Lessons on optimizing DNA design, watermarks and synthesis for mRNA therapeutics shared by Dr. Axel Trefzer, R&D Leader at Thermo Fisher Scientific
Biotech writer Silvia Hnatova recalls this key webinar on DNA synthesis for mRNA therapeutics by Dr. Axel Trefzer, R&D Leader at Thermo Fisher Scientific. The session was part of TIDES: Oligonucleotide & Peptide Therapeutics Digital Week in May 2021.
Dr. Axel Trefzer, R&D Leader at Thermo Fisher Scientific delivered a fascinating presentation on synthetic DNA and biological engineering.
He started his presentation by highlighting the exponential advances in our understanding of DNA in the past two decades, and the drive towards applied biology, in contrast to the history of biology focused on observations.
Synthetic biology makes biotechnology more efficient: historically this was done through breeding, but the recent developments in molecular biology allow genetic engineering of new organisms.
Our ability to produce biological organisms with desired phenotypes has been accelerated at an unprecedented rate.
Dr. Trefzer pointed at the role of synthetic biology in biological engineering, which led to developments in multiple areas of applied biology: vaccine research, agriculture, drug discovery, and antibody engineering.
The main focus of Dr. Trefzer’s talk was on synthetic DNA. There are different types of synthetic DNA, including cloned and ready-to-use plasmid; DNA provided in a functional vector or a linear fragment.
Additional services can include plasmid production or directed evolution.
The right piece of DNA needs to be designed using bioinformatics tools, leading to the desired design of a molecule, followed by oligo synthesis, gene assembly of a short fragment, followed by cloning into a larger system, sequencing, QC, and final documentation.
Bioinformatics plays a pivotal role in the right sequence design and ensuring that a sequence with the best biological function is obtained.
The next part of Dr. Trefzer’s presentation talked about embedded watermarks in DNA reading frames and coding regions.
The research group in Thermo Fisher Scientific asked what information they can encode without changing the biological function, using the codon redundancy.
The group managed to encode messages in heterologous constructs, proving that synthetic genes and GMO-engineered organisms can be watermarked, enabling tracing and design ownership.
It could enable distinguishing the synthetic sequences from any natural sources.
Elaborating upon the points presented, Dr. Trefzer turned the attention towards mRNA therapeutics and mRNA vaccines and how DNA synthesis enables these.
DNA synthesis is an important enabler of mRNA therapeutics production, serving as a template for gene synthesis or as template plasmids.
Dr. Trefzer presented two examples of DNA synthesis enabling mRNA therapeutics, with contrasting requirements.
The first was an example of mRNA vaccines for COVID-19, where the main challenge is the massive scaling: large quantities of template DNA (kilograms) are needed to generate mRNA vaccines for the entire world.
The second example was personalized medicine, designing epitopes for cancer neoantigen immunotherapy, where individual synthesis for every patient is necessary.
The challenge is to generate individual solutions for each patient on a short timescale (~2 weeks), so that treatment can be created in a short timeframe for the patient to benefit from the treatment since the biopsy.
To enable fast and reliable delivery, compression of workflows is necessary to synthesize DNA in a short timeframe, delivering high-quality products and high-reliability meeting standards for therapeutics.
To summarize the talk, Dr. Trefzer concluded that engineering contributions to biology allow us to design the best functional molecule for biological applications.
The secret of synthetic DNA gives us control over complex processes enabling new approaches, and enables new applications such as mRNA therapeutics, embedding specific technologies in the process.
The talk was followed by a Q&A session that sparked various interesting debates. There was a discussion about minimizing dsRNA contaminants in the final product.
Dr. Trefzer responded that further work was needed to understand the mechanisms behind the production of dsRNA contaminants, to tackle these.
There was an interesting discussion about directly synthesizing RNA instead of first synthesizing DNA. Dr. Trefzer said that it is possible to synthesize short sequences of RNA similarly to DNA synthesis.
However, if the sequence is longer than 100bp, usually, a dsDNA template would be advised to be created first, as there is no shortcut to directly create large RNA oligonucleotides.
Dr. Trefzer was asked if large quantities of the template are needed for gene therapy. He replied that the amount of template DNA would depend on the size of the patient population and the number of treatments required which would correlate with the amount of RNA required for the application.
Another question from the audience asked, “How can sequence optimization improve performance, and is this limited by length?” It was concluded that this depends on the starting point – typically, this would be a 3-10x improvement.
Dr. Trefzer said that the longer the sequence and the associated proteins are, the more complicated the process is. For most standard protein sequences up to 5-10kbp, optimization improves the chances of success of the desired protein being expressed.
The last question to the speaker enquired about the key criteria for the design of sequences for therapeutic applications. Dr. Trefzer said customers typically require higher quality and reliability, which is typically time-sensitive because the application affects people’s lives.