After learning about the intense and financially taxing drug discovery process and the dire need for an improved glioma model, we recognized the demand for a high-throughput drug screening platform. To implement our design and deploy an engineering product, we conducted key stakeholder interviewers to consider the needs of a wide range of target users, including patients, researchers, and pharmaceutical companies. We developed a reporter system enabling drug discovery researchers to analyze the impacts of different therapeutic candidates by measuring a key oncometabolite D-2HG. We also demonstrated that our co-culture system along with the reporter has the potential to quantify drug effects in vitro. In addition, we will expand upon current transfection protocols to determine how to optimally introduce our genetic constructs into cell lines that are relevant to our model system, especially primary glioma cell lines, as the lack of such a transfection protocol currently presents a barrier in evaluating new constructs. Our system satisfies the need for a physiologically accurate and effective model system to screen cancer drugs, mitigating the expensive and often ineffective use of animal models to test the effectiveness of novel drug treatments against this lethal disease.
Current drug development pipelines rely on animal testing for safety and efficacy screening, but this process is costly, time-consuming, and often disappointing. Billions of dollars are spent on animal drug screening experiments every year, only for many drugs to show poor efficacy in animals [1]. Currently, there is no way around this dilemma as animal testing is the best way to validate novel treatments. We designed our reporter system with the goal of creating a model that would allow drug development researchers to gain a better understanding of the exact molecular and metabolic impacts of their compounds on disease samples. After choosing to target IDH1 mutation-specific gliomas, we concentrated our efforts on quantifying the amount of the oncometabolite D-2HG in the cell. Due to the downstream effects of D-2HG accumulation, which include genome-wide histone and DNA methylation alterations and an increase in HIF-1α levels, monitoring its levels within the system and observing how those levels change in response to novel therapeutics may lend a hand in finding a cure for this lethal disease [2] [3]. We hope that our system can help address the ongoing problem in cancer research of lacking a scalable and physiologically-relevant model system, and expect that this system can be implemented into multiple research settings to facilitate advancements in this field.
We have successfully performed initial drug testing on our co-culture systems, which has followed expected trends. We used an established fluorescently labeled cancer cell line for this preliminary co-culture study and tracked fluorescence over the course of 36 hours of treatment as an indication of cancer cell survival in co-culture. The drugs and their dosage used in this preliminary screening were all concentrations commonly recommended for verified chemotherapy drugs for brain cancers. We were able to observe a continual decrease in signal intensity of the fluorescence in treated groups, suggesting that the drugs inhibited cancer cell growth. Furthermore, between different doses of the drugs, we saw differential rates of fluorescence decrease, with higher doses corresponding to a faster killing, matching the expected trend. We demonstrated that our co-culture system can be used to easily and effectively determine the effects of applied treatments on tumor invasion and development. This proof-of-concepts suggests that our final co-culture system, with the integrated reporter system, is capable of meaningfully quantifying drug effects in vitro. Moving forward, we hope to offer NODES as a drug screening platform in the preclinical phase to eliminate drugs that are harmful or ineffective before they enter clinical trials.
There are several well-established cell lines, like HEK293T, utilized in synthetic biology to test plasmid constructs in vitro and assess how they might act in a cellular system. The human body is composed of over 200 cell types, each with a unique physiology and function [4]. Thus, it is crucial to verify engineered plasmid constructs in relevant cell lines to gain a more comprehensive understanding of plasmid expression in specific cell types and their impact on distinct areas of the body. However, current transfection protocols are not optimized for nonstandard cell lines, which acts as a barrier for thorough evaluation of new constructs and their function in their relevant setting.
With this in mind, our team wanted to expand upon current transfection protocols to determine how to optimally introduce our constructs into cell lines that are relevant to our model system, specifically primary glioma cell lines. We tested several electroporation protocols, tuning it to produce the most efficient method that results in optimal gene expression with high transfection efficiency and low cell death. In this phase, we have successfully developed a protocol to introduce recombinant plasmids via non-viral delivery into patient-derived glioma cells. Alternatively, we recognize that stable introduction of our reporter system using adeno-associated viruses (AAV) may be the optimal delivery method due to its efficiency and ability to target specific serotypes [5]. However, due to time restraints and necessary safety protocols in place at Duke, we were unable to explore this avenue. We hope to examine the potential of AAV delivery to our co-culture system in the future and determine if this is the best delivery method. Ultimately, we hope to contribute new insight on how to successfully and efficiently deliver our constructs to primary cells, which will advance new synthetic biology applications in primary glioma cells and other specialized cell types.
Our team also considered safety concerns that would need to be addressed during the ultimate real-world implementation of our device. We envision that our products will be mostly applied in a clinical research setting, with an extremely low chance of being introduced into the environment. We acknowledge, however, that there will be a possibility that the final product we deliver will be assessed to pose a potential threat when in contact with the outside environment. Before launching the product to the public, we plan to implement a kill-switch into our system upon need. Our guiding principle is that we will limit our products from contacting the environment as much as possible, and if contact is necessary, an intrinsic kill-switch will eventually be installed to prevent the escape of recombinant DNA.