Abstract

Cancer remains one of the deadliest diseases. Survival rates of patients with gliomas, a type of brain tumor, are extremely low and intraoperative diagnostics is a key factor for increasing patients’ lifespans. Our project ASTERISK combines intraoperative diagnostics via Nanopore sequencing with mRNA treatment. Our innovative therapy uses an mRNA molecule (extending DART VADAR technology) that, upon detection of a tumor-specific mutation or amplification, results in the translation of a toxic protein, killing the tumor. To facilitate broad usage in iGEM projects, we present our composite part ASTERISK and our software DVSensor for automated design of new DART VADAR systems. Furthermore, we recognize the unique challenges of cancer treatment, including limited resources for sufficient patient information. For this reason, we have created a children’s book as a reliable resource that can help explain the concept of brain cancer to children of all abilities and ages.

Why Cancer diagnosis and therapy?

We directed our focus toward our team's core interests: diagnosis and therapeutics. Our mRNA-based treatment not only addresses tumors but also chronic diseases. We utilized the Oxford Technologies Nanopore sequencing platform to advance diagnostics and RNA therapeutics. Gliomas, particularly glioblastoma, are highly lethal brain tumors, albeit less common, requiring significant attention. Current treatments fall short of achieving complete remission due to the tumor's deep infiltration and drug-resistant stem cells [12]. Our project tackles this challenge by enabling rapid tumor subtyping during surgery and tailoring mRNA-based treatments [5] to the tumor's molecular profile, selectively targeting glioma cells while sparing healthy brain tissue.

Why Glioma?

Every year, 10 million people die from cancer globally, with gliomas accounting for 80.9% of malignant tumors in adults [6] [29]. Glioma patients endure not only common cancer symptoms but also neurological complications such as seizures and blindness, while those with glioblastoma have a 5-year survival rate of only 6.8% [9]. Current treatments, including surgery, radiation, and chemotherapy, have seen little improvement in the past 30 years and rarely lead to a better quality of life, highlighting the urgent need for more targeted therapeutics [11] [12] [13] [14] [15] [16]. Find out more about glioma here .

Why now?

Recent advancements in nanopore-based sequencing have shown promise in clinical diagnostics, but wider adoption is needed. Our project aims to demonstrate its utility for rapid cancer diagnosis, promoting its use in clinical labs and enhancing diagnostics. Additionally, the progress in RNA-based therapeutics, exemplified by mRNA vaccines, has created opportunities for targeted disease treatment [3] [17] [18]. We leverage these advancements to combine quick cancer cell profiling with a unique treatment approach based on cancer-specific mRNA detection.

Our Technologies

The diagnostics

Due to the development of 3rd generation sequencing technologies, fast generation and analysis of sequencing data are becoming more accessible. Numerous studies refer to the potential for tumor diagnosis by implementing nanopore sequencing developed by Oxford Nanopore Technologies [19]. Because of the simple workflow and short data acquisition time, we will integrate nanopore sequencing for diagnostic purposes [20]. By utilizing reusable, pocket-size sequencing devices, we generate long reads of DNA and RNA in real-time, as opposed to 2nd generation sequencing techniques [21]. Nanopore sequencing enables the identification of tumor-specific mutations and genomic alterations such as characteristic DNA-methylation patterns that modulate transcriptional activity [22]. Analyzing the transcriptome-specific differences between tumor and non-tumorigenic cells, the identification of tumor biomarkers can be performed and utilized for therapeutic approaches. Therefore, we aim to improve existing cancer diagnostic procedures by utilizing long-read nanopore sequencing technology.

The therapy

We plan to create synthetic RNA constructs using an innovative RNA-sensing technology called DART VADAR [25]. These constructs will selectively activate the expression of a toxic payload when they bind to specific tumor-related mRNA, such as IDH1 R132H, a common mutation in gliomas and EGFR [27] [28], a frequently overexpressed gene in glioblastoma. The conditional protein expression relies on RNA-editing in our construct, mediated by endogenous ADAR [25]. Initially, a stop codon prevents continuous translation of the toxic payload [25]. However, when the construct binds to the target mRNA, it recruits endogenous ADAR, which then disables the stop codon allowing translational readthrough [25]. The construct includes a positive feedback loop. Alongside the payload, additional MCP-ADAR is expressed [25]. Flanking the stop codon, there are two hairpin structures that are recognized by the MCP domain, which then leads ADAR to the UAG stop codon [25]. There, ADAR conducts an A-to-I conversion, turning the codon into UIG, which is read as UGG [25]. This enables expression of both the payload and more MCP-ADAR, concluding our positive feedback loop.

Our Vision

Our project aims to revolutionize glioma diagnosis and treatment by combining Nanopore sequencing and mRNA therapy. We will identify targetable genetic alterations using this integrated approach, paving the way for precise, personalized interventions. Our vision is to make Nanopore sequencing a standard tool in tumor diagnosis. This technology also has the potential to detect other diseases, addressing the increasing incidence of tumors and providing a more cost-effective alternative to existing antibody technologies.