Programmable Transcription Factor Recruitment for Reversible Gene Control
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Solution Overview
Problem
Current methods for modulating gene expression, such as gene editing and synthetic transcription factors, often result in irreversible changes and do not effectively respond to physiological signals, leading to unintended side effects and limited therapeutic efficacy.
Innovation Solution
A programmable gene modulator (PGM) system using endogenously produced transcription factors activated by environmental signals, comprising a dCas polypeptide and a chimeric guide nucleic acid (sgCNA) to reversibly modulate gene expression at specific genomic locations in response to cellular cues.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gene editing is used to modulate gene expression, then permanent therapeutic effect is achieved, but off-target effects and irreversible harmful modifications occur
Solution Approach 1:
The patent employs a dynamic, reversible gene modulation system using dCas9-bound transcription factors that can be activated or deactivated in response to physiological signals. This allows the system to adapt its activity level rather than permanently altering the genome, thereby achieving therapeutic effects while minimizing irreversible off-target damage.
Solution Approach 2:
The patent introduces an intermediary system where dCas9 (a catalytically inactive Cas9) serves as a mediator that binds to guide RNA and recruits endogenous transcription factors to target genes without causing DNA cleavage. This intermediary approach enables precise gene modulation without the harmful DNA cutting and permanent editing associated with traditional gene editing tools.
2Duration of action of moving object
If synthetic transcription factors are used to modulate gene expression, then reversible control is achieved, but the factors are non-native and do not respond to physiological signals
Solution Approach 1:
The patent merges two previously separate concepts: the programmable DNA-binding capability of dCas9-guide RNA complexes and the physiological signal-responsiveness of endogenous transcription factors. By combining these elements into a single functional system, the invention achieves both reversible control and adaptability to physiological signals simultaneously.
Solution Approach 2:
The system utilizes the cell's own endogenous transcription factors that are naturally activated by physiological signals, rather than introducing entirely foreign proteins. These endogenous factors self-regulate in response to cellular conditions, providing the system with automatic adaptability to the physiological state of the cell.
3Reliability
If pharmaceutical agents are administered to promote recovery, then therapeutic effect is achieved, but the activity is not restricted to the time and place where it is needed
Solution Approach 1:
The patent implements local quality by directing the dCas9-transcription factor complex to specific genomic loci through guide RNA sequences that are complementary to target gene regions. This ensures that gene modulation occurs only at the precise location in the genome where it is needed, rather than systemically throughout the organism as with pharmaceutical agents.
Solution Approach 2:
The system incorporates feedback mechanisms by using endogenous transcription factors that are activated by physiological signals specific to the tissue and time of injury or disease. The system automatically responds to cellular conditions, activating gene modulation only when physiological signals indicate that therapeutic intervention is needed, thereby avoiding unnecessary side effects.
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
Enables targeted and reversible gene expression in response to physiological signals, minimizing off-target effects and ensuring gene modulation occurs only when and where it is needed for therapeutic benefit.
Implementation Method 1
a ribonucleoprotein complex comprising the Cas9 (CRISPR-associated protein 9) endonuclease and a guide RNA can bind to and cleave DNA genomic sequences specified by the guide RNA
Implementation Method 2
each transcription factor binding site in the PGM binds to at least one endogenous transcription factor that is activated in a cell comprising the PGM in response to the environmental signal(s)
Data Source
AI summary
The disclosure provides a method for modulating gene expression in a cell-specific manner in response to an intracellular or extracellular stimulus. The disclosure provides a platform entitled the PROTEGE platform, which comprises a DNA binding module and a transcription factor binding module, referred to herein as PGM. The PGM binds the promoter region of a target gene with sequence specificity through the DNA binding module and also binds a TF through the TF-binding module. When the TF is activated in response to an intracellular or extracellular stimulus, it binds the PGM and, due to its close proximity to the promoter of the gene, modulates expression of a target gene in response to the stimulus.