In Oncoligo, our goal is to checkmate cancer by combining multiple biological strategies into a unified therapeutic framework.
Just like in chess, every move in our wet-lab work represented a deliberate step toward victory - each experiment validating a different piece of our modular design.
Our computational model served as the strategist, guiding the sequence of experiments that tested antisense oligonucleotides (ASOs), synthetic-lethality targets, antibody optimization, and epitope-driven immune activation.
Our experiments validated the core pillars of Oncoligo’s modular therapeutic platform — from RNA knockdown to cell death and antibody optimization- demonstrating efficacy across molecular, cellular, and protein levels.
Every experiment was performed under strict biosafety guidelines and only after receiving approval through the iGEM Safety Check-In form. We worked exclusively with non-pathogenic model systems and used reagents in accordance with institutional and iGEM safety regulations. For more details, visit our Safety Page.
To provide a clear picture of our progress, we mapped our experimental workflow into a timeline that highlights both completed and ongoing experiments. Each stage represents a critical step in validating our therapeutic strategy: from early ASO knockdown trials, through synthetic lethality assays and yeast-based testing platforms, to antibody design and epitope integration (Figure 1).
This progression reflects how our project has unfolded step by step—building confidence in each component of the platform before moving forward.
By following this roadmap, we ensure that every element of our design - ASOs, synthetic lethality partners, antibodies, and epitopes-has been systematically tested, optimized, and integrated into the final therapeutic framework.
Our first step in investigating ASOs was to examine their mode of action. We designed 18 different unmodified ASO sequences targeting various regions of the GFP mRNA and tested them in HEK293-GFP cells. No significant reduction in GFP expression was observed for any of the 18 unmodified ASOs, suggesting either inefficient transfection or rapid ASO degradation due to the absence of chemical modifications.
See - Results - ASO - ״Unmodified ASOs in HEK293-GFP: The Importance of Chemical Modifications”
We continued working with the unmodified ASOs, but to track and monitor their cellular uptake, we added a fluorescent label and delivered them using Oligofectamine. Flow cytometry confirmed that the ASOs successfully entered the cells; however, they still failed to significantly reduce GFP levels. These results suggest that the issue lies in the stability or activity of the ASOs. The lack of stability and activity is likely due to the absence of chemical modifications, and therefore, in our subsequent experiments, we incorporated the necessary modifications into the ASOs.
See - Results - ASO - “Fluorophore-Labeled ASO Cell Uptake Assay: Successful Oligo Entry”
Our next step requires a confirmation of the success of our system and a reduction in mRNA levels. To achieve this milestone, we use MALAT1 - a valid ASO that is currently being used in the industry as a validated method. The sequence is control provided by IDT. The system check was performed using HEK293 and A549 - to keep up safety protocol a check in form was submitted and team briefed.
In this experiment we achieved a conformation of a successful system and delivery methods done by our team.
See - Results - ASO - “IDT Reference MALAT1 ASO: Validated Knockdown in HEK293 and A549”
Now that we have learned that our system works we start generating novel ASOs targeting MALAT1 mRNA for testing our pipeline. Our model generated 3 ASOs that were tested in A549 cells, confirming successful knockdown that outperformed the industry standard sequence.
See - Results - ASO - “Computationally Designed MALAT1 ASOs: Knockdown in A549 Cells”
Now that we have learned that our system works we start generating novel ASOs targeting GFP mRNA for testing our pipeline.
In this experiment we used A549-GFP cell line. We used RT–qPCR and flow cytometry to evaluate the novel ASOs success rate which indicate a high level of GFP reduction in both methods. We show that our pipeline is able to generate novel and effective ASO with a high rate of reduction as the reference sequence.
See - Results - ASO - “Computationally Designed GFP ASOs: Knockdown in A549-GFP Cells”
Once we confirmed our system and our ability to create effective ASOs our goal now is to apply our work into a synthetic lethality system. We focused on specific known MTAP-deletion 3 synthetic lethality gene partners, and tested 2 of them. With our model we generated new ASO sequences which caused a significant and dose-dependent cell death, proving once again our ability to create high efficient ASO with the power to eliminate cells with sequence specificity.
Another aspect we investigated was whether ASOs could serve as a new gene silencing tool in yeast. For that, we engineered yeast to constitutively express human-optimized GFP. Our aim was to create a cross-species comparative platform for the study of ASO. The system went under several testing, optimizations and adjustments. And so we are now able to effectively test ASO against GFP mRNA in yeast.
See - Results - ASO - “Yeast: ASO as a New Tool for Gene Silencing”
Followed by the establishment of a yeast with expression of human optimized GFP, we tested different transformation ways to deliver ASOs into the yeast cell and measured GFP expression level using RT-PCR. This provided significant results in the difference between transfected yeast to wild type yeast proving a conformation for a successful engineering.
See - Results - ASO - "ASO Uptake and RT-PCR: Yeast Transformation Methods”
As part of our ongoing effort to further develop our computational prediction model and follow another iteration of DBTL, we generated a second generation of ASOs. Although this generation did not outperform the first, it produced results comparable to those of the initial version.
See - Results - ASO - “Computationally Designed MALAT1 ASOs: Second Iteration”
As part of our design, we aim to develop a specific and efficient antibody to serve as a delivery vehicle for our ASOs. Our goal is to enhance targeted delivery to tumor tissue while minimizing off-target effects on healthy cells. We selected a clinically validated monoclonal antibody, Erbitux (Cetuximab), as the basis for optimization using two bioinformatic tools: MNDL Bio and the ESO system. Antibody production will be carried out in CHO cells, and this part of the project is currently ongoing.
Off-target effects are an important factor that must be carefully evaluated. Therefore, we designed a dual-plasmid reporter assay to assess the off-target activity of our ASOs, both with and without the BROTHER strand, and to monitor system performance to ensure a high level of safety and specificity.
This experiment is currently in the design and preparation phase- experimental results will be added once testing begins!
See - Results - ASO - “Off-Target Analysis: ASOs and BROTHERs” for experimental design!
After establishing a yeast expressing humanized GFP, our goal now is to be able to confirm that our ASOs generated by our model is maintaining the ability to reduce GFP mRNA expression level similar to mammal cells.
This experiment is currently in the design and preparation phase- experimental results will be added once testing begins!
Our therapeutic strategy incorporates the immune system through the use of a cancer-associated neoepitope. To evaluate this approach, we designed an assay to confirm three key processes: antibody–epitope internalization, epitope presentation on MHC-I, and subsequent CD8⁺ T-cell activation, in collaboration with Prof. Cyrille Cohen.
This experiment is currently in the design and preparation phase- experimental results will be added once testing begins!
Once all steps of our design are completed, we will be able to integrate the different components of our system into a single multifunctional therapeutic complex — a conjugate consisting of an optimized antibody carrying an embedded epitope and an ASO. This structure represents the culmination of our design and optimization efforts to create an efficient, precise, and safe platform. Our goal is to help reinvigorate ASO research, ushering in a new era empowered by big data and advanced computational models such as our own.
This experiment is currently in the design and preparation phase- experimental results will be added once testing begins!
Our wet-lab journey was not just about data - it was about teamwork, creativity, and discovery.
Each experiment - from cloning and qPCR analysis to antibody optimization - deepened our practical understanding of molecular biology and advanced our journey toward developing a modular therapeutic platform.
Working together in the lab gave us hands-on experience with techniques such as cell transfection, qPCR, flow cytometry, yeast transformation, and RNA handling, while strengthening our appreciation for the interdisciplinary nature of synthetic biology.
In addition to our experiments, we contributed a rich set of BioBrick parts to the iGEM Registry - expanding the toolbox for RNA-based therapeutics, antibody engineering, and yeast platform development.
Our parts include promoters, coding sequences, protein domains, terminators, antisense oligonucleotides, and composite constructs, all designed to support modular and cross-organism research.
Our library includes 21 basic parts, 1 composite part, and 2 part collections, all designed to support modular, interoperable, and cross-organism research.
We developed new sequences for ASO validation (targeting MALAT1 and GFP), optimized antibody expression (Cetuximab heavy and light chains), synthetic lethality testing (human MTAP CDS), and a dual-reporter plasmid for assessing ASO off-target activity.
In yeast, we created an inducible GFP expression cassette combining the GAL10 promoter, GFP with a flexible linker and degron, and the CYC1 terminator - enabling dynamic gene expression and degradation control.
Altogether, these contributions form a complete genetic toolkit aligned with our project’s “chessboard” vision — where each part represents a distinct piece in our therapeutic strategy.
Explore our full list of parts on the Parts Page.
