The structural properties of ssDNA contribute to its superior cellular uptake and stability compared with dsDNA. ssDNA adopts flexible conformations that facilitate passive diffusion and receptor-mediated endocytosis, enhancing intracellular delivery efficiency.
Structural stability elements such as inverted terminal repeats (ITRs) and hairpin motifs can protect ssDNA from nuclease degradation while helping with intracellular transport and supporting episomal stability.
In addition to these stabilising elements that can only be implemented in ssDNA, formulation strategies such as encapsulation in lipid nanoparticles (LNPs) and conjugation with biodegradable polymers further improve ssDNA delivery.
LNPs have been successfully used in RNA-based vaccines and are now being explored for ssDNA-based therapies to enhance bioavailability and systemic circulation times.
Self-folding nanocarriers for gene delivery
By applying the principles of programmable DNA nanofabrication, a nanoscale technique that folds DNA into programmable structures with precise spatial control, highly customisable nanocarriers can be engineered and evaluated for therapeutic gene delivery.
Using Watson-Crick base pairing, three-dimensional scaffolds are created to encapsulate a therapeutic cargo, such as ssDNA payloads, and enable their controlled release in target cells. This technique enhances gene therapy by improving stability, specificity and biocompatibility.
The advantages of self-folding nanocarriers for gene delivery include the following.
- Enhanced structural stability: DNA nanostructures resist enzymatic degradation and immune clearance, preserving therapeutic cargo integrity.
- Targeted delivery: Ligand-functionalised DNA strands enable selective binding to cellular receptors, ensuring precise therapeutic targeting.
- Controlled gene expression kinetics: Engineered DNA nanostructures allow fine-tuned gene expression, minimising off-target effects and optimising therapeutic efficacy.
- Reduced immunogenicity: Unlike viral vectors, DNA nanostructure delivery elicits minimal immune responses to improve patient safety.
Precise and targeted gene delivery
Engineered DNA nanocarriers exhibit remarkable stability under physiological conditions, thereby protecting the encapsulated ssDNA from enzymatic degradation and immune clearance.
Their engineered design allows them to fold into stable shapes that resist degradation by nucleases; this prolongs the duration for which the payload remains intact until delivery.
The structural stability of these nanocarriers may be further enhanced by chemical modifications that improve resistance to mechanical stress, pH variations and temperature fluctuations.
By incorporating ligand-functionalised DNA strands, these nanostructures can selectively bind to specific cellular receptors, enabling precise delivery to diseased tissues while sparing healthy or off-target cells.
The ability to modify nanocarrier surfaces by targeting ligands such as antibodies, peptides or small molecules ensures a high degree of specificity in drug delivery.
This targeted approach reduces systemic toxicity and increases therapeutic efficacy by ensuring that the genetic payloads reach their intended cellular destinations.
The tuneable nature of DNA nanocarriers allows for the precise control of gene-expression kinetics, ensuring transient and localised expression to minimise off-target effects.
Engineered nanocarriers release their genetic payloads in response to specific biochemical stimuli such as changes in pH, enzyme activity or redox potential, and this level of control enables therapeutics to be engineered for specific cellular conditions such as disease progression.
Based on results seen in a variety of in vivo studies (and unlike viral vectors), DNA nanocarrier delivery promises to elicit minimal immune responses; this reduces the risk of adverse reactions and improves patient safety.
Viral vectors, although effective for gene delivery, often trigger immune responses that can lead to unwanted side-effects or limit the effectiveness of repeated dosing.
The immune system recognises engineered DNA nanocarriers as biocompatible materials with low immunogenicity, making them an attractive alternative for gene therapy applications.
Further, lipid nanoparticles (LNPs) face significant challenges in terms of achieving their therapeutic potential, particularly when targeting specific tissues while minimising adverse effects.
The predominant challenge stems from the inherent liver tropism of LNPs: 80–90% of intravenously administered LNPs accumulate in the liver via LDL receptor interactions, leaving less than 5% available for intended extrahepatic targets.
This limitation creates substantial off-target effects, potentially triggering toxic reactions and inflammatory responses and modulating immune responses unintentionally.
Immunogenicity presents another barrier as LNPs are recognised as foreign materials by the body and stimulate innate immunity through multiple pathways.
Their lipid components activate the NLRP3 inflammasome, IL-6 receptor and MyD88, whereas PEGylated lipids can elicit anti-PEG antibodies, potentially causing anaphylaxis, complement activation-related pseudoallergies and autoimmune reactions.
In contrast, self-delivery DNA nanocarriers offer a more targeted approach with reduced immunogenicity as they can be engineered with precise molecular recognition features, such as antibodies or peptide ligands, enabling cell-specific binding and uptake while bypassing non-specific liver accumulation.
This targeted delivery enhances therapeutic efficacy and minimises off-target effects and adverse immune responses.
Mechanisms of action
DNA nanocarriers operate through a series of well-defined steps to achieve controlled gene delivery. Ligand-functionalised DNA nanostructures first recognise and bind to specific surface receptors on target cells, thus ensuring high specificity in therapeutic targeting.
By integrating receptor-binding domains into their structures, DNA nanocarriers can exploit natural cellular uptake mechanisms to enhance intracellular delivery efficiency.
Following receptor binding, these nanostructures are internalised via receptor-mediated endocytosis, which facilitates controlled cellular entry and cargo transport.
Once inside the intracellular environment, DNA scaffolds undergo pH-sensitive or enzyme-responsive disassembly, releasing the ssDNA payload at the desired location.
The technology has been developed such that DNA nanocarriers are programmed to disassemble in response to specific intracellular conditions, ensuring that therapeutic genes are released only within target cells.
The delivered ssDNA functions as a template for transient gene expression, enabling controlled therapeutic effects with minimal genomic integration risks.
Unlike viral vectors, which often integrate their genetic payloads into the host genome, DNA nanocarrier delivery ensures the temporary expression of therapeutic genes; this reduces the risk of insertional mutagenesis and unintended genetic alterations.
Compared with lipid nanoparticle (LNP) delivery, self-delivery DNA nanocarriers may eventually offer superior targeting precision because of their programmability, receptor-mediated uptake and flexible controlled intracellular release options while also avoiding the liver accumulation and immunogenicity associated with LNPs.
Future perspectives and challenges
The future of ssDNA-based therapeutics is highly promising; rapid advances are paving the way for innovative solutions to the production, development and regulatory challenges confronting new therapeutic approaches.
Achieving high-fidelity synthesis of long ssDNA sequences has been a key focus and cutting-edge methods such as phage-based bacterial expression are driving remarkable progress. These breakthroughs are expected to enhance scalability, precision and cost-effectiveness.
Beyond manufacturing, expanding research efforts are unlocking insights into the pharmacokinetics and long-term safety of ssDNA therapeutics.
With rigorous preclinical and clinical validation, ssDNA-based therapies will be well-positioned to meet and exceed regulatory standards and accelerate their path to clinical translation.
Continued work will improve stability, targeted cellular uptake and therapeutic efficacy.
As the field advances, the integration of multidisciplinary approaches — including chemical biology, nanotechnology engineering and computational modelling — will further refine ssDNA therapeutics, making them more accessible, effective and personalised.
Read part I here.
