Disorders like muscular dystrophies are difficult to treat using gene therapies because of a size problem. The dysfunctional genes in these conditions are often very big, and current methods used in gene therapies are unable to deliver genetic loads large enough to treat them. Now scientists from the University of Rochester, CANbridge Pharmaceuticals, and elsewhere have developed RNA-based technology that could be the key to developing gene therapies that work for these diseases.
Full details of the technology, dubbed stitch RNA or StitchR for short, are published in a Science paper titled, “Ribozyme-activated RNA 1 Trans-ligation Enables Effective Gene Therapies for Muscular Dystrophies.” StitchR solves the problem of delivering the full gene in a single vector, which is currently impossible, by delivering the two halves of a gene separately using an efficient dual vector system. Once in a cell, both segments generate messenger RNAs that join together to restore missing or inactive protein expression in the disease.
According to the paper, StitchR restored expression of large therapeutic muscle proteins to normal levels in two different animal models of muscular dystrophy. Specifically, StitchR enabled expression of the dysferlin protein, which is lacking in individuals with limb girdle muscular dystrophy type 2B/R2, as well as the dystrophin protein, which is absent in patients with Duchenne muscular dystrophy.
Scientists discovered StitchR several years ago in the lab. They noticed that two separate mRNAs that were cut by ribozymes became seamlessly ligated and translated into full-length protein. What seems to happen is that when ribozymes cleave or cut RNA, they leave ends that are recognized by a natural repair pathway.
“Similar to when CRISPR enzymes are used to cut DNA, the CRISPR enzymes are just the scissors, and it’s a cell’s natural repair enzymes that glue the DNA back together,” explained Douglas Anderson, PhD, lead study author and assistant professor of medicine in the Aab Cardiovascular Research Institute at the University of Rochester School of Medicine and Dentistry. Something similar could be happening here but for RNA. “The ribozymes are acting as the scissors and the cell’s natural repair pathways are able to join the two RNAs back together. It’s remarkable that two separate mRNAs are able to find themselves and that the process can be so efficient.”
Furthermore, Anderson and his team report that the stitched mRNAs behave essentially the same way as their natural full-length counterparts and are able to produce functional full-length protein. The scientists have also optimized the efficiency of the process over 900-fold from their initial experiments.
“Other dual vector approaches have been in development for decades but have been plagued by lack of efficiency and the production of less than full-length products,” Anderson noted. “Because StitchR occurs at the level of RNA, we can control and ensure that only the full-length protein product gets made. This differentiates StitchR from other dual vector technologies, for example, inteins, a protein-ligation technology, which can be efficient but requires the expression of smaller protein fragments that may have unknown effects in a cell.”
Anderson’s lab is now in the process of forming collaborations with other research labs and generating StitchR vectors for use in therapies for various diseases associated with large genes. The data shows that StitchR can be coupled with different vector types and it seems to work efficiently with any mRNA sequence. “StitchR is really plug and play at this point. The sequence requirements for StitchR are minimal, and we’ve now tested this with many different genes and sequences,” Anderson said.
