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What are Circular RNAs (circRNAs)

Circular RNAs (circRNAs) are endogenous RNA molecules that form covalently closed continuous loops. They are named after their circular shapes [1-2].

CircRNAs are mainly divided into three categories based on their components:

• Exonic circular RNAs (ecircRNAs)- formed by exons
• Intronic circRNAs - (ciRNAs) formed by introns
• Exon-intron circular RNAs (EIciRNAs) formed by both exons and introns

Key Milestones in circRNA Discovery and Research

This timeline reflects the major milestones in the discovery and in vitro development of circRNAs, highlighting their journey from initial discovery to potential clinical applications [3].

What are the advantages of developing therapeutics based on circRNA?

CircRNAs offer numerous advantages for developing therapeutics due to their unique properties and versatile functions.

Here are some key benefits:

• Stability: CircRNAs are highly stable and resistant to degradation due to their covalently closed loop structure. This stability ensures prolonged expression of engineered constructs in therapeutic applications, such as CAR T cells.
• Expression Patterns: Specific and tissue-related expression patterns aid in precise targeting and reduced side effects.
• Efficient Gene Regulation: The ability to regulate gene expression effectively through microRNA sponging is crucial for therapies like CAR T cells.
• Improved Transfection Efficiency: CircRNA-based constructs have shown higher transfection efficiency compared to linear mRNA constructs, leading to more effective and consistent production of therapeutic cells.
• Reduced Immunogenicity: CircRNAs are less immunogenic than viral vectors or linear mRNAs, reducing the likelihood of adverse immune reactions and enhancing patient safety.
• Precision Targeting: Design flexibility allows for precise targeting of specific antigens, improving therapeutic specificity and reducing off-target effects.

CircRNA therapy vs traditional methods

Overall, circRNAs offer a promising platform for developing innovative therapies across a wide range of medical conditions, including cancer, infectious diseases, autoimmune diseases, and genetic disorders. Their unique properties make them ideal for next-generation RNA-based therapies.

Therapeutic potential of circRNAs in cell therapy and immunotherapy

Circular RNAs (circRNAs) offer significant therapeutic potential due to their unique properties and versatile functions. Recent research shows that circRNAs play key roles in many cellular processes, and their dysregulation is linked to various diseases. CircRNAs have broad potential in developing innovative cell and gene therapies for a wide range of medical conditions. In particular, circRNAs could significantly impact cancer treatments in the future.

Promising application of circRNAs in cell therapy

1. CircRNA for Base Editing in Primary T Cells

Base editing is an advanced gene-altering method that allows precise changes to the genome without causing harmful DNA breaks. Typically, base-editing enzymes are delivered using a modified linear messenger RNA (mRNA), which is expensive to produce. Martin Pule et al. [5] at Autolus Therapeutics in the UK introduced a new approach using circRNA for base editing in primary T cells. They found that circRNA outperformed the linear mRNA in efficiency. They developed a protocol to manufacture a clinical dose (1 × 10^8 cells) of base-edited CAR T cells that lack the inhibitory receptor PD-1. They achieved editing rates as high as 86% using just 0.25 μg of circRNA per 1 × 10^6 cells. Compared to linear mRNA, circRNA enabled an 8-fold reduction in the amount of RNA needed, potentially lowering the cost of base-edited cell therapies. This method is scalable and could be used to produce large quantities of base-edited anti-CD19 CAR T cells with complete gene disruption, impacting both personalized and off-the-shelf cell therapies.

2. CircRNA-based TCR-T Therapy

Lianghua Shen and colleagues [6] explored the use of circular mRNA to develop T cell receptor-engineered T cell (TCR-T) therapy. This pioneering study marks the first use of circRNA technology to discover antigen-specific TCRs and produce TCR-T cells with therapeutic potential. Their evaluation demonstrated that circRNAs can be a more effective approach for TCR isolation and identification. Their non-viral, non-integrating method offers a new treatment avenue for cytomegalovirus (CMV) infection following hematopoietic stem cell transplantation (HSCT).

CircRNA platforms are recognized for their high safety, excellent stability, and ease of production. They show potential for treating a range of diseases beyond CMV infection, such as cancer therapy by identifying TCRs targeting tumor neoantigens or immunogenic self-antigens. This method could streamline the creation of TCR-T cells, potentially advancing personalized medicine and immunotherapy efforts.

3. Immune Regulation by circRNAs

Growing evidence suggests that circRNAs are involved in regulating both adaptive and innate immunity. Certain circRNAs act as miRNA sponges or interact with proteins, influencing the expression of immune-related genes including crucial immune checkpoint molecules. This regulation shapes the tumor microenvironment and significantly impacts the response to immunotherapy by affecting macrophage, NK, and T cell function. The specificity of circRNAs also makes them ideal biomarkers in cancer, providing essential support for diagnosis, prognosis, and treatment guidance [7].

Broad Applications of circRNAs

CircRNAs' high stability, specific expression patterns, and diverse regulatory functions make them a promising platform for developing innovative therapies for many medical conditions. Their broad potential applications include treatments for infectious diseases, rare diseases, hematological disorders, neurological disorders, cardiovascular diseases, autoimmune diseases, gene therapy, and cancer therapy.

What considerations should developers keep in mind when outsourcing circRNA synthesis?

When outsourcing circRNA synthesis, developers should ensure the success of their projects by considering several critical factors:

• Quality and Purity
  • Ensure the provider uses advanced techniques, such as HPLC purification, to achieve high purity and quality of circRNA.
  • Check for quality control measures and consistency in product batches.
• Efficiency of Circularization
  • Assess the efficiency of the circularization process to ensure high yields and minimal precursor residue.
  • Ask about the methods used for circularization (chemical, enzymatic, or a combination).
• Customization Capabilities
  • Evaluate the provider's ability to customize circRNAs to specific requirements, including sequence design, length, and modifications.
  • Confirm that they can engineer circRNAs with the desired properties for your specific biological roles.
• Turnaround Time
  • Consider the provider’s turnaround time for delivering synthesized circRNAs.
  • Ensure that the timeline aligns with your project’s deadlines.
• Scalability
  • Check if the provider can scale up production to meet your needs, from small-scale research quantities to larger batches for clinical or industrial applications.
• Cost
  • Compare costs among different providers, but also consider the quality and reliability of the service.
  • Look for transparent pricing and any additional fees that may apply.
• Regulatory Compliance
  • Verify that the provider complies with relevant regulatory standards and guidelines, especially if the circRNA will be used in clinical applications.
• Technical Support and Expertise
  • Assess the level of technical support and expertise offered by the provider.
  • Ensure they have a team of knowledgeable scientists who can assist with troubleshooting and optimizing protocols.
• Intellectual Property
  • Clarify the ownership and rights to the synthesized circRNA, especially if custom sequences or proprietary methods are involved.
  • Ensure that any intellectual property agreements protect your interests.
• Reputation and Track Record
  • Research the provider’s reputation in the industry, including reviews and testimonials from other clients.
  • Check their track record for successful delivery of similar projects.

By considering these factors, developers can make informed decisions when outsourcing circRNA synthesis, ensuring they receive high-quality, customized products that meet their research and development needs.

Customized circRNA Synthesis Solutions with GenScript

Preparing circRNA in vitro is crucial for future advancements in research, clinical development, and industrial applications. As the need to model circRNA biology grows, engineering custom circRNAs with specific properties becomes increasingly important.

Recognizing the unique functions of individual circRNAs, GenScript focuses on customizing synthesized circRNAs for specific biological roles. The in vitro generation of circRNA involves synthesizing a linear RNA precursor and then ligating its ends to form a covalently closed loop. This process can be achieved using various chemical and enzymatic methods, which can be combined as needed based on the desired circRNA product and the available technologies.

GenScript’s proprietary platform offers enhanced circularization efficiency and increased purity. Our optimized circRNA preparation method leads to higher protein expression compared to linear mRNA and ensures high circularization efficiency with minimal precursor residue. Additionally, with our proprietary HPLC purification method the quality of circRNA is significantly higher. Our custom circRNA in vitro synthesis service provides numerous options to meet diverse research needs. We are committed to offering reliable and efficient services, ensuring reduced turnaround times and lower costs.

References:

1. Liu, J., et al. Circles reshaping the RNA world: from waste to treasure. Mol Cancer. 16, 58, 2017.

2. Caba, L. et al. Circular RNA—Is the Circle Perfect?. Biomolecules. 11, 1755, 2021.

3. Pisignano, G. et al. Going circular: history, present, and future of circRNAs in cancer. Oncogene. 42, 2783, 2023.

4. Meiling Sun et al., Biological functions and applications of circRNAs—next generation of RNA-based therapy. Journal of Molecular Cell Biology. Volume 15, Issue 5, mjad031, May 2023.

5. Martin Pule et al., Large-scale manufacturing of base-edited chimeric antigen receptor T cells. Molecular Therapy - Methods & Clinical Development. Volume 31, 101123, ISSN 2329-0501, 2023.

6. Shen L et al., Circular mRNA-based TCR-T offers a safe and effective therapeutic strategy for treatment of cytomegalovirus infection. Mol Ther. 3; 32(1), 168, 2024

7. REF He et al., Targeting circular RNAs as a therapeutic approach: current strategies and challenges. Signal Transduct Target Ther; 6: 185, 2021.