Split Luciferase Reporter System for Specific Cell Tracking
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Solution Overview
Problem
Current PET/SPECT reporter genes for tracking cells in gene and cell therapies are limited by their size, ease of cloning, and expression specificity, as well as short imaging windows due to natural expression in non-target tissues and tracer efflux.
Innovation Solution
A novel reporter gene system comprising a fusion protein with a transmembrane domain and a split luciferase reporter domain that associates with a high-affinity reporter peptide, allowing for both PET/SPECT and bioluminescence imaging, enabling specific and prolonged tracking of cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the NIS reporter gene is used for PET/SPECT imaging, then imaging capability is provided, but the gene is naturally expressed in multiple tissues causing non-specific signal and tracer efflux
Solution Approach 1:
The reporter system is divided into two separate components: a membrane-anchored reporter protein expressed in therapeutic cells and a circulating radiolabeled peptide probe. This segmentation allows the reporter to be selectively displayed on cell surfaces while the probe circulates freely, enabling specific binding only at the target location and eliminating background signal from tissues expressing the reporter gene.
Solution Approach 2:
The invention extracts the reporter function from the NIS system by using a custom-designed membrane-anchored protein domain that binds specifically to the radiolabeled peptide. This extracted reporter system removes the harmful non-specific binding characteristic of NIS while retaining the imaging capability.
2Reliability
If the NIS reporter gene is used, then imaging is enabled, but the gene is relatively long making cloning difficult
Solution Approach 1:
The invention extracts only the essential reporter function from the full NIS gene, using a minimal membrane-anchored protein domain (approximately 100-200 amino acids) that provides the necessary binding capability. This extracted fragment is significantly smaller and easier to clone into therapeutic vectors while maintaining imaging functionality.
Solution Approach 2:
The reporter system is segmented into a small membrane-anchored protein component and a separate radiolabeled peptide, allowing the genetic material to be minimized to only the essential binding domain while the imaging function is provided by the small peptide probe.
3Reliability
If NIS reporter gene is used, then cell tracking is possible, but tracer probes are not trapped and can efflux resulting in short imaging windows
Solution Approach 1:
The membrane-anchored reporter protein is pre-positioned on the cell surface before administration of the radiolabeled peptide probe. This preliminary anchoring ensures that when the probe circulates, it binds immediately and specifically to the reporter, creating a stable complex that remains trapped at the target location, thereby extending the imaging window.
Solution Approach 2:
The membrane-anchored reporter protein acts as an intermediary that captures and holds the radiolabeled peptide probe on the cell surface. This intermediary mechanism prevents probe efflux and ensures prolonged retention of the tracer, extending the imaging window compared to direct NIS-mediated trapping.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The system provides highly specific and prolonged imaging capabilities, allowing for effective monitoring of cell therapies like CAR T-cell therapy, with minimal background signal and easy cloning into therapeutic vectors, enhancing diagnostic and prognostic imaging.
Implementation Method 1
the reporter domain comprises, preferably consists of, the large polypeptide subunit of a split luciferase, and wherein said reporter peptide comprises, preferably consists of, the small peptide subunit of said split luciferase, wherein both subunits associate by complementation to assemble into a (preferably luminescent) luciferase complex
Implementation Method 2
a reporter peptide labeled with a radiolabel; wherein said reporter domain comprises, preferably consists of, the large polypeptide subunit of a split luciferase, and wherein said reporter peptide comprises, preferably consists of, the small peptide subunit of said split luciferase
Data Source
AI summary
The present invention provides a reporter system comprising (i) a gene expression construct for expression in a cell of a reporter gene, said reporter gene encoding a fusion protein comprising a transmembrane domain fused in-frame to a reporter domain, wherein said transmembrane domain upon insertion of the fusion protein into the cell membrane anchors the fusion protein in the cell membrane while expressing the reporter domain at the cell surface, and (ii) a reporter peptide labeled with a radiolabel, wherein said reporter domain comprises the large polypeptide subunit of a split luciferase, and wherein said reporter peptide comprises the small peptide subunit of said split luciferase, wherein both subunits associate by complementation to assemble into a luciferase complex.