Synthetic introns enable splicing factor mutation-dependent targeting of cancer cells

Area of Science:

• Oncology
• Molecular Biology
• Gene Therapy

Background:

• Recurrent mutations in RNA splicing factors are common in many cancers.
• Aberrant RNA splicing in tumors presents a potential therapeutic vulnerability.
• SF3B1 mutations are frequently observed in various cancer types, including leukemia and melanoma.

Purpose of the Study:

• To develop a method for selectively eliminating cancer cells by exploiting tumor-specific RNA splicing defects.
• To engineer synthetic introns that are differentially spliced based on the presence of SF3B1 mutations.
• To evaluate the therapeutic potential of mutation-dependent gene expression for cancer treatment.

Main Methods:

• Engineered synthetic introns designed for differential splicing in SF3B1-mutant versus wild-type cells.
• Conducted a large-scale screen of 8,878 introns to optimize size and identify splicing elements.
• Utilized herpes simplex virus-thymidine kinase (HSV-TK) as a reporter gene for mutation-dependent protein production.
• Tested the efficacy of ganciclovir (GCV)-mediated cell killing in vitro and in vivo xenograft models.

Main Results:

• Synthetic introns demonstrated efficient splicing in SF3B1-mutant cancer cells and minimal splicing in isogenic wild-type cells.
• Mutation-dependent expression of HSV-TK led to selective killing of SF3B1-mutant leukemia, breast cancer, uveal melanoma, and pancreatic cancer cells in vitro.
• In vivo studies showed significant suppression of tumor growth and improved survival in mice bearing SF3B1-mutant xenografts after treatment with synthetic intron-containing constructs and GCV.
• Wild-type cells remained unaffected by the therapeutic intervention.

Conclusions:

• Synthetic introns can be engineered to harness aberrant RNA splicing for targeted cancer gene therapy.
• This approach enables mutation-dependent gene expression, leading to selective tumor cell elimination.
• Exploiting SF3B1 splicing mutations offers a promising strategy for treating a range of SF3B1-mutant cancers.