Marcus Graf, Thomas Schoedl, David Raab and Ralf Wagner at GENEART consider recent advances in synthetic biology, which represent an attractive alternative to DNA templates for research
The design of recombinant bioproducts for R&D of novel therapeutics critically depends on the availability of DNA templates. The induction of the polymerase chain reaction in the mid-1980s made cloning and sequencing technically very easy; and working with naturally occurring templates became standard. Following this first wave of innovation, the availability of biological sequence data exponentially increased by the end of the 20th century, primarily due to the availability of novel fluorescence dyes and sequencing hardware (for example, capillary sequencing promoted by ABI).
Today, as a result of this second wave of innovation, the most extensively studied mammalian genomes (human, mouse and rat) have been fully sequenced, genotypes of various pathogens have been characterised and most of this sequence data is available online.
Unfortunately, the speed at which sequence data became available was not paralleled by the development of genetic engineering technologies. As a consequence, access to specific physical DNA templates by retrieving genes, cDNAs or splice variants thereof from libraries or clones remains discommodious, and errors due to PCR amplification are widespread. Downstream applications to improve such DNAs, for instance, heterologous in vitro screening systems (for instance, phage or ribosomal display technologies used to improve stability and binding capacity of the protein) may be well suited to select the best performers.
However, upon switching the expression system from prokaryotic cells (that is, E coli, used to propagate selected bacteriopages displaying an antibody Fab fragment) to mammalian cells (driving the production of a genetically engineered antibody), the encoding DNA inherits an increased risk of improper or insufficient expression for downstream assays.
Another challenge arises during the retrieval of crucial antigenic targets from new and emerging diseases, such as avian flu or SARS, using patient material or isolated pathogens. This method can be highly dangerous and is limited to laboratories with a sufficient biosafety clearance.
Moreover, conventional genetic engineering technologies that rely on natural templates – even PCR-based methodologies – are largely inflexible regarding the optimal adaptation of coding sequences for desired downstream applications. Although the introduction of desired sequence modifications and the removal of sequence repeats and inhibitory motifs is technically trivial and attainable, they are not straightforward processes, and can cause delays in research progress. Recent advances in synthetic biology are now providing an economical and reliable alternative to DNA templates for biopharmaceutical research: rationally designed de novo constructed DNA (see Figure 1).
Although the principal techniques of constructing genes from synthetic oligonucleotides were developed many years ago, recent advances in synthetic biology allow users to rely on gene synthesis as a quick and economically attractive source of DNA optimised for downstream applications. The price for a de novo constructed gene dropped from more than $15.00 USD in 1999 to less than $1.00 USD per basepair today.
The delivery times also decreased from several weeks for a 1.5kB gene in 2000, to 10 days or less today. By providing quick access to any sequence at very attractive conditions, gene synthesis is well on its way to triggering a third wave of innovation in genetic engineering; this will have a substantial impact on the discovery, development and production of novel therapeutics. Consequently, an increasing number of biopharmaceutical companies turn to gene synthesis specialists as their main source for DNA templates tailored to their specific research programmes.
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