Tremendous excitement continues to build around the potential of genetic medicines to change the way diseases are managed and treated, reports Grant Boldt Chief Operating Officer at CPTx.
Growing numbers of approvals for gene therapies and adoptive cell therapies are contributing to a rapidly expanding market. One estimate pegs the value of the global genetic medicines market at $36 billion in 2024 and rising to $118 billion by 2032 at a compound annual growth rate of 16%.1
Continued growth is not a given; challenges to both manufacturing and the safe, targeted delivery of gene and adoptive cell therapies must be overcome if these promising medicines are to be successfully commercialised for a broader array of diseases.
Current delivery systems, predominantly viral vectors and lipid nanoparticles (LNPs), suffer from several limitations; a major challenge is inefficient delivery to the right cell/tissues/organs and off-target effects.
Other delivery concerns include immunogenic reactions, physiological clearance issues, extracellular and intracellular obstacles, toxicity and safety issues, and the complexity created by significant genetic diversity amongst patients with the same diseases.
The administration of high doses to overcome inefficient delivery can lead to inflammation and greater immunogenic responses. The need for repeat dosing can also be an issue.
Furthermore, both types of delivery vehicles present manufacturing challenges ranging from limited raw materials availability to issues with developing robust, scalable and cost-effective production processes.
Interestingly, a so far underappreciated modality — single-stranded DNA (ssDNA) encoding entire genes — may hold great potential to overcome the genetic medicine delivery challenge.
Not only can ssDNA be manufactured in a rapid, robust and cost-effective manner, but it can also be developed into targeted therapeutic interventions that effectively and efficiently deliver genetic payloads to target cells with enhanced transduction efficiency and improved safety.
ssDNA for gene delivery
Leveraging more than 15 years of research and development work at the Technology University of Munich, CPTx spun out to develop innovative therapeutics built from and around ssDNA.
Using this approach, transient gene expression, gene editing and the controlled delivery of therapeutic payloads can be achieved more simply and with less cost.
The effectiveness and adaptability of these ssDNA-based therapies are directly tied to advances in ssDNA manufacturing, which have been made possible by the scalable production of high-fidelity and application-specific long-chain ssDNA molecules.
Progress in ssDNA manufacturing techniques has enhanced the potential for use in programmable therapeutics.
Unlike dsDNA synthesis, which benefits from mature technologies such as robust polymerase-based amplification and sequencing methods, ssDNA synthesis requires specialised processes that are less developed to ensure high yield, purity and functional integrity.
Phage-derived bacterial expression systems
Several approaches have emerged for scalable ssDNA production, including asymmetric PCR, rolling circle amplification (RCA) and chemical synthesis. We believe, however, that the phage-derived bacterial expression of ssDNA represents the most promising and most scalable solution for the production of ssDNA for therapeutic applications.
This technology uses bacteriophage-derived replication mechanisms to generate high-fidelity ssDNA at an industrial scale. Filamentous bacteriophages and phage-like particles such as M13 serve as efficient vehicles to produce long ssDNA sequences.
By integrating a target ssDNA sequence into the phage genome, bacterial hosts such as Escherichia coli can be engineered to continuously produce and secrete ssDNA into the culture medium. Key advantages of this method include the following.
- High yield and scalability: Unlike enzymatic methods that are constrained by expensive reagents and starting materials, require significant development times and offer limited flexibility, the CPTx phage-based system enables rapid scale-up using existing microbial fermentation infrastructure, thereby making it ideal for industrial applications.
- Superior purity and fidelity: Bacteriophage replication naturally produces ssDNA with minimal impurities and unwanted secondary structures, simplifying downstream processing.
- Cost-effectiveness: The ability to propagate phage-based expression systems in bacterial cultures reduces manufacturing costs compared with purely synthetic or enzymatic methods.
- Ease of genetic modification: CPTx phage vectors allow for efficient sequence modifications, functional element incorporation and chemical conjugations, offering high levels of flexibility when designing programmable therapeutics.
Compared with asymmetric PCR, RCA and chemical synthesis, this approach to phage-based bacterial expression is the most effective and scalable method, supporting both structural and functional integrity for therapeutic applications.
Advantages of ssDNA in therapeutics
Developing ssDNA as programmable therapeutics provides advantages compared with conventional nucleic acid-based modalities, particularly in gene delivery, transient expression and cellular reprogramming.
By leveraging promoter sequence optimisation, it is possible to achieve the precise control of gene expression in specific cell types and tissues to enhance both selectivity and therapeutic efficacy.
Additionally, the integration of scaffold/matrix attachment regions (S/MARs) extends the duration of gene expression by addressing a key limitation of transient gene delivery strategies.
For example, the ssDNA platform allows for controlled, transient and tissue-specific expression, which may reduce the risks commonly associated with plasmid-based dsDNA (such as unpredictable expression patterns and genomic integration).
This level of control is particularly beneficial in chimeric antigen receptor (CAR)-T therapy, where transient CAR expression in T cells can help to fine-tune immune activation to reduce the risk of prolonged toxicity and immune exhaustion.
Furthermore, the incorporation of S/MAR sequences enables extended but non-permanent gene expression, offering a middle ground between transient and stable expression.
This is critical for applications that require prolonged yet reversible therapeutic effects, such as cancer immunotherapy and regenerative medicine.
The flexibility of ssDNA also allows for precise dose control and repeatable administration, enabling iterative dosing strategies that are tailored to patient responses.
Unlike viral-based gene therapies that rely on permanent genomic integration, ssDNA thus promises a safer and more adaptable approach for precision medicine applications requiring dynamic gene modulation.
Reduced immunogenicity and toxicity
A well-known limitation of nucleic acid-based therapeutics is their potential to activate innate immune responses, subsequently reducing efficacy and increasing adverse effects.
ssDNA demonstrates lower immunogenicity than dsDNA as it avoids the activation of cytosolic DNA sensors such as cyclic GMP-AMP synthase (cGAS) and stimulators of interferon genes (STING).
Additionally, toll-like receptor 9 (TLR9) responds differently to ssDNA than to dsDNA, opening new options to enhance the safety profile of ssDNA-based therapeutics (particularly for in vivo delivery).
Further, the absence of viral components eliminates any concerns associated with insertional mutagenesis and immunogenic reactions that are commonly observed with viral-based therapies.
The ability to deliver genetic material without triggering strong innate immune responses significantly expands the therapeutic potential of ssDNA, particularly for conditions requiring repeat dosing, such as chronic diseases or autoimmune disorders.
Reference
- www.prnewswire.com/news-releases/gene-therapy-market-to-hit-usd-36-55-billion-by-2032-with-19-4-cagr--marketsandmarkets-302381166.html.
Read part II here.
