20/15 Visioneers, Leaders in Science and Technology
Change can be good, change for the better is great! - John F. Conway
Introduction and Problem Statement
The world is needing great science more than ever! Over the last year, the world has been significantly impacted by the COVID-19 pandemic and with it we have witnessed a global movement to develop vaccines to combat its spread in record time. While a relatively new technology, mRNA vaccines produced by Pfizer/BioNTech and Moderna are prime examples of the evolution of advanced therapeutic approaches and our increasing reliance on genetic materials and their engineering technologies.
The rapid progression of SARS-CoV-2 underscores not only the importance but the criticality of being able to get drugs to market as quickly as possible, while minimizing risk to quality, safety, and efficacy. When working toward the development of biologics, the industry still relies heavily on PCR and cell-based cloning for the manipulation of genetic information, both of which are decades old technologies. While PCR and cell-based cloning are deeply rooted in virtually all aspects of genetic engineering, more efficient and flexible alternatives could greatly accelerate development timelines for a wide variety of modalities and indications.
As with vaccines, cell and gene therapies continue to gain traction with several methods already in mainstream use (gene editing, CAR-T, and AAV therapies). A look at the history and potential future of therapeutics in these areas further highlights the importance of genetic engineering in the drug development process. Fig 1
Figure 1 Evolution of Therapeutics
Given the exponential rise of applications requiring genetic manipulation, we also see increasing demand for genome scale DNA synthesis to support use cases in synthetic biology and genome editing.
Let us emphasize that these techniques are used in both early and late biologics research and development. Target identification and validation can be a microcosm of what goes on in the project-oriented research. So, weigh the options carefully when choosing techniques as an overall level of effort may end up costing you more time and hence money.
PCR provides an efficient method of synthesizing linear DNA in vitro, but is limited in DNA strand length, DNA sequence composition, and can lead to sequence biases introduced during the replication cycle. Cloning is often used when trying to amplify larger sequences, however, this approach also comes with challenges, including equipment access, expertise, and iterative experimentation to create viable cell banks. Once the vehicle for amplification has been established, the appropriate filtration process to isolate the target genetic material still needs to be developed. Both of PCR and cloning involve hands-on experimentation and do not lend themselves to automation given the time, materials, and process optimization required to produce the amplified product cost effectively.
There will continue to be enhancements to these technologies, but it makes sense to think differently if we are to accelerate the research, development, and the manufacturing lifecycle of a new class of drugs
An Overview of DNA Amplification Technologies
Since the discovery of DNA by Miescher in 1870, the interest and complexity of applications for DNA have continued to increase.
The advent of polymerase chain reactions (PCR) in 1983 further accelerated the field of molecular biology, giving scientists the ability to amplify target DNA in vitro dramatically reducing the time required to generate genetic material. As an essential technology for genetic engineering, scientists have been able to sequence and begin understanding the role DNA plays in the development and
function of biological systems. This, in turn has resulted in the development of a multitude of diagnostics and therapeutics.
As critical as PCR has become to genetic engineering, nearly 3 decades since its introduction, there remain challenges that must be overcome depending on the sequence to be amplified. Sequences with high GC-content are often difficult to amplify due to the strong bonds that can form between these bases, while longer sequences generally require added experimentation to optimize cycle temperature conditions and risk creation of incomplete fragments. PCR is also more prone to amplification errors that will continue to propagate during every amplification cycle that takes place. The thermal processes and the challenges highlighted also result in an inability to effectively scale this process.
The illustration in Fig. 2 highlights the impact to errors during PCR cycles.
Figure 2 Error Impact with PCR
This is not to discount the significant impact and importance of this technique. As noted earlier, the advent of PCR has resulted in our ability to perform multiple studies, including genotyping, paternity testing, forensics, detection of mutations, sequencing, etc.
Where PCR has not been viable, scientists have often used cloning strategies. Cloning techniques have been developed over the last 50 years and generally result in more accurate DNA amplification at longer lengths (up to 1Mbp using chromosomal vectors). Numerous variations of cell-based cloning have been developed, though they generally follow a standard approach. Fig. 3
Figure 3 Cell Based Cl oning Example
Each of these steps can take a significant amount of time to optimize and require scientific expertise to assess, preventing the automation of much of this process. In cloning specifically, there are often limitations based on size of plasmid and target genes to amplified (e.g., genes coding for toxic proteins are difficult, if not impossible to clone using E. coli). Further, when using these plasmids in the context of drug development, the purification process is critical and needs to be well characterized to ensure that byproducts of cloning are removed to ensure the biosafety of end product(s) and there is not an increased risk for patients. Where speed is of the essence, each of these steps ultimately results in delayed patient care.
As we look forward, synthetic biology, next generation sequencing, DNA identification analyses continue to gain traction. Synthetic biology specifically requires flexible and high-performance technologies for the artificial synthesis of genome scale DNA. We are seeing progressive and innovative companies such as Ginkgo Bioworks focused on the build of entirely customized organisms, applications of which have the potential to impact all industries where biology plays a part (e.g., agriculture, space exploration, global warming). Accurately engineering genetic material is a critical success factor to getting the right medicines to the right patients; it is also becoming just as important across industries.
Addressing the Challenge
We should expect that there will continue to be incremental improvements to PCR and cloning techniques, however, the time, cost, quality of these approaches remain on the critical path of the drug development timeline. Imagine, if you will, a new approach that accurately, consistently, and quickly amplifies DNA and drastically reduces the development timeline; not by improving on these existing technologies, but provides a fundamentally different approach.
In 2019, OriCiro was founded to address these challenges and aims to revolutionize our approach to genetic engineering. By leveraging the “tools” used within E. coli during the DNA replication process, they have successfully developed a rapid DNA amplification platform that overcomes challenges associated with PCR and cloning techniques to enable rapid advancements in synthetic biology and gene therapies. Having isolated the critical DNA replication components used in E. coli, they have effectively created a cell-free amplification technology that results in circular DNA amplification that is not hindered by sequences with high-GC content nor cell-toxic sequences. As with cloning, amplification takes place at a constant temperature eliminating the need for thermal cyclers.
Figure 4 How DNA amplification technologies compare and highlights the benefits offered by OriCiro’s platform.
An amplification technology that does not require thermal cycling, nor the setup of a lab to support cloning and the corresponding filtration steps, lending itself to high-throughput automation opportunities to drastically speed up the development process.
In contrast to the standard E. coli cloning process, OriCiro’s cell-free approach, has the potential to revolutionize this space by drastically reducing costs and increasing throughput, while also improving the safety profile of the end-product using a simpler, more efficient process. Fig 6
Figure 6 Comparison of methods
To further illustrate the potential for this new capability, we’ve illustrated the difference in process between these two approaches in Fig 7.
DNA is the central building block that enables the production of next-generation gene/cell therapies. Where speed to market is critical, optimizing the approach to designing, constructing, and producing high quality plasmids with minimal error rates and high levels of purity remain critical success factors.
Focusing on cell-free DNA technology, OriCiro is eliminating the time-consuming process of growing and purifying DNA from cellular vehicles (e.g., E. Coli) while still maintaining the fidelity of amplification for which this approach is often used. By having isolated the components necessary for chromosomal replication in E. coli, they have enabled large sequence length amplification (>50kbp) in hours instead of days and have eliminated the challenges that are posed by cytotoxic products that ultimately cause cloning approaches to fail.
OriCiro has also eliminated the challenges arising from amplifying high GC-rich regions that pose challenges in PCR cycling while still enabling amplification that does not require the purification processes of traditional methods. Like PCR, however, the cell-free approach remains a viable option for high-throughput 96-well experimentation.
Thinking differently might accelerate development, reduce costs and equipment needed for experimentation, and increase the safety profile of the next generation of cell and gene therapies; if you are interested in finding out more about OriCiro’s capabilities, please contact:
Ram K. Gupta, John F. Conway
Shagin, D.A., Shagina, I.A., Zaretsky, A.R. et al. A high-throughput assay for quantitative measurement of PCR errors. Sci Rep 7, 2718 (2017). https://doi.org/10.1038/s41598-017-02727-8