Trans-splicing RNA therapies

Trans-splicing is the biological process where pieces of premature mRNA from multiple different molecules are joined together to form a functional mRNA. Normally cells use cis-splicing, which is when one pre mRNA transcript produces one strand of mRNA. Trans-splicing involves multiple exons from different mRNA transcripts joining together to form one large chimeric strand of mRNA. This is also possible at the protein level in polypeptides with inteins and exteins. It has been observed in microbes and vertebrates, suggesting an early origin of it in bacteria and viruses. RNA trans-splicing is seen in genes coding for certain enzymes, transcription factors, cancer biomarkers, gene expression regulators and more. 

Trans-splicing is able to do targeted repair at the RNA level instead of permanently altering the DNA. Unlike CRISPR, trans-splicing works on the messenger RNA after it is made. This allows it to potentially be safer since it avoids permanent mutations that CRISPR could possibly cause. It is also, depending on the treatment given, reversible and adjustable. It is able to selectively correct mRNA by only replacing or repairing mutant transcripts and preserves the natural gene. It can replace point mutations, deletions and large defective regions of a genome. It has applications in repairing and curing genetic disorders like cystic fibrosis, hemophilia and sickle cell disease. It is able to eliminate viral infections by targeting viral RNA. Additionally, it is able to replace oncogenic transcript and add toxic payloads to cancerous tumor cells weakening cancer. 

There are three major strategies to trans-splicing, spliceosome-mediated, ribozyme-mediated and protein trans-splicing. Spliceosome-mediated RNA trans-splicing uses the cell’s natural spliceosome machinery. It introduces a designed RNA molecule called a pre-trans-splicing molecule which binds to a target pre-rRNA and replaces a defective portion during splicing. This method either starts replacing at the beginning (5’ replacement), end (3’ replacement) or in the middle where it starts by swapping a middle exon. Ribozyme-mediated trans-splicing uses engineered catalytic RNA molecules which recognize a specific RNA sequence, cut it and attach it to a new segment. Despite it being less efficient compared to using spliceosomes it is more specific and targeted due to the sequence recognition. Protein trans-splicing works at the protein level instead of the RNA level. It uses inteins which are protein segments that can self-splice. These protein fragments come together and splice into a functional protein. This method is usable for rebuilding proteins and delivering therapeutic protein fragments.

References:

1. Hong, Elizabeth M, et al. “Therapeutic Applications of Trans-Splicing.” British Medical Bulletin, U.S. National Library of Medicine, 15 Dec. 2020, pmc.ncbi.nlm.nih.gov/articles/PMC7737522/