StitchR is a novel gene therapy method that addresses muscular dystrophies by delivering large genes in two segments, allowing the restoration of essential proteins in animal models.
Gene therapy shows promise in treating various diseases, but a significant challenge exists for severe conditions like muscular dystrophies: the size of the genes involved. The genes causing muscular dystrophies are typically very large, making it difficult for current delivery methods to transport these substantial genetic materials into the body. A new approach called “StitchR” overcomes this challenge by separately delivering two halves of a gene. Once inside a cell, these DNA pieces produce messenger RNAs (mRNAs) that easily combine to re-establish the expression of a protein that is either missing or not functioning properly due to the condition.
As reported in the journal Science, StitchR—short for “stitch RNA”—successfully restored normal levels of large therapeutic proteins in two distinct muscular dystrophy animal models. This technique enabled the expression of Dysferlin, which is deficient in those with limb girdle muscular dystrophy type 2B/R2, as well as Dystrophin, a protein lacking in patients with Duchenne muscular dystrophy.
Duchenne muscular dystrophy (DMD) is the most prevalent early-onset form of muscular dystrophy, often leading to young boys requiring wheelchairs by their teenage years and facing life expectancy limitations in their twenties. Limb girdle muscular dystrophy causes muscle weakness and atrophy, particularly in the shoulders, hips, and thighs, hindering individuals’ ability to stand, move, and perform daily activities.
“Gene therapy is an effective method for reintroducing a healthy gene copy to a patient’s cells to address genetic disorders, but the small vectors for delivering this genetic information limit their capability to treat many diseases linked to large gene mutations,” stated Douglas M. Anderson, PhD, lead author and assistant professor of Medicine at the Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry (SMD). “Instead of sending the entire gene in one vector, we’ve crafted an efficient dual vector system that transmits two gene halves separately, which then merge to form the complete mRNA in the targeted tissues.”
This technology originated from an unexpected discovery made in the lab years ago, where they observed that when two distinct mRNAs were cleaved by small RNA segments known as ribozymes, they fused seamlessly and translated into a full-length protein. The researchers found that ribozymes cut RNA and produce ends that are recognized by the cell’s natural repair mechanisms.
“Much like how CRISPR enzymes sever DNA, where the CRISPR functions as scissors and the cell’s repair enzymes reconnect the DNA, we propose a similar mechanism might be happening for RNA using ribozymes as the scissors, with the cell’s repair pathways stitching together the two RNA fragments. It’s impressive how efficiently two separate mRNAs can locate each other,” noted Anderson, who also works with the University of Rochester Center for RNA Biology.
The lab significantly improved the efficiency of this method (over 900 times greater than earlier tests) and turned the technique into a robust gene delivery mechanism. When both halves of a large therapeutic gene are included in adeno-associated virus (AAV) vectors—which are widely used in gene therapy due to their safety and non-pathogenic traits in humans—the ribozymes cleave the ends of the mRNAs, allowing them to combine into a single, continuous mRNA that can produce protein in the target tissue.
The research team, including co-first author Sean Lindley, who recently completed his PhD in the Anderson lab, determined that the assembled mRNAs function comparably to their natural full-length versions, effectively translating genetic instructions into working proteins.
Self-cleaving ribozymes, which are crucial for StitchR’s operation, naturally exist in various species and belong to several families, each displaying different cleavage activities. After testing multiple ribozyme families and sequences, they finally found a combination that achieved high levels of protein production, nearly matching that from genes expressed from a single vector.
“Doug’s creativity and meticulousness in discovering how two distinct mRNA segments can seamlessly join in a cell to create functional mRNA is truly exhilarating,” commented Lynne E. Maquat, PhD, director of the UR Center for RNA Biology. “Though the idea seems straightforward, optimizing the involved molecules for stability and maximum efficiency required significant hands-on experimentation.”
Anderson mentioned that StitchR could be adapted with a variety of vectors used for gene delivery or expression and appears to work effectively with any mRNA sequence, which could broaden its potential applications across numerous diseases. “StitchR is quite flexible now. Its sequence requirements are minimal, and we’ve successfully tested it with various genes and sequences,” he added.
Another benefit of this technology is that it ensures only full-length proteins are produced.
“While other dual vector strategies have been explored for years, they often struggle with efficiency and the risk of producing incomplete protein products. StitchR’s operation at the RNA level allows for precise control, ensuring only full-length protein products are generated, distinguishing it from other dual vector methods like inteins, which require the production of smaller, fragmentary proteins that may have unforeseen cellular effects,” explained Anderson.
“It’s been a long, rewarding journey from initial lab observations to potential therapeutic uses, which has always been a key aim of our research. With StitchR and other innovations, we are making strides toward treatments for some of the most severe genetic disorders today, many of which currently lack effective therapies or cures,” Anderson added.
The lab is currently working on partnerships with other research groups and developing StitchR vectors to address various diseases caused by large genes, of which there are thousands.
In addition to Anderson and Lindley, other contributors from SMD include Kadiam C. Venkata Subbaiah (co-first author), Pornthida Poosala, FNU Priyanka, along with technicians William Richardson and Tamlyn Thomas, as well as scientists Yijie Ma, Leila Jalinous, and Jason A. West from CANbridge Pharmaceuticals. This research received funding from the University of Rochester, Jain Foundation, CANbridge Pharmaceuticals, and Scriptr Global, Inc. Anderson is among the co-founders of Scriptr Global, Inc., and has several pending US and international patents related to StitchR and other RNA technologies, with Scriptr licensing StitchR from the University of Rochester.
