Cancer research would benefit from flexible technologies enabling early detection of cancer events. Bioluminescence imaging (BLI) presents a powerful tool for noninvasive in vivo imaging. Its application to cancer research requires the development of sensitive probes that detect molecular changes occurring in cancer progression. This proposal describes a technology that senses these changes through cell-specific cis-acting sequences on a transcript that in turn regulate the alternative splicing pattern of target transcripts. This technology will be built on a flexible, modular platform such that specific probes can be generated for different cancer types and stages. This technology will have a widespread impact on cancer research and will provide a sensitive tool for simultaneous early detection, treatment, and monitoring disease progression.
The specific aims under the R21-phased proposal are designed to show proof of principle for the development of this technology and include: (1) Generating test constructs to select for cancer-specific cis acting control elements that regulate alternative splicing; (2) Selecting cis-acting intronic splicing elements that act as cell-specific silencers of an alternative exon inclusion event; (3) Demonstrating functional transfer of these sequences to verify independence of this regulatory sequence on the protein-specific sequences; (4) Building a kinetic model of the alternative splice-site selection processes useful in guiding experimental design. The research design in this phase utilizes an iterative selection process with a two-step in vivo functional screen to identify sequences that act as cell-specific intronic silencers of an exon-inclusion event.
The specific aims under the R33-phased proposal are designed to examine the feasibility of this technology for noninvasive cancer detection, treatment, and monitoring and include: (1) Expanding libraries of cis-acting control elements to include those that differentially regulate alternative splicing in response to different stages of cancer; (2) Altering constructs for noninvasive imaging strategies in mouse models; (3) Determining utility for in vivo BLI of cancer cells using subcutaneous injection; (4) Determining the ability to target specific cancer locations in mouse cancer models for detection, treatment, and monitoring by in vivo imaging. The research design in this phase uses the constructs developed in the previous phase as novel probes for BLI applications and develops their utility for simultaneous detection and treatment strategies.
