Synthetic introns can be used to effectively kill tumor cells with spliceosomal mutations.

  • Major Finding: Synthetic introns can be used to effectively kill tumor cells with spliceosomal mutations.

  • Concept: Mutation-dependent protein production occurs in cancer cells with SF3B1 mutations but not wild-type cells.

  • Impact: Mutations affecting RNA splicing can feasibly be harnessed for development of cancer gene therapy.


The pan-cancer nature of mutations in RNA splicing factors as well as their occurrence early in tumor development make them a compelling therapeutic target. Additionally, previous studies have suggested cancer cells with mutations in their spliceosome are more susceptible to further splicing alterations. Therefore, to exploit these splicing aberrations for therapeutic development, North, Benbarche, and colleagues developed synthetic constructs of genes differentially spliced in cancer cells with mutations in SF3B1, which is the most commonly mutated spliceosomal gene in tumors. Missplicing was found in diverse SF3B1-mutated cells, with six introns being used for synthetic intron development and subsequent further studies. Using the herpes simplex virus-thymidine kinase (HSV-TK) system, which induces cytotoxic metabolite production upon treatment with ganciclovir (GCV) in HSV-TK–expressing cells, a synthetic intron derived from MTERFD3 was transduced and revealed significant loss of viability upon GCV treatment in only the SF3B1-mutant cells, with this effect being observed across multiple cancer cell types. To reveal critical elements that govern synthetic intron function, a massive parallel screen was used and showed the ideal intron size as well as that the 5′ splice site (ss) was needed for intron recognition, the 3′ ss was required for mutation responsiveness, and far distal consensus branchpoints improved splicing in SF3B1-mutant cells. Further evaluation of these synthetic introns in in vivo cancer models confirmed cancer cell specificity, with the use of these synthetic introns in a mixed cell population of wild-type and SF3B1-mutant hematopoietic stem and progenitor cells resulting in depletion of only the SF3B1-mutant cells after GCV treatment. Use of this treatment in solid tumors, specifically breast cancer and uveal melanoma, indicated suppressed tumor growth and improved survival post–GCV treatment. Thus, this study reveals the potential of engineering new therapies to target protumorigenic splicing aberrations through the use of synthetic introns that can be applied to a wide range of cancer-related alterations in multiple tumor types.

North K, Benbarche S, Liu B, Pangallo J, Chen S, Stahl M, et al. Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells. Nat Biotechnol 2022 Mar 3 [Epub ahead of print].

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