Myostatin regulation with rna?

Ok so there's been bits of talk here and there about gene doping and some gene projects but I feel it's time we give one a shot. The idea is fairly simple. See if we can down-regulate myostatin via rna interference or other fairly harmless methods. I'd rather avoid viral vectors for now as they are tricky and permanant. I want this to be a temporary thing. Something that will interfere with the genetic pathways for a short while and then eventually either be broken down or be treatable with a counter agents to turn it off. I felt like rna interference would be ideal as you could use a small dosage so the affect could be fairly localized as once the rna is in use it shouldn't move anywhere. Or a larger dose could spread but eventually be broken down and allow normal function again.

That's the plan. Thoughts?



  • I'm down. What part do you need help with?
  • Well there's a few bits that need to be considered. First, which part of the mystatin synthesis are we targeting? Do we target the actual mRNA sequence carrying the myostatin code? or do we target something that triggers that mRNA transcription? How do we deliver the vector? should it be local delivery? or full body
  • Have you thought about epigenetic targeting? If you could increase the methylation of the myostatin gene, you could really reduce its transcription. I think if you targeted the transcription factors that regulate myostatin expression, you'd end up with a lot of off-target effects, which could be really nasty (though I guess the same could be said for epigenetic modification, unless your targeting is good...) Targeting your treatment to the right sequence is, I guess, the first major hurdle.

    Whatever you do, I'd do it locally...
  • Ya given this a bit of thought lately but been swamped with other projects. I was thinking of just using RNA interference. So all we'd have to do is figure out what controls myostatin regulation and then either shut down the enzyme that produces it or boost whatever down regulates it. Should be simple enough to do once we figure out the  biosynth of myostatin
  • I hear RNA is pretty short lived. Like <2 hrs? So if it is something we need to hit every two hours then I'm hoping it's cheap. If memory serves, ACVR2B is the knockout target for the DNA route. PPAR delta is also something I had in my notes, but I forget if that was related to myostatin or not. I'll dig around in the morning and see if I can find the target.
  • From wiki:

    Human myostatin consists of two identical subunits, each consisting of 109 (NCBI database claims human myostatin is 375 residues long) amino acid residues. Its total molecular weight is 25.0 kDa. The protein is made in an inactive form. For it to be activated, a protease cleaves the NH2-terminal, or "pro-domain" portion of the molecule, resulting in the now-active COOH-terminal dimer.

    Myostatin binds to the activin type II receptor, resulting in a recruitment of a coreceptor called Alk-3 or Alk-4. This coreceptor then initiates a cell signaling cascade in the muscle, which includes the activation of transcription factors in the SMADfamily - SMAD2 and SMAD3. These factors then induce myostatin-specific gene regulation. When applied to myoblasts, myostatin inhibits their differentiation into mature muscle fibers.

    Recently, myostatin has also been shown to inhibit Akt, a kinase that is sufficient to cause muscle hypertrophy, in part through the activation of protein synthesis. However, it is notable that Akt does not responsible for all of the observed muscle hyperthrophic effects which are mediated by myostatin inhibition[7

    TL:DR myostatin is a protein dimer encoded by the gene MSTN. Once active it binds to the ACVR2B protein and causes muscle growth to be suppressed.

    So they've identified a mutant that can be controlled by microRNA. When that gene is transcribed into mRNA there's a bit at the end, part that doesn't get encoded that is a target for the miRNA and inhibits translation. This isn't super helpful for us since that would mean adding in a gene to humans which is tricky. What'd be easier is look at either the MSTN or ACVR2B sequence and decide which is better for the RNA interference and then design a miRNA that would interfere with it. This is a super temporary thing thought since as soon as you transcribe more mRNA the blocked ones become irrelevant. So we need to interfere with whatever causes MSTN to be transcribed in the first place. Which my guess may be PPAR delta

  • Also if the RNA is that short lived we could make a small plasmid instead that all it does it crap out miRNA. It'd still break down but it'd last a lot longer than the RNA on its own. 
  • Also I found out that a local option won't be possible. It is exported from skeletal muscles and circulated in the bloodstream. So it'd have to be knocked out in the full body. 
  • PPAR-delta is unrelated I guess. It is pretty cool though
  • Ya ok so question becomes, do we want to just target the myostatin itself? or the thing it activates? Maybe the activin has other roles and myostatin doesn't? if that's the case it'd be ebst to leave it in place. Or do we target both for good measure?

    Once we decide that we need to design the plasmid that'll produce the miRNA. The sequence should be repeated a bunch of times on the plasmid so that lots of copies are formed. Ideally we make it so that the repeats are the sequence we buy so we can just stick the copies onto themselves and then select for plasmids of the length (has the number of copies) we want. If it's got a bunch of repeats we would only need one spot for rna polymerase to bind and then it can just go along the loop and make a bunch of copies before hoping off. Make it so that the bit between each copy is where it will get cut. SO the plasmid would basically be:


    The RNA that results will get processed by a protein to remove the poly A tail and cap. Then exported to the cytosol. Finally it will be cut up by Dicer and will then separate to become active and useful. The plasmid itself should breakdown in a couple weeks to months and since the RNA will only bind to myostatin mRNA or whatever we pick, it shouldn't matter if it's transcribed in odd places if the nanoparticles accidentally deliver it to the wrong cells.
  • Could we mutate the MSTN gene several times in lab rats and see the potential effects in order to find the exact mutation that would decrease the GDF-8 levels for a more permanent modification? The only problem I see is that if the gene mutation goes wrong it could start producing some other protein and potentially have negative effects.
  • Few reasons. First either way we're looking for either no myostatin present or non functional versions present. If we were looking at the mutations that means a lot of sequencing and to get that into a person would require far more complicated methods of delivery which rack up pricing. All we want to do with this project is temporarily disable myostatin's production so you have a month to bulk before you go back to normal. This way if they are able to test for it, it'll be out of your system eventually.
  • Also point mutations as you suggest are a pain in the ass.
  • Ahh ok, it dose seem a bit more difficult than what you guys suggest.
  • "do we want to just target the myostatin itself? or the thing it activates? " 

    I think just inhibiting it before it is produced would be easier, wouldn't it? Or did I misunderstand?

    A myostatin inhibitor has been talked about a lot but there might be a few other targets that might be easier to pull off (like the PPAR-delta). I've collected a small list of potentials. I don't want to derail the thread though. I could make a new thread for general discussion of feasibility if you think that is wise.
  • Well since myostatin itself is a protein it is easy to interfere with it's production by the way of messing with the mRNA carrying it's structure. As soon as the rna binds to it no myostatin is produced and that message will break down. So if we wanted to block it from being produced at all we'd have to stop the mRNA from leaving the nucleus which is harder.
  • ok, gotcha. Have you done much with morpholinos?
  • Ah the reasons I love this forum, you learn all the interesting and useful stuff with non of the fluff. I've never heard of them until now but I like them for this project as well. If they are easier to design and make they may work well for this
  • They are sturdier. I don't know about easier to make.
  • K read up on them. So they do almost exactly the same thing, the difference is they don't break down easily so once the thing they're bound to breaks down they pop off and keep working. Whereas normal miRNA would degrade as well. Hence why I suggested a plasmid since it will continue to replace the degrading RNA and would only become non active when the cell carrying it dies. SO you'd only need an injection of new plasmids every few months. I think morpholinos will have the same problem in that they're only in the cells we deliver them too and as soon as that cell dies it ends up getting flushed from your system so either way you'll need to re-dose occasionally. So the question is of the two methods which is easier to produce and deliver and which has the greater affect.
  • An advisor just dropped some Intel on me and recommends checking into this before we decide. The bleeding edge of RNA tech is wielded by ModeRNA Therapeutics.

    They have some proprietary techniques that are laid bare in these instruction manuals....err...patents

    One of them is supposed to help us. I'll start digging through them in the morning.
  • I found this website quite useful in explaining mRNA processing.  Just look around for a while and you should get a pretty good idea on how its done.

  • I love patents. Big kid instruction booklets. I'll read through them when I can, probably tomorrow at some point. @kingofrandom ya that's the basics of how mRNA works. Basic biology really. The fun bit is what happens when you start screwing with that, which is what we're trying to do.
  • So tried reading through one of those patents. HOLY HELL that's a lot of bloody information. That said, they have one specifically for nano particle design that'll deliver our package to specific cell types. So we can just borrow that.

    Also they're focus seems to be more on mRNA delivery rather than interference so we may still be shit out of luck and on our own for that.
  • Yeah. I was looking into their preRNA stuff and I don't get it. It is a lot of info.
  • I think we're better off on our own. You'd need a team of highly trained monkeys to get through that patent clusterfuck
  • Agreed. I haven't given up on figuring them out, but it's a side project at this point. So are you thinking plasmids?
  • Ya in some form. I feel it's the easiest way to go really. Build a plasmid that will make as many copies of the micro rna as possible. Pick a promoter that will be fairly consistently making copies, maybe repeat the sequence a couple times. It's short so it shouldn't be hard to cram in some extra copies of the gene. We'll have to pick the rna polymerase binding site and any enhancers and such that we need. 
    So this is really in two-three parts. 1 design the actual rna sequence. 2 design the control mechanism. 3 pick the delivery method

    which would you like to tackle first?
  • edited March 2015

    well that's helpful

    EDIT: never mind. It either only works for plants or i can't get it to work properly
  • it's been a genetics-y night. 
    ok so here goes. 
    that is the mrna sequence for mysotatin. If you scroll down you'll see a list of highlights and points of note of the gene. If you click CDS it'll highlight an area of extreme importance, i.e the start codon onwards. So our microrna needs to interfere in that region. I'd say give it a 20bp gap from the start codon, that way anything consistent with another gene in terms of signalling is out of the way. We set the length of our microrna (and adjust as we start testing possibilities) and start picking sequences from the area and run them through blast. see if anything pops up as a match. if it does, then it'll likely interfere and we need to try again. repeat until no matches.
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