StitchR technology delivers large genes for muscular dystrophy treatment

gene therapy Although a variety of diseases can be effectively treated, size poses a major problem for some debilitating diseases, such as muscular dystrophy. Genes that are malfunctioning in muscular dystrophy are often very large, and current delivery methods are unable to carry such large gene loads into the body. A new technology named 'StitchR' overcomes this obstacle by delivering each half of the gene separately. Once inside the cell, both DNA segments produce messenger RNA (mRNA), which combine seamlessly to restore expression of proteins that are missing or inactive in the disease.

Published in a magazine scienceStitchR-;abbreviation for “stitch RNA”-; restored expression of therapeutic large muscle proteins to normal levels in two different animal models of muscular dystrophy. StitchR enabled expression of the protein dysferlin, which is missing in patients with limb-girdle muscular dystrophy type 2B/R2, and the protein dystrophin, which is missing in patients with Duchenne muscular dystrophy.

Duchenne muscular dystrophy is the most common early-onset form of muscular dystrophy. Many boys end up in wheelchairs in their teens and die in their 20s. People with limb-girdle muscular dystrophy often experience muscle weakness and weakness in the shoulders, hips, and thigh muscles, making it difficult to stand, move, and perform daily activities.

“Gene therapy is a powerful tool for delivering healthy gene copies into a patient's cells to cure inherited diseases, but the vectors used to deliver this information are small, so gene therapy “Gene therapy cannot be used to treat many diseases caused by large genetic mutations,” said study lead author Douglas M. Dr. Anderson said. “It is not possible to deliver a complete gene in a single vector, but instead we can deliver half of the gene separately and efficiently combine them to reconstitute a large mRNA in the affected tissue. We have developed a unique dual vector system.

The technology first emerged from a chance observation made in a lab several years ago. This means that when two separate mRNAs are cleaved by small RNA sequences called ribozymes, they are seamlessly ligated (joined) and translated into a full-length protein. The researchers found that when ribozymes cut or cleave RNA, they leave behind ends that are recognized by natural repair pathways.

“Just as we use the CRISPR enzyme to cut DNA, the CRISPR enzyme is just a pair of scissors, and it's the cell's natural repair enzymes that reattach the DNA,” said Anderson, who is also a member of the University of Rochester Center. said. RNA biology. “A similar thing seems to be happening here with RNA: ribozymes act as scissors, and the cell's natural repair pathways can recombine the two RNAs. Two separate mRNAs can find themselves and the process can be very efficient.”

The lab has optimized the efficiency of the process (more than 900 times greater than initial experiments) and adapted the technology to a powerful gene delivery mechanism. Once half of a large therapeutic gene is encoded in an adeno-associated virus (AAV) vector, it is safe and does not cause disease in humans, making it the most commonly used vector for gene therapy. However, ribozymes cut the ends of the gene. The mRNAs are then combined to form a single, seamless mRNA that can produce proteins in the desired tissue.

The research team, including co-lead author Sean Lindley, who recently received his PhD from Anderson's lab, found that the stitched mRNA behaves essentially the same as its natural full-length counterpart, converting genetic information into functional molecules. They found that it appears to be effectively converted into other proteins.

Self-cleaving ribozymes essential for StitchR activity occur naturally throughout the animal kingdom and are comprised of different families that exhibit distinct cleavage activities. After testing numerous ribozyme families and sequences, we ultimately identified a formulation that yields high levels of protein production and approaches levels achieved by genes expressed from a single vector.

Doug is a very creative and detail-oriented person, and the fact that he was able to figure out how two different mRNAs find each other in a cell and combine seamlessly to create a functional mRNA is really exciting. is. Although the concept sounds simple, considerable benchwork was required to optimize the molecules involved, ensure they were stable within cells, and make the process as efficient as possible. ”

Dr. Lynne E. Maquat, Director, UR Center for RNA Biology

Anderson said StitchR can bind to many different types of vectors used to deliver or express genes in cells and appears to work efficiently with any mRNA sequence, making it useful in a wide range of diseases and conditions. This will open the door to its use. application. “At this point, StitchR is truly plug-and-play. The sequence requirements for StitchR are minimal, and we are currently testing this with many different genes and sequences,” he said.

Another feature of this technology is that it produces only full-length proteins.

“Other dual-vector approaches have been developed for decades but have suffered from a lack of efficiency and production of less than full-length products. StitchR occurs at the RNA level, so ” This allows StitchR to control and reliably generate other duals. It is distinguished from vector technologies, such as inteins, a protein ligation technique that is efficient but requires the expression of small protein fragments that may have unknown effects within the cell,” Anderson added.

“It has been a long but fulfilling process to go from initial research observations to therapeutic applications, but it has always been a primary goal of our laboratory and one that I believe is the promise of basic research. Using StitchR and other tools, we are working towards cures for some of the most debilitating genetic diseases on the planet, many of which have current treatments and treatments. There is no,” Anderson said.

The lab is currently collaborating with other research institutions to generate StitchR vectors to treat numerous diseases caused by thousands of large genes.

In addition to Anderson and Lindley, SMD staff scientists Kadiam C. Venkata Subbaiah (co-first author), Pornthida Poosala, FNU Priyanka, engineers William Richardson and Tamlyn Thomas, and CANbridge Pharmaceuticals' Scientists Yijie Ma and Leila Jalinous contributed to this research. , Jason A. West. This research was funded by the University of Rochester, the Jain Foundation, CANbridge Pharmaceuticals, and Scriptr Global, Inc. Anderson is a co-founder of Scriptr Global, Inc. and has filed multiple U.S. and foreign patents for research related to StitchR and others. RNA-based technology. Scriptr has licensed StitchR technology from the University of Rochester.