Myostatin inhibitors again

[Bryan Bishop]

Here's a project proposal from yashgaroth.

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by Yashgaroth » Wed Feb 29, 2012 10:58 pm

Out of the many possibilities in DIY gene modding, myostatin inhibition to augment muscle growth is the most immediately doable. For a thorough and current review on the topic, I highly recommend S.J. Lee's recent paper, available at http://www.jhu.edu/sejinlee/downloads/Lee%20IEMAMC%202010.pdf .

If you don't feel like reading that first, here's a brief overview: myostatin is a protein which, in skeletal muscle, is part of the network that negatively regulates muscle mass. By inhibiting myostatin and/or its related proteins, up to a quadrupling of muscle mass has been observed in transgenic mice, with naturally inhibiting mutations in the gene leading to the “double-muscled” phenotype found in certain breeds of dogs, cattle, and that one kid in Germany you may have read about.

My hope is to express a protein called follistatin in muscle tissue, as follistatin is a highly potent inhibitor of myostatin. Currently, follistatin is receiving much interest for treating muscular dystrophy; a clinical trial starts this month, based on research that culminated in this paper:http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852878/ . Basically, they're doing what I plan to do, although they will be using a viral vector.

Since viral vectors require a great deal of expense to produce, as well as a regimen of immuno-suppressive drugs, they don't really work for the DIY approach right now. Among the non-viral vectors, plasmid electrotransfer (or electroporation) is the most powerful, and, as a bonus, it requires no reagents other than electricity.

The plasmid backbone is fairly simple: standard CMV promoter, synthetic 5' intron, SV40 polyA and enhancer, all to increase expression of the follistatin transgene as much as possible. Beyond that, I'm thinking a couple of short S/MAR insulators flanking the antibiotic resistance gene, which should be CpG-depleted to prevent chromatin condensation.

Anyway, I'm not too good at writing long and coherent statements, so the rest of this is some of my notes detailing the execution of the project. I welcome any comments and critiques.


On the topic of which gene to express, a few alternatives to follistatin do exist:

• Anti-myostatin monoclonal antibody (developed by Wyeth), perhaps the simplest method, from the old “raise an antibody against it” school of thought. Made it to clinical trials, but was not pursued as it caused skin rashes.


• Fusion of a mutant myostatin propeptide to an antibody Fc region (fusion to increase the half-life at least 100-fold). The propeptide normally gets produced with myostatin, and blocks its acivity until cleaved at a later point, which “activates” the mature myostatin; the mutant propeptide is resistant to said cleavage, and sequesters myostatin. Showed some efficacy in a canine model, but has not yet been pursued.


• ACVR2B solubilized receptor (Acceleron Pharma and Amgen), again fused to Fc for half-life extension like the propeptide above. ACVR2B is the “main” receptor for myostatin in muscle, so the soluble extracellular receptor was intended to soak up circulating myostatin, similar to the previous two approaches. This worked incredibly well in mice, but triggered an increase in hematocrit in human trials (due to its non-muscle effects), so it got shelved.

All of these options were pursued by injecting recombinant proteins into the bloodstream, rather than a gene therapy approach. This means that off-target effects on organs other than muscle must be carefully controlled. With gene therapy, the transgene can be targeted to a specific location, where expression will occur along the length of the muscle fiber, but not spread to the heart, skin, gonads, etc. This also opens up a number of other possibilities, including intracellular protein expression or RNA interference, which would not be possible with a protein-injection-based therapy. For example:

The ACVR2B soluble receptor comes a close second to follistatin as a choice of transgene. One simply removes the intracellular kinase domain, with which the receptor would normally transmit the myostatin signal. The extracellular myostatin-binding domain is all that remains, which then soaks up myostatin. Note that this would retain the transmembrane anchoring region, safely limiting it to the injected muscle. Unlike the Fc fusion protein mentioned above, this should not affect RBC count.

Please note that I say 'myostatin' here, but ACVR2B and follistatin appear to bind additional muscle-limiting ligands beyond myostatin. Myostatin-knockout mice still exhibit increased muscle growth with administration of either transgene, indicating at least one other factor at play.

Plasmid production:

Plasmids can be grown in a standard e. coli culture, and purified either with the commercial endofree giga-prep kits (Qiagen or a cheaper competitor) or, with some investment but a lower $/mg cost, column chromatography: standard alkaline lysis protocols, followed by a Triton-X114 precipitation (to remove endotoxin), and a DEAE + hydrophobic interaction column setup should give acceptable purity of plasmid for injection.

Electroporator:

The parameters for large mammals haven't really been nailed down yet, so I'd like one with variable settings that cover the range of the previous experimental techniques. This would be a unipolar square-wave generator capable of 20-200 volts, 0.1-0.5 amps, with pulses of 10-50 milliseconds in duration, with a variable number and spacing of said pulses. Optimal settings for humans are hard to pin down due to the difficult in assaying a reporter gene in humans, so I am aiming for the maximum tolerable settings that do not produce unnecessary tissue damage.

My favorite applicator so far is a carriage enclosing two 1mL needles, which simultaneously inserts the needles while depressing the plunger, ensuring an even distribution of plasmid along the length of the needle. Once injection is complete, the needles act as the electrodes.

The only other modern technique in practice involves multiple electrodes positioned around an injection needle, which then fire in different combinations. This is seen in most clinically available electroporation devices, and while I prefer the two-needle technique, the pulse generator should be easily adaptable to an arbitrary arrangement of needles.

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http://transhumani.com/topic61.html

- Bryan
http://heybryan.org/
1 512 203 0507

irc.freenode.net ##hplusroadmap

[Nathan McCorkle]

How would the plasmid target muscle cells only, not including the heart?

Mouse liver glowed when intranasal plasmid was administered, so electroporation may not be necessary

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[mad_casual]

On Mar 1, 11:30 am, Nathan McCorkle <nmz...@gmail.com> wrote:
> How would the plasmid target muscle cells only, not including the heart?

Myostatin and MyoD are skeletal muscle genes and electroporation is
local. IMO, the concern is the other way; non-genetic DIYBio muscle
growth has been going on for 60 yrs., there's plenty of non-clinical
evidence to suggest that rapid growth of muscle and decades of walking
around with tons of extra muscle (pharmacologically induced or not)
doesn't do your heart or connective tissues any favors. If I were 20
yrs. old, 6'2" and bulletproof, I'd probably try this, once my kids
are out of the house and I'm having trouble keeping up in the gym, I
might try something like this. Right now, it just seems like a bad
idea.

[Max Berry]

Hi fellas, original author here.

Nathan, the overlapping area of the electrical field and the plasmid
injection site is what determines the location of expression; In this
case, the major muscle groups. Some immune cells passing through the
injection site at the time may also get transfected, but the vast
majority will be local muscle fibers.

Generally speaking, making a mouse inhale a plasmid will generate some
gene expression, perhaps even in the liver, but mice are much more
receptive to naked DNA transfection than larger mammals. While the
amount of transgene required for a visible effect here isn't super
high, the efficiency increase of at least 100x that I expect from
electroporation will likely be necessary, as I prefer as few
injections/electrocutions as possible.

Mad_casual: it's true that this could cause just as much wear & tear
on the heart and tendons as muscle gained from weightlifting, though
possibly less since the growth occurs without the strenuous exercise.
I am in fact 20(-something), 6'2", and consider myself bulletproof, so
I'm willing to accept the risks there :P

[Nathan McCorkle]

On Thu, Mar 1, 2012 at 9:21 PM, Max Berry <maxb...@gmail.com> wrote:
> Hi fellas, original author here.
>
> Nathan, the overlapping area of the electrical field and the plasmid
> injection site is what determines the location of expression; In this
> case, the major muscle groups. Some immune cells passing through the
> injection site at the time may also get transfected, but the vast
> majority will be local muscle fibers.
>
> Generally speaking, making a mouse inhale a plasmid will generate some
> gene expression, perhaps even in the liver, but mice are much more
> receptive to naked DNA transfection than larger mammals. While the
> amount of transgene required for a visible effect here isn't super
> high, the efficiency increase of at least 100x that I expect from
> electroporation will likely be necessary, as I prefer as few
> injections/electrocutions as possible.

Yeah but how often would you have to inject/electroporate? I think
most people would rather inhale a few milligrams of DNA aerosol
through their nose every once in a while. It also seems like with
electroporation of muscle you'd get uneven expression, and thus
develop muscle deformities/disfigurement.

--
Nathan McCorkle
Rochester Institute of Technology
College of Science, Biotechnology/Bioinformatics

[Max Berry]

Since muscle fiber nuclei aren't turned over in the same manner as
most other cells, due to the syncytium they form, the plasmid keeps
expressing the protein for quite a while, barring promoter methylation
or other forms of downregulation. In most other organs, transfected
cells will eventually die and be replaced, so you lose the plasmid
over time. I had considered using CpG depleted and/or muscle-specific
promoters, both of which might increase the duration of expression,
but the unbridled power of a wild-type CMV promoter has never been
exceeded. I can't say for sure how often/many these injections would
need to be, that's why I want to try it out.

As another exploitable characteristic of muscle syncytium, the
transfected nuclei spread along the entirety of the fiber's length,
and the protein will diffuse into the immediate area to sequester any
local myostatin, in a paracrine fashion. As there will likely be a
need for multiple injections per muscle group, those can be spread
laterally to reach most of the fibers.

I would be totally supportive of huffing the plasmids instead, but I
can guarantee that the expression levels will be extremely low. Also,
I'd like to avoid any off-target effects from follistatin, unlikely as
they may be.

[mad_casual]

On Mar 1, 8:21 pm, Max Berry <maxbe...@gmail.com> wrote:
> Mad_casual: it's true that this could cause just as much wear & tear
> on the heart and tendons as muscle gained from weightlifting, though
> possibly less since the growth occurs without the strenuous exercise.
> I am in fact 20(-something), 6'2", and consider myself bulletproof, so
> I'm willing to accept the risks there :P

I wasn't concerned about wear and tear. Let me say it this way, I have
some experience gaining muscle without exercising; or to a level
higher than what exercising was inducing. I'm sure you're aware that,
myostatin knockout and mutant infants die more often than their wild-
type counterparts. I'm sure you know that anti-sense myostatin alone
makes muscular dystrophy worse because it enhances contractile
elements without enhancing connective and structural elements. (It is
touted/suspected/makes sense that)Humans hypertrophied in excess of
what they can do exercising suffer ill effects along the same pattern.
Hell, at 6'2" you or someone you know at the same height probably had
tendinitis/tendinosis simply from growing bigger naturally.

Not telling you not to try it, it sounds fun and it sounds like now
(or soon) is the time to try it; I'm just saying my kids are impressed
with what I can lift, I'm thankful that I can lift them (most of the
time). You sound like you know the harm you could be doing to yourself
and I couldn't stop you if I wanted to; it was the proliferation on
the internet (with associated optimism) that gave me reservations. Am
I a muscle/bio geek or just way over-parenting? Either way, I look
forward to results or publications as they become available.

[Max Berry]

Regarding myostatin knockout animals, the protein has a wide range of
functions outside of muscle; in fact, I'm surprised they survive as
well as they do, since very few gene knockouts are able to be shrugged
off with such relative ease. If one considers the scenario of
myostatin inhibition specifically in the muscle, and after the onset
of adulthood, many of the side-effects could potentially be avoided.
Just like with anabolic steroids, the off-target effects are what
really limit you.

I am in agreement with your skepticism on its use in treating muscular
dystrophy; it's akin to filling up the tank on a burning car. Sadly,
MD is the only excuse by which researchers can get funding, since
enhancement isn't really the focus of the clinical trial system, at
least these days. I'll keep you updated if it works.

[Matthew Avant]

Well, if you need a guinea pig, I'm 20 and bulletproof and think this sounds incredibly cool.