The design and optimization of RNA trans-splicing molecules for skin cancer therapy

Area of Science:

• Molecular Biology
• Cancer Research
• Gene Therapy

Background:

• Targeting tumor-specific genes with RNA trans-splicing molecules (RTMs) offers a novel strategy for inducing cancer cell death.
• SLCO1B3 is a marker gene identified in epidermolysis bullosa-associated squamous cell carcinoma (EB-SCC).

Purpose of the Study:

• To design and validate RTMs that specifically bind SLCO1B3 pre-mRNA for targeted cancer therapy.
• To assess the efficacy of RTMs in inducing apoptosis in SLCO1B3-expressing cancer cells.

Main Methods:

• Development of a screening system to identify SLCO1B3-specific RTMs.
• Utilizing a minigene system (SLCO1B3-MG) to analyze RTM trans-splicing potential.
• Employing RT-PCR and Western blot to detect chimeric mRNA and protein expression.
• Conducting cell viability and apoptosis assays to confirm RTM-induced cell death.

Main Results:

• SLCO1B3-specific RTMs were successfully designed and demonstrated trans-splicing activity.
• Expression of the chimeric SLCO1B3-tk fusion gene was confirmed.
• RTMs induced apoptosis in SLCO1B3-MG expressing cells via the suicide gene (HSV-tk).
• The lead RTM facilitated trans-splicing into endogenous SLCO1B3 pre-mRNA in EB-SCC cells, leading to tk-mediated apoptosis.

Conclusions:

• The developed RTMs effectively target SLCO1B3 and induce tumor-specific apoptosis in skin cancer cells.
• The inducible cell-death system accelerates the design of optimized RTMs for cancer therapy.
• This RNA trans-splicing approach holds significant promise for developing targeted skin cancer treatments.