Why eteplirsen should be approved and what it is.

Posted by: Christine McSherry on March 14, 2014

Why eteplirsen should be approved and what it is.
Jon D. Moulton,Ph.D.

I'll start with my main message, that the drug eteplirsen for treatment of Duchenne muscular dystrophy (DMD) should be approved. I am excited by the amazing technology the drug is based on, so I'll finish with a discussion of how Morpholino oligos work. I am a molecular biologist working at Gene Tools LLC for Dr. Jim Summerton, who conceived of and co-invented the Morpholino oligos in the 1980s and founded the first antisense company, the company that became Sarepta Therapeutics; my work involves targeting Morpholino sequences for researchers exploring many aspects of biology.

DMD is a genetic disease caused by a mutation in the gene encoding dystrophin. Dystrophin is a protein that links an internal network of protein fibers called the cytoskeleton to a raft of proteins bound in and on the cell membrane; it is particularly important for maintaining integrity of muscle cells. The dystrophin protein has additional functions, including binding to an enzyme that makes nitric oxide. If someone has DMD, they make vanishingly little to no functional dystrophin in most of their cells. The disease is a progressive muscle-wasting condition that often leads to death in the teens or early twenties, typically from heart or respiratory failure. There is no approved medication that restores production of functional dystrophin in those with DMD; until recently the technologies that could do that did not exist.

Sarepta Therapeutics is a small biotech company that has developed an innovative experimental drug called eteplirsen. The drug is a Morpholino antisense oligo, an analog of natural nucleic acids that differs from DNA by its unnatural backbone. Morpholinos bind to complementary sequences of RNA, allowing their use as a sort of molecular masking tape to cover specific sequences of RNA. A Morpholino can be designed to bind in a particular place and interfere with a specific process occurring on RNA. I'll describe in more detail later how eteplirsen can cause functional dystrophin to be produced in the muscles of someone with DMD.

The Food and Drug Administration controls what drugs are available for sale in the USA. From the original mandate of preventing dangerous compounds from being sold as drugs, the FDA's job now includes ensuring that drugs are effective prior to approving their sale. This has prevented much suffering due to sale of bad medicine, but has also created a stiff barrier for entry into the US drug market, with clinical trials commonly costing hundreds of millions of dollars and the approval process taking many years. Eteplirsen has undergone several clinical trials, though these trials have had few participants compared to the trials conducted by the big pharmaceutical companies.

The boys in the ongoing eteplirsen trial are doing well in terms of their muscular abilities and respiration. Eteplirsen has shown no signs of toxicity. If other DMD boys do not receive treatment, their life expectancy is drastically less than that of the non-DMD population and, as they lose ambulation and the ability to care for themselves, they require heroic supportive care. Eteplirsen shows promise to extend lifespans and decrease the personal and social burden of providing supportive care by helping those with DMD retain more strength to care for themselves.

Eteplirsen has been through several Phase I and II clinical trials, with dosing currently ongoing in an extension study that has lasted well over two years. Assessment of muscle biopsies taken before the start of treatment and compared with biopsies taken after 12 weeks of treatment (at 50 mg/kg eteplirsen) and after 24 weeks of treatment (at 30 mg/kg eteplirsen) have shown small increase in the concentration of dystrophin and considerable increase in dystrophin-positive fiber counts. However, because Morpholino oligos do not degrade when they are in tissues and because excretion of Morpholinos from cells is very slow if it occurs at all, we can expect the concentration of Morpholino oligo in the cells to increase over repeated weekly doses. But while dystrophin measurement is important, the proof is in the pudding: The 10 boys treated over two years in the eteplirsen trial that maintained ability to walk have on average improved in muscular abilities, some have ceased toe-walking, and the boys in the trial, even the two who lost ambulation early in the trial, have maintained their level of respiratory function.

Eteplirsen should be considered under the accelerated approved regulatory pathway in the USA now. The FDA protects Americans from the sale of bad drugs. But shouldn't the FDA also protect Americans from the worst of diseases? If an unexpected deleterious side-effect arises, the US FDA has the option to withdraw approval. Every day that a dystrophin-restoring drug awaits approval, DMD boys' muscles deteriorate, some lose the ability to walk or brush their teeth or roll over in bed or breathe. Toxicity of eteplirsen has not been detected in the trials, the benefit of the drug has been shown already in the ongoing small trial, and the cost of waiting is too high.

Without approval of a dystrophin-restoring drug these outcomes are certain: those with DMD will be living with difficulty and dying young, parents and other caregivers will be devoting their lives and fortunes to supporting them, and those with mutations treatable by skipping exon 51 will live with the knowledge that the federal government has made the choice to withhold access to the drug that has so dramatically improved the quality of life of the eteplirsen trial participants. With approval the DMD kids will retain more abilities and might live longer lives, their parents will not face as great a burden though the teenage years of a DMD child, and the families and physicians will see the FDA as their ally in the struggle against this disease.

The drug: how our cells make proteins, one way a Morpholino can change that, and why a Morpholino makes a better drug.

When a cell makes a protein, it starts with the instructions for the order in which to assemble the amino acids. These instructions are in the DNA, and the region of DNA that codes the instructions for a protein is called a gene. The cell makes a copy of the information in the gene by making a strand of RNA. In an organism more complex than a bacterium, that RNA is initially called a pre-mRNA and parts of the RNA are removed in the cell nucleus; that process is called splicing. The parts that are removed are called introns, the parts that are left are called exons and these exons form the mature mRNA. The mature mRNA leaves the nucleus and goes to a ribosome, the site of protein synthesis. The ribosome uses the information coded in the RNA to direct the synthesis of a new protein from amino acids.

A Morpholino can be targeted to change the way that pre-mRNA is spliced in the nucleus of a cell. There are several stages in splicing that depend on other molecules binding to the pre-mRNA, some to serve as signposts marking the edge of introns or as switches to turn splicing on or off at particular exons. Morpholinos can be designed to cover the binding sites of these other moelcules, which changes the way that the pre-mRNA is spliced. A Morpholino can be designed to cause the cell to splice out a particular exon in a particular gene. The DMD drug Eteplirsen causes exon 51 of the gene for dystrophin to be removed during splicing; we call that skipping exon 51.

Some mutations that cause DMD cause a frameshift mutation. When a ribosome directs synthesis of a protein, it hooks onto one end of an mRNA, called the upstream end. The ribosome moves downstream along the RNA. When it reaches a start site encoded in the mRNA it starts assembling amino acids into a new protein. Every three bases on the RNA calls for a new amino acid. A frameshift mutation is a change in the number of bases in the mRNA that changes the 3-base groupings of RNA so that they no longer specify the same amino acids. If this kind of mutation occurs, all the amino acids are assembled incorrectly once the ribosome moves downstream from the mutation and that end of the protein doesn't work right.

Often it is possible to skip an exon near a mutation so that the reading frame is restored and the amino acids are assembled in the correct order downstream of the mutation and skipped exon. For instance, if one base were deleted in the original mutation, removing two more bases would restore the original clusters of three bases downstream of the changes and the clusters of bases would again call in the right amino acids at the ribosome. Usually it is larger chunks of the gene that are missing and the exons being skipped are much bigger than two bases, but by using a Morpholino to skip an exon that has an appropriate number of bases, in many cases the reading frame can be restored.

Eteplirsen can't help all of the people with DMD, but some have frameshift mutations that can be helped by skipping exon 51 and restoring a reading frame that makes a functional protein. Different oligos will be needed to treat mutations that can't be treated by exon 51 skipping. Sarepta has announced its intention to bring Morpholinos into clinical trials to skip other DMD exons; approval of eteplirsen would help fund these clinical trials of other exon skipping Morpholinos needed to treat other DMD mutations.

The lack of adverse effects (e.g. toxicity) might be expected based on the widespread use of Morpholino oligos for research in embryonic zebrafish. The developing vertebrate embryo is a very sensitive system for detecting gene modulation, which can lead to birth defects (teratogenesis). While changes in off-target gene expression are sometimes detected when using Morpholinos in embryos, such changes are a sequence-specific effect and some sequences have very little effect on gene expression (except on their intended target RNA). Part of the reason for the specificity of Morpholino compared to most other antisense types is that, unlike natural nucleic acids (RNA and DNA) and phosphorothioate-linked oligos, the Morpholino backbone has no electrostatic charge and so the oligos are not recognized by the proteins that normally bind nucleic acids. For a chemical structure and more background on the molecules, see http://en.wikipedia.org/wiki/Morpholino

You might have realized that the flexibility in Morpholino sequence means that other diseases might be treated using Morpholino-based drugs. If so you are right. Studies are ongoing developing therapeutic oligos for spinal muscle atrophy, Hutchinson-Gifford progeria, Marburg virus and a broad range of genetic, parasitic, bacterial and viral diseases. Papers describing research using Morpholinos can be found using the Morpholino publication database at pubs.gene-tools.com. The approval of eteplirsen will encourage clinical development of Morpholino-based drugs for many other diseases. This is an exciting time to be involved with this technology.

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