Messenger RNA (mRNA) acts as the molecular instruction manual, carrying genetic information from DNA in the cell nucleus to the cytoplasm where proteins are manufactured. Standard mRNA is a single-stranded, linear molecule designed for temporary use, serving its purpose before being rapidly broken down by the cell’s defense systems. This linear blueprint features a protective cap structure at one end and a long poly-A tail at the other, both common targets for degradation enzymes. This transient nature of linear mRNA has driven the search for more durable alternatives for therapeutic applications.
The Unique Structure of Circular mRNA
Circular mRNA (circRNA) is a form of single-stranded RNA distinguished by its structure as a covalently closed continuous loop. Unlike linear mRNA, which possesses free 5′ and 3′ ends, the ends of circRNA are joined together, forming a seamless ring. This unique configuration means the molecule lacks the 5′ cap and the 3′ poly-A tail that characterize standard linear mRNA.
The closed-loop structure is naturally produced within cells through a process called backsplicing, where a downstream splice site is joined to an upstream splice site on the precursor RNA molecule. This mechanism skips the conventional splicing pathway that creates linear mRNA. For therapeutic purposes, scientists engineer synthetic circRNA that maintains this superior stability for prolonged protein expression.
How Circular mRNA Works
Circular mRNA is translated into protein using a distinct, cap-independent mechanism. Engineered circRNA typically incorporates a specific sequence known as an Internal Ribosome Entry Site (IRES) to begin translation. This IRES sequence serves as a landing pad for the ribosome, allowing it to attach directly to the RNA loop rather than scanning from a capped end. The ribosome complex assembles on the IRES and begins reading the genetic code to synthesize the desired protein.
An alternative initiation method involves incorporating $N^6$-methyladenosine ($m^6A$) modifications into the circRNA sequence. These modifications recruit specific proteins that help guide the ribosome to the correct starting location, enhancing the efficiency of the cap-independent translation process.
Key Advantages Over Linear mRNA
The primary advantage of circular mRNA lies in its exceptional stability compared to linear mRNA, which is rapidly degraded by cellular enzymes. Linear RNA is vulnerable to exonucleases, enzymes that dismantle the molecule by chewing away nucleotides from its exposed ends. Since circRNA is a closed loop with no free ends, it is resistant to this exonuclease-mediated degradation.
This resistance increases the molecule’s half-life within the cell, extending it from hours (typical for linear mRNA) to several days or even weeks. The prolonged persistence of the circRNA template allows for sustained protein production, leading to a higher total yield of the therapeutic protein over time. The circular structure also tends to elicit a lower innate immune response from the body, which is an undesirable side effect with some linear mRNA treatments. This combination of durability and reduced immunogenicity suggests that circRNA could be administered in lower doses or less frequently to achieve the same therapeutic effect.
Therapeutic Applications
The sustained and robust protein expression offered by circular mRNA makes it an appealing platform for developing next-generation therapeutic strategies. A primary area of focus is next-generation vaccines, where circRNA is being explored to create more durable immune responses against infectious diseases like COVID-19. The prolonged presence of the antigen-encoding circRNA means lower doses may be required, potentially reducing manufacturing costs and simplifying logistics.
Circular mRNA is also relevant for protein replacement therapies aimed at treating genetic disorders where patients lack a functional protein. By delivering a circRNA encoding the missing protein, researchers can achieve sustained, high-level expression of the therapeutic factor, addressing the need for frequent dosing. Early research has also explored using circRNA in cell-based therapies, such as in situ chimeric antigen receptor (CAR) T-cell therapy, to temporarily program immune cells to target cancer. Furthermore, the stability of circRNA makes it a promising delivery vehicle for components of gene editing systems, offering a transient but highly effective method to introduce editing tools into target cells.