Using Viruses to Rebuild Damaged Nerves

As much as we take it for granted in our everyday life, human history is built upon the landmarks of self-assembly — a process in which a system’s components organize into a beautifully ordered and functional structure, without external direction.

One popular example is DNA, where nitrogenous bases interact with each other via hydrogen bonding, forming an antiparallel, double-helix structure. The marvelous thing is such small molecules make up the 1.5 Gigabytes of human genome, which stores all information about your appearances, athletic ability, or even political inclinations!

What’s amazing about self-assembly is, if you understand the chemical and physical properties of naturally self-assembling molecules, you can manipulate them to potentially engineer high-impact solutions.

Inspired by this natural phenomenon, I want to propose how we can genetically engineer phages, specifically M13 viruses, to self-assemble and rebuild damaged nerves for billions of patients with neurological diseases.

How self-assembly works in nature

To recapitulate the self-assembly process for virus, we first need to understand how it works in nature. As M13 has a filamentous structure, let’s look at how similar natural helical — such as collagens, chitins, cellulose — assemble!

It turns out that these structures undergo a process called morphogenesis, where they are secreted in a timely manner, inducing flow force and organizing themselves according to the flow direction. The local environments, such as confined spaces, curvatures, and pre-existing structures, also influence the orientation of helical molecules.

Like the pieces of bricks that build on top of one another to create a pyramid, the helical nano fibers assemble to form hierarchical, macroscopic structures that plays a specific role within the organism’s body. It can be the orthogonally arranged corneal tissues in the eyes, the cholesterically rotating matrices on colored mammalian skins, or helicoidally arranged bone structures.

Pattens in the human eye, bone marrow, and leopard’s skin

All of these structures we observe today, in fact, fall under Alan Turing’s “Theory of Morphogenesis” in 1952, which states that “a system of two different interacting molecules, called morphogens, which could establish chemical gradients through a “reaction-diffusion system.” The results of these substance interactions are dependent on just four variables per morphogen — the rate of production, the rate of degradation, the rate of diffusion and the strength of their activating/inhibiting interactions.

Although biological processes often include more than two morphogens, Turing’s mathematical model introduced a predictable mechanism for pattern formation with a clear idea of the four elements that come into play. We will keep these four variables in mind as we recapitulate the morphogenesis process for M13 viruses.

Understanding the physical and chemical properties of M13 viruses

M13 phage possesses a fibrous shape with a helical surface, which is covered by 2,700 copies of the pVIII major coat proteins with a fivefold helical symmetry with an additional five copies of the minor coat proteins pIII and pIX located at either end. Phage has a huge potential for the construction of nanomaterials at our will as the proteins on their surface can be easily engineered by modifying their DNA.

Due to their filamentous structures, M13 phages can assemble liquid crystals — a state of matter between that of a liquid and a solid. High concentrations of filamentous phage will spontaneously align and form a liquid crystal, due to the intermolecular forces between neighboring phage particles. As the substrate is slowly pulled from phage suspensions, a liquid crystal phase transition of the phage solution is induced at the air/liquid/solid. Basically, M13 phages have the ideal properties to self-assemble into various nanostructured films and scaffolds that can benefit tissue engineering.

The liquid-crystal proper of phages — Engineered M13 viruses moving upwards on the substrate to form different patterns and scaffolds

Programming M13 phages for neural regeneration

Step 1: Genetically engineer M13 phage to display different proteins on its capsid

By using CRISPR to edit its genome, we can engineer M13 phage to display signaling motifs on their major protein coats, specifically RGD and IKVAV peptides at the N-terminus of protein VIII.

RGD is a type of cell adhesion molecule called integrin, which provides essential links between outside of the cell and the signalling pathways inside of the cell. Such molecule can play important roles in cell behavior, such as apoptosis, differentiation, survival, and transcription.

On the other hand, IKVAV is a protein network called laminin that resides outside of the cell and is critical in promoting neural cell adhesion and neurite extension. Both RGD and IKVAV engage in an intimate relationships, and they work in harmony to provide a platform for cells to grow.

Step 2: Amplify the number of phages

Bacteria host bursting releasing numerous phages

The collected phages can then be amplified using bacterial cultures and purified through polyethylene glycol precipitation. Due to their large number and physical properties, phages will assemble into a liquid crystal scaffold where neural cells can grow.

Step 3: Modify the phage solution to control morphogenesis

We can control the thermodynamic (phage solution concentration or ionic concentration of solution) and kinetic factors (pulling speed or surface chemistry) to modulate the interaction of the phages and build phage structures for tissue engineering at large scale.

For example, by controlling the phage concentration, we can control the number of particles engaged in self-assembly. By controlling the ionic concentration, we can tune the electrostatic forces bringing phage particles together. Through control of the pulling speed, we can tune the force field that interacts with the substrate and particles adsorbed onto the surface. In addition, the wettability (i.e., hydrophilic or hydrophobic surface functionalization) of the solid substrate that is supposed to pull the phages up can be controlled to selectively deposit phage at precise locations on the substrate.

Overall, M13 phages provide a scaffold where neural cells can grow, differentiate, and replicate effectively. Unlike current methods of generating scaffolds, such as ink-jet printing or dip-pen nanolithography, M13 phages provide a bottom-up approach that is 10X cheaper, faster, and easier to manipulate. We can easily scale it up to form aligned nanofibrilar structures to control the macroscopic behaviors of neural cells. The small virus can lead to a huge impact in tissue engineering and treating neurological diseases in years to come!

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