The key to the tomb
How we Learn to Treat Stephen Hawking's Disease
Polina Loseva, N+1
Every year about six thousand people hear from a doctor the diagnosis of "amyotrophic lateral sclerosis" (ALS). This means that their bodies will literally become a tomb, the walls of which will gradually shrink: eventually the paralysis will spread so much that a person will no longer be able to breathe. That's how Lou Gehrig, Mao Zedong and Stephen Hawking died. Over the past hundred years, we have gone from a complete misunderstanding of why this is happening to clinical trials of drugs: yesterday preliminary reports were published on new successes – while, however, still questionable – but they already allow scientists to talk about "the beginning of gene therapy for ALS". N+1 it is clear what exactly they are trying to "fix" new medicines and explains what was the use of medicine from the fact that a few years ago the whole world cheerfully overturned buckets of ice water.
"There is no cure. The chances of keeping me the way I am are 50-50. Perhaps in 10 years I will need a cane," The Iron Horse, an American baseball legend, wrote to his wife from the Mayo Clinic in 1939. The athlete was only 36, and two years later he died, having lost the ability not only to walk, but even to sign documents without the help of his wife. At that time it was already known that the cause was the progressive death of motor (motor) neurons of the brain and spinal cord, but its cause remained unclear. "It's probably some kind of microbe," he said in the same letter. "But no one has ever heard of it being passed between spouses."
35 years later, the 80-year-old dictator received the same diagnosis. By that time, the version about pathogen microbes was no longer seriously considered, and neurodegeneration was considered one of the manifestations of aging of the nervous system. It was clear that some plaques appear in the brain tissue - as in Alzheimer's or Parkinson's disease – which cause progressive paralysis. It was these plaques, then of an obscure nature, that in ten years turned a cheerful old man, who swam across the Yangtze at the age of 72, into a hopeless patient who could breathe only lying on his side. From diagnosis to death, as in the case of a baseball player, it took about two years.
The British astrophysicist was more fortunate. He heard his verdict even before his fellow sufferer crossed the Yangtze for the last time, but the disease stopped half a step from the lethal finish. Slowly losing the mobility of his head, the scientist lived for another 55 years, became an icon of scientific pop and a new symbol of his illness. During this time, it became clear that aggregates in the brain consist of proteins, these proteins have names, and possible culprit genes loomed on the horizon. Fifty clinical trials of drugs started and left the race ahead of schedule. Millions of people poured buckets of ice water on themselves, raising money for research on the disease. But no one has figured out how to treat it.
Amyotrophic lateral sclerosis (ALS), which killed Lou Gehrig, Mao Zedong and Stephen Hawking is just one of many neurodegenerative diseases against which humanity is still powerless. And although ALS does not always end with dementia, it has been compared with Alzheimer's and Parkinson's diseases for good reason. The obstacles that separate us from the medicine are about the same in all these cases: it is impossible to predict the disease, it is not always possible to diagnose it in the early stages, the causes are manifold, and their list is not complete.
In addition to this, the damage quickly becomes irreversible, because neurons, unlike many other cells, are not so easy to replace with new ones. As in the case of Alzheimer's and Parkinson's diseases, the only medications that exist for amyotrophic lateral sclerosis only help to compensate for the symptoms. In the case of ALS, they block either the excessive release of glutamate, briefly delaying the death of neurons, or suppress oxidative stress – but how it works, they don't even know in the FDA (American Ministry of Health), where they approved the corresponding drug. No one has managed to stop neurodegeneration yet.
And here in the latest issue of the magazine The New England Journal of Medicine has published the results of two new clinical trials and a review that marks the beginning of a new era in the fight against the recalcitrant disease. According to the authors of the review, we were finally able to aim at the very root of amyotrophic sclerosis. And, therefore, there was a glimmer of hope for success. It is all the more surprising that both the target and the weapon for its destruction have been known for a long time: the first was discovered more than 25 years ago, the second is known for more than half a century. But they were able to meet only now.
Invent a weapon
The transmission of information in the cell is based on the principle of Velcro. Double-stranded Velcro-DNA is divided into two halves, and copying enzymes add a paired (complementary) Velcro-RNA to one of the halves. It turns out an RNA copy of the gene, which is sent from the nucleus to the cytoplasm for protein synthesis. Transport RNAs carrying amino acids swim up to the informational RNA Velcro in turn. Sticking in the correct sequence allows you to build a protein chain from amino acids.
There are many different ways to "block" a gene or stop its expression, but the easiest way is to complementarily seal it so that no one else sticks to it anymore. Even bacteria can do this, and this method of regulating expression was discovered in them back in 1967, just a few years after the discovery of informational RNA. Then it turned out that non-coding RNA is produced in Escherichia coli cells, which adheres to certain sections of information RNA and prevents them from transmitting information further.
The principle of antisense therapy (Robinson R / PLOS Biology, 2004).
After 10 years, people also learned to do this, already for their own purposes. Harvard scientists created a short chain (oligonucleotide) of DNA that was complementary to the site of the Raus sarcoma virus. Sticking to the viral RNA, the oligonucleotide prevented the virus from multiplying, and the cells infected with the virus from turning into tumor cells. This is how the technology of antisense oligonucleotides appeared – short chains that are complementary to the "meaningful" parts of the gene and, thanks to this, can stop its work. Since then, our idea of "Velcro" and "anti-Velcro" has become much more complicated. Among non–coding RNAs, many different types have been identified - they differ in length, origin and function. It turned out that they can act at a variety of levels: block the creation of RNA, its maturation and subsequent protein synthesis, or simply force cellular enzymes to cut it into pieces. And all this could be used for antisense therapy - to learn how to build complementary DNA or RNA strands to the right gene to control its work.
It was a small matter – to find where to shoot.
Find a target
The first medicine, which included antisense oligonucleotides, appeared in 1998. It exploited the very idea with which the history of antisense therapy began - to block the reproduction of the virus. The drug was hunting for cytomegalovirus, which is usually not terrible for healthy people, but it spreads uncontrollably through the body of AIDS patients.
However, it quickly turned out that it was easier to prevent AIDS itself than to deal with its consequences – and in 2006, citamegalovirus medicine disappeared from the market, giving way to antiretroviral drugs. Trials of oligonucleotides against other viruses have begun, but have not been crowned with success so far. And there was a lull.
It is relatively easy to shoot at a virus – it is enough to read its, as a rule, a small genome, and select the desired section of the gene. It is much more difficult with a person in this sense. Among the twenty thousand of his genes, it is not easy to aim at the right one, especially if you do not know for sure which one is causing problems – and this is exactly the case with most neurodegenerative diseases.
By the time the antisense drug against cytomegalovirus appeared, the human genome had not even been read in its entirety. And there were still four years left before the first genome-wide association search (GWAS research), with the help of which today they are looking for the genetic causes of anything (from diseases to their alleged connection with behavior).
The technology of antisense therapy was ahead of its time. The weapon appeared before the target was found – the cytomegalovirus in this sense was just a warm-up.
In this sense, we were a little more lucky with amyotrophic lateral sclerosis than with other diseases – we knew something about it even before the first genome-wide searches. Despite the fact that it is not always inherited (which means that not all of its cases can be correlated with a specific mutation), about one in five cases is associated with a mutation in the SOD1 gene. It encodes the protein superoxide dismutase, one of the main cellular antioxidants. However, the participation of SOD1 in the development of ALS has nothing to do with its main functions: apparently, the mutant protein behaves like a prion - in the motor neurons of the spinal cord, it forms protein aggregates that grow like a snowball. They disrupt intracellular transport in motor neurons, gradually leading to their death.
The structure of the SOD1 protein (Emw / wikimedia commons / CC BY-SA 3.0).
Scientists made an accusation against SOD1 back in 1993. It's a small matter: it remains only to read the gene, collect the antisense thread and block its work. But new difficulties arose in the process. For example, it turned out that there are at least two hundred mutations in SOD1, and not all of them are equally associated with the development of ALS. In addition, antisense RNAs turned out to be quite unstable: in the cell they are quickly cleaved by RNases, and in the blood immune cells mistake them for viruses – and everything ends with inflammation and destruction of the drug.
It took another twenty years to find the right antisense chain, make it stable and work on mice. During this time, promising examples have appeared in the field of antisense therapy - drugs against spinal muscular atrophy and some forms of Duchenne myodystrophy. Now it is much easier to enter clinical trials with antisense RNA – we know for sure that such a treatment can work.
Warning shot
The two research groups that published their results in NEJM did the same thing, but in different ways. The first group, from Massachusetts, followed the path of gene therapy, that is, they placed an RNA strand inside the adenovirus in order to "infect" motor neurons with it. Only two patients participated in the trial – the first step towards more serious tests – with confirmed mutations in the SOD1 gene.
One of them received experimental therapy a few months after the diagnosis. The treatment did not cause significant clinical improvement, and he died 15 months later. However, at the autopsy, the researchers found that the amount of mutant SOD1 in his spinal cord was 90 percent lower than the average in ALS patients. This may mean that the therapy works, although it does not save at the stage of neurodegeneration at which the subject was at the time of the start of treatment. The second patient has stabilized after therapy and is still alive, so the amount of SOD1 in his spinal cord remains unknown.
Another research group represents a large pharmaceutical company Biogen, which, among other things, produces antisense oligonucleotides against spinal muscular atrophy. This is not their first study of ALS antisense therapy, the results reflect the 1-2 phase of the trial, and they expect the results of phase 3 in a year.
This trial involved 50 patients with mutations in SOD1: four groups received different doses of the drug, the fifth served as a control. First of all, scientists were interested in safety: it turned out that many side effects are associated with an injection into the spinal cord and do not depend on whether a placebo or an oligonucleotide was injected into it. In addition, they measured the concentration of SOD1 in the cerebrospinal fluid (it serves as an indirect marker of how actively the protein is formed in neurons) – the highest doses of the drug reduced it by an average of 32 percent. At the same time, when the researchers post factum divided the patients into groups with rapid and slow development of the disease, it turned out that treatment could be more effective for the former: external signs of degeneration began to appear slower in them, and there were fewer markers of neuronal death in the cerebrospinal fluid.
Graph of the effect of antisense therapy of ALS in the first two phases of clinical trials (T. Miller et al. / The New England Journal of Medicine, 2020).
What was brought in a bucket of water
The authors of both articles try to avoid loud statements and predictions – especially considering that no one could be cured to the end, and even there was no such goal (as is usually the case in the first phases of research). But let's imagine for a moment that the third phase will be successful and the destruction of neurons in patients will be possible, if not reversed, then at least stopped. Will this mean that amyotrophic lateral sclerosis is defeated, and Parkinson's and Alzheimer's are next?
On the example of even these modest tests, it is clear how well the oligonucleotide gun hits. Even when patients are specially selected so that they carry a mutation in the same gene, the group still splits into two with different treatment effects. But mutations in SOD1 are no more than 20 percent of cases of hereditary ALS.
In 2014, Facebook was filled with millions of videos with people pouring icy water on themselves. The Ice Bucket Challenge flash mob has raised more than $200 million in a couple of months for amyotrophic lateral sclerosis research, patient support and drug search. With this money, among other things, it was possible to conduct GWAS and replenish the list of targets with three new mutations, and now it "closes" almost all cases of hereditary ALS. Each of the mutations will need its own antisense oligonucleotide, and possibly more than one.
From the point of view of "one mutation – one pill", we can say that we are not dealing with one disease, but with many. Now we can't even be sure that the three most famous victims of ALS were sick with the same thing. Judging by what Lou has Gehrig's disease manifested itself early, Mao Zedong's – late, and Stephen Hawking was suspended for decades, they could carry different mutations in their genes. But then it didn't occur to anyone to check it out, and now we won't know if we could save any of them now.
The same problem will arise in the case of other neurodegenerative pathologies. Neither Alzheimer's disease, nor Parkinson's disease, nor many other lesser-known syndromes have yet found a single culprit gene. The lists are multiplying, the stakes are rising. And despite the fact that we know for sure that antisense therapy works, and individual medications are possible, it's scary to imagine how many buckets of ice water we will have to pour over ourselves before we find a pill for everyone.
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