Myotonic dystrophy type 1 (DM1), an inherited disease affecting muscles, was one of the first described “expanding repeat” disorders. In these 50 or so conditions, symptoms may appear earlier and worsen from generation to generation, as the mutant gene grows, adding copies of a 3- or 4-base DNA sequence. For many expanding repeat disorders, forty copies seems to be a threshold, causing symptoms when crossed.
In a family with myotonic dystrophy type 1, a grandfather might experience mild weakness in his forearms, while his daughter may have more noticeable arm and leg weakness, slurred speech, and a flat facial expression. Her children have even weaker muscles that contract for too long, creating limitations like being unable to unclench a fist or release a grip.
In MD1, skeletal muscle fibers that contract for too long impair balance and coordination, called ataxia. The condition also causes cataracts, small gonads, frontal balding, fatigue, sleepiness, digestion problems, and cognitive and behavioral impairment. Life may be shortened. MD1 affects about one in 7,500 people, or more than 40,000 people in the US.
A repurposed arrhythmia drug, mexiletine, is available in some countries. Although it improves symptoms by increasing the ability to relax muscles, it does not affect the underlying cause of the disease.
Now Avidity Biosciences has developed a candidate drug that weds a monoclonal antibody (MAb) to a short piece of RNA – a small interfering RNA (siRNA). The duo is designed to correct the mutation, the MAb targeting the siRNA to the gene behind MD1, where it snips out DNA repeats. This novel class of RNA therapeutics is called Antibody Oligonucleotide Conjugates (AOCs™).
For many years, clinicians called the worsening of symptoms in a family with MD1 “anticipation,” merely kids imitating a parent or grandparent’s movements. Then, with the ability to sequence DNA, researchers found that the gene expands. The gene that causes MD1, on chromosome 19, has too many repeats of the DNA sequence CTG. Typical repeat number is 5 to 37; people with the disease have 50 to thousands of copies.
The disease is autosomal dominant, caused by inheriting a single expanded gene copy from one parent, more severe if from the mother. Yet some people can inherit many extra copies of the gene and never develop symptoms – perhaps other genes provide protection.
Huntington’s disease is another expanding repeat neurological genetic disorder. DNA Science ran the story of a 6-year-old diagnosed in 2002 six weeks before her 35-year-old father was diagnosed, whose mother had it. Father and daughter died within weeks of each other in early 2010. The child was doubly unlucky – the triplet repeat mutation inherited from her dad looped around itself in the sperm cell, generating a double-sized repeat.
Understanding Expanding Repeat Disorders Mirrors Evolving DNA Sequencing Technology
MD1 first came to medical attention in 1909, when German neurologist Hans Gustav Wilhelm Steinerti identified scar tissue in the muscles of six patients on autopsy. Then in the 1980s, when genes began to be assigned to chromosomes based on inheritance patterns in families – linkage studies – the causative gene was narrowed down to chromosome 19.
In 1992, more definitive gene maps were assembled based on how mutations alter the sites at which DNA-cutting enzymes, called restriction enzymes, cut. The gene behind MD1 had a mutation that led to an unusually sized piece – aka a restriction fragment length polymorphism, or RFLP. Fragile X syndrome and Huntington disease are two other conditions for which the genes were narrowed down with RFLP maps.
Next, DNA microarrays revealed repeats behind spinocerebellar ataxia SCA31, similar to ALS. Then next-generation sequencing clarified two more types of spinocerebellar ataxia. Most recently, long-read sequencing filled in tiny genome gaps to reveal the repeats behind a form of epilepsy, another ataxia, and neuronal intranuclear inclusion disease, which impairs balance, movement, cognition, and communication.
In “30 years of repeat expansion disorders: What have we learned and what are the remaining challenges?” in the American Journal of Human Genetics, Christel Depienne and Jean-Louis Mandel include a graphic that superimposes identification of expanding repeat disorders upon evolving DNA sequencing technologies. It’s fascinating.
The Glitch in Myotonic Dystrophy Type 1
The gene behind MD1 is dystrophia myotonica protein kinase (DMPK). A kinase is an enzyme that transfers high-energy phosphates from ATP molecules to other molecules, and is therefore critical to cell energetics, especially in skeletal and cardiac muscle. The expanded gene causes a “toxic gain-of-function” because it doesn’t remove an action but provides an atypical one. The repeat is in the initial part of the gene that is not transcribed into mRNA.
The triplet repeat mutation in MD1 is in intron 7a, which is part of the gene in the fetus but is typically spliced out as the pre-mRNA forms after birth. In MD1, the retained expanded repeats form classic hairpin loops as complementary parts bind and bulge from the linear configuration, dislodging various proteins. The muscle phenotype arises from the repeats gumming up the chloride channels that festoon skeletal muscle cells. Signals to cease contraction are blocked and the cells’ proteasomes, which degrade excess or abnormal proteins, are impaired. In MD1, the result of this complex genetic signaling gone awry is that muscle cells contract for too long.
Monoclonal antibody technology was born in the 1970s, and short interfering RNAs (siRNAs) described in 1999. Arthur A. Levin, from Avidity Biosciences, describes the strategy for the pairing in a Clinical Implication of Basic Research article in The New England Journal of Medicine.
The strategy uses a MAb as a molecular drone of sorts to deliver an siRNA designed to bind the extra repeats in the mRNA corresponding to the MDPK gene. The binding of the single-stranded siRNA to the single-stranded mRNA signals other molecules to snip the now double-stranded short molecule. Interim results of the clinical trial, called MARINA, were presented at the American Academy of Neurology annual meeting in Boston in April, with final results of the phase 1/2 randomized, double-blind, placebo-controlled trial due in September.
Synthesizing a bit of RNA, Levin writes, is the simple part. “Reality, however, is more complex.”
An initial hurdle is delivering the RNA into cells without it getting hung up crossing the cell membrane, or chopped up by enzymes once inside. Attaching a nanoparticle that eases transport can make lower doses possible.
Once enough of an siRNA is delivered and it functions, the next challenge is reaching enough muscle cells to counter symptoms and improve quality of life. That’s long been a hurdle in developing gene therapy for Duchenne muscular dystrophy; initial attempts were tested on patients’ toes.
In 2016, researchers in Japan published in The Journal of Controlled Release on an siRNA bound to part of an antibody that targeted it to the transferrin receptor, directing it to skeletal and cardiac muscle. The siRNA-antibody combo to treat MD1 binds the transferrin receptor.
The Clinical Trial So Far
The phase 1/2 clinical trial of AOC 1001 on 38 patients showed “functional improvement, DMPK reduction, splicing improvements and a favorable safety and tolerability profile,” according to a company news release.
The patients were evaluated with standard tests of muscle strength, such as hand opening time, how long it takes to walk 10 meters, and other measurements of various muscle groups.
“The AOC 1001 topline data demonstrated directional improvement across a variety of functional assessments in patients with DM1, including myotonia and muscle strength in a six-month period. This result is more than we could have anticipated in such a short time. These AOC 1001 data are remarkable and could make a real impact for people living with DM1,” said Nicholas E. Johnson, from Virginia Commonwealth University and lead investigator in the trial.
“Our vision is to profoundly improve people’s lives by revolutionizing the delivery of RNA therapeutics. These AOC 1001 data further demonstrate the broad and disruptive potential of our proprietary AOC platform to address targets and diseases previously unreachable with existing RNA therapies,” added Sarah Boyce, president and chief executive officer at Avidity.
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