Synthetic DNA sequences with fewer constraints on sequence complexity are crucial to drive cell and gene therapies forward. Here’s why.
The promise of cell and gene therapy
With an increasing number of cell and gene therapies vying for approval, and three already given the green light in 2024, this new wave of therapeutics is set to change millions of lives.
Cell and gene therapies are advanced medical treatments that involve modifying or replacing cells or genes to treat or cure diseases. They hold enormous promise for treating many diseases with poor or no effective intervention. However, the development and production of cell and gene therapies is a long, laborious, and complex process.
Delays, disruptions, and backlogs are common when establishing supply chains for materials in development pipelines, clinical trials, and later, scaling up for entry into the commercial market. In particular, DNA synthesis methods are currently unable to meet the rising demands for high-quality, complex, gene-length DNA.
Design and manufacture of a cell and gene therapy
Cell and gene therapy design employs an iterative Design-Build-Test-Learn (DBTL) framework. The design stage has been propelled by significant improvements and cost reductions to DNA sequencing. This cost-effectiveness, coupled with developments in computation (like machine learning), has allowed us to amass and probe vast amounts of genomic information to inform the test, learn, and design phases of the DBTL cycle.
The build phase of a cell and gene therapy requires synthetic DNA. Since development pipelines typically go through many build phases, vast amounts of custom DNA are required to develop a cell and gene therapeutic. Not to mention, once a therapy has been developed synthetic DNA is needed at scale for manufacturing.
As cell and gene therapies continue to be developed, approved, and manufactured, the demand for scalable production of synthetic DNA is only increasing. However, DNA synthesis technologies are struggling to keep pace with the rate of cell and gene therapy innovation.
Until now.
Cell and gene therapies need complex DNA
Whether you’re editing T cells to produce surface proteins for CAR-T therapies or inserting therapeutic genes into AAV vectors, the development of cell and gene therapies hinges on access to scalable, accurate, full-length, synthetic DNA sequences.
Many synthetic sequences commonly used in cell and gene therapies contain complex elements that are challenging for most DNA providers to produce. For example:
- Regulatory elements: These are essential components of synthetic DNA needed for cell and gene therapy applications since they regulate RNA and protein abundance. However, they are notoriously challenging to produce since they often contain arrays of repeats, hairpins, and regions of very high or very low GC content.
- Inverted terminal repeat sequences (ITRs): ITRs are a crucial component of AAVs. Each AAV has two ITR sequences (145 bp each) and the DNA sequence between the ITRs is what gets packaged into the AAV molecule. Due to their secondary structure, repeat sequences and high GC content make them challenging to synthesise and lead to side products in assembly and amplification.
- Therapeutic payloads: Some gene therapies aim to introduce a new or modified copy of a gene. For example, Roctavian carries the factor-VIII gene into the liver cells, enabling patients with haemophilia A to produce the blood clotting factor. Therapeutic payloads are extremely variable in length and complexity but are often beyond the limits of chemical DNA synthesis technology.
CRISPR sequences: CRISPR-Cas is a gene editing tool used extensively in cell and gene therapies. Synthesising and assembling CRISPR sequences can be challenging due to repetitive sequences and an increasing demand for multiplexed guide RNAs.
Complexity in sequences can result in rejection before you even set foot in a DNA provider’s proverbial front door. You may be forced to decrease complexity through codon optimization, spend huge amounts of time and resources assembling your sequence in-house, or even abandon your desired sequence altogether.
Having your sequence accepted is just the first hurdle.
Assembling error-prone sequences
To make long stretches of double-stranded DNA, DNA providers start with short oligonucleotides (typically less than 300 bp) and assemble into longer sequences. Sequence assembly is time-consuming and becomes increasingly challenging with complex DNA sequences.
DNA complexity |
Assembly challenges |
| High GC content | Oligos with high GC content may contain mismatches and still hybridise due to the strength of GC bonding. They can form unstable secondary structures, such as hairpins and G-quadruplexes, which can obstruct assembly. |
| Low GC content | Oligos with low GC content may match perfectly, yet dissociate from the target. They may form unstable secondary structures, such as AT hooks, which can obstruct assembly. |
| Long repeats | Repetitive regions can hybridise with themselves or other repetitive regions. This can cause polymerase slippage, secondary structure formation, homologous recombination, and hybridization problems, all of which lead to increased error rates |
| Hairpins | Hairpin structures can block DNA polymerase, cause misalignment of primers, and complicate the assembly process, leading to increased error rates and inconsistent amplification |
Additionally, starting oligonucleotides often contain synthesis errors. These errors are carried forward and can proliferate during the assembly process. Even a single deletion can shift the reading frame, resulting in unusable DNA. Because of this, cell-based cloning is used to isolate a perfect DNA molecule.
Challenges of cell-based cloning
In cell-based cloning, the final assembled sequence is cloned into plasmids and transformed into bacteria. The resulting colonies are picked and screened using sequencing to find an exact match to your submitted sequence.
However, cells can generate further errors, particularly in more complex sequences. Combined with errors carried forward from oligonucleotides and assembly, the synthesis of longer genes often requires multiple rounds of cloning and repeated assembly steps.
gSynth: DNA without compromise
Cell and gene therapies are changing people’s lives and are certain to improve many more. However, this change comes at a cost.
The three new cell and gene therapies granted FDA approval this year come with a price tag of up to $4.25 million. This cost is largely associated with laborious and complex manufacturing processes, including the difficulty scaling synthetic DNA production.
“We're giving people a little extra chance of making a life-saving medicine.”
Steve Harvey, CEO Camena Biosciences
The gSynth platform is opening up the bottleneck of DNA synthesis in cell and gene therapy and helping pave the way for more accessible therapeutics. Using TdT-free, enzyme-based technology, gSynth bypasses the need for error-prone oligonucleotides and error-magnifying assembly and cloning processes – all while addressing the challenges of accuracy, length, scale, and speed.
Any sequence
Codon optimisation is often the only option if your sequence doesn’t meet complexity criteria. But codon optimisation can have huge consequences for your research and product. We’ve eliminated the need for codon optimisation just to make synthesis work. With gSynth, the only optimisation you need to do is that which enhances your research, application, or therapeutic outcomes.
Any length
As DNA sequences increase in length, the yield of error-free DNA decreases significantly. With the current gold standard, phosphoramidite synthesis, typically limited to less than 200 bases, long and laborious assembly and cloning become the only viable option.
We’re changing the way you make DNA. Rather than error-generating assembly and cloning processes, the gSynth platform can directly synthesise accurate, gene-length DNA.
Any complexity
No one likes rejection. But that’s just what you’ll face if your sequence is considered too long or too complex. If DNA providers do accept your sequence, long delays are common as complexity increases.
We’ve removed the limits on complexity—regardless of length, GC content, homology, repeats, or overall complexity score, gSynth can meet your synthesis needs.
Expedite your cell and gene therapy
