As the potential of mRNA vaccines to meet a significant, unfulfilled medical demand becomes increasingly clear, we’ll need greater access to gene-length DNA far beyond what’s available right now.
Enabling mRNA vaccines at scale
As the potential of mRNA vaccines to meet a significant, unfulfilled medical demand becomes increasingly clear, we’ll need greater access to gene-length DNA far beyond what’s available right now.
The promise made by mRNA vaccines
Cancer stands as one of the most formidable challenges to human health globally, claiming countless lives each year. Considering the high morbidity and mortality rates, the need for better and more effective treatments is clear.
Despite advancements, traditional treatments often fall short. Pancreatic cancer in particular remains stubborn in the face of new therapeutics, leaving patients with a dismal prognoses. Survival rates remain staggeringly low, with only 7 out of 100 individuals surviving five years post-diagnosis.
However, hope glimmers on the horizon. Results from early clinical trials of mRNA vaccines targeting tumours, including pancreatic cancer, have ignited newfound optimism
A recent, albeit small, clinical trial, showed that a personalised mRNA vaccine activated T-cells targeted to cancer neoantigens in half the patient cohort. In addition, in the 8 patients who responded, more than 80% of the vaccine-induced T-cells persisted from 2 to 3 years after treatment.
In 6 out of the 8 patients displaying an activated T-cell response, their cancer did not return. In contrast, cancer returned in 7 out of the 9 patients whose immune systems did not respond to the treatment. As such, researchers will be investigating whether the mRNA vaccine is responsible for the delay in cancer recurrence in the ongoing phase 2 clinical trial.
How to build a personalised mRNA vaccine
Developing a personalised mRNA vaccine involves a meticulous process aimed at harnessing the body’s own immune system to combat cancer.
The search for neoantigens
Total RNA, known as the transcriptome, is extracted and sequenced from patient tumour and healthy tissue samples.. Through a detailed comparison of the RNA datasets, specific proteins expressed solely on the surface of tumour cells, known as neoantigens, are identified. Neoantigens that are predicted to elicit the strongest immune response are selected for vaccine development.
Creating the genetic blueprint
mRNA vaccine development begins with the design of a plasmid DNA template that contains sequence elements essential for efficient translation and stability, as well as the neoantigen sequence. The DNA template is inserted into a plasmid, cloned into a bacteria, and amplified by bacterial fermentation. The relevant DNA fragments are isolated from the cells and plasmids and transcribed in vitro into mRNA. The mRNA is purified by a filtration process to remove product-related impurities.
Encapsulation, delivery and response
The mRNA is combined with lipid nanoparticles (LNP) and delivered to the patient. The vaccine is taken up by cells, and the mRNA sequences are translated into neoantigens. By presenting these tumour-specific antigens to the immune system, the therapeutic vaccine helps train immune cells to recognise and attack the cancer cells.Template DNA: The bottleneck
Creating an effective, efficient and safe mRNA vaccine involves tweaking the mRNA sequences to optimise stability, expression levels, and efficacy. As mRNA vaccines are developed and trialled for personalised cancer therapies that target neoantigens unique to the patient, demand for unique DNA templates will skyrocket.
The current methods for generating plasmid DNA templates for vaccine development and manufacturing encounter many challenges, resulting in costly downstream processing, prolonged development timelines, and ultimately, delays in delivering potentially life-saving treatments to patients.
High error rates
The required sequences for template DNA for mRNA vaccine production are typically produced by phosphoramidite synthesis. It is well known that over long stretches of nucleotides, the phosphoramidite synthesis method is error prone.
Even with the best nucleotide coupling efficiencies, when phosphoramidite synthesis reaches 200 nucleotides, only ~40% of the material is the correct full-length product, and at 1kb, the error-free yield is <0.01%.
The impact? With mRNA vaccine development demanding DNA templates ranging from upwards of 1kb, the utility of phosphoramidite synthesis quickly becomes obsolete.
Long lead times
To overcome the error rates associated with synthesising gene-length DNA, suppliers will typically divide longer DNA constructs into smaller overlapping segments that are short enough to yield sufficient error free quantities. The resulting overlapping synthons are then assembled into larger pieces of DNA.
Screening for error-free sequences involves cloning into a vector, transformation into bacteria, isolation of individual clones, and sequence validation by Sanger sequencing. In turn, the time it takes to receive a validated DNA sequence increases with length of the DNA product.
The impact? Lengthy lead times to produce large-scale clinical grade material that is GMP compliant, creating a significant bottleneck on mRNA vaccine development pipelines.
Lack of stability
At the 3’ end of mRNA used in vaccines is a sequence element known as the polyA tail. The poly-A tail is a long chain of adenine nucleotides that is essential for translation and stability of mRNA.
The phosphoramidite synthesis method faces challenges with repeat sequences due to inherent biases in the building blocks, affecting the accuracy and efficiency of synthesising gene-length DNA constructs with repetitive elements.
Moreover, replicating plasmids commonly results in polyA deletions and partial deletions. Alternatively, post-transcriptional polyadenylation of mRNA is possible but is costly, challenging to control, and can lead to heterogeneous poly(A) lengths in the final mRNA product.
The impact? Unstable mRNA vaccines with poor translational efficiencies and reduced effectiveness.
Downstream purification
One of the fundamental issues with using plasmid template DNA for mRNA synthesis is the presence of contaminants including antibiotic resistance genes, endotoxins and the plasmid backbone sequence.
While rolling circle amplification (RCA) can be used to amplify plasmids without the need for bacteria eliminating bacterial contaminants, the amplification of the plasmid backbone and antibiotic resistance genes will still need to be enzymatically digested.
The impact? Risk of contaminants being incorporated into the final product and expensive and lengthy downstream purification processes, increasing cost and development timescales.
The codon compromise
The coding sequence of the neoantigen can be optimised by selecting codons efficiently translated in human cells and by avoiding immunogenic ones. However, phosphoramidite synthesis constraints may necessitate sacrificing optimised codon usage to ensure successful synthesis. Furthermore, if the resulting sequence is inserted into a plasmid and amplified in bacteria, codons may need to be optimised for bacterial translation, rather than for their ultimate destination – the patient.
The impact? Reduced translational efficiency of the mRNA vaccine leading to reduced vaccine effectiveness.
gSynth: The potential to empower vaccine development
While the potential of mRNA vaccines is vast, current methods face significant challenges, particularly in DNA template synthesis. Traditional approaches are plagued by high error rates, lengthy lead times, and stability issues, jeopardising the efficiency and effectiveness of vaccine development pipelines. Enter gSynth – a platform with the potential to empower the future of mRNA vaccine production.
