Circular DNA in diagnosis and disease models

Informations projet

CIRCULAR VISION

N° de convention de subvention: 899417

DOI

10.3030/899417

Projet clôturé

Date de signature de la CE 28 Juillet 2020

Date de début 1 Octobre 2020

Date de fin 31 Mars 2024

Financé au titre de

• EXCELLENT SCIENCE - Future and Emerging Technologies (FET)

Coût total

€ 4 195 697,50

Contribution de l’UE

€ 3 998 822,50

Coordonné par

KOBENHAVNS UNIVERSITET
Denmark

Periodic Reporting for period 3 - CIRCULAR VISION (Circular DNA in diagnosis and disease models)

Période du rapport: 2022-10-01 au 2024-03-31

Résumé du contexte et des objectifs généraux du projet

Circular DNA is emerging as a novel biomarker for many types of cancer and inflammatory disorders. However, we lack the technology for detecting circular DNA and for generating appropriate disease models.

Many human diseases, including cancers and inflammatory disorders, are associated with the production of ‘circular DNA’ from chromosomes. These molecules have potential as biomarkers, and in the case of cancers are potential drivers of disease progression. However, the technology for detecting circular DNA and for creating disease models is lacking, meaning we cannot explore their potential use in diagnostics.

CIRCULAR VISIONs goal is to explore the new opportunities circular DNA creates in early diagnosis and monitoring of disease, particularly screening, and to create an experimental system to understand the link between disease and circular DNA.

CIRCULAR VISION explores this brand new territory by creating highly sensitive whole genome screens for circular DNA that correlate with disease, using lung cancer (LC) and inflammatory bowel disease (IBD) as model examples. We develop and combine novel methods in molecular biology, microfluidics, DNA sequencing and bioinformatics in order to identify new diagnostic markers. Such markers for IBD and LC will then be adapted to clinical diagnosis and prognosis using advanced image analysis and cytometry methods. Finally, CIRCULAR VISION will show the causal link between circular DNA and cancer, by producing disease models with oncogenes on circular DNA.

CIRCULAR VISION assembles key pioneers in the emerging field of circular DNA with leading clinical experts and key commercial players in cytometry and genomics. We are convinced that our technology has broad applications in early noninvasive diagnosis of cancer and monitoring of inflammatory diseases. We believe our technology will lay the foundation for future research into circular DNA biology and spur future drug development.

Travail effectué depuis le début du projet jusqu’à la fin de la période considérée dans le rapport et principaux résultats atteints jusqu’à présent

The consortium finalized an efficient protocol for the purification of circular DNA and tested the protocol on plasma in a study of patients with lung cancer and another study of patients with inflammatory bowels disease.
A computational biology study of the inter-laboratory analysis of the samples was performed and an array of tools for mining of circular-DNA data was also developed. The project further developed algorithms for identification of circular DNA, especially those that have an impact on phenotype.
We have also implemented and developed new methods for enumeration of nucleic acids, e.g. circular DNA, in cells and in cell-free samples. Protocols and proper workflows for fabricating microfluidic chips were established and optimized.
The purification of circular DNA from pilot plasma was carried out and we also purified, sequenced and mapped circular DNA from plasma of hundreds of lung cancer patients and patients with other cancers that serve as controls.
We enrolled over 200 IBD patients in a prospective study, and followed them over a year. We sampled biopsies and plasma from hundreds of samples and started the sequencing and analysis of circular DNA on the first hundred samples, to identify circular elements that can be used to mark different stages of the disease.
Several strains with reporter genes on circular DNA were generated in fission yeast, and we observed that endogenous circular DNA behaves differently from circular DNA coming from the environment around the yeast. Lastly we developed an optimized CRISPR-C method for generating circular DNA in human cells.
We published our findings in 26 publications, 16 in high impact journals (IF 5<), and 7 in very high impact journals IF 10<), with several manuscripts are still in progress. We disseminated information through podcasts, videos and newsletters with stories and documentaries about our research and what it means to be a patient with IBD or cancer, on the project website and social media channels.

Overview of results:

Alternative circular DNA purification methods
- IPR obtained, new product Circle-Pure, TM, on the market.
- Methods published in three articles.

Detection of circular DNA biomarkers for cancer
BHRI:
- Bioinformatic pipelines for analysis of circular DNA.
- Tested on cancer and non-cancer illness, such as Systemic Lupus Erythematosus and Amyotrophic Lateral Sclerosis.
- Results published.
UCPH:
- IPR applied for.

Prototype of microfluidic (small amount of fluid) systems for purification of circular DNA
- Results published.

Cytometric (the counting and measuring of cells) solution for quantification of mutant allelic fraction and of circular DNA levels
- IPR applied for.
- Manuscript for quantifying allelic fractions in progress.
- Two new products planned.

Profiles (unique patterns) of circular DNA identified in colon and lung cancer
- Lung cancer results published.
- Manuscript for colon cancer results in progress.

Circular DNA in inflammatory bowels disease
- Potential circular DNA biomarkers identified.
- Results published, another manuscript in progress.

Yeast cell models with specific circular DNA
- Manuscript in progress.

Mammalian cell models with specific cancer related genes on circular DNA
- IPR applied for.
- Results published.

Progrès au-delà de l’état des connaissances et impact potentiel prévu (y compris l’impact socio-économique et les conséquences sociétales plus larges du projet jusqu’à présent)

We hope to revolutionize the detection of circular DNA by producing efficient and accurate technologies demonstrated in relevant environments for circular DNA visualization, purification, sequencing and computation processing methods.
We will exploit the potential of circular DNA as an early biomarker for blood-based diagnosis and efficient prognosis of IBD and cancer.
By establishing model cell lines where circular DNA can be removed, inserted and monitored at will we want to find out how circular DNAs form in vivo, how some of them contribute to oncogenesis in human cells and how most cells eliminate these molecules.

Beyond state of the art, we have;
- The first system for eccDNA on the purification of microfluidic systems.
- Created a whole pipeline for the bioinformatic analysis of circular DNA, from circles to clustering.
- The first cancer cell lines, where we have reconstituted circular DNA.
- Developed new yeast strains that look a lot like human cells, to analyze circular DNA in a genetic model of schizosaccharomyces pombe.
- Created the first fully automated cytometric (the counting and measuring of cells) software for analysis of circular DNA.
- Shown the first example of circular DNA from inflammatory IBD.
- Shown in our cancer study that areas of the genome is amplified by circular DNA, because importantly, small circular DNA can carry small genes from the genome and have very high transcription.

Impact of note, is we;
- Created have created a company, CARE-DNA Aps.
- Can potentially employ more people in both Chemometec and CARE-DNA Aps.
- Can potentially increase income for the companies based on project results.

There is a socio-economic impact for the patients that can potentially live longer, based on further research/development of the results of the project.
The project has been important to place Europe in the field of circular DNA technology, where China and USA are already strong.
The project is also important for early detection, which we are aiming at, which will have a great impact on mortality for the cancer types researched. This is millions of euro made, for EU countries, who do not lose people from the work market, when they get cancer.