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Periodic Report Summary 1 - REPAIR-HD (Human pluripotent stem cell differentiation, safety and preparation for therapeutic transplantation in Huntington’s disease)

Project Context and Objectives:
There are compelling reasons for considering cell replacement therapy in a wide range of, currently untreatable, neurodegenerative conditions. For most of these conditions targeted pharmacological treatments are a long way off, as the detailed pathogenesis is not yet described, thus making targeted treatments difficult or impossible. However, even if pathogenesis is obscure, a condition can still be amenable to cell replacement therapy if the anatomy and distribution of neuronal cell loss is characterised. There is clinical ‘proof of principle’ that primary foetal precursors that are specified through normal development can repair circuitry in both Parkinson’s (PD: specified to become dopaminergic neurons) and Huntington’s disease (HD: specified to become medium spiny neurons: MSNs), and with preliminary evidence of sustained alleviation of some of the functional deficits. Nevertheless, although transplantation of primary foetal cells is important to establish the validity of cell replacement therapy in a particular condition using appropriately specified “gold standard” cells, the scarcity of foetal tissue of sufficient quality and the impossibility of standardisation of such grafts severely limit clinical application. Human pluripotent stem cells (hPSCs) present an attractive potential alternative source, as they can be expanded indefinitely in vitro, can be stored by cryopreservation, and can be differentiated into mature somatic cells. However, controlling their proliferation and differentiation sufficiently to provide cells suitable for clinical transplantation is key and has been challenging.
Repair-HD proposes to establish all the components necessary to take human pluripotent stem cell-derived neuronal cells through to the point of ‘first-in-man’ clinical trial in Huntington’s disease (HD). The concept is to use HD as a ‘model’ disease to test the principle that an in vitro differentiated stem cell product can yield clinically useful structural and functional repair, and that this will pave the way for clinical translation of hPSC-based products in a broad spectrum of neurodegenerative conditions. Given the complexities inherent in both the hPSC-derived donor cells and neurodegenerative conditions it is important to use a single well-defined target condition to provide a pathway to clinical translation. HD provides an excellent test-bed for several reasons, not least that, donor cells must be placed homotopically into their normal position within the striatum, in contrast to PD where they are placed ectopically, thus allowing restoration of normal anatomical circuitry and restoring striatal synaptic plasticity at the host-graft interface.
Specific objectives:
• Select and standardise the optimum protocol for in vitro differentiation of hESCs to an authentic MSN phenotype whilst controlling the production of non-neuronal and non-striatal populations.
• Test and rank the lead protocols selected from the in vitro work for reliability of MSN differentiation and lack of tumour formation in rodent HD animal models according to behavioural profile of the animals, electrophysiological assessments, and anatomical parameters.
• Identify surrogate markers of the safety and therapeutic potential of hPS-MSNs for HD cell therapy
• Determine parameters for neurosurgical scale-up, to optimize in vivo imaging protocols, to extend sophisticated functional analysis, and to determine tumorigenic potential of the existing differentiation protocols in the primate HD lesioned brain using macaque iPS-MSNs (induced pluripotent stem-MSNs) allografted into macaque as the closest match to allografting in man.
• Xenograft (human-to-monkey) hPS-MSNs into immunosuppressed HD-lesioned macaques as the final stage of characterisation of phenotype-specific differentiation, integration, function, and safety of the lead hPS-MSN product prior to clinical application.
• Adapt and scale up the most promising hES-MSNs for medicinal (GMP) grade production.
• Generate and test GMP-compatible hiPSCs up to the stage of GMP production of hiPS-MSNs as an alternative donor cell source should the ethical and/or regulatory hurdles associated with hESCs prove prohibitive.
• Establish all elements for the first in man proof of concept clinical trial of hPS-MSNs. This will be designed to assess safety (in particular absence of overgrowth/tumour), and functional efficacy and will require the prior development and validation of a sensitive assessment battery, an effective trial design, and complete ethical and regulatory permissions ready to commence a first in man clinical study of differentiated hPS-MSNs by the end of the study.
Project Results:
The first task of WP1 was to procure and distribute all the key resources to all the labs involved in the development and standardisation of protocols for the generation of MSNs from hPSCs. To this end, master banks of the target hESC line and banking of one of the two back-ups is in place, with banking of the third underway, and full characterisation, including electrophysiology and calcium imaging, is nearing completion. Working banks of target lines are now established in the relevant partner’s labs and the partners have discussed and exchanged SOPs to ensure commonality. Early on in the course of the project it became clear that one of the three protocols selected for comparison was not suitable, and thus the partners are working together to select or converge on a final common protocol from the two remaining. The regulation is also in place to ship the target line for transplantation into rodents and into monkeys. Adaptation of the protocol to be GMP compliant has begun and work is ongoing to define the target specification of an hPS-MSN for GMP production, with a preliminary definition in place. A gap analysis has been completed for compliance with GMP and EMA requirements.
During the course of the project, a GMP-compliant hiPSC, representing a back-up hPSC target, has become available and will be available for distribution by its due date (M24). In addition, work has commenced to generate a hiPSC by a GMP-compatible genome non-integrating method. Authorisations and legal legal agreements are in place to transfer foetal cells from P1 to P5 to generate foetal derived iPSCs as an additional backup.
Anatomical and functional assessment of the differentiation protocols following transplantation into rodent models of HD is established and validated in two of the partner labs. Screening for overgrowth has demonstrated safety in 4 out of 5 cell protocols, with increasing graft maturity with progressively longer survival times. Functional screening has been undertaken up to 16-20 weeks post transplantation to date, and has revealed the first signs of motor recovery at this time point. Once the lead target protocol has been agreed, longer-term anatomical and functional assessment will be undertaken. Work is underway to identify the best strategies for facilitating long-term xenograft survival, refine behavioural outcome measures, and to set up slice electrophysiology for the next phase of work.
In parallel a quinolinic acid primate model of HD has been set up, with demonstration that the lesions can be detected non-invasively by PET and MRI and that they correlate with behavioural deficits. A parallel transplant study of a monkey GFP tagged iPS line unexpectedly showed poor survival, which appeared to be due to an immune response to GFP and possibly also an MHC class II mismatch between donor and host. This is being addressed by a pilot study to test the need to HLA match the donor and host. The regulatory documentation to allow progression to human-to-monkey xenografts has now been prepared.
Work is underway for a full revision of CAPIT-HD, the existing battery for assessment of patients with HD undergoing surgical interventions. An analysis of published CAPIT-HD data has been performed and an analysis of existing unpublished data (previously generated by the partners) is underway with preliminary data now available to the consortium. On the basis of this, the most useful tasks from CAPIT-HD have been selected for inclusion in the preliminary revised battery which will go forward for beta testing. In addition, a number of novel tools to assess motor, cognitive, and behavioural outcomes have been developed, along with new functional assessments. The protocol for beta testing is nearly complete and will be submitted shortly for ethical approval. A review of existing post-operative rehabilitation literature has been performed and work is underway to assess the timing and nature of interventions to enhance the function of grafts postoperatively.
To regulate the consortium, a regulatory oversight committee and ethical advisory committees have been established and convened.

Potential Impact:
This programme is expected to yield a major impact on the creation of new knowledge and the development of new techniques controlling differentiation and proliferation of human stem cells and reprogrammed cells for therapeutic purposes. It will also develop a framework for the safe and effective clinical delivery of cells into the brain in HD. Together, these advances will facilitate progression to ‘first in man’ trials of stem cell-based therapy in HD. Specifically, this program will:
i. Enrich our understanding of how to produce authentic striatal MSNs – the cells required for transplantation in HD. This will be of fundamental importance for both neural and non-neural differentiation for regenerative medicine, making in vitro cell models, and especially for planning of GMP strategies for regenerative medicine. It also likely that additional knowledge and innovations, which can be made generally available to the research community, will emerge from the detailed cellular profiling undertaken during this work.
ii. Validate the concept of proliferation through complete differentiation. Based on existing data, the principle strategy in the consortium for controlling proliferation of cells post-grafting is through achieving complete differentiation to neural cell subtypes. The advantage of this strategy for translation is that it avoids additional unnecessary steps involving regulated procedures.
iii. Development of tools for quality control. A specific set of tools for quality assessment of differentiating cells intended for use in HD and HD animal models will be developed. They will be applicable to any cell source intended for use in HD, but will also define the principles for developing similar batteries for other neuronal subtypes.
iv. Establish objective criteria for the selection and validation of lead candidate hPSCs for clinical trials. The parameters will be transferable to other cell lines for HD and will also lay down standards for a wide range of cell lines designed for therapeutic use. Moreover, ultimately, once the work proceeds to first in man studies, it will be possible to test how accurately the preclinical data predicts success in the patients. This will have major implications for the development of similar programs for other neurological diseases and also more generally for the use of rodents and primates in the translation of regenerative medicine.
v. Further develop knowledge and guidelines for the production, storage, and characterisation of GMP grade hESCs and their neuronal differentiation. This includes testing the Pathways set up for the transfer of knowledge and materials between research labs and GMP.
vi. Establish and validate protocols for safety management of a range of risk factors for specific cell protocols destined for clinical use. On the basis of this information, safety monitoring assessments will be selected for clinical use in the future clinical trial.
vii. Through the monkey allograft studies, Repair-HD will clarify the immunological interaction between graft and host following intracerebral stem cell transplantation, determining the influence of an inflammatory environment on graft survival and functionality. This will facilitate identifying the optimal immunosuppressive strategies important for successful clinical application of novel cellular therapies.
viii. Provide a new and more sensitive clinical assessment tools that will be suitable for assessment of a range of neurosurgical interventions in HD. The new tool will be validated for use in multiple centres and in multiple European languages, and will be suitable for studies where, by necessity, relatively small cohorts of patients will be followed longitudinally.
ix. Establish a European framework for delivery of complex trials in neurodegeneration. This will benefit not only HD, but will also provide a road map for a wide range of other neurodegenerative conditions.
x. Although Repair-HD is primarily focussed on translation of novel cell therapies into clinical application, the process is also expected to advance understanding of the role of the neostriatum in providing a neural substrate for neuroplasticity and recovery of function, in particular as it relates to learning, relearning and adaptation of motor skills and habits.
Provide advanced training in stem cell and developmental biology, and in experimental and translational medicine in leading European laboratories, with opportunities for sharing of expertise and movement between centres encouraged, enhancing employability and mobility.
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