Single-stranded DNA (ssDNA) has been until recently largely ignored as a source of genetic material for use in gene therapies, largely due to a lack of cost-effective, scalable manufacturing methods. The numerous benefits to using ssDNA, including increased safety and efficiency, compared with viral vectors and double-stranded DNA, have driven interest in developing practical production solutions. Currently, several companies are offering research and GMP-grade ssDNA in different formats in support of the development of gene and gene-editing therapeutics.
Understanding the Role and Potential of Single-Stranded DNA
Single-stranded DNA (ssDNA) is a less stable version of the more commonly known double-stranded DNA (dsDNA) comprising complementary DNA sequences in a helical shape. In the body, ssDNA is involved in DNA replication and repair. In the biopharmaceutical industry, it has a wide range of applications, including in fundamental biological research, the construction of nanoscale DNA scaffolds for use in molecular machines and drug delivery systems, and as the genetic material in gene, gene-editing, and ex vivo and in vivo gene-modified cell therapies, cancer vaccines, and vaccines against infectious diseases.
Advantages of ssDNA in therapeutic and vaccine applications include its biocompatibility, low immunogenicity and toxicity, lack of concerns about pre-existing immunity, and superior cellular uptake. In addition, ssDNA with customized sequences and modifications can be engineered and synthesized to support targeted delivery and extended expression without integration and insertional mutagenesis concerns.
Lack of access to robust, scalable manufacturing processes has been a major hindrance to the development of ssDNA-based therapeutics and vaccines despite these potential advantages. Delivery of ssDNA can also be challenging. Several companies are working to address these issues. As a result, ssDNA is emerging in numerous forms as a promising new modality and delivery solution.
Applications of ssDNA in Gene Therapy: A Safer, Nonviral Alternative
Most gene and gene-modified cell therapies on the market today are produced using viral vectors for delivery of the genetic material, including adeno-associated viruses (AAV), lentiviruses (LVs), retroviruses (RVs), and a few others. There are concerns with the use of viral vectors, however.1-3 AAV vectors have a limited genetic cargo capacity, while LV vectors, because they integrate into the genome, present a risk of insertional mutagenesis. Both can generate undesired immune responses and have potential toxicity issues, particularly when administered at higher doses. Repeat dosing can be an issue due to development of immunity in patients. Vector manufacturing is also complex and costly.
Nonviral dsDNA-based approaches, largely leveraging plasmid DNA (pDNA), present their own sets of challenges.1-3 In addition to difficulties with achieving targeted, highly efficient delivery without the use of specialized delivery technologies, naked pDNA can be highly toxic and immunogenic, only supports transient expression, and suffers from physiological clearance issues. Lipid nanoparticles (LNPs) can be used as delivery vehicles, but they tend to be less efficient than viral vector for gene delivery.
For these reasons, interest is growing in the use of ssDNA for delivery of genetic material to cells, both in vivo and ex vivo. A few ssDNA-based (and RNA-based) therapeutics have been approved, the first of which received marketing authorization from the U.S. Food and Drug Administration in 1998 (formivirsen, Isis Pharmaceuticals).4 Continued advances in the design and engineering of these antisense oligonucleotides (ASOs) and immune stimulatory oligonucleotides (ISOs), which are short sequences with chemically modified backbones, are enabling the development of drugs with higher potency and stability, improved pharmacokinetics, and more targeted binding to mRNA or pre-mRNA species for enhanced modulation of gene expression.
Long ssDNA strands are currently being explored for use in gene therapies due to their ability to deliver genetic material up to 10 kb in length combined with their reduced immunogenicity and toxicity and ability to generate specific sequences and with various, controlled modifications.1 For instance, promoter sequence optimization can be used to achieve gene expression in target cells and tissues, while the addition of scaffold/matrix attachment regions (S/MARs) lengthens the period of gene expression without integration.3 Incorporation of inverted terminal repeats (ITRs) and hairpin motifs increases resistance to nuclease degradation and increases intracellular transport.5 There is also no concern for development of immunity, making repeat dosing possible.
In vivo delivery of ssDNA-based gene therapies does require the use of some type of delivery vehicle. LNPs are the most widely explored approach, but other systems, including ssDNA-based nanocarriers (vide supra), are also under investigation.
Enhancing Gene Editing Efficiency with ssDNA Templates
One of the most promising applications for ssDNA in the biopharma industry is in gene editing, both for research purposes and the production of therapeutics. Many of the same attributes are advantageous in these applications as in traditional gene therapy, including reduced immunogenicity, toxicity, and off-target integration compared with viral vectors and pDNA.6 In addition, studies have shown that ssDNA is a much more effective template for creating gene knock-ins using CRISPR (clustered regularly interspaced short palindromic repeats) homology-directed repair (HDR) owing to its high editing efficiency and specificity. Furthermore, ssDNA with specific chemical modifications allows for successful editing of a wide variety of cell types.7 Circular ssDNA, meanwhile, has been shown to improve the gene editing efficiency of both the TALEN (transcription activator-like effector nuclease)8 and CRISPR9 gene-editing systems. The latter was shown to support genetic sequences up to 20 kb with precise and controlled integration, creating opportunities for use of this technology in the development of adoptive cell therapies.10,11
Nanoscale Delivery Systems Leveraging ssDNA Structures
Efficient gene delivery — and delivery of small molecule drugs as well — is a primary challenge facing developers of these advanced therapies. Naked DNA (and RNA) molecules are fairly large and high negatively charged, making it difficult for them to pass into cells without assistance. Added to this issue is the need to protect the DNA sequences from enzyme degradation and ensure that they reach only desired cells. LNPs overcome instability issues and provide improved cellular uptake, but targeting outside of the liver remains challenging.
Nanoscale systems appear to be effective solutions for overcoming many drug delivery problems for all types of drug substances with many different release profiles. They have been explored for the delivery of a wide variety of payloads, including chemical drug substances, proteins, peptides, antibodies, and DNA/RNA formulated as both oral dosage forms and injectables (including long-acting, sustained-release products).11,12
Nanocarriers derived from ssDNA help overcome the challenges to successful, targeted delivery of genetic payloads for both in vivo gene therapies and adoptive cell therapies.5,13 Programmable DNA fabrication (akin to the synthesis of DNA origami) is applied to generate structures with specific shapes, sizes, and functionalities (including attachment of highly specific binding ligands) and reduced immunogenicity. These DNA nanocarriers protect the encapsulated ssDNA and enable highly targeted delivery of ssDNA with release of the payload in response to specific biochemical stimuli such as changes in pH, enzyme activity or redox potential, allowing controlled expression kinetics. In one example, an-icosahedral-shaped DNA framework functionalized with virus-specific binding agents was shown in vitro to effectively trap viruses and inhibit their infectious ability.14
In a similar vein, self-assembled DNA-origami-based, programmable T cell engagers comprising DNA origami nanocarriers with attached, precisely spaced IgG antibodies, F(ab) and scFv fragments have also been reported. These systems were shown to target and lyse tumor cells in a mouse model.15 Self-assembled, stable ssDNA nanotubes have also been used to cross the blood–brain tumor barrier and selectively deliver the anticancer agent doxorubicin to glioblastoma cells in a disease model.16 Bispecific circular ssDNA aptamers have also been explored for targeted delivery of treatments for various cancers and neurodegenerative disorders.17
Emerging and Exploratory Uses of ssDNA in Therapeutics
In addition to gene therapies of various types, ssDNA is being explored in a few other therapeutics applications, Researchers have developed ssDNA aptamers directed against various snake venom toxins as treatments for snake bites.18 Circular ssDNA is being investigated in various theragnostic applications in the form of aptamers and miRNA inhibitors and as templates for production of DNA origami, DNA nanoflowers, and DNA hydrogels with therapeutic and diagnostic applications.17
Key Innovators Advancing ssDNA Therapeutic Platforms
Given that ssDNA-based therapeutics and vaccines represent a nascent field within the biopharmaceutical industry, only a limited number of companies are active in this area. Examples include Moligo Technologies, CPTx/GXstrands GenScript, Takara Bio, Kano Therapeutics, Touchlight, and Full Circles Therapeutics.
Moligo Technologies uses a patented, PCR-free “injection molding” enzymatic technology to produce ultrapure long ssDNA strands. The company is partnering with various groups on the development of gene therapies based on its ssDNA. One such collaboration with Nationwide Children’s Hospital focuses on gene therapies for cystic fibrosis and other airway diseases.19
Originating from work with DNA origami at the Technical University of Munich (TUM), GXstrands (part of CPTx) spun out from TUM in 2024 to offer high-quality, custom-designed ssDNA to gene editing companies and other therapeutic innovators. The company produces ssDNA in a range of lengths using a phage-based fermentation process that it says is a more cost-effective and scalable process than enzymatic synthesis methods and provides high fidelity and allows for easy sequence modification.4 CPTx (Composite Programmable Therapeutics) uses the ssDNA GXstrands produces as a raw material in its proprietary non-viral vector platform based on unique DNA nanocarriers. This technology allows for safe and precise gene delivery for in vivo CAR-T cell therapies and many other innovative genetic medicines.
GenScript produces GenExact™ ssDNA (200–6,000 nucleotide insert length capacity) in both research and GMP grade, focusing on CRISPR gene editing for the generation of gene-modified cell therapies.20 Takara Bio’s Guide-it Long ssDNA Production System, according to the company, allows for on-demand production of ssDNA donor templates up to 5,000 nucleotides for use in CRISPR gene editing with minimal off-target integration and precise site-specific insertion.21
Kano Therapeutics is developing circular ssDNA-based gene therapies using its own ssDNA produced by a fermentation-based process.22 The company says its circular DNA is more readily manufactured and enables longer DNA insertions, which supports gene editing of novel targets. It offers design, manufacturing, and screening services in support and is indication, disease, and technology agnostic.
Touchlight’s MegaBulb ssDNA is also a circular ssDNA template with a unique structure that the company says provides enhanced stability and efficacy.23 It can incorporate multi-kilobase sequences in CRISPR gene-editing applications, and the proprietary enzymatic synthesis method yields high purity, high-fidelity ssDNA suitable for not only gene editing applications but also other types of therapeutics. The product is available in research and GMP grades.
Full Circles Therapeutics produces circular ssDNA (C4DNA™) intended for gene therapy appplications.24,25 Its GATALYST platform then allows optimization of complexes comprising the ssDNA and nuclease editor, leading to enhanced gene editing efficiency. The company says it has reduced the cost and time required for CAR-T cell development by 60% and 80%, respectively, and has shown increased editing efficiency in multiple cell types.
References
1. “Why Use ssDNA?” Moligo Technologies. Accessed 21 Jul. 2025.
2. Ducani, Cosimo. “The Emerging Role of ssDNA in Gene Therapy.” Gen. Eng. News. 29 Apr. 2024.
3. Boldt, Grant. “Opening new doors to precision medicine with single-stranded DNA (ssDNA): part I.” Manufacturing Chemist. 24 Mar. 2025.
4. Scharner, Juergen and Isabel Aznarez. “Clinical Applications of Single-Stranded Oligonucleotides: Current Landscape of Approved and In-Development Therapeutics.” Mol. Ther. 29: 540–554 (2020).
5. Boldt, Grant. “Opening new doors to precision medicine with single-stranded DNA (ssDNA): part II.” Manufacturing Chemist. 26 Mar. 2025.
6. “Efficient production and application of ssDNA for CRISPR/Cas knockins up to 5 kb.” Takara Bio. Accessed 21 Jul. 2025.
7. Kanke, Karen L, et. al. “Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.” Nucleic Acids Res. 52: 13561–13576 (2024).
8. Palmgren, Gorm. “Circular ssDNA boosts TALEN editing efficiency.” CRISPR Medicine News. 13 May 2025.
9. Evangelou, Christos. “Circular Single-Stranded DNA Enables Efficient Non-Viral Immune Cell Engineering: Implications for CAR-T Cell Therapy.” CRISPR Medicine News. 11 Dec. 2024.
10. Wu, Hao. “Efficient non-viral engineering of immune cells for cell therapy using circular single-stranded DNA.” Cell & Gene Therapy Insights. 11: 7–11 (2025).
11. Challener, Cynthia. “Achieving Targeted Delivery with Nanoscale Systems.” Pharm. Tech. 9 Jun. 2025.
12. Challener, Cynthia. “Enhancing Solubility and Bioavailability with Nanotechnology.” Pharm. Tech. 7 Apr. 2025.
13. Kretzmann, JA, et al. “Gene-encoding DNA origami for mammalian cell expression.” Nat. Commun. 14: 1017 (2023).
14. Sigl, C, et al. “Programmable icosahedral shell system for virus trapping.” Nat. Mater. 20: 1281–1289 (2021).
15. Wagenbauer, KF, et al. “Programmable multispecific DNA-origami-based T-cell engagers.” Nat. Nanotechnol. 18: 1319–1326 (2023).
16. Harris, Michael A, et al. “ssDNA nanotubes for selective targeting of glioblastoma and delivery of doxorubicin for enhanced survival.” Science Advances. 1 Dec. 2021.
17. Shen, Tingting, Yu Zhang, Lei Mei, Xiao-Bing Zhang, and Guizhi Zhu. “Single-stranded circular DNA theranostics.” Theranostics. 12: 35–47 (2022).
18. Alomran, Nessrin, et al. “Exploring the utility of ssDNA aptamers directed against snake venom toxins as new therapeutics for tropical snakebite envenoming.” Toxins. 14: 469 (2022)
19. Ducani, Cosimo. “The Emerging Role of ssDNA in Gene Therapy.” Gen. Eng. News. 29 Apr. 2024.
20. “Single-Stranded DNA (ssDNA) and Double-Stranded DNA (dsDNA) services for next generation gene analysis.” GenScript. 8 Sep. 2022.
21. “Efficient production and application of ssDNA for CRISPR/Cas knockins up to 5 kb.” Takara Bio. Accessed 21 Jul. 2025.
22. Chowdhry, Amit. “Kano Therapeutics: $7.1 Million Raised To Create ssDNA As Flexible Biomolecule For Gene Insertions.” LinkedIn Pulse. 13 Aug. 2024.
23. Adie, Tom and Elena Stoyanova. “MegaBulb DNA: a Non-Viral ssDNA Breakthrough Revolutionizing Gene Editing.” Pharma’s Almanac. 30 Sep. 2024
24. “Full Circles Therapeutics, Full Circles Therapeutics Secures U.S. Patent Allowance for Targeted Genome Modification Using Circular Single-Stranded DNA.” Full Circles Therapeutics 7 Mar. 2025.
25. Evangelou, Christos. “Circular Single-Stranded DNA Enables Efficient Non-Viral Immune Cell Engineering: Implications for CAR-T Cell Therapy.” CRISPR Medicine News. 11 Dec. 2024.