Harnessing synthetic biology for advancing RNA therapeutics and vaccine design

The paper addresses how synthetic biology can be harnessed to overcome key challenges in RNA therapeutics and vaccine design. It situates the work in the context of rapid advances in RNA platforms (highlighted by COVID-19 mRNA vaccines) and the limitations of traditional small-molecule drug discovery for complex and genetic diseases. The goal is to review synthetic biology tools—from component-level RNA devices to systems-level circuit engineering—and explain their potential to improve RNA-based diagnostics, therapeutics, and vaccines in terms of speed, safety, scalability, efficacy, and cost. The introduction traces enabling advances from genetic engineering and PCR to genomics and the emergence of modular, composable biological parts and circuits, motivating a focus on RNA as a safer, fast-acting substrate for therapeutic engineering.

The review synthesizes prior work on synthetic biology’s evolution from DNA-centric engineering (artificial genes, regulatory networks, and synthetic genomes) to RNA-focused devices and circuits (aptamers, ribozymes, switches) that regulate cellular behavior. It summarizes applications of RNA devices in diagnostics (e.g., toehold switches for viral detection; aptasensors for cancer, cardiovascular, and neurological biomarkers), living therapeutics (e.g., RNA-controlled CAR-T safety and activation switches), and gene regulation modalities (ASOs, siRNA/RNAi, aptamers, CRISPR). It catalogs FDA-approved RNA therapeutics (e.g., ASOs: mipomersen, inotersen; siRNAs: patisiran, givosiran, lumasiran) and highlights CRISPR control via ligand- and light-responsive guide RNA designs, riboswitches, and tissue restriction using miRNA response elements. The literature also covers RNA vaccine advances across infectious diseases and oncology, while noting limitations for bacterial antigens and complex posttranslational modifications.

This is a narrative/conceptual review that aggregates and organizes published evidence and design strategies at three layers: (1) RNA-based diagnostics and therapeutic control devices (aptamers, ribozymes, switches, logic circuits); (2) RNA therapeutic modalities (ASOs, siRNA, aptamers, CRISPR-based editing/regulation) and vaccines; and (3) engineering strategies to advance RNA vaccines via synthetic biology. For vaccine design, the review details sequence-level engineering (codon optimization; multi-antigen strategies via chimeric proteins, IRES elements, or 2A self-cleaving peptides), construct architectures (modRNA, saRNA with replicases, circRNA formed by self-splicing or enzymatic ligation, RNA origami), and delivery systems (AAV with synthetic control elements and hybridization strategies, bacterial vectors like engineered E. coli, and chemical carriers such as liposomes/LNPs with targeting ligands). It integrates mechanistic rationale, design trade-offs, and reported performance from the cited literature without conducting new experiments.

- Synthetic biology provides modular RNA devices and circuits that enable sensitive, low-cost diagnostics (e.g., toehold switches for SARS-CoV-2, Zika, Ebola; aptasensors for HER2, CRP, Aβ) and controllable living therapeutics (e.g., small-molecule- or temperature-responsive switches to modulate CAR-T activity). - Multiple clinically validated RNA therapeutics demonstrate modality maturity: FDA-approved ASOs (mipomersen, inotersen) and siRNAs (patisiran, givosiran, lumasiran). Aptamers (pegaptanib) show extracellular targetability advantages. CRISPR systems can be precisely regulated using ligand- or light-responsive gRNAs, riboswitch exposure of spacer sequences, and tissue-restricted editing using miRNA-repressible anti-CRISPR elements. - mRNA vaccines offer rapid design, scalable cell-free manufacturing, strong immunogenicity, and safety; COVID-19 mRNA vaccines progressed from conception to EUA in under one year. Research indicates potential across influenza, dengue, HSV-2, rabies, Zika, and oncology. - Synthetic biology can expand antigen breadth and expression: • Codon optimization (aided by deep learning) boosts translation but is context-dependent; overly rapid translation can impair folding. • Multi-antigen strategies include chimeric proteins and polycistronic expression. IRES elements (>500 bp) enable internal initiation but can yield gene-order-dependent inefficiency; 2A peptides (<100 bp) support efficient multi-gene expression via ribosome skipping, though cleavage is incomplete. • Advanced constructs enhance stability and potency: modRNA (nucleoside and structural optimization), saRNA (in vivo amplification reduces dose), and circRNA (closed-loop architecture resists exonucleases). A circRNA SARS-CoV-2 RBD vaccine elicited more potent and prolonged responses than modRNA at equal dose and remained stable for up to 2 weeks at room temperature. RNA origami scaffolds can improve delivery and stability. - Delivery engineering is critical given mRNA’s size (~1.7 MDa), negative charge, and RNase susceptibility. Tools include: • AAV vectors with synthetic switches to control receptor activation, transgene expression, and trafficking; regulatory cassette optimization; hybridization with other vectors and exosomes. • Engineered bacterial vectors (e.g., attenuated E. coli) leveraging abundant genetic tools and inherent adjuvanticity; endosomal escape proteins (e.g., listeriolysin O) can enhance antigen presentation, though bacteria are unsuitable for mRNA delivery and add biological complexity. • Liposomes/LNPs protect mRNA and can be functionalized with targeting ligands (e.g., antibodies) to direct tissue-specific delivery.

The review argues that synthetic biology directly addresses key challenges in RNA therapeutics and vaccines by enabling precise control over antigen choice and form (chimeras, multi-antigen constructs), expression efficiency (codon optimization, IRES/2A design), transcript stability and dosing (modRNA, saRNA, circRNA, RNA origami), and delivery specificity and safety (engineered AAVs, microbial vectors with controlled features, LNP functionalization). These advances collectively improve immunogenicity, safety profiles, manufacturing speed, and distribution feasibility (e.g., circRNA heat stability). For therapeutics beyond vaccines, RNA devices allow spatiotemporal control of living therapeutics and gene editing, potentially reducing adverse events (e.g., CAR-T toxicity) and increasing tissue specificity (miRNA-regulated anti-CRISPR). The synthesis indicates that integrating RNA platform flexibility with synthetic biology’s design-build-test-learn paradigm can broaden disease coverage, including emerging pathogens and cancer, and pave a path toward complex antigen production and context-aware therapeutic regulation.

Synthetic biology offers a powerful toolkit to elevate RNA-based diagnostics, therapeutics, and vaccines through modular device design, multi-antigen engineering strategies, stability-enhanced constructs (modRNA, saRNA, circRNA), and programmable delivery systems. The review highlights promising evidence—such as circRNA’s improved potency and thermal stability and clinically validated RNA drugs—while outlining design trade-offs (e.g., IRES vs 2A, codon optimization context). Future work should: (1) integrate AI/ML to optimize codon usage, UTRs, and structural elements; design aptamers, encoded antibodies, and complex circuits; and predict immunogenicity; (2) enable in-host production of complex antigens (multidomain proteins, polysaccharides) and pathogen-specific posttranslational modifications via multi-gene RNA systems; (3) refine delivery vectors for targeted, safe, and efficient in vivo delivery; and (4) develop responsive, disease-state-triggered RNA circuits for precise therapeutic control.

As a narrative review, the work does not present new experimental data and is limited by the scope of cited literature. Several technologies discussed remain early-stage or context-dependent: codon optimization can impair folding if over-accelerated; IRES elements are large and can show gene-order-dependent inefficiency; 2A peptides have incomplete cleavage; biological vectors (bacteria, viruses) add complexity and potential immunotoxicity; and bacteria are unsuitable for mRNA delivery. Current RNA vaccines struggle to express complex bacterial polysaccharides and certain multidomain antigens due to folding and modification requirements. Stability, delivery specificity, and manufacturability constraints persist, and generalizability across pathogens and patient populations requires further validation.