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Towards the development of an effective enzyme replacement therapy for human alpha- mannosidosis

Final Report Summary - HUE-MAN (Towards the development of an effective enzyme replacement therapy for human alpha-mannosidosis)

The aim of the HUE-MAN project was to set the ground for the introduction of the effective treatment of the rare disorder human alpha Mannosidosis using enzyme replacement therapy (ERT) as the generally accepted and most promising therapy for lysosomal storage disorders. Based on our initial observation that correction of storage in many tissues of a mouse model for this disease including brain was found after administration of lysosomal acid a-Mannosidase (LAMAN) from bovine kidney, and human and mouse recombinant LAMAN our major goals were to investigate and establish clinical parameters in the mouse model and a natural history study of the human disease in order to define clinical endpoints for the future clinical trials in a-Mannosidosis. Furthermore, in parallel, HUE-MAN wanted to establish conditions for the production of rhLAMAN that can pave the way for a first clinical trial in man.

HUE-MAN brought together cell and molecular biologists, clinicians with regular patient contact, neuropharmacologists with expertise in behavioural analysis, epidemiologists and biochemists with experience in large scale enzyme production and toxicology testing to set up the conditions and knowledge for eventually introducing the reLAMAN drug into first clinical trials. The combination of top level European scientists and clinicians was aimed to facilitate the integration of research capacities across Europe, increasing coherence and providing critical mass of investigators. The integrated multidisciplinary research enabled direct interactions between technology and biology and will provide the knowledge base essential for the rational design of therapeutic interventions. The unique composition and collection of expertises of this consortium was of major importance to pave the way for a successful introduction of ERT for human patients suffering from a-Mannosidosis. The ultimate aim was to push forward the development a high quality, safe and effective drug.

The present project is of great value for the introduction of recombinant human lysosomal a-Mannosidase into the first clinical trials of ERT in patients. In line with our plans we have been able to evaluate the benefit of a-Mannosidase ERT in the mouse model and we have made significant advances to get a suitable protocol for treatment of this lysosomal storage disorder. Importantly, the correction of storage has been followed over time in visceral organs but also in the central nervous system. A possible improvement of the brain associated symptoms has been analysed by sophisticated behavioural monitoring of treated and non treated mice, respectively. We have initiated a study in the mouse model of the hitherto unrecognised role of epigenetic factors contributing to the manifestation of the disease. On the other hand, a systematic clinical and diagnostic natural history study of the disease in human patients was successfully started. This study enables us to define the critical parameters and hallmarks of the disease which will eventually be used in phase I and phase II clinical trials to evaluate the efficacy of the ERT in patients. Apart from providing sufficient enzyme for the preclinical studies the optimisation of the production of the human recombinant LAMAN enzyme has been pushed forward and will be of major importance for the successful introduction of this enzyme into clinical phase trials. We could successfully develop the enzyme production and the replacement therapy in the mouse model. We have obtained important information about dose and interval of ERT treatment, about the mode of correction in peripheral neurons and neurons of the CNS, about behavioural profiles of the mouse model with and without ERT, about differentially expressed genes in treated and untreated mice. We could further analyse in detail the oligosaccharide storage by newly developed mass spectrometry analysis and started to establish this methodology for the analysis for clinical use.

A direct prerequisite for an effective enzyme replacement therapy in patients is the development of a suitable therapeutic protocol aiming to reduce the storage burden in peripheral organs and the central nervous system of the mouse model. Injection experiments using different doses of recombinant human LAMAN and administration of the drug at different intervals have revealed the best and successful therapeutic strategy. The biochemical and histological analysis of storage material in the brain and other tissues has been accompanied by a detailed behavioural analysis of a-Mannosidase knockout mice which have undergone ERT treatment. These examinations revealed significant improvements after enzyme replacement therapy in all parameters tested. Preliminary long term experiments with an injection schedule of 16 weeks (injection every other week) revealed that the chosen conditions reduced the storage in visceral (e.g. spleen) tissues but not in the central nervous system. However, the mice developed a severe immune response against the injected enzyme followed by a high mortality, therefore serious long term studies were precluded. To circumvent the immunological reaction, we have generated an immune-tolerant a-Mannosidosis mouse model which will allow chronic long-term ERT-studies.

It was a major achievement that a sensitive neuromotor performance test was developed during this project - the use of an adaptation of this method enabled us to reveal subtle neuromotor impairment in Mannosidosis mice. The detailed behavioural assessment also provided further support to the definition of a-Mannosidase-deficient mice as a valid preclinical model of human Mannosidosis. The behavioural defects described in the Mannosidosis mice mimic the emotional, neuromotor and cognitive alterations of Mannosidosis patients. The prominent storage-related alterations in hippocampal CA3-CA1 cells and pathways could at least partly underlie the functional changes in the mouse model, and could have important pathophysiological consequences.

The dissemination of knowledge was most prominently expressed by a number of press releases, publications, communications and presentations at international conferences and approvals by different authorities.

The ultimate impact of the proposal is in providing the basis for therapeutic strategy to treat the rare disorder a-Mannosidosis. The principle feasibility of ERT in the mouse model of a-Mannosidosis was shown. In the treated mice corrections of lysosomal storage in viceral organs but also -most importantly- brain was demonstrated making the introduction of ERT for human a-Mannosidosis into clinical trials a very promising project. The HUE-MAN project was of substantial importance to establish preclinical and clinical therapy protocols, to set out the conditions for a large scale and toxicity monitored drug production, to better understand and define the typical hallmarks of this disease and to evaluate epigenetic factors influencing the disease progression. All these goals of HUE-MAN have added new important information which will pave the way to realise a causative cure of this still untreatable disease. The major technological prospect of this research has been the achievement of a medicament for curing a-Mannosidosis. This research project was intended to decrease the economical burden, and to improve the prospects of a new medicament, to the point where there is an incentive among biotechnological companies to produce this drug.