Harnessing synthetic biology for advancing RNA therapeutics and vaccine design

Abstract

Recent global events have drawn into focus the diversity of options for combatting disease across a spectrum of prophylactic and therapeutic approaches. The recent success of the mRNA-based COVID-19 vaccines has paved the way for RNA-based treatments to revolutionize the pharmaceutical industry. However, historical treatment options are continuously updated and reimagined in the context of novel technical developments, such as those facilitated through the application of synthetic biology. When it comes to the development of genetic forms of therapies and vaccines, synthetic biology offers diverse tools and approaches to influence the content, dosage, and breadth of treatment with the prospect of economic advantage provided in time and cost benefits. This can be achieved by utilizing the broad tools within this discipline to enhance the functionality and efficacy of pharmaceutical agent sequences. This review will describe how synthetic biology principles can augment RNA-based treatments through optimizing not only the vaccine antigen, therapeutic construct, therapeutic activity, and delivery vector. The enhancement of RNA vaccine technology through implementing synthetic biology has the potential to shape the next generation of vaccines and therapeutics.

Fig. 1. Overview of RNA-based diagnostics, therapeutics, and living therapeutics.

A Schematic representation of RNA-based diagnostics, highlighting toehold switches detecting a trigger RNA to activate fluorescent protein expression, and aptamers recognizing a specific target molecule to generate a fluorescent signal. B Illustration of the cellular mechanism of action of various RNA therapeutics and vaccines, including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 with guide RNA, antisense oligonucleotides (ASOs), aptamers, small interfering RNAs (siRNAs), ribozymes, and mRNA vaccines. C Depiction of engineered RNA devices or circuits transforming cells into living factories producing therapeutic outputs, such as the expression of cell surface molecules like chimeric antigen receptors (CARs), the production of therapeutic proteins, or the initiation of cell migration. These engineered cells can also be designed to respond to small molecules as a switch to regulate therapeutic output. “Created with BioRender.com”.

Fig. 2. Synthetic biology tools for engineering RNA.

A Codon optimization tackles issues with non-optimized codons that can lead to misfolded antigens or low yield when expressing antigens from non-human organisms in human cells. By substituting non-optimized codons (red) with synonymous codons optimized for human expression (green), this strategy significantly enhances the yield of correctly folded, functional antigens. B internal ribosome entry sites (IRES) elements function as a novel entry point for ribosomes, allowing them to bind at locations beyond the traditional 5’ Untranslated Region (UTR) on the mRNA. This feature facilitates the expression of multiple distinct antigens from a single mRNA construct. C The strategy for the creation of chimeric proteins fuses two distinct peptides to form a single, functional protein, enhancing the diversity of potential antigenic constructs. D Inclusion of 2A self-cleaving peptides serve as a molecular ‘comma’ during protein synthesis. They induce a ‘ribosome skipping’ event during translation, leading to the cleavage of the polypeptide at the 2A site. This allows for the independent expression of multiple antigens from a single open reading frame, thereby enabling multi-antigen expression from a single construct. “Created with BioRender.com”.

Fig. 3. Comparative overview of linear mRNA, saRNA, and circRNA constructs.

A For traditional mRNA vaccines, the antigenic or immunomodulatory sequence is present between 5’ and 3’ UTRs and translated directly from linear, non-replicating mRNA transcripts. B saRNA encodes additional replicase sequences within their construct that facilitate in vivo replication of entire mRNA construct. C circRNA constructs display a covalently closed loop structure created via back-splicing events that connect the 3’ end of an upstream exon to the 5’ end of a downstream exon. D CircRNA constructs can also be constructed enzymatically using enzymes that catalyze the formation of covalent bonds between moieties located at the 5’ and 3’ ends of the sequence. These constructs are notably stable due to their resistance to exonucleases, leading to potential prolonged antigenic protein expression within the cell. “Created with BioRender.com”.