DNA Technology: Overcoming Challenges in the Development of Vaccine, Cell, and Gene Therapies

Updated: Aug 17


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

Evolution of Therapeutics

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.

Error Impact with PCR

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

Cell Based Cloning Example

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.<