Chimeric T-Cell Promoter for Stable High-Activity Gene Expression
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Solution Overview
Problem
Existing promoters used in tumor immunotherapy face challenges with transient expression due to epigenetic modifications or low activity in non-immune cells, limiting the stable and efficient expression of foreign genes in activated immune cells.
Innovation Solution
A promoter composed of a CMV enhancer, an IFNγ promoter, and a long terminal repeat (LTR) sequence from HTLV is constructed, exhibiting high activity in activated immune cells and low activity in other non-immune cells, enhancing gene expression in immune cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If virus derived constitutive promoters (such as CMV promoter) are used, then transient expression activity is high, but expression is easy to be turned off due to epigenetic modification
Solution Approach 1:
The patent combines multiple promoter elements (CMV enhancer, IFNγ promoter core sequence, and HTLV LTR) into a chimeric promoter structure. This merging of different functional elements allows the promoter to achieve both high transient expression activity from the CMV enhancer and stable long-term expression through the IFNγ promoter core and HTLV LTR components, resolving the contradiction between productivity and reliability.
Solution Approach 2:
The invention creates a composite promoter system by integrating sequences from different viral and human sources (CMV, IFNγ, HTLV) into a single functional unit. This composite structure leverages the strengths of each component: CMV enhancer for high transcriptional activation, IFNγ promoter for T-cell specific stable expression, and HTLV LTR for strong viral-driven transcription, thereby achieving both high productivity and reliability.
2Reliability
If human natural constitutive promoters are used, then expression is stable, but expression activity is relatively weak
Solution Approach 1:
The patent merges the human IFNγ promoter core sequence (which provides stable T-cell specific expression) with the CMV enhancer (which provides high transcriptional activity) and HTLV LTR (which provides strong viral-driven expression). This combination allows the promoter to achieve both the stability of human natural promoters and the high activity of viral promoters.
Solution Approach 2:
The invention modifies the promoter's functional parameters by replacing or supplementing the natural human promoter's enhancer regions with more potent viral enhancer elements (CMV enhancer and HTLV LTR). This parameter change in enhancer strength while retaining the human promoter core sequence achieves both stability and high expression activity.
3Productivity
If artificial chimeric promoters are constructed, then expression efficiency is enhanced, but promoter structure becomes complex
Solution Approach 1:
The chimeric promoter is segmented into distinct functional modules: CMV enhancer region, IFNγ promoter core sequence, and HTLV LTR. Each segment performs a specific function (transcriptional activation, T-cell specificity, and strong viral-driven transcription), making the complex structure manageable and functionally optimized while achieving high expression efficiency.
Data Source
AI summary
Provided is a promoter having high activity in an activated T-cell. The promoter comprises, from 5′-end to 3′-end, a CMV enhancer, an IFNγ promoter, and a long terminal repeat sequence from human T-cell leukemia virus that are connected in sequence. The promoter exhibits greater activity in an activated immune cell than the existing promoters and is low in activity or inactive in other non-immune cells.