Duchenne muscular dystrophy — a novel genetic treatment

Could the innumerable genetic defects underlying Duchenne muscular dystrophy all be treated the same way?  Possibly.  Paradoxically, the treatment involves actually making the gene  even worse.

Understanding how and why this might work involves a very deep dive into molecular biology.  You might start by looking at the series of five background articles I wrote — start at https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/ and follow the links.

I have a personal interest in Duchenne muscular dystrophy because I ran such a clinic from ’72 to ’87 watching young boys and adolescents die from it.  The major advance during that time, was NOT medical or anything I did, but lighter braces, so the boys could stay ambulatory longer.  Things have improved as survival has improved by a decade so they die in their late 20s.

So lets start.  Duchenne muscular dystrophy is caused by a mutation in the gene coding for dystrophin, a large (3,685 amino acids) protein which ties the contractile apparatus of the muscle cell (actin and myosin) to the cell membrane. Although it isn’t the largest protein we have — titin, another muscle protein with 34,350 amino acids is, the gene for dystrophin is the largest we have, weighing in at 2,220,233 nucleotides.  This is why Duchenne is one of the most common diseases due to a defect in a single gene, the gene is so large that lots of things can (and do) go wrong with it.

The gene comes in 79 pieces (exons) which account for under 1/200 of the nucleotides of the gene.  The rest must be spliced out and discarded.  Have a look at http://www.dmd.nl.  to see what can go wrong — the commonest is deletion of parts of the gene (60 – 70% of cases), followed by duplication of other parts (10% of cases) with the rest being mutations that change one amino acid to another.

Duchenne isn’t like cystic fibrosis where some 600 different mutations in the causative CFTR gene were known by 2003 but with 90% of cases due to just one.  So any genetic treatment for that young boy sitting in front of you had better be personalized to his particular mutation.

Or should it?

Possibly not.  We’ll need to discuss 3 things first

l. Nonsense Mediated Decay (NMD)

2. Nonsense Induced Transcriptional Compensation (NITC).

3. The MDX mouse model of Duchenne muscular dystrophy

Nonsense mediated decay.  Nonsense is a poor term, because the 3 nonSense codons (out of 64 possible) tell the ribosome to stop translating mRNA into protein and drop off the mRNA.  That isn’t nonsense.  I prefer stop codon, or termination codon

An an incredibly clever piece of business tells the ribosome (which is after all an inanimate object) when a stop codon occurs too early in the mRNA when there are a bunch of codons afterwards needed to make up the whole protein.

Lets go back to dystrophin and its 79 exons, and the fact that 99.5% of the gene is made of introns which are spliced out.   Remember the mRNA starts at the 5′ end and ends at the 3′ end.  The ribosome reads and translates it from 5′ to 3′. When an intron is spliced out, a protein complex of several proteins is placed on the mRNA some 20 – 24 basepairs 5′ to the splice site (this happens in the nucleus way before the mRNA gets near a ribosome in the cytoplasm).  The complex is called the Exon Junction Complex (EJC). The ribosome then happily munches along the mRNA from 5′ to 3′ knocking off the EJCs as it moves, until it hits a termination codon and drops off.

Over 95% of  genes do not have introns after the termination codon.  What happens if it does? Well then it is called a premature termination codon (PTC) and there is usually an EJC 3′ (downstream) to it.  If a termination codon is present 50 -55 nucleotides 5′ (upstream) to an EJC then NMD occurs.

Whenever any termination codon is reached, release protein factors (eRF1, eRF3, SMG1) bind to the mRNA.  It there is an EJC around (which there shouldn’t be) the interaction between the two complexes triggers phosphorylation of one of EJC proteins, triggering NMD.

So that’s how NMD happens, when there is a PTC.  Clever no?

Nonsense Induced Transcriptional Compensation (NITC).  I realize that this is a lot to throw at you, but a treatment for Duchenne is worth the effort (not to mention other genetic diseases in which the mechanism to be described also applies).

NITC is something I never heard about until two papers appearing in the 13 April Nature (vol. 568 pp. 179 – 180 (editorial), 193 – 197, 259 – 263).  Ever since we could knock out by placing a PTC early (near the 5′ end) of the gene we’ve been surprised by some of the results –e.g. knocking out some genes thought to be crucial had little or no effect.  Other technologies which didn’t affect the gene, but which decreased the expression of the mRNA (such as RNA interference, aka Post-Transcriptional gene silencing — PTGS) did have big phenotypic effects.

This turns out to be due NITC, which turns out to be due to increased transcription of genes which are ancestrally related to the mutant. Gene.  Hard to believe.

Time to go back to NMD.  It doesn’t break mRNA down nucleotide by nucleotide, but fragments it.  These fragments get into the nucleus, and bind to complementary genomic sequences of the gene containing the PTC, and also to genes ancestrally related to the mutant gene (so they’ll have similar nucleotide sequences). Then epigenetics takes over because the fragments recruit the COMPASS complex which catalyzes the formation of H3K4Me3 which is part of the histone code which helps turn on transcription of the gene.  The sequence similarity of ancestrally related genes, allows them and only them to be turned on by NITC.  Even cleverer than finding a PTC by the ribosome.

Something so incredible needs evidence.  Well heterozygotic zebrafish can bemade to have one normal gene and one with a PTC. What do you think happens?  The normal gene is upregulated (e.g. more is made).  Pretty good.

Finally the Mdx mouse.  I’ve been reading about it for years.  It has a PTC in exon 23 of the dystrophin gene, resulting in a protein only 27% as long as it should be.  All sorts of therapeutic maneuvers have been tried on it.  Now any drug development chemist will tell you that animal models are lousy, but they’re all we’ve got.

The remarkable thing about the mdx mouse, is that they don’t get weak.  They do have muscle pathology.  All the verbiage above probably explains why.

So to treat ALL forms of Duchenne put in a premature termination codon (PTC) in exon #23 of the human gene. It should work as there are  4 dystrophin related proteins scattered around the genome — their names are — utrophin, dystrophin related protein 2 (DRP2), alpha dystrobrevin, and beta dystrobrevin

There is an even better way to look for a place to put a PTC in the dystrophin gene.  Our genomes are filled with errors — for details see — https://luysii.wordpress.com/2018/05/01/how-badly-are-thy-genomes-oh-humanity-take-ii/.

There are lots of very normal people around with supposedly lethal mutations (including PTCs) in their genomes.  Probably scattered about various labs are at least 1,000,000 exome sequences in presumably normal people.  I’m not sure how much clinical information about them is available (other than that they are normal).  Hopeful their sex is.  Look at the dystrophin gene of normal males (females can be perfectly healthy carrying a mutant dystrophin gene as it is found on the X chromosome and they have 2) and see if PTCs are to be found.  You can’t have a better animal model than that.

At over 1,000 words this is the longest post I’ve written, and hopefully the most useful.

How little we know

Who would have thought that a random mutagenesis experiment throwing Ethyl Nitroso Urea (ENU) at unsuspecting mice looking for genes using a mutagenesis strategy to identify novel immune regulatory genes would point to a possible treatment for muscular dystrophy? When the experimenters looked at the mutated offspring, they found that the muscles appeared unusually red.

What happened?

You need to know a bit more about muscles. On a very simplistic level there are only two types of muscle fibers, red and white. Carnivores eating chicken know about dark meat and white meat. The dark meat is composed of red fibers, which have that appearance because of large numbers of mitochondria (which are full of iron) giving them the same red appearance as blood (which is also full of iron). In both cases the iron is bound by porphyrin rings. As one might expect, these muscles consume a lot of energy, being postural for the most part. The white meat made of white fibers has muscle which can contract very quickly and strongly, for flight and fight. They don’t have nearly the endurance of red muscle, because they can’t produce energy for the long term.

Humans have the two types of muscle fibers mixed up in each of our muscles.

The ENU had produced a mutation in something called fnip1 (Folliculin INteracting Protein 1). What’s folliculin? It prevents a gene transcription factor (TFE3) from getting into the nucleus. Folliculin prevents an embryonic stem cell from differentiating. It is mutated in the Birt Hogg Dube syndrome which is characterized by many benign hair follicle tumors. What in the world does this have to do with muscular dystrophy? It’s not something someone would start investigating looking for a cure is it? Knock out both copies of folliculin and the embryo dies in utero.

It gets deeper.

What does Fnip1 do to folliculin? It, and its cousin fnip2 form complexes with folliculin. The complex binds an enzyme called AMPK (which is turned on by energy depletion in the cell. AMPK phosphorylates both fnip1 and folliculin. Folliculin binds and inhibits AMPK.

So animals lacking fnip1 have a more activated AMPK. So what? Well AMPK activates a transcriptional coactivator called PGC1alpha (you don’t want to know what the acronym stands for). This ultimately results in production of more mitochondria (recall that AMPK is an energy sensor, and one of the main functions of mitochondria is to produce energy, lots of it).

This ultimately means more red muscle fibers. There is a mouse model of Duchenne dystrophy called the mdx mouse (which has a premature termination codon in the dystrophin protein, resulting in a protein only 27% as long as it should be. That still leaves a lot, as normal dystrophin contains 3,685 amino acids. Knocking out fnip1 in the mdx mice improved muscle function. Impressive !!

I’m quite interested in this sort of work, as I ran a muscular dystrophy clinic from ’72 to ’87 and watched a lot of kids die. The major advance during that time wasn’t anything medical. It came from engineering — lighter braces using newer materials allowed the kids to stay out of wheelchairs longer.

You can read all about it in Proc. Natl. Acad. Sci. vol. 112 pp. 424 – 429 ’15 ] Clearly we know a lot (AMPK, dystrophin, PGC1alpha, fnip1, fnip2, folliculin, TFE3), but what we didn’t know was how in the world they function together in the cell. We’re sure to learn a lot more, but this whole affair was uncovered when looking for something else (immune regulators) using the bluntest instrument possible (throw a mutagen at an animal and see what happens). No one applying for a muscular dystrophy grant would dare to offer the original work as a rationale, yet here we are.

So directed research isn’t always the way to go. Although we know a lot, we still know very little.