Aptazyme-Regulated AAV Vectors for Controlled Gene Expression
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Solution Overview
Problem
Current gene therapy systems face challenges in achieving controlled and efficient expression of therapeutic proteins, particularly due to immunogenic risks from DNA-binding proteins and limited dynamic range, which hampers their clinical applicability in treating proliferative diseases like cancer.
Innovation Solution
The development of a nucleic acid construct comprising a transgene encoding a therapeutic protein, a tetracycline-responsive aptazyme, and inverted terminal repeats (ITRs), delivered via an adeno-associated virus (AAV) vector, allowing for tightly controlled expression through tetracycline induction, reducing immunogenic risks and enabling broad dynamic range expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If transcriptional control systems (Tet-ON/OFF promoter systems) are used to control gene expression, then gene expression can be induced, but DNA-binding proteins are required which pose immunogenic risks
Solution Approach 1:
The patent replaces the protein-based transcriptional control system with an RNA-based aptazyme system. Instead of using DNA-binding proteins that require ligand activation, the invention uses an aptamer-ribozyme fusion where the aptamer binds the ligand (tetracycline) and this binding event directly modulates the ribozyme's catalytic activity, leading to mRNA cleavage or stabilization. This substitution of protein-based control with RNA-based control eliminates the need for additional protein components that could be immunogenic.
Solution Approach 2:
The patent introduces an RNA intermediary (the aptazyme) that mediates between the ligand (tetracycline) and the gene expression control. The aptazyme acts as a molecular switch where ligand binding to the aptamer domain causes conformational changes that regulate the ribozyme domain's cleavage activity. This RNA intermediary system allows for indirect control of gene expression without requiring direct protein-DNA interactions, thereby avoiding immunogenicity issues.
2Productivity
If conventional gene therapy systems are used, then therapeutic proteins can be expressed, but the dynamic range of expression control is limited
Solution Approach 1:
The patent implements a dynamic gene expression control system using the aptazyme riboswitch. The system transitions from a static or limited dynamic range control to a highly dynamic system where gene expression can be continuously adjusted by varying the concentration of the inducing ligand (tetracycline). The aptazyme provides graded response where different ligand concentrations produce different levels of mRNA stabilization and thus different levels of protein expression, enabling fine-tuned control over a broad dynamic range.
Solution Approach 2:
The patent utilizes parameter changes in the RNA structure upon ligand binding to control gene expression. The aptamer domain undergoes conformational changes when binding tetracycline, which are transmitted to the ribozyme domain to modulate its catalytic activity. By changing the ligand concentration parameter, the system achieves variable degrees of mRNA cleavage inhibition, thereby controlling the level of therapeutic protein expression across a wide dynamic range.
3Reliability
If gene expression is continuously active, then therapeutic effect is maintained, but toxic side effects occur due to narrow therapeutic window
Solution Approach 1:
The patent implements periodic or inducible gene expression using the aptazyme system instead of continuous expression. The therapeutic gene is under the control of the aptazyme riboswitch that remains in the cleavage-competent state by default, preventing expression. Upon administration of the inducing ligand (tetracycline), the aptazyme undergoes conformational change that stabilizes the mRNA and enables protein expression. This allows for periodic induction of therapeutic effect only when needed, avoiding continuous expression and its associated toxic side effects.
Solution Approach 2:
The patent employs preliminary suppression of gene expression through the aptazyme's default cleavage activity, which prevents therapeutic protein production until induction is required. The ribozyme is designed to be constitutively active in cleaving the mRNA, thereby preemptively blocking any potential toxic effects of uncontrolled expression. The therapeutic effect is activated only when the ligand is administered to counteract this preliminary suppression, ensuring safety by preventing premature or uncontrolled protein production.
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 achieves dose-dependent and dynamic induction of therapeutic proteins, such as IL-12, with repeated ON-OFF switching capability, demonstrating clinical relevance by inducing significant cytokine levels and achieving near complete remission in a hepatocellular carcinoma model without toxicity.
Implementation Method 1
Allosteric control over ribozyme cleavage is achieved by fusing the ribozyme to an aptamer domain, whose structural re-arrangement upon binding of its cognate ligand alters the global riboswitch architecture.
Implementation Method 2
autocatalytic ribozyme self-cleavage results in 5'-cap or 3'-poly(A) tail loss, respectively, thereby inducing mRNA degradation and shutdown of gene expression
Data Source
AI summary
This invention generally relates to the field of somatic gene therapy. The invention provides a nucleic acid construct comprising a transgene encoding a therapeutic protein, a tetracycline-responsive aptazyme sequence, and inverted terminal repeats (ITRs). The nucleic acid construct can be transferred to a subject in need thereof in the form of a viral vector, in particular an adeno-associated virus (AAV) vector. The Tet-responsive aptazyme sequence allows for a tightly controlled expression of the transgene in the subject, thereby avoiding toxic side effects. The nucleic acid construct and the viral vectors comprising same are particularly useful in the treatment of proliferative diseases like cancer.