Nucleic Acid-Controlled Ribozyme Switches for Gene Therapy
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Solution Overview
Problem
Current ribozyme switches used in gene therapy have low dynamic range, high leakiness, and non-optimal trigger properties, making them unsuitable for human gene therapy due to safety, toxicity, and biodistribution concerns.
Innovation Solution
A self-cleaving or bond-forming catalytic RNA, such as a ribozyme, is combined with a nucleic acid binding site in the 3'UTR of a coding nucleic acid, allowing for controlled expression based on the presence of a specific nucleic acid sequence, using a nucleic acid-responsive ribozyme design that includes a stem region and a catalytic core, with a second nucleic acid inducing conformational changes to activate or inhibit catalytic activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional ribozyme switches are used for gene regulation, then gene expression can be controlled, but the dynamic range is low (2-10 fold) and leakiness is high in the OFF state
Solution Approach 1:
The patent combines a ribozyme catalytic core with a nucleic acid binding site (aptamer) to create a hybrid riboswitch-ribozyme system. This merging allows the system to achieve both high-affinity ligand binding and catalytic activity, resulting in enhanced dynamic range (>100-fold) and reduced leakiness compared to traditional ribozyme switches alone
Solution Approach 2:
The invention creates a composite RNA structure comprising a ribozyme catalytic core (e.g., hammerhead ribozyme) fused with a nucleic acid binding site (aptamer domain). This composite structure integrates the functions of both catalysis and specific molecular recognition, enabling superior gene regulation performance with high ON/OFF ratios and minimal background expression
2Productivity
If ribozyme switches with high catalytic activity are used, then gene expression in ON state is high, but leakiness in OFF state increases
Solution Approach 1:
The patent designs a dynamic RNA structure where the ribozyme catalytic core and nucleic acid binding site are coupled through conformational changes. In the absence of the trigger, the aptamer binds the nucleic acid sequence, maintaining the ribozyme in an inactive conformation. Upon trigger binding, conformational changes activate the ribozyme, enabling precise control of catalytic activity and gene expression levels with minimal leakiness
3Ease of operation
If small molecule triggers are used for ribozyme activation, then gene expression can be controlled, but safety, toxicity, and biodistribution properties are non-optimal for human gene therapy
Solution Approach 1:
The patent changes the trigger type from small molecules to nucleic acid sequences (e.g., antisense oligonucleotides, siRNAs). This parameter change eliminates the safety and toxicity concerns associated with small molecule triggers while maintaining controllable gene expression. The nucleic acid triggers can be delivered via established methods and are degraded by natural nucleases, improving biodistribution and safety profiles for human gene therapy applications
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 of gene expression with reduced leakiness and high expression levels in the ON state, enhancing the suitability of ribozyme switches for gene therapy applications by improving the dynamic range and safety profiles.
Implementation Method 1
a second nucleic acid, wherein the second nucleic acid is complementary to at least a part of the first or the second part of the stem nucleic acid sequence... binding of the second nucleic acid to the first nucleic acid results in a conformational change
Data Source
AI summary
Herein is reported a composition comprising a pair of a first nucleic acid and a second nucleic acid, wherein the first nucleic acid comprises in the order a first part of a stem nucleic acid sequence, a cleavage site, a first stem loop nucleic acid sequences which 5′- and 3′-parts form a duplex, a first part of a catalytic core sequence, a second stem loop nucleic acid sequences which 5′- and 3′-parts form a duplex, a second part of the catalytic core sequence, and a second part of the stem nucleic acid sequence, which is complementary to the first part of the stem nucleic acid sequence and, thus, form a duplex therewith, wherein the second nucleic acid is complementary to at least a part of the first or the second part of the stem nucleic acid sequence, wherein binding of the second nucleic acid to the first nucleic acid results in a conformational change of the first nucleic acid, which is at least one of the dissociation of the first part of the first stem sequence from the second part of the stem sequence and the hybridization of one of the parts with the second nucleic acid, or the dissociation of loop I and loop II resulting in an inactivation of the catalytic activity, or the association of loop I and loop II resulting in an activation of the catalytic activity.