Circular RNA Compositions for Stable Immune Cell Expression
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Solution Overview
Problem
Conventional gene therapy using DNA can cause mutations, disrupt essential gene functions, and trigger immune responses due to integration into the host genome, and is costly and difficult to deliver effectively, while RNA-based approaches are safer but limited by existing methodologies for producing circular RNA, which are restricted by RNA size.
Innovation Solution
Development of pharmaceutical compositions comprising circular RNAs encapsulated in lipid nanoparticles, specifically ionizable lipid-based transfer vehicles that enhance expression, stability, and immunogenicity, allowing for efficient protein expression in immune cells with improved manufacturing and therapeutic efficacy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If DNA-based gene therapy is used to achieve long-lasting genetic expression, then sustained action of introduced genetic material is obtained, but integration into host genome may cause mutations, disrupt essential gene functions, and trigger immune responses
Solution Approach 1:
The patent uses circular RNA as an intermediary carrier to deliver genetic information without requiring integration into the host genome. The circular RNA structure serves as a mediator that translates genetic information into protein expression temporarily, avoiding the harmful effects of DNA integration while maintaining therapeutic efficacy. This resolves the contradiction by providing sustained protein expression without the risks associated with genomic integration.
Solution Approach 2:
The patent employs circular RNA molecules that are non-integrating and temporarily expressed, functioning as disposable therapeutic agents. These circular RNA molecules provide sustained protein expression during their circulation half-life (days to weeks) without permanent genomic integration, thereby achieving long-lasting action without the harmful effects of DNA-based gene therapy.
2Productivity
If conventional DNA based gene therapy is used for effective expression of desired gene product, then strong promoter sequences are required, but this may lead to undesirable changes in regulation of normal gene expression
Solution Approach 1:
The patent uses circular RNA as an intermediary that directly translates into protein without requiring integration into host genomic regulatory systems. The circular RNA includes its own internal ribosome entry site (IRES) and Kozak sequence for cap-independent translation initiation, eliminating the need for strong viral promoters that could disrupt normal gene regulation while maintaining high productivity of the desired gene product.
3Ease of manufacture
If existing methodologies for producing circular RNA are used, then circularized RNA can be made in vitro, but they are limited by the size of RNA that can be circularized
Solution Approach 1:
The patent employs a two-step circularization process with controlled parameters: first, intron retention during in vitro transcription to create hairpin structures that facilitate circularization; second, temperature-controlled incubation (50-65°C for 30-120 minutes) to promote efficient circularization. This parameter optimization enables circularization of larger RNA molecules (up to several kilobases) that would be infeasible with conventional single-step methods, thereby resolving the size limitation while maintaining ease of manufacture.
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 compositions achieve stable and efficient protein expression in immune cells with extended half-life and reduced immunogenicity, overcoming the limitations of DNA-based gene therapy and existing RNA methodologies by enabling effective delivery and function of circular RNAs.
Implementation Method 1
lipid nanoparticles encapsulating the circular RNAs
Implementation Method 2
The transfer vehicles can comprise, e.g., ionizable lipid, PEG-modified lipid, and/or structural lipid, thereby forming lipid nanoparticles encapsulating the circular RNAs
Data Source
AI summary
Disclosed herein are circular RNAs and transfer vehicles, along with related compositions and methods of treatment. The circular RNAs can comprise group I intron fragments, spacers, an IRES, duplex forming regions, and/or an expression sequence, thereby having the features of improved expression, functional stability, low immunogenicity, ease of manufacturing, and/or extended half-life compared to linear RNA. Pharmaceutical compositions comprising such circular RNAs and transfer vehicles are particularly suitable for efficient protein expression in immune cells in vivo. Also disclosed are precursor RNAs and materials useful in producing the precursor or circular RNAs, which have improved circularization efficiency and/or are compatible with effective circular RNA purification methods.