JPND - Multicellular organoids: modeling, mechanisms and therapy development for C9ORF72-associated neurodegeneration.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are devastating disorders. The most genetic cause of ALS and FTD is linked to mutations in C9ORF72 gene. While significant discoveries have recently been made in the genetics of ALS/FTD, patients still have no real therapeutic treatments. The lack of any cure for ALS-linked C9ORF72 is attributable primarily to: (i) Poor understanding of the molecular pathogenesis of neurodegeneration; (ii) Lack of reliable animal models mimicking the multicellular and multi-mechanism complexity of the human disease; (iii) Delivery of therapeutically attractive molecules has been hampered by inefficient delivery methods including factors like the blood-brain barrier; (iv) Ineffective targeting of therapeutic agents specifically to the diseased sites of the brain and spinal cord. Research efforts aimed at understanding how mutations in ALS causative genes leads to motor neuron injury are of the utmost importance to enable therapeutic development for these disorders. The limitations associated with existing C9orf72 models can be overcome by using specific patient cells - derived 3D models named organoids. Thus, organoids provide unique opportunities as a system for the development of pharmacological or tailored gene therapies for C9ORF72-linked neuronal injury. Here, we assembled a multidisciplinary research team with complimentary expertise to examine strategies to overcome some of these challenges. The overall aims of our research programme are: (1) Generate 3D in vitro multicellular organoids models from iPSCs derived from healthy and patients with C9ORF72 ALS/FTD; (2) Fully characterise the newly generated models using established assays in the consortium (e.g. molecular markers and electrophysiology); (3) Explore how neuronal injury happen in ALS; (4) develop treatment for C9orf72 linked ALS..

The delivery of therapeutic agents to the central nervous system remains a major challenge. This is especially the case for the devastating disorders at the focus of the current proposal [amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)]. The most common genetic form of ALS/FTD is caused by expansion of a hexanucleotide repeat (G4C2) in the non-coding region of chromosome 9 open reading frame 72 (C9ORF72). The lack of any cure for ALS-linked C9ORF72 is attributable primarily to: i) Poor understanding of the molecular pathogenesis of neurodegeneration, the contribution of each cell type to the disease and the underlying causes of the variability that characterises the patient population; ii) Lack of reliable animal models mimicking the multicellular and multi-mechanism complexity of the human disease; iii) Delivery of therapeutically attractive molecules has been hampered by inefficient delivery methods including factors like the blood-brain barrier; iv) Ineffective targeting of therapeutic agents specifically to the diseased CNS site and/or cell type. Research efforts aimed at understanding how mutations in ALS causative genes affect various cell types and pathways, thus leading to motor neuron degeneration are of the utmost importance to enable therapeutic development for these disorders. The limitations associated with existing C9orf72 animal models can be overcome by using specific patient induced pluripotent stem cells (iPSC)- derived 3D models. Here, we assembled a multidisciplinary team with complimentary expertise to overcome some of these challenges. The overall aims of our research programme are: 1) Generate 3D in vitro multicellular organoids models from iPSCs derived from healthy and patients with C9ORF72 ALS/FTD; 2) Fully characterise the newly generated models using established assays in the consortium; 3) Explore mechanistic pathways (e.g. pro-inflammatory, anti-oxidant); 4) Therapeutic screening in collaboration with AstraZeneca.

The current proposal will use multidisciplinary approaches to achieve 3D modeling, investigate mechanistic pathways and therapy development for C9ORF72-linked ALS/FTD. The expected outcomes at completion are:

Short- and mid-term
Generation of the 3D organoid system would provide a great toll for:
1) Drug screening: the 3D model generated from healthy and C9ORF72 patients will be used to test a range of compounds. This will allow establishment of partnerships with other pharma companies [in addition to AZ (see MICA)]. The promising targets will be tested in animal models of disease. We are already in conversation with our patent office to discuss IP on the use of this 3D tool for drug screening.
2) The 3D system will be useful for 3Rs: Replacement, Reduction and Refinement. A Pubmed search using the terms: spinal cord, neurodegeneration and either mouse or rat returned 703 studies in 2017 alone. Considering each study reports data from an average of 44 animals, we estimate that >30,100 rodents were used in 2017 alone to study the neurodegeneration process in the spinal cord. Once proven functional and relevant to complex CNS studies, we expect other groups to set up this system. To facilitate the process, we will deposit our cells, protocols and data in public repositories. If this system will replace even only 1% of the animals used in 1 year, this would correspond to replacement of about 3,000 animals/year. In addition, the spinal cord organoids (SCO) generated here will be more relevant to study disease mechanisms and achieve patient stratification, one of the major challenges in the treatment of neurodegenerative diseases.
3) Data from WP3 will allow identification of new pathways and therapeutic targets

Long term and implementation
Successful generation of efficacy proof-of-concepts based on drug and gene therapies (WP4 and WP5) will likely lead to clinical development of the best targets. This will be done as follows:
1) Data generated from WP4 and WP5 will allow initiation of clinical development: a) presentation of the preclinical package to regulatory bodies as part of scientific advisory meetings (EMA, MHRA); b) GLP regulatory safety studies in rodents; c) GMP manufacturing of clinical products; d) Preparation and submission of Clinical Trial Application (CTA), e) Recruitment and initiation of clinical trials in C9ORF72 patients.
2) IP generation and potential commercialisation: commercialisation of our potential products can be achieved: a) establishment of start-up to take the product forward; b) or partnership with biotech or pharma companies; c) or licencing the product to a private entity.