Being one of the earliest focused upon diseases for CRISPR to treat other than retinitis, Duchenne muscular dystrophy has seen a lot of scientific effort be put into finding a cure or at least a palliative treatment to restore some amount of strength. Previous attempts have involved various types of viral vectors to help remove the nonfunctional and mutated dystrophin gene, while possibly inserting a working copy.
But proper delivery of the CRISPR components into the required cells has remained a challenge to this day. For a complex eukaryotic group of organisms like mammals, achieving operating gene expression even after CRISPR application has remained elusive. It works to some extent, but at a much lower level than desired. And that’s saying something when talking about diseases where even a 5-10% expression level would relieve most of the symptoms of the condition.
The Golden Answer
Now, researchers at the University of California, Berkeley have decided to try an entirely new vector mechanism, one that doesn’t require viruses as a delivery instrument. Instead, they’ve made a sort of call-back to the old gold nanoparticle “gene gun” technology, one of the original gene editing methods. Though without the necessity of particle bombardment of cells like that method utilized.
This novel technique, which they have taken to naming CRISPR-Gold, uses the aforementioned gold nanoparticles that have been conjugated with thiol-modified oligonucleotides (DNA-Thiol) that help to give the entire thing structure. This allows the gold to bind with the donor DNA that has the gene of interest in a single stranded form. The Cas9 proteins and the guide RNA can then be complexed with the donor DNA, binding them together.
With all of that done, the medley of DNA, gold nanoparticles, and Cas9 proteins are encapsulated in a polymer specifically designed to induce target cells to take the entire framework inside of them. This is done by the polymer causing endocytosis that has the formation moved inside of the cell within a vacuole, which the polymer then breaks open to release itself and everything it contains within it.
A Well-Timed Delivery
The gold nanoparticle is then able to deliver all of the components to the cellular genome, allowing the Cas9 proteins to get to work. The major benefit to this scheme is that it keeps every part together and activates it all at once. An issue with prior delivery methods is that the Cas9 would be used to cut out the offending mutated dystrophin sequence, but would be unable to successfully insert the correct sequence due to the timing of when every part reaches the genome.
If the genome doesn’t have the gene ready for insertion when the other material is removed, it cannot start the process of homology-directed repair (HDR) that would use the donor DNA as a template for correction. With CRISPR-Gold, the components all arrive simultaneously, allowing the cutting and inserting/repairing to happen at the same time. Being able to induce HDR is a big step toward increasing the efficiency and success of gene insertion.
This improvement in the delivery system is shown from the experimental results. The mice in the study had an 18 times increase in gene uptake as compared to methods like just injecting the CRISPR and donor DNA by themselves into a cell. This resulted in 5.4% of the genes in the area uptaking and expressing the corrected dystrophin gene, leading to a visible enhancement in the mice affected by Duchenne muscular dystrophy. Overall, they saw a two-fold gain in muscle development and total strength.
A Localized Issue
The researchers also checked to ensure that there wasn’t an immune system response that might complicate the delivery system. But after two weeks of immunogenicity testing, they found no escalation of immune factors, even after multiple injections with CRISPR-Gold, implying that it has a high capability for use as a therapeutic treatment.
The one major downside with the approach is that, due to it being done via injection, the cells affected are only contained within a localized area around that point. For diseases that have a confined area of activation, this won’t be too much of a concern. But for systemic disorders, CRISPR-Gold won’t be as effective.
The scientists are still looking into ways to change this problem and to also increase gene uptake further. For now, however, this current process is helping to solve one of the final outstanding obstacles to CRISPR success as a medical remedy. It may be no time at all before we have active human trials leading to cures for Duchenne muscular dystrophy and more.
Photo CCs: Embryonic smooth muscle cell from Wikimedia Commons