Multi-circRNA AAV Constructs for High Tissue Expression
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Solution Overview
Problem
Current designs for synthetic circRNA therapeutics lack rational approaches for therapeutic use, particularly in delivering multiple circRNAs effectively for treating diseases.
Innovation Solution
The development of nucleic acid molecules encoding for at least two circular RNAs (circRNAs) flanked by intronic elements, with internal ribosome entry sites (IRES) and translation regulating regions, packaged into adeno-associated virus (AAV) particles for targeted delivery and enhanced expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple circRNAs are delivered using current synthetic designs, then therapeutic coverage is limited, but delivery efficiency and expression levels remain insufficient
Solution Approach 1:
The patent combines multiple circRNA-encoding sequences into a single nucleic acid molecule, allowing co-delivery of multiple circRNAs through one AAV particle. This merging approach enables simultaneous expression of multiple therapeutic circRNAs (achieving versatile therapeutic coverage) while maintaining high expression levels through coordinated design of intronic elements and IRES sequences within the unified construct.
Solution Approach 2:
The nucleic acid molecule is designed with universal functional elements including promoter regions, intronic elements, and IRES sequences that can accommodate multiple different circRNA-encoding sequences. This multi-functional design allows the same backbone structure to deliver various therapeutic circRNAs for different diseases, achieving both broad adaptability and consistent high expression through standardized regulatory elements.
2Productivity
If circRNA expression is enhanced using traditional methods, then therapeutic efficacy improves, but delivery to specific tissues remains inefficient
Solution Approach 1:
The patent uses AAV particles as intermediary vectors to deliver the nucleic acid molecule encoding multiple circRNAs. The AAV capsid serves as a mediator that enables targeted delivery to specific tissues (liver, muscle, brain) while the packaged nucleic acid molecule ensures high circRNA expression through its optimized design with intronic elements and IRES sequences, thus resolving the contradiction between targeted delivery and expression enhancement.
3Ease of manufacture
If synthetic circRNA designs are simplified for ease of manufacture, then production becomes easier, but therapeutic effectiveness is reduced
Solution Approach 1:
The nucleic acid molecule is segmented into distinct functional modules including promoter regions, intronic elements, IRES sequences, and circRNA-encoding sequences. This segmentation allows for standardized, modular construction that simplifies manufacturing (easier to assemble and produce) while maintaining therapeutic effectiveness through the preserved integrity of each functional element and their coordinated interactions.
Solution Approach 2:
The patent optimizes specific parameters of the circRNA design including the arrangement of intronic elements, positioning of IRES sequences, and structural organization of the nucleic acid molecule. These parameter changes enhance therapeutic effectiveness by improving circRNA stability, expression levels, and protein production, while the overall modular architecture maintains ease of manufacture through systematic design principles.
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 significantly increases circRNA expression in tissues like heart, liver, and skeletal muscle, offering a 2- to 50-fold enhancement compared to baseline, facilitating effective therapeutic intervention.
Implementation Method 1
the intronic elements flanking the first circRNA-encoding sequence of interest and the second circRNA in tandem are backspliced yield the at least two circRNAs without forming a scar at a site wherein the circRNAs are covalently closed
Implementation Method 2
a first circRNA disclosed herein can further have an internal ribosome entry site (IRES), a promoter region in the 5′ untranslated region (UTR) and outside of the intronic elements
Data Source
AI summary
The present disclosure provides nucleic acid molecules encoding for at least two circular RNA (circRNAs), adeno-associated virus (AAV) particles including nucleic acid molecules encoding for at least two circRNAs, pharmaceutical compositions, and methods for delivering such to a subject.