn’t go via the liver. It was also targeting a virus, and only in one self-contained part of the body, so there wasn’t much risk of widespread interference with human genes.
All of which sounds peachy, so why did the manufacturer stop selling it in 2004? This drug was developed for severely immunocompromised patients, of whom the vast majority were people suffering from AIDS. By 2004, there were drugs available that were pretty good at keeping HIV, the causative virus, under control. The patients’ immune systems were in much better shape, and they simply weren’t succumbing to viral infections in the retina anymore.
More recent developments have also shown that there is still life in the use of antisense junk DNA for therapy. There is a serious condition called familial hypercholesterolaemia. In the UK it is predicted that there are about 120,000 people with this disorder, although many of them may not have been diagnosed. These people have genetic mutations that prevent their cells from taking up bad cholesterol and dealing with it properly. As a consequence, between a third and half of all such patients will have serious coronary artery disease by their mid-50s.{401}
For some patients with this condition the standard lipid-lowering drugs, known as statins, work really well to lower their risk of cardiovascular disease. This is often the case for people who have one mutant copy of a particular gene, but in whom the other matching copy is normal. But there are some severely affected patients, especially those in whom both copies of the specific gene are mutated, for whom statins are ineffective. These patients often have to undergo plasmapheresis once or twice a week, where their blood is passed through a machine and the dangerous cholesterol is removed.
If you want to stop a bathtub from overflowing, you have two options. You can keep letting water out via the drain, or you can turn down the taps to stop adding more water.
A company called Isis developed an antisense molecule which targets the primary protein in low-density lipoproteins, the so-called ‘bad cholesterol’.[67] This antisense therapy for familial hypercholesterolaemia works by turning off the taps. The anti-sense drug binds to the messenger RNA for the bad cholesterol protein and suppresses it, resulting in lower expression and lower levels of bad cholesterol. Isis licensed this to a larger company called Genzyme, in a deal costing hundreds of millions of dollars.