Long, Pure, Complex, Scalable and Multifunctional Single-Stranded DNA – A Close-up Look at Moligo Technologies

Our patented technology is also distinct from de novo enzymatic DNA synthesis based on terminal transferases, which has been adopted by many new companies. In fact, rather than writing DNA base-by-base, our method can be compared to injection moulding, whereby we assemble a sequence-verified monoclonal production template (our mould), and single-stranded DNA molecules are mass-produced directly from this mould. This is a crucial difference with our approach, since it allows us to truly produce DNA at industrial scale for large DNA studies and to meet the demands of clinical applications. We currently provide DNA in batches at multi-milligram scale and we are optimising our scale-up to provide batches at gram scale.

Unmatched sequence quality within the DNA synthesis field

- So, what typically happens when a customer approaches Moligo for DNA products?

Our customers usually know the exact sequence they need, although we can help with DNA sequence design if necessary. In most cases, a project begins when a customer requests a certain quantity of DNA molecules containing their chosen sequence.

We start by analysing the sequence in silico with our algorithm and by assembling a production template. This means that we make a double-stranded DNA sequence which contains short proprietary sequences that are recognised by our enzymes. This template is then used for rolling circle amplification, which results in the generation of millions of copies of single-stranded DNA molecules.

Within the DNA synthesis market, we believe that the purity of our products, combined with all the sequence complexity we can provide, is unmatched. The purity is guaranteed both by the error-free production templates and the high fidelity of the polymerases we use. This is critical for any clinical- or molecular diagnostics application that requires ultra-pure DNA sequences, which cannot currently be produced using chemical methods.

Long, complex and functionalised DNA molecules for cell- and gene therapy applications

- You mentioned the urgent need for long, pure and scalable DNA sequences for clinical applications. What sequence lengths can Moligo currently provide and can that be pushed further?

Right now, we routinely produce DNA molecules that are 10 Kb in length or slightly longer. Moligo produces the longest DNA molecules on the market and we will soon be able to offer molecules longer than 15 Kb.

For genetic diseases caused by mutations in very large genes, e.g., muscular dystrophies and cystic fibrosis, limitations in the length of DNA molecules, combined with the limited packaging capacity of the most adopted viral particles, have impeded the development of therapies which are based on the delivery of the entire, long therapeutic gene. Our vision at Moligo is to produce ultra-long DNA sequences so that the experimental goal is never limited by the DNA tools available.

- Let’s talk more about the clinical applications. Besides scalability, purity and length, what else can Moligo offer to customers in the cell- and gene therapy fields?

Firstly, we can incorporate any type or level of complexity into the DNA molecules we produce. This is very significant for viral and non-viral based cell- and gene therapy constructs, where the required DNA sequences often include high GC content, as well as inverted terminal repeats and strong secondary structures.

In addition to complexity, we have identified ways to produce highly functionalised or modified DNA. We can introduce chemical functionalities during synthesis to modulate the properties of the resulting DNA molecules depending on the downstream application. For example, we can make DNA molecules that are less immunogenic, more resistant to endonucleases or more permeable. Thanks to these unique selling points, Moligo is not only offering customised synthesis of DNA molecules but we have also established co-development therapeutic programmes with major companies in the cell- and gene therapy fields, who are advancing non-viral therapeutic programmes.

Killing two birds with one stone: highly-modified single-stranded DNA as end products for non-viral gene therapies

- You mentioned Moligo working with some companies that are willing to move away from viral-based therapies. How are the donor DNA molecules packaged for delivery in such cases?

Most of the ‘non-viral’ companies we are working with are developing protocols to encapsulate the DNA molecules into some kind of particle, for example, lipid nanoparticles. These are attractive because they circumvent the capacity limitation of viral vectors, e.g., AAV, which has a capacity of 4.7 Kb.

- There’s a lot of hype about non-viral delivery in the gene- and gene-therapy fields, but none of the non-viral approaches seem to offer the perfect solution either, especially with respect to organ-specific delivery. Can Moligo’s technology help to solve those issues?

Yes, we believe so and I will elaborate on that in a moment. But firstly, the capacity limitation of AAV is not the only barrier to delivering longer DNA molecules. Another major barrier has been the difficulty in synthesising the long DNA molecules themselves. We have solved that part of the problem with our long, pure, complex DNA molecules.

We are also beginning to see a shift from the use of double-stranded DNA to single-stranded DNA because the latter is less cytotoxic, associated with fewer off-target effects and can lead to more efficient knock-in outcomes.

With our expertise in synthesising long, highly-modified, single-stranded DNA, we envision one day that it will be possible to treat diseases using a highly-modified, single-stranded DNA molecule that encodes both the required therapeutic information and the properties required for its targeted delivery in the body, without the need for any delivery vehicle.