Interview with Jörg Kutter
Interview with Partners
The consortium of 18 partners has successfully finalized work within the NaDiNe (Nanosystems for early Diagnosis of Neurodegenerative diseases). The project’s main goal was to provide diagnostic tools to determine the onset of a neurodegenerative diseases (such as Alzheimer’s disease or Parkinson’s diseases) by using blood or serum samples, a procedure that is potentially amenable to regular screening of a certain segment of the population, as it is very little invasive, works on a small sample volume and can be performed at a cost level that is not prohibitive for public health systems. This was an extremely ambitious goal, as the concentration level of biomarker molecules indicative of these neurodegenerative diseases can be expected to be very low in the early (onset) stages of the diseases. Furthermore, to be able to distinguish between different types of neurodegenerative diseases and to improve the fidelity of the diagnosis, it is not sufficient to rely on a single biomarker, but on a group of biomarkers, providing a signature or fingerprint.
During the project period, a wealth of approaches utilizing miniaturization and novel nanotechnologies have been developed and tested in combination with advanced biochemical protocols. It was a chosen strategy from the start to pursue several avenues, both to be able to explore different possibilities, but especially also to reduce the risk of failure of one single approach. In the project period, some of the chosen technologies (e.g., evanescent wave detection, self-assembled bar-coded particles) were demonstrated, but deemed to not provide sufficient performance or sensitivity to be useful for NaDiNe’s main applications, but could certainly be used for other (medical) applications. In the end, novel magnetic nanoparticles with custom- made antibodies and fine-tuned assay protocols were used for an immunoprecipitation-based enrichment step, to be followed by either a protein array with fluorescence detection or a mass spectrometric approach with nanopillar arrays for further up-concentration. Also, in parallel, an electrochemical assay again using antibody-coated magnetic microparticles was used as a further risk-reducing measure. Further refinements are possible by incorporating miniaturized separation systems as well to aid in the fingerprinting. Microdevices were fabricated from polymer materials and operated using sophisticated fluidic control hardware and software, established by one of the project partners. This led ultimately to integrated lab demonstrator devices for the above-mentioned processes. These were fully functional, albeit not yet so user-friendly as to be used directly be the medical partners.
Instead, validation of the developed systems was performed in the labs of the developing partners, using non-clinical and clinical samples provided by the medical partners. The medical partners also provided guidance for handling and preparation of the samples, and also joined in critically evaluating the data, together with bioinformatics experts. In particular, data from clinically relevant samples was compared to previously obtained results with the current gold standard method. Overall, while on a limited data set, the data produced by NaDiNe technology aligns very well with established methods, and has the potential to provide diagnostic answers with less sample, at earlier times in the disease stage and at significantly reduced costs. In conclusion, the results of the NaDiNe underline the significance of using sophisticated biochemistry combined with nanotechnology to provide next-generation medical diagnosis tools.
NaDiNe has addressed areas of technologic development and diseases with continuously increasing market forecasts. The achievements of the project have an important economic impact in: 1) analytical instrumentation; 2) diagnostics and monitoring of dementia patients. The achievement in the NaDiNe project is summarized in Fig. 1 below. The graph shows an intricate network of output in relation to R&D and wider societal impact. Overall the NaDiNe project has mostly worked in the R&D field with small activities in commercialization and end user product development. A number of new techniques developed under NaDiNe are sensitive enough to detect very low expressed biomarkers in blood such as those expected for neurodegenerative disease. Unfortunately, we did not find suitable biomarkers for diagnosing Alzheimer’s disease (AD) in blood. However, neurofilament measured in blood provided diagnostics for amyotrophic lateral sclerosis (ALS). The NaDiNe project has achieved a large knowledge base for various kinds of diagnostics and future advanced diagnostics devices. These techniques can be used for ultrasensitive detection of, e.g., cancer and infections in humans and animals. The technologies are also applicable to environmental control such as analysis of drinking water.
Fig. 1: Overview of impact of the NaDiNe project. NaDiNe has been firmly planted in the R&D sphere (light blue) with a few activities going into the general societal sphere (commercialization or usage in the clinic). Dashed lines indicate projections with further additional R&D funding.
According to the World Health Organization, the number of people with dementia worldwide in 2010 is estimated at 35.6 million and is projected to nearly double every 20 years, to 65.7 million in 2030 and 115.4 million in 2050. The total number of new cases of dementia each year worldwide is nearly 7.7 million. Treating and caring for people with dementia currently costs the world more than US$ 604 billion per year. AD is the most common form of dementia and thus one of the main causes of disability and dependence in elderly people, with great socioeconomic impact. On this ground, AD has been recognized as a public health priority by WHO which has recently recommended the development of national programs to address dementia, focused on improving early diagnosis and disease management, and providing better support to caregivers.
NaDiNe supports this societal need fully by proving validated assays, biobanking for future biomarker discovery, and a whole range of viable technologies for detecting biomarkers in CSF and blood. Some of these technologies (microfluidics, nanoparticles, microarray, sensing) can be packages in a point of care device serving developing countries, while developed countries may opt for automated processes like the mass spectroscopy methods developed in NaDiNe. The foundation is thereby laid in NaDiNe to improve disease management of neurodegenerative diseases. Even small improvements of disease management may have large positive economic consequences.
Some of the developed techniques in NaDiNe proved to be so sensitive and powerful that they are better used for cancer diagnostics as compared to neurodegenerative diagnostics. Preliminary tests performed in the NaDiNe project indicated that some of the technologies could be converted to cancer diagnostics tests for breast cancer and leukemia. Oncology is the second fastest growing segment of the molecular diagnostics market (infectious diseases being the fastest growing). The technologies proposed here is equally ground breaking for infectious diseases, as they also typically require long procedures in order to amplify few infectious agents from a large volume of patient samples. Furthermore, other preliminary experiments in the NaDiNe project showed ultra sensitive detection of bacteria in milk using a highly efficient sample preparation method. Hence, the proposed technology under NaDiNe could carve a large share of the molecular diagnostics market after future maturation (Fig 1).
The total economical impact on cancer treatment of NaDiNe achievements is likely much larger than just getting a part of the diagnostic market as cancer-related costs in the EU were about 126 billion Euro in 2009. 40% were directly associated with health care costs while the rest were lost work, care leaves for relatives etc. The cancer burden is projected to increase just like AD over time given EU’s ageing population. Diagnosing disease efficiently leading to more efficient treatment will have a large economic impact. A 10% efficiency boost would give the EU about 12 billion Euro annually in savings (and 120 000 saved lives per year) using 2009 annual cancer cost numbers. NaDiNe has provided state of the art technologies and knowledge to strengthen Europe in a highly competitive and large market, and the potential products are poised to reduce the economic burden of cancer.
Digital analysis of samples has a strong future because of its sensitivity and ability to process large sample volumes. Some digital analysis was explored in the NaDiNe project and a so-called droplet array was the most successful. It is expected that digital analysis will have a large impact on molecular diagnostics.
Sales of molecular diagnostics totaled $4.1Billion (app. €3B) in 2010 and $4.5B (app. €3.4B) in 2013 (Ref1, Ref2, Ref3). Estimates of annual growth rates vary from 8.7 to 11%. This shows that NaDiNe provides tools and technology into a fast growing field.
Much of molecular diagnostics is driven by polymerase chain reaction (PCR) and protein assays. Due to its ability to screen large sample volumes, the droplet arrays platform can compete efficiently with PCR for a number of applications including cancer diagnostics and infectious disease diagnostics. This indicates that the platform has the possibility to take a significant part of the diagnostics market as droplet arrays have a number of practical, economical and analytical advantages compared to PCR. When super sensitive detection is needed for identification of protein markers in blood, droplet arrays will be useful and have a chance to compete with existing de facto gold standards, such as enzyme linked immune assays (ELISA) as well as existing digital protein analysis platforms (Qanterix). Therefore, we strongly believe that droplet arrays will produce job opportunities in the EU as well as enable better disease management through streamlined and highly sensitive assays.
Point of care diagnostics encompasses many variants and implementation modes, but most techniques are typically based on simple flow control. These devices are not compatible with advanced diagnostics such as those demonstrated in NaDiNe. The prototypes developed under NaDiNe demonstrate that advanced molecular diagnostics can be packaged into devices that can be further developed into point of care devices. We expect that these types of advanced diagnostics devices will enable new types of diagnostics in medicine but also in other areas.
Providing a highly skilled and educated work force to relevant industries
It is without doubt that future molecular diagnostics devices will be very complex, but at the same time, as easy to operate for the users as modern mobile phones. In the NaDiNe project we have had numerous students working with the development of advanced systems and characterization and usability of potential biomarkers. NaDiNe has therefore provided Europe with a number of people that are knowledgeable in instrument development, usage of these instruments, developing molecular diagnostic kits, and their application in the clinical setting.
NaDiNe has assembled a large biobank for the research in the project. However, the biobank is equally usable in the future for further studies. Having a large available biobank is really important for rapid biomarker discovery. The concrete impact of the biobank is not easy to predict and depends very much on the research it is going to enable. There are, however, numerous examples where biobanks provide competitive advantages in research for biomarkers. In this context, it is noteworthy that any new biomarkers together with suitable detection technologies are patentable intellectual properties. We thus see this effort as an investment for the future.
Analytical tools and techniques developed in the project.
The technological development in NaDiNe lays the foundation for further studies and later possible commercialization.
There has been a large development of innovative nanoparticles in the NaDiNe project. Specifically, we specialized on their biofunctionalization. All the achievements obtained in this project constitute the starting point for further inventions in the field of preparation and application of biofunctionalized magnetic particles. Most of the developed approaches are versatile and applicable in variety of therapeutic and diagnostic contexts. A large part of existing (and, arguably, future) biomedical methods are particle-based, and thus there is a substantial commercial interest. Particles have furthermore large binding capacity and can be extended to other usages, such as, for instance, nano particle cleanups and other environmental applications.
Many of the microfluidics techniques developed in NaDiNe are modules that are going to be used in compound instruments or work flows. For instance, a - microfluidic fluidized bed was developed to extract and pre-concentrate analytes from large samples volumes (1-1000µL). This is important and useful for a number of down-stream analytical techniques such as mass spectrometry, protein microarrays (both tested in NaDiNe), but also other detection or identification techniques. There is an unmet need for highly automated and miniaturised sample pre- concentration in order to improve detection limits and specificity of a range of analytical instruments existing in labs today. This technology has therefore a large commercial potential if connected to down stream analysis equipment.
NaDiNe has explored a whole range of different detection technologies including electrochemistry, quantum dots enhanced detection, enzymatic amplifications, fluorescence and absorbance. The detection methods were furthermore combined with various techniques to obtain identity of the biomarker such as on chip capillary electrophoresis, isoelectric focusing and sandwich immune assays. In the end common fluorescent based microarrays, enzymatic amplified droplet array detection were superior combinations. However, the other techniques and combination may have suitable application is other areas where low detection limit is not required. The importance of this exhaustive effort is the knowledge, which are the viable alternatives for future instrumentations.
The new concepts for ultra-sensitive matrix-assisted laser desorption ionization (MALDI) mass spectrometry, as developed in this project, could have a considerable impact for the discovery of unknown biomarkers, not only in relation to Alzheimer’s disease, but also to other diseases such as different forms of cancer for which the number of reliable clinical diagnostic tools is very limited or not available yet.
Therefore, a scenario could be foreseen that the technology platform developed in this project could have a large socio-economic impact, and a great benefit for society as a whole. However, it should be pointed out that thus far, the project has only shown the feasibility of the concepts. A more focused, large effort is required to develop the technology into a clinical tool for routine use (Fig. 1). Apart from further basic improvements, such work should include the development of automated handling of patient samples, as well as a sample work-up system, using a high throughput robotized system.
Especially with the recently investigated markers Ubiquitin, Neurofilaments and SerpinA1 disease management of an individual patient can be better organized. This is far beyond a proof of principle study, but will have a direct differential diagnostic value. For markers like tau and abeta1-42 the diagnostic value was already established. This was not the case for the recently investigate markers. Ubiquitin will directly help us to define subtypes of AD patients and may help to restratify therapeutic trails. The same hold true for SerpinA1. This is the first neurochemical marker, which helps to predict the development of a dementia in Parkinsons´s disease. The measurement of NF has the impact to be included in the diagnostic criteria for Amytrophic lateralsclerosis (ALS). Instead of waiting until all clinical criteria are fulfilled, patients under the differential diagnosis of an ALS can be directly treated if NF levels are elevated or included in therapeutic trail at a time point which a therapeutic effect is more realistic. The economic impact must be evaluated in further studies and is not within the scope of NaDiNe.
Fluigent – fluidics control instruments.
Three different products have emerged from the NaDiNe project and are now commercialized by Fluigent:
The different products enable the company to increase its sales revenue, its market share and also to penetrate new markets. One such market is microfluidics driven by syringe pumps, the most popular solution for microfluidics at research labs. The thousands of microfluidics labs constitute a large commercial opportunity. However, Fluigent also developed an application programming interface (API) which significantly broadens Fluigent’s potential customer base.
Already today, almost all instrumentation in the general practitioner’s office is based on microsystems and some are even microfluidics based. With the NaDiNe project, Fluigent is well positioned to go into further collaboration to solve the next step in diagnostics – namely molecular diagnostics that is usually not found in the practitioner’s office (or in bed side diagnostics) yet. In fact, the prototype developed by Fluigent is capable of controlling automated and highly advanced molecular analyses, as was successfully demonstrated in the project. The billion USD molecular diagnostics market will likely increasingly incorporate more miniaturized analysis systems. The NaDiNe project therefore has strengthened Fluigent in particular and Europe in general to address this fast growing market.
AK Diagnostics, a new NaDiNe spinoff company.
One of the techniques developed in the project was the droplet array digital platform where very small concentrations of biomarkers could be detected. The platform has a potentially large commercial value in molecular diagnostics in general. Therefore, we have investigated patenting and commercialization. The result is a Danish spinoff company (AK-Diagnostics), possibly a patent application (still in preparation) and, at a later stage, scientific journal publications. The goal of the company is molecular diagnostics on serum samples. Areas of interest are cancer and infection diagnostics. AK Diagnostics is looking into point of care diagnostics devices but the technology is also highly suitable for single cell proteome or transcriptome analysis. Recently, talks with a medium sized European company have been initiated to explore collaborations to put the technology into research instruments for various purposes such as epigenomics.
Diagoswiss has had the opportunity to develop assays for neurodegenerative diseases, which will be considered to be commercialized. They further improved upon their equipment (both from a hardware and software point of view) and, together with the new assays, are now able to offer their customers advanced solutions for medical diagnostic applications.
In a broad sense, the reagents we have produced are expected to contribute to further understanding of the (neurodegenerative) diseases, allowing for improved diagnostics and potentially identifying novel therapeutics, with the corresponding socio-economic benefits. With specific respect to Moravian Biotech (MB), the reagents that have been produced will be further commercialised to allow their wider use across the scientific community, also providing MB with a stable income. This future impact relies on the dissemination of data relating to the reagents, which is available on the MB website and is accessible through the reports and the website of the NaDiNe project.