Thanks to Maura Davis at Northwestern for sending me a version of the submitted manuscript, mine among the many requests Prof. Stupp’s lab is receiving for this important study.
It’s not a question of if, but when. We will enable people with spinal cord injuries to regain function, and to walk again. Paralysis may not be doomed just yet, but it’s starting to get a little worried.
We’re to the point where we know a fair amount about stimulating the regeneration of human neurons. We just have to figure out how to do it better in situ, in a real spinal cord. And we really are getting there. Before I go on too long, just watch this six-second video of mice with spinal cord injuries, with and without a single dose of a brand new kind of treatment:
Duuude! That mouse can move his legs again!
The key to stimulating the regeneration of neurons is to trigger particular receptors on their surfaces, not merely once but over a period of time. We all possess proteins known to do just that. But injecting proteins into an injured spinal cord can be very clumsy, because the key proteins aren’t very stable, the body clears them out, and they often have difficulty accessing the receptors they are supposed to stimulate.
Now, researchers at Northwestern University, with lead author Zaida Álvarez and principal investigator Samuel Stupp, have made serious inroads toward defeating this problem. By applying principles of synthetic chemistry, physiology, materials science, and protein engineering, they’ve developed a gel-based delivery system that holds the most promise we have seen in some time for those with spinal cord injuries and neurodegenerative disorders. Their findings are reported in the November 12 issue of Science.
For some brief context:
“Currently, there are no therapeutics that trigger spinal cord regeneration,” said Stupp, an expert in regenerative medicine. “I wanted to make a difference on the outcomes of spinal cord injury and to tackle this problem, given the tremendous impact it could have on the lives of patients. Also, new science to address spinal cord injury could have impact on strategies for neurodegenerative diseases and stroke.
"It's the most important paper I've ever written, because I have never integrated so deeply so many parts of science.”
Stupp and his team have been working on this problem for many years, with incremental successes, but this kind of breakthrough is viscerally different. It’s one thing to see neurons growing under a microscope, but it’s quite another to see a paralyzed animal move its legs. So what was the last nudge that put them over the top? It all came down to a string of just four amino acids.
The great coach Vince Lombardi appreciated the fine line between winning and losing. He used to say, “Football is a game of inches, and inches make the champion.” Biology is like that too, but it’s much finer than that: Biology is a game of Ångströms.
We’ve had some tantalizing pieces of knowledge about neuron regeneration for a while now. We know, for example, that neuron growth can be stimulated by a protein that occurs in the body called laminin. Laminin forms scaffolds by linking to itself, giving the neurons a framework to grow on. But it also triggers receptors on the surfaces of neurons called integrins, and this tells the neurons to turn on their growth pathways.
Here we see what happens when an adult neuron is stimulated in culture by adding laminin. It sprouts “arms”, or neurites:
We even know the specific short sequence of the laminin protein that triggers the neuron’s integrins: IKVAV. (That’s isoleucine-lysine-valine-alanine-valine, for those keeping score at home.)
There is another stimulatory protein called fibroblast growth factor (FGF-2), and this one is especially important for angiogenesis (the formation of new blood vessels), which is also critical for regenerating tissue. It can induce cells to grow and change their shape, like this shot of corneal cells elongating when exposed to FGF-2:
And guess what? We also know the specific short section of FGF-2 protein that does the stimulating: YRSRKYSSWYVALKR. (I’m not typing that one out, so if you’re curious, check out the code.) The cell receptor that senses this snippet of FGF-2 is called FGFR.
So we know the switches we need to turn on (integrin and FGFR), and we know a short protein sequence for each that will get the job done. Great! But now how do we deliver those short little sequences to the proper receptors, right inside damaged nerve tissue? And again, it’s not a matter of flipping them on for a second and being all set; we have to keep the stimulating signal on, to give the tissue a chance to regenerate and heal. You can’t just squirt some protein in there and expect success.
Knowing this, Stupp’s lab has been working since at least 2004 on nanofiber-shaped “supramolecular” polymers to improve delivery of these key protein snippets. Here’s an early example of that which lays out the principles visually very well. The key sequence — here IKVAV — sits atop a lollipop-like molecule, at the bottom of which is a hydrophobic (water-hating) segment. The hydrophobic parts flock to each other and hide from the water around them, and so these “lollipops” self-assemble into nanofibers with IKVAV all over their surfaces. The fibers form a network, imparting a gel-like quality to the fluid they are in:
In the 2021 version, they’ve doubled it up by also including YRSRKYSSWYVALKR (FGF-2 mimic) display “lollipops” within the same nanofiber structure. Those will self-assemble right along with the IKVAV “lollipops” because they share the same hydrophobic center. So now you have a combo fiber that displays both of those signals all over its surface and forms a mesh to create a gel. There’s no reason you couldn’t add a third signal, or change the signals up completely, to be applied to other therapies.
So NOW we get to those four key amino acids that made all the difference. Several important structural tweaks have been made since the 2004 version shown above, but that figure still illustrates the concept very nicely. (And we don’t want to publish new figures we don’t have copyright clearance on, do we?)
Looking at the figure above, you can see that each “lollipop” has a red-colored section in the middle, made up of amino acids. That section obviously connects the bioactive tip to the hydrophobic center and gives the fiber some width, but it does much more than that.
The red section has two parts. The outer part, nearer the tip, provides water solubility to help with self-assembly and also to make the fiber compatible with its water-rich surroundings. That outer part, in its current iteration, is EEEEG (four glutamic acids and a glycine).
And the inner part, nearer the center, has been retooled here in 2021 to provide what turns out to be the big breakthrough: MOTION.
Let’s say it’s dark and you’re trying to find your glasses on the end table. What do you do? You keep feeling around until you hit them. Well, up to now, these bioactive tips weren’t feeling around. They were kind of static. If they happened to land right upon a receptor, great, but if not, then not so effective. They’ve got to be in the right position and orientation to make contact, much like another well-known docking procedure where movement was key…
So to improve that inner part, eight different short amino acid sequences were tried. All were combinations of valine (V), alanine (A), and glycine (G). It’s known that those three are especially pivotal in the ability of proteins to form structures called beta-sheets, which tend to be rigid, and that the order of good beta-sheet formers goes V > A > G. So the sequences they tried, they hoped, would give a range of rigid to flexible: VVAA, AAGG, AVGG, VAGG, GGGG, VAAA, AVAA, and AAAA.
The eight different nanofiber types (displaying only IKVAV at the surface, for now) were tested outside of animals first, and three spectroscopic techniques (along with mathematical simulations) all seemed to agree that AAGG and GGGG were allowing especially good motion of the IKVAV tips. Fibers containing these two were also the best at stimulating neural cells in a glass dish. It did seem, then, that motion was important for effectiveness.
They tested that idea further by adding calcium ions to the best gels. That would tend to suppress motion by electrostatically holding fiber “lollipop” segments together. Indeed, the addition of calcium did suppress motion and reduced the nanofibers’ ability to stimulate cells.
So then it was time for the big test: injection of the gel into mice with spinal cord lesions. For combo fibers (the ones with both IKVAV and YRSRKYSSWYVALKR on their surfaces), AAGG turned out to be better than GGGG because it was better at making the combo fibers form nice water-soluble gels. So they tried three kinds of fibers: (1) IKVAV (AAGG) only; (2) IKVAV (AAGG) plus YRSRKYSSWYVALKR (AAGG); and they also threw in (3) IKVAV (AAGG) plus YRSRKYSSWYVALKR (VVAA), just to see how a relatively rigid VVAA segment would compare.
They found that in terms of observable neural regeneration, plain saline solution did nothing as expected, (1) was just OK, (2) was pretty good, and surprisingly, (3) was twice as good as (2)! It’s not entirely clear why (3) was the best, although the authors did gather crude evidence that it has to do with the interaction between the two different types of tips within the same fiber.
Sometimes it’s all about knowledge and prediction, but sometimes it’s also about simply trying different things. As Albert Einstein is often quoted as saying (and probably apocryphally, but we’ll let that slide), “If we knew what we were doing, it wouldn’t be called research.”
Here is an injured spinal cord treated with (3), and you can see, in red, axons that have regenerated within the tissue after a single dose, in about 4 weeks:
Not only did axons and vasculature regenerate, but they did so in a productive way. GEN sums that up nicely:
By sending bioactive signals to trigger cells to repair and regenerate, the breakthrough therapy dramatically improved severely injured spinal cords in five key ways: (1) the severed axons regenerated; (2) scar tissue was significantly diminished; (3) myelin reformed around cells; (4) functional blood vessels formed to deliver nutrients to cells at the injury site; and (5) more motor neurons survived.
And there you have a mouse who can move his legs again.
To top it off, the gel is bioresorbable and is metabolized by the body within 12 weeks.
Encouragingly, human stem cells in a dish showed the ability to differentiate into neurons with the treatment, and obviously human clinical trials will start as soon as is practicable. Stupp, for his part, is “extremely confident and excited” that the main features of the therapy will hold up in humans.
I know I’m not a regenerative expert like Stupp, but I must say I do share his optimism on this. The pieces keep on falling into place. Many of us will live to see the day…..