Impact

Socio-economic impact and the wider societal implications of the project


Contribution to Community and social objectives


Improving Health of European citizens


The improvement of health of European citizens as well as the increase of competitiveness of innovative capacities of European health-related industries and businesses are the key aims of the European Health theme. The DEM-CHILD project has a significant impact on key EU social and economic issues by increasing and reinforcing European Brain Research and its competitiveness in this domain. Moreover, the DEM-CHILD project clearly addresses the main goals of the International Rare Disease Research Consortium (IRDiRC) to team up scientists from academia and industry and organisations investing in rare disease research in order to achieve two main objectives by the year 2020, namely 200 new therapies for rare diseases and means to diagnose most rare diseases. In addition, the DEM-CHILD project addresses the recommendations of the Consensus document on European brain research issued by the European Brain Council (2006), both in its design (multidisciplinary effort of scientists from academia and industry together with clinicians) and its expected impacts: improved understanding of molecular mechanisms of the neurodegenerative diseases, innovative options to treat or prevent these disorders and increased innovation and competitiveness of European brain research.

The DEM-CHILD project has focused on a group of neurodegenerative diseases, the neuronal ceroid lipofuscinoses (NCLs). As the most frequent cause for childhood dementia in Europe, they represent an important problem for society. Even though the NCLs fall under the definition of “orphan diseases”, they are nevertheless the most common neurodegenerative diseases of childhood leading to dementia, blindness, motor deterioration and epilepsy. To date, none of the ten different NCL diseases can be cured, and as such they all lead to an early death of the patients, usually after a protracted period of disability. In this respect these diseases place a burden of long-term suffering upon the affected patients and their families, as well as a major socio-economic burden upon health care systems.

In order to advance the development of treatment options for the NCL diseases, the DEM-CHILD project has increased knowledge of the biological processes and mechanisms involved in the NCL diseases, especially CLN3 disease (WP4) and CLN1 disease (WP5) and their impact on the clinical spectrum of these diseases. This knowledge has been successfully transferred into clinical applications for prevention and early detection of these diseases (WP1, WP2, WP3), and ensuring that clinical (including epidemiological) data guide further research towards the establishment and evaluation of innovative therapies (WP3).

The new therapeutic options for NCL patients arising from the DEM-CHILD project (WP6) have the potential to make a substantial impact on improving the health of children suffering from childhood dementia, retinal and neurodegeneration in Europe. In addition, DEM-CHILD research has a significant effect on the understanding of biological processes leading to visual and brain dysfunction and the establishment of innovative therapies of neurodegenerative diseases in general, including age-related diseases, e.g. Alzheimer disease, which affect an even larger proportion of the European population.

 

Methods for health promotion and prevention including promotion of child health


Prevention of paediatric neurodegenerative diseases in Europe and India


With one exception, all NCLs are inherited in an autosomal recessive fashion, which means that there is a significant risk of multiple children being affected in one family. For most families there will be no prior clue that both unaffected parents carry an NCL disease-causing mutation. Moreover, affected children appear to be completely healthy during the first years of life. So by the time an affected child shows the first clinical symptoms and the right diagnosis is finally made, younger brothers and sisters might have already been born, each carrying a 25% risk of also being affected by this NCL disease. As such, a family may find that several or all children are affected by an incurable neurodegenerative disease, leading to immense suffering and the early death of these children.

However, the sooner a genetic diagnosis of NCL is made in a family, the lower is the risk of multiple children being affected, because the family can be provided with genetic counseling and the opportunity of prenatal testing for NCL to ensure delivery of an unaffected child. In addition, relatives can be tested for being carriers and also obtain genetic counseling as well.

DEM-CHILD project has aimed at preventing the risk of multiple affected children with NCL in one family, and at reducing the risk of occurrence of NCL disease in general by reducing the time and associated costs for making a genetic diagnosis. Multiple actions have been taken to achieve this aim:

(1)    Increase awareness of NCLs

(2)    Development and dissemination of a new diagnostic algorithm for NCLs

(3)    Development of new testing and screening methods applicable to the wider community

 

(1) Increase awareness of NCLs

 

As described in WP08, members of the DEM-CHILD consortium have given numerous talks and presented posters about NCLs in general and the DEM-CHILD project in particular to NCL experts as well as non-expert child neurologists, geneticists, other clinicians and scientists across the world in various cities including those in the UK, Italy, Germany, India, Finland, Ireland, and USA. Over 2000 international professionals have participated in these conferences. These efforts have significantly increased awareness of NCLs in the scientific and also medical community.

In order to increase awareness for the general public, several interviews with public newspapers as well as during TV-shows have been performed by various DEM-CHILD partners. For a detailed list of DEM-CHILD dissemination activities to the general public, please refer to the dissemination list in ECAS.

A total of six DEM-CHILD teaching workshops have been organised during entire project, specifically one in Hyderabad, India, in order to improve awareness of NCLs in multiple countries including India.

 

(2) Development and dissemination of a new diagnostic algorithm for NCLs


As the number of known NCL genes had increased from eight to at least thirteen, a new diagnostic algorithm had to be established for NCLs. A DEM-CHILD diagnostic algorithm for NCL diseases had been developed in collaboration with all NCL clinical experts in the consortium and published as part of a review on clinical presentation of NCL diseases (Schulz A. et al, BBA 2013). Dissemination of this algorithm was performed by different means:


Workshops

 

Six DEM-CHILD teaching workshops focussing on this diagnostic algorithm have been organised during the entire project period (Table 5). During these workshops, not only the diagnostic algorithm but also performance and evaluation of diagnostic procedures such as electroretinogram (ERG), electroencephalogram (EEG), light microscopy of blood smears (LM), electron microscopy (EM), and cMRI have been taught by the respective specialists. Each workshop has been attended by approximately 80-100 participants such as paediatricians, child neurologists, adult neurologists, care givers, lab technicians etc.


Handouts to medical professionals


A flyer illustrating both, this diagnostic algorithm for NCL diseases as well as providing information where to send diagnostic samples, has been designed and distributed not only during the respective DEM-CHILD teaching seminaries but also at various international congresses (see list of dissemination activities). The flyer can also be downloaded for free from the DEM-CHILD website.


Project website

 

A project website has been created and can be accessed under http://www.dem-child.eu. The website provides information regarding the new diagnostic algorithm (handout can be downloaded for free), as well as information regarding addresses, contact persons, institutions to where and under which conditions diagnostic samples should be sent in the specific country.


Teaching video

 

A teaching video on NCL has been created with the support of the German NCL Family Association. For this purpose, German NCL patients gave permission to be recorded during neurologic exams, EEG, MRI etc. when they visited the NCL clinic at the University Medical Center Hamburg-Eppendorf, Germany (partner 01). The video has already been given to NCL families at the Annual Meeting of the German NCL Parents Association in September 2012. Once translated, it will be distributed during the project’s next diagnostic workshops (see above) and can also be ordered for free via the project’s website.

 


(2) Development of new testing and screening methods applicable to the wider community


Two WPs (WP1 and WP2) of DEM-CHILD have successfully developed new testing and screening methods applicable to the wider community:

 

(a) Development of a diagnostic gene chip


The novel Next-Generation-Sequencing (NGS) -based diagnostic tool designed and developed by partners 11 and 12 in WP01 has the potential to become a significant extension of the existing gene-based NCL diagnostics. Within the DEM-CHILD funding period we could establish a state-of-the-art technology which will allow, within a few weeks, to scan the currently known NCL related gene loci plus a number of NCL candidate genes base by base. This is a clear improvement of the current situation, where no NCL diagnostic tool like this is available to our knowledge.  In contrast to yet existing methods like PCR and/or classical Sanger sequencing, all relevant genomic regions are covered by the new diagnostic tool in one step and with the highest possible resolution. As of today, the NGS-tool is comparable in terns of costs to microarray-based methods, but superior in terms of resolution and precision. Since the NGS-technology is still advancing very fast, it can be expected that the costs will go down further and a price level of less than 500 Euros per sample will be possible very soon. Partners 11 and 12 are able and willing to offer the tool as a full service to all researchers and physicians in the NCL field and the wider community. The service can be delivered from today on, without further technology developments. All necessary equipment, know-how and software tools are available in the partners companies.

In terms of novel gene mutation detection the studies carried out within DEM-CHILD resulted in a significant number of mutations, which were not known before and which are subject to further screenings and studies with the project partners (1,2,3,4,5).

All data generated by the diagnostic tool are and will be available to the project partners via the ftp-servers of partners 1, SME partners 11 and 12 have successfully developed a new genetic testing method for NCLs, which significantly reduces time and costs for diagnosis and which is readily available to the wider community.


(b) Development of automated enzyme testing for CLN1, CLN2, and CLN10


Three forms of NCL are caused by a deficiency in soluble lysosomal enzymes (CLN1, CLN2, CLN10). These three NCL forms can be easily diagnosed using fluorometric enzyme activity measurements in dry blood spots. These assays had already been established before the beginning of the DEM-CHILD project. However, they are quite time consuming, because measurements cannot be automated and single assays have to be run for each of the three enzymes.

 


Early detection of paediatric neurodegenerative diseases in Europe and India


Even though all NCL diseases are still beyond remedy, for three forms of NCL caused by a deficiency in soluble  lysosomal enzymes (CLN1, CLN2, CLN10), significant progress has been made recently in preclinical studies in mouse models of these diseases using a variety of approaches to deliver the missing gene product into the brain. As such there have been relatively successful attempts at enzyme replacement therapy, viral-mediated gene transfer, and more recently neural stem cell transplantation, but these effects are greatest if the therapy is delivered early in disease progression. Based on first promising effects upon behavioural and neuropathological phenotypes, two phase I / II clinical trials for CLN2 disease for the first two approaches are open for enrollment. This demonstrates that therapy options for NCL forms caused by the deficiency of single soluble lysosomal enzymes (CLN1, CLN2, and CLN10) are within reach.

These three NCL forms can be easily diagnosed using enzyme activity measurements in dry blood spots. Dry blood spots are also used for newborn screening for a variety of metabolic diseases. But newborn screening in European countries is only legal, if a treatment option exists for such diseases. Therefore, to date, the NCL diseases are excluded from such newborn screening programs. Once a therapeutic option becomes feasible for the CLN1, CLN2, and CLN10 forms of NCL, the opportunity of newborn screening for these diseases will have to be discussed again, as early detection of the disease will be crucial for the therapeutic success. Taking into consideration that phase I / II clinical trial for one of these diseases, CLN2, have started already, it is crucial to prepare testing methods applicable for future newborn screening efforts once treatment options are available.

The successful development of automated multiplex enzyme activity testing for CLN1, CLN2, and CLN10 by SME partners 7 and 13 (WP02) will ensure, that a method for newborn screening of these diseases is readily available once therapeutic options do exist. In addition, even before being used for newborn screening, this technique will significantly reduce time and costs for diagnosis, and support early detection of NCL diseases in Europe and India.

In addition, extensive collection and analysis of natural history data of these diseases in WP03 has led to the identification of early risk factors for certain forms of NCL such as CLN2. These will be implemented in targeted population screening programs in order to improve early diagnosis of NCLs This will also have a scientific impact as it will enable the study of prevalence and incidence for these diseases in large patient populations as well detect high risk populations.

In terms of novel gene mutation detection the studies carried out within DEM-CHILD resulted in a significant number of mutations, which were not known before and which are subject to further screenings and studies with the project partners (1,2,3,4,5). All data generated by the diagnostic tool of WP01 are and will be available to the project partners via the ftp-servers of partners 1, 11, 12. This will improve the interpretation of diagnosed sequence variations in NCL genes and reduce the time towards a clear genetic diagnosis for patients and families.

WP7 has contributed the identification of novel mutations, and added to the data on patients with known mutations. This will enable faster diagnosis in families, lowering the risk of multiple children being affected as genetic counselling can be provided and the opportunity to test prenatally to ensure delivery of an unaffected child. Relatives can be tested for carrier status. It has also contributes to knowledge on the genotype-phenotype variability of NCL, as the number of affected individuals has been substantially increased.

 


Development and validation of new therapies


As called for in the work program of HEALTH.2011.2.2.1-4, the DEM-CHILD project is expected to have a major impact on the creation of tools for experimental therapy in NCL diseases which are currently incurable. Four out of nine work packages of the DEM-CHILD project aim at the development and validation of new tools for experimental therapies for these diseases.

Three out of the nine work packages of the DEM-CHILD project have a significant impact for the development of new rationale therapies for NCLs. One work package has focused on validating therapies in patients

WP4 and WP5 have studied modifier and biomarker genes of CLN3 and CLN1 disease, respectively, which represent two of the three most common NCL forms in Europe. WP6 has focused on the development of therapies for NCL forms caused be defects in transmembrane proteins, CLN3 and CLN6. The extensive collection of longitudinal natural history data in WP3 represents an important tool for the evaluation of experimental therapies in patients.


WP4: Biomarkers and modifiers of CLN3

WP4 has contributed to future development of therapies for CLN3 disease by

(1) extending the broad spectrum of model organisms available for studying CLN3, which will enable better understanding of gene function and disease mechanism;

(2) identifying a novel signalling pathway and potential therapeutic targets, together with three small molecules in the yeast model

(3) identifying regulatory components and mechanisms capable to alter the gene expression profile in CLN3-defective mouse brain cells;

(4) evaluating the dual specificity phosphatase 2 (DUSP2) as  a highly potential biomarker of CLN3 disease

(5) performing the first comprehensive analysis of the lysosomal CLN3 proteome which identifies subsets of lysosomal enzymes impairing lysosomal homeostasis and lysosomal targeting routes that might represent novel targets for therapeutic approaches.

These results will provide important targets for future alternative therapeutic strategies that lead to the development of new treatments.


WP5: Biomarkers and modifiers in CLN1

WP5 characterized molecular basis and pathophysiology in CLN1 cell and animal models with the goal to identify modifiers and biomarkers of CLN1. WP5 has utilized induced pluripotent stem (iPS) cells from CLN1 patients to show that CLN1 has a role in neuronal extension and migration. WP5 could also show that enhanced apoptotic cell death and defects in mitochondrial function are part of the CLN1 cell pathology. Furthermore, results derived from proteomic profiling using MALDI-MS in CLN1 knockout mouse brain tissue showed that - depending on the disease stage  - different metabolic processes in specific brain structures are affected. The findings of WP5 indicate that CLN1 disease has a major neurodevelopmental component and therapeutic trials will have to be performed very early in life.

The results of DEM-CHILD WP5 regarding CLN1 disease modifiers and biomarkers as targets for future therapies will have a clear impact on future therapeutic strategies of CLN1 disease since in this programme we have confirmed two defective pathways - originally detected in the Cln1/Ppt1-/- mouse model – now also in human iPSderived neurons as well as human CLN1 fibroblasts. Proteomic profiling further confirmed the earlier results obtained in the mouse model by other methods. Altogether, the future research programmes have now the possibility to follow-up certain metabolic pathways defective in the CLN1 disease. Currently, the major treatment studies are performed in the mouse model and this study provides clear advances to follow up the treatment of mouse models. However, the fact we could here show for the first time that some of the CLN1 defects observed in the mouse model replicated also in the human CLN1 cells, will open up a new avenue to develop imaging and spectroscopic technologies to follow up the outcome of treatment in the human patients.


WP 6: Innovative therapies for NCLs caused by mutations in transmembrane proteins

The central aim of this WP6 was to develop novel therapies for the treatment of NCL caused by deficiency in transmembrane proteins. In contrast to NCL forms caused by a deficiency in a soluble lysosomal protein (CLN1, CLN2, CLN10), where significant progress has been made recently in preclinical and clinical studies using a variety of approaches to deliver the missing gene product into the brain, to date the therapeutic outlook for those individuals with mutations in transmembrane proteins still remains very bleak. In addition, the number of patients being diagnosed with NCL forms caused by defective NCL-related transmembrane proteins (CLN3, CLN6, CLN7, and CLN8) is increasing. Therefore, there is a significant and unmet need to embark on the challenging task of developing therapies for these NCL forms.

In order to address this need, WP6 has investigated the feasibility of two therapeutic protocols in mouse models of disease:

1. AAV-mediated delivery of the missing CLN6 or CLN3 genes to the retina and brain, respectively, and

2. neural stem cell-mediated delivery of neuroprotective factors to the retina 

The retinal CLN6 gene therapy study has shown that gene supplementation of the CLN6 gene to the mouse photoreceptor cells is insufficient to attenuate photoreceptor cell degeneration. Future experiments will aim to target the bipolar cells specifically.

The CLN3 gene therapy study analysed the delivery of CLN3 to the CNS showing that AAV2/9 vectors are capable of widespread and long lasting transduction of the CNS.

The study using stem cell grafts to deliver neurotrophic factors to the retina has shown that expression of CNTF can attenuate the loss of the photoreceptor cells in the nclf (CLN6) mice. These results encourage further research into a factor that rescues the photoreceptor cells without negatively affecting their function.

Taken together, WP6 has a major impact on the challenging subject of the development of therapies for NCL diseases caused by mutations in intracellular transmembrane proteins, in particular the juvenile CLN3 and the variant late infantile CLN6 disease.


WP3: Epidemiology and natural history of NCLs

The NCL Registry (WP3) will have a significant impact on the validation of current of future experimental therapy studies in NCLs. To date the DEM-CHILD NCL patient database contains the largest set of natural history data for all NCL forms worldwide which will continue to grow with the participation of the new database consortium members from a total of 12 countries. Results from the analysis longitudinal natural history data describe well that data derived from the DEM-CHILD NCL patient database represent a valid tool for the evaluation of current and future experimental therapy studies. In fact, CLN2 natural history data collected in WP3 are already used as control data in a phase I/II clinical trial on intraventricular enzyme replacement therapy in CLN2 disease.

As to date there is still no cure for any form of NCL, palliative treatment and supportive care are an important part of current treatment in all NCL forms. Therefore information on current medication to treat the most severe symptoms in all NCL forms such as epilepsy, myoclonus and spasticity has been collected in the DEM-CHILD database including the respective positive and negative effects, analysed and published. These data will help to improve palliative medical care for NCL patients and thus help to reduce the burden of disease and improve quality of life in these patients. In addition, this approach will also enhance the delivery of cost-effective care, helping to reduce the costs of health care provision.

 


International cooperation


European cooperation of DEM-CHILD

 

The DEM-CHILD project has proven that working at a European level in the field of the NCLs is indispensable:

Since the NCLs are complex disorders, caused by defective soluble lysosomal enzymes or defects in transmembrane proteins, affecting both the retina and brain, there is an increasing need to concentrate research efforts by reaching a critical mass of complementary competencies (genetics, neurology, child neurology, paediatrics, ophthalmology, molecular biology, biochemistry etc) that are spread out around Europe, creating a real synergistic effect from a technical and financial point of view. Without this concentrated effort, there would not be such synergy, which would subsequently slow down research and considerably delay the discovery of new treatments and their delivery to patients. Therefore, in all basic research work packages (WP4, WP5, and WP6), several DEM-CHILD partners from different European countries have collaborated within the same work package. Exchange of data, mouse models, research samples has been routinely implemented without any problem. Moreover, PhD students were given the opportunity to spend a certain time in another laboratory to learn new research techniques. Moreover, using the “virtual biobank” implemented in the DEM-CHILD NCL patient database, exchange of patient material for different research projects has become a routine within the consortium and beyond.

Since the NCLs are rare disorders, studying a significant number of patients is a crucial point to gain statistically significant data in order to accurately understand each NCL form and its natural course. Because the prevalence of the different NCL forms varies in different European countries, only a collaborative effort of several European countries can guarantee that enough NCL patients of each NCL form are studied. Therefore, all partners pf the DEM-CHILD project (Germany, UK, Italy, Finland) have successfully collaborated in WP3 in extensively collecting natural history data on all NCLs and in establishing prevalence figures.


Close cooperation with India and Europe


The cooperation between the European partners and the Indian partner has been very close throughout the entire DEM-CHILD project despite the fact that the project had to overcome some challenging regulatory obstacles:

Due to the late joining of our Indian partner in month 13 of the project and due to a restriction that only 10% of patient material was allowed to leave India and to be entered into the project, the number of samples (n=63) the project has received so far for diagnosis is too small to be used for any prevalence studies for India. The 10% restriction rule had been enforced by the Indian Council of Medical Research (ICMR) after the project started and was a pre-condition that partner 05 got permission from the ICMR to sign the DEM-CHILD grant agreement and be part of the consortium. The new restriction rule for India had been included in amendment no. 1 and accepted by the European Commission.

In order to face this challenge, we have tried to overcome these difficulties in obtaining reliable prevalence figures for India by increasing diagnostic capabilities for India: All partners have developed independent contact to Indian clinicians not connected with this project, and to clinicians of other countries in which Indian families were living. Moreover, we have organised one of our DEM-CHILD teaching workshops during the Annual Meeting of the Association of Child Neurology in Hyderabad, India, where also flyers with the newly developed diagnostic algorithm were distributed. Since then the number of samples transferred for diagnosis has increased, especially during the 4 weeks after project end (n=45). For those samples, diagnosis is still pending.

To conclude, the cooperation with India has achieved to improve awareness and diagnosis of NCLs in India despite national regulatory challenges.


Cooperation with other international research activities


In order to extensively study the natural course and clinical spectrum of all NCL forms, the DEM-CHILD database consortium has expanded to a total of twelve participating countries by now (Germany, UK, Finland, Italy, France, Norway, Denmark, Turkey, India, US, Brazil, Argentina) with the aim to collect the world’s largest, clinically and genetically best characterized, set of NCL patients. Funding for the new partners Norway, Denmark, France, Turkey, Brazil, and Argentina was obtained through two ways: i) a successful research grant application to the American NCL Family Association BDSRA (153 000 USD) coordinated by partner 1; ii) financial compensation for transfer of anonymous natural history data of CLN2 patients collected by DEM-CHILD partners to the company BioMarin to serve as historical control data for a phase 1 enzyme replacement study for CLN2 disease (100 000 €). The DEM-CHILD consortium had voted unanimously to use the entire newly obtained funding to finance database managers for each new partner country of the DEM-CHILD Database Consortium.

Patient numbers provided from all current DEM-CHILD database partners from the twelve participating countries add up to a total of 558 NCL patients

Participant 3 who coordinates the International Rare NCL Gene Identification Consortium (RNGC), offered the research opportunity offered by DEM-CHILD to identify new genes to clinicians in the RNGC as an encouragement to submit new families. In addition, participant 3 became involved in a novel Consortium focusing on adult onset NCL, although in this case little of this work was directly relevant to DEM-CHILD.

 


Boosting the innovative capacity of European health-related industries (SME participation)


Strong SME participation


At the beginning of the project, the DEM-CHILD consortium involved three SMEs. During the project, additional three SMEs have been added in order to boost the innovative capacity of several work packages.

Two WPs highly depended on the innovative work of SMEs, WP1 and WP2 which should develop new diagnostic tools for the consortium.

In WP1 there was and is a close cooperation between SME partners 6,11,12 and the academic partners 1,2,3,4 and 5. It turned out that the flexibility and expertise of the SMEs was ideal for the challenges and needs of the project, e.g. the adoption of essential methods and even changes of technologies within project run time was completely managed by the SMEs.

 

In WP2, SMEs partners 7 and 13 successfully developed the multiplex automated enzyme assay for CLN1, CLN2, and CLN10.

SMEs (partners 6,11,12,) were also very involved in the basic research projects of WP4, and WP5 (detection of biomarkers) where they provided technique and expertise for large-scale egene expression analyses.

 

Also WP7 has benefited a lot from the capabilities, which SMEs (partners 11,12) can offer by the diagnostic tool developed in WP1, since the classification of NCLs needs a strong and differential diagnostics.

The SME GABO:mi has led WP9 and taken full responsibility for contractual and communication management, project controlling and reporting, finance controlling and reporting, meeting organisation, and monitoring of IPR regulations. This expertise in project management has ensured that the DEM-CHILD project has not encountered problems due to mismanagement in any of these areas and that researchers had sufficient support in these areas so that they could focus on their research activities.


Innovation through SME participation


The SMEs partners 6,11,12) in WP01 developed a new sequence-based diagnostic tool, which offers for the first time a routine application, i.e. detection of yet classified NCL-relevant mutations, plus screening of mutations in a number of potentially relevant genes (candidate genes). The diagnostic tool in its present form did not exist before and will allow a much better genetic diagnostics of NCLs and also help a lot to find new markers (WP04, WP05) and even mutations (WP07) not yet known before. The SMEs partners 11,12) have the infrastructure and technologies in place to offer the NCL gene-based diagnostics to project partners and all interested researchers and physicians,

The SMEs partners 7 and 13 have successfully developed a MS-based automated multiplex enzyme for CLN1, CLN2, and CLN10. Such multiplex assay has never existed before and will significantly reduce time and costs for diagnosis of these NCLs.

These tools will not only be used by the DEM-CHILD project, but are now available to clinicians in general. These diagnostic tools significantly reduce time and costs for the diagnosis of all NCL forms and therefore have a major impact on the prevention and early detection of these diseases worldwide.

 


Main dissemination activities and exploitation of results


WP01: Development of a diagnostic gene chip

The diagnostic tool can be used as is by NCL researchers and physicians after the funding period, a first concepts of commercialization is under discussion. It was agreed between the partners that the companies of partners 11 and 12 are well prepared and are willing to offer the tool as a full service to the NCL community. A respective price model will be negotiated shortly. For partners 11 and 12 the tool will be a service component, which is well integrated in their yet existing facilities.

There is strong interest by the project partners to continue the use of the NGS-based diagnostic tool after end of DEM-CHILD and the SMEs (11,12) are supporting the initiatives to make the tool available to the world. Especially in terms of finding novel biomarkers and yet unknown NCL-relevant mutations the diagnostic tool is planned to be used by the partners after end of DEM-CHILD.  


WP02: Automated enzyme analysis for CLN1, CLN2, and CLN10

Similar to WP01, this new diagnostic tool can be used as is by NCL researchers and physicians after the funding period, a first concepts of commercialization is under discussion.

 


Outlook and future research


WP01: Development of a diagnostic gene chip

NCL diagnosis will benefit significantly from the possibility to perform deep and comprehensive genetic testing (sequencing) of patients within a very short time period (a few weeks ). In addition, the diagnostic tool will allow a quick and substantial scan of yet undetected mutations and their relation to NCL. The more analysis runs are performed the more the findings of identical mutations will increase their score to be real and to be related to NCL. Similar tools based on target sequencing are currently developed for a number of disease loci, so that we believe that our NCL diagnostic tool will be built on state-of-the-art technology for the next years.


WP2 Automated enzyme testing for CLN1, CLN2 and CLN10

The newly developed MS-based multiplex enzyme assay will be used for targeted populations screening in order to pbtain incidence figures for NCLs in various countries.

Moreover, the new diagnostic tool will be made available to the international community for rapid and cost-effective diagnosis of CLN1, CLN2, and CLN10.


WP3 Epidemiology/ Natural history study

Work of the DEM-CHILD NCL database consortium will continue beyond the DEM-CHILD project time. As previously indicated, the DEM-CHILD database consortium has expanded to a total of twelve participating countries by now (Germany, UK, Finland, Italy, France, Norway, Denmark, Turkey, India, US, Brazil, Argentina) with the aim to collect the world’s largest, clinically and genetically best characterized, set of NCL patients. Funding for the new partners Norway, Denmark, France, Turkey, Brazil, and Argentina was obtained through two ways: i) a successful research grant application to the American NCL Family Association BDSRA (153 000 USD) coordinated by partner 1; ii) financial compensation for transfer of anonymous natural history data of CLN2 patients collected by DEM-CHILD partners to the company BioMarin to serve as historical control data for a phase 1 enzyme replacement study for CLN2 disease (100 000 €). The DEM-CHILD consortium had voted unanimously to use the entire newly obtained funding to finance database managers for each new partner country of the DEM-CHILD Database Consortium.

Patient numbers provided from all current DEM-CHILD database partners from the twelve participating countries add up to a total of 558 NCL patients


WP4 Biomarkers and modifiers of CLN3

The work using yeast will provide background for new applications for further work on understanding CLN3 gene function and developing new therapies. The studies on the dysregulated gene network and the lysosomal protein composition in CLN3-defective brain cells will provide new insights into pathogenesis and modulatory processes affecting the course of the disease which might be novel targets for therapeutic approaches.


WP5 Biomarkers and modifiers of CLN1

The results of DEM-CHILD WP5 regarding CLN1 disease modifiers and biomarkers as targets for future therapies will have a clear impact on future therapeutic strategies of CLN1 disease since in this programme we have confirmed two defective pathways - originally detected in the Cln1/Ppt1-/- mouse model – now also in human iPSderived neurons as well as human CLN1 fibroblasts. Proteomic profiling further confirmed the earlier results obtained in the mouse model by other methods. Altogether, the future research programmes have now the possibility to follow-up certain metabolic pathways defective in the CLN1 disease. Currently, the major treatment studies are performed in the mouse model and this study provides clear advances to follow up the treatment of mouse models. However, the fact we could here show for the first time that some of the CLN1 defects observed in the mouse model replicated also in the human CLN1 cells, will open up a new avenue to develop imaging and spectroscopic technologies to follow up the outcome of treatment in the human patients.


WP 6 Innovative therapies for NCLs caused by mutations

The analysis of treatment outcome after AAV2/9-mediated gene delivery to the brain of the CLN3-deficient mice is still ongoing, but the level, spread and longevity of CLN3 expression show promise for the future. If a positive effect of treatment on neuronal survival is found, further research will be needed firstly to resolve the variation in transduction efficacy that was detected and secondly to compare the efficacy of treatment in the striatum with that in other CNS structures, as part of the process of translating this work to the clinic. Furthermore, the identification of AAV2/9 as an efficient vector for gene delivery to the CNS also promotes gene therapy as a promising treatment approach for a range of recessive disorders of the brain, including other transmembrane (and soluble) forms of NCL, other lysosomal storage diseases, and certain forms of Parkinson’s disease.

Despite considerable effort, the development of retinal gene therapy for CLN6-deficiency has thus far been failed to show a positive treatment effect. However, the data does provide us with pointers for future studies. The fact that a successful supplementation of the CLN6 gene to the photoreceptor cells at an early age fails to provide a benefit suggests that the photoreceptor cells are not the primary cell type affected, or not the only retinal cell type affected by the disease. This conclusion is supported by new data that show high levels of CLN6 protein in the inner nuclear layer, probably the bipolar cells, in the human retina. Future studies aiming to treat this condition should therefore focus on the delivery of CLN6 to the bipolar cells, either in isolation or in combination with transduction of the photoreceptors.

The use of stem cell grafts to deliver neurotrophic factors to the retina has shown that expression of CNTF can attenuate the loss of the photoreceptor cells in the Cln6nclf mice for at least 6 weeks. As CNTF delivery has failed to be efficacious in a clinical trial for the treatment of retinitis pigmentosa, there are some doubts whether the work is directly applicable in the clinic. However this study does show that the photoreceptor cells in the CLN6 retina are responsive to sustained exposure to a neurotrophic factor and encourages further research into a factor or combination of factors that rescues the photoreceptor cells without negatively affecting their function. Moreover, as the principle behind the method is independent of the disease mechanism and target cell, the results can help in the development of similar therapeutic approaches for other retinal dystrophies and potentially other neurodegenerative diseases.


WP7 Identification of new NCL genes

The data on prevalence for each NCL type in Europe will influence the priority of therapeutic development for these individual diseases. For example, enzyme replacement therapy for CLN2 disease is in phase 1 clinical trial, and lays the groundwork for similar therapy for other NCL forms caused by mutations in soluble lysosomal enzymes - CLN1, CLN10, CLN13.  However, the most common type of NCL in Europe is CLN3 disease. CLN6 and CLN7 diseases are more common in Mediterranean countries and were a significant portion of Indian families that were genetically identified. These three genetic types are caused by mutations in membrane proteins, which are beyond current therapeutic technologies. Work in DEM-CHILD on CLN3 disease, and the use of the mouse model for CLN6 disease for the development of retinal gene therapy, has inspired the formation of a new consortium, BATCure, in response to a Horizon20:20 call that involves three participants of DEM-CHILD with novel partners, to work towards new therapies for these very challenging NCLs.


WP8 Dissemination

Many children in India in whom NCL was neither suspected nor diagnosed have been examined for the disorder. Several of them have also been diagnosed with NCL through the project partners. This has clarified that all types of NCL are prevalent in India but are not really being diagnosed. The creation of the genetic analysis and hopefully its availability at a reasonable price in the near future is expected to help in the genetic diagnosis of NCL in India in a big way.

The project has also opened gateways for further international research. Younger scientists from India would be able to find research positions with various EU partners in future. Also international researchers interested in NCL would be assured that there is a lot of potential in India to conduct research on these disorders.

The DEM-CHILD database consortium will continue its work and also continue to update the DEM-CHILD webiste and diagnostic algorithm in order to ensure and improve awareness of NCL disease internationally.