Capped-sgRNA Complex for Targeted Protein Translation Control
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Solution Overview
Problem
Current methods for enhancing gene expression and regulating protein translation face challenges such as inefficient delivery of large RNA molecules, inaccurate reproduction of spliced isoforms, and immunogenicity issues with engineered RNA binding proteins, which limit their therapeutic applicability.
Innovation Solution
The development of complexes comprising a Cas polypeptide and a capped-sgRNA, where the capped-sgRNA includes an m7G cap, a spacer for specific hybridization with a target RNA sequence, and a direct repeat for binding to the Cas polypeptide, allowing for targeted recruitment of translational pre-initiation complexes to enhance protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If messenger RNAs are delivered to enhance gene expression, then protein production is increased, but delivery efficiency is low and technical challenges arise
Solution Approach 1:
The patent uses an engineered RNA binding protein as an intermediary to recruit endogenous translation machinery to target mRNAs. Instead of directly delivering large amounts of mRNA or complex translation initiation complexes, the invention introduces a smaller RNA binding protein that acts as a mediator to bridge the gap between endogenous cellular components and target mRNAs, thereby enhancing protein production while avoiding the delivery challenges associated with direct mRNA therapy
2Productivity
If viral-encoded gene products are delivered via AAV or lentivirus, then gene expression is enhanced, but accurate reproduction of spliced isoforms is compromised
Solution Approach 1:
The patent employs endogenous cellular translation machinery to translate target mRNAs, allowing the cell's own splicing and translation systems to naturally produce the correct isoforms. By using endogenous resources rather than viral-encoded components, the system automatically preserves the native splicing patterns and isoform ratios without requiring external intervention or complex viral vector design
3Adaptability or versatility
If engineered RNA binding proteins are used to regulate translation, then translational control is achieved, but immunogenicity issues arise
Solution Approach 1:
The patent uses an engineered RNA binding protein with a structure and function that mimics natural RNA binding proteins, making it more compatible with the cellular environment. The engineered protein maintains homogeneity with endogenous proteins in terms of its interaction mode with RNA and cellular machinery, thereby reducing the likelihood of triggering immune responses while preserving translational control capabilities
4Productivity
If engineered RNA binding proteins are expressed in fusion protein context, then translation initiation is enhanced, but stoichiometry of translation machinery is disrupted
Solution Approach 1:
The patent uses engineered RNA binding proteins that bind to specific mRNA sequences with high affinity and specificity, allowing for localized and partial enhancement of translation initiation at target sites without globally disrupting the stoichiometry of translation machinery. The engineered proteins act in a targeted manner rather than uniformly across all mRNAs, thereby avoiding imbalance in the overall translation system
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
This approach enables precise control over protein translation by bringing the m7G cap closer to the start codon, enhancing protein production efficiency and overcoming existing delivery and immunogenicity challenges.
Implementation Method 1
a spacer capable of specifically hybridizing with a target sequence in an RNA molecule
Implementation Method 2
an m7G cap or an analog thereof... bringing the m7G cap closer to the start codon, enhancing protein production efficiency
Data Source
AI summary
Provided herein are compositions and methods for regulating protein translation. The compositions include a Cas polypeptide and a capped-sgRNA that includes (i) an m7G cap or an analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide. The disclosure further provides methods of regulating translation of an mRNA in a cell, the method comprising contacting the cell with a nucleic acid comprising (a) a sequence encoding a Cas polypeptide; and (b) a sequence encoding a capped-sgRNA comprising (i) an m7G cap or analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide.