Gene therapies have tremendous potential in treating a variety of diseases, but ones that use adeno-associated viruses (AAV) as their delivery vehicle are often too small to deliver a full human gene. While there have been many attempts at using multiple AAVs to deliver gene fragments into the cell where they can assemble together once inside, these efforts often proved inefficient.

Researchers at the University of Rochester recently found that they could use ribozymes — self-cleaving RNA sequences — to bring two gene fragments into a cell where an endogenous enzyme can reassemble them into a functional gene (1). First, the researchers put the gene fragments on two different extrachromosomal DNA plasmids. Then, by placing ribozyme sequences on either side of the gene fragments, the researchers control precisely where the enzymes cut to release the gene fragments.

“[We] ultimately developed that finding into a technology called StitchR, which stands for stitch RNA,” said Doug Anderson, a molecular biologist at the University of Rochester and senior author of the study.

As a proof-of-concept, the researchers split the gene for the green fluorescent protein into two pieces, placed ribozyme sequences adjacent to the gene fragments on two separate plasmids, introduced them into cells, and found that the cells fluoresced. The team then went through four rounds of optimization, which included testing out different ribozymes, the ribozyme location relative to the gene fragments, and even splitting a gene in the middle of an intron. In the end, they arrived at StitchR 4.0, which is over 900 times more efficient than the initial version of StitchR.

“It's quite impressive what they were able to do,” said Samie Jaffrey, a RNA biologist at Cornell University who previously designed RNA aptamers that self-circularize after ribozyme cleavage (2). Jaffrey, who was not involved in the StitchR study added, “What they introduced were all these interesting tricks and improvements that really made it work very efficiently.” 

Amelia Cervera, a molecular biology researcher at the Polytechnic University of Valencia who was also not involved in the study agreed. "The authors seem to have put together quite an effective system to get a full-length mRNA from two different vectors,” she wrote in an email. “Although the technique will be limited to joining only two fragments of RNA, it still doubles the cargo that can be delivered by AVV, which is a big advantage for gene therapy."

What they introduced were all these interesting tricks and improvements that really made it work very efficiently.
- Samie Jaffrey, Cornell University

To test their application in therapeutic settings, the researchers packaged StitchR 4.0 sequences into AAV vectors for two applications: to deliver the dysferlin gene and the dystrophin genes. Loss of function mutations in the dysferlin protein results in three types of muscular dystrophy while mutations in the dystrophin gene results in a mild form of Duchenne muscular dystrophy. These two genes are each far too large to fit inside one AAV, making them great candidates to test out StitchR’s capabilities to deliver gene fragments and reassemble the gene in vivo.

“When we were able to get endogenous [levels] or above, we were thrilled,” said Anderson. “We were excited to see [a] very broad distribution across the muscle, so almost all of the myofibers were expressing the dysferlin protein.” Further studies into the amount of virus delivered could help fine tune expression levels of these proteins.

Aside from muscular dystrophy, StitchR could have implications for other diseases. However, Jaffrey cautioned that getting enough gene expression after AAV delivery might not be feasible for genes that require high expression levels. “There are hundreds, if not thousands, of monogenic diseases that are too big to be packaged into a single AAV,” said Anderson. “Being able to utilize this technology to be able to create therapeutics for those diseases, I think, would be really important.”

References

1. Lindley, S.R. et al. Ribozyme-activated mRNA trans-ligation enables large gene delivery to treat muscular dystrophies. Science 386, 762-767 (2024).
2. Litke, J.L. & Jaffrey, S.R. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat Biotechnol  37, 667–675 (2019).