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Periodic Report Summary 2 - 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 welldefined 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
• 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 ofdifferentiated hPS-MSNs by the end of the study.

Project Results:
In the first 18 months of the project the partners evaluated research grade stocks of two GMP-hESC lines (RC9 &RC17), and set-up in vitro quality-control assays to identify and quantify the appearance of the target cell therapy product (CTP: medium spiny neuron precursors) in response to cell differentiation protocols. Using these assays RC9 cells were selected over RC17 and existing MSN differentiation protocols were compared to our newly optimized protocols that have been designed to be compatible with GMP requirements. Where appropriate, the new protocols were also compared to primary foetal human MSN precursors as these represent “genuine” MSNs. In the M19-M36 period we developed and documented additional in vitro quality-control assays based on next generation sequencing, electrophysiology and calcium imaging technologies to better characterize the accuracy, reliability, reproducibility, safety and functionality of the CTP. We also locked down and fully documented the protocol for CTP production and this protocol has now been translated to a GMP environment with subsequent research being based on cells generated under GMP conditions. This is currently ongoing as small scale GMP production, and in parallel,
generation of GMP banks, sufficient for the planned first-in-man trial, will shortly be generated. Work is also underway to generate a GMP-compliant human induced pluripotent cell line (iPSC), representing a back-up target.
In the first half of the project, we established effective transplantation of CTP into the rat brain following excitotoxic lesions to model the core pathology of human HD. Transplantation worked well, and the preparation protocols were modified to ensure good cell survival. During the second reporting period (19-36 months) there has been an iterative process whereby systematic comparison of the survival, growth, phenotypic differentiation and preliminary functional screens of the lead protocols has informed modification and development of the differentiation protocols developed under GMP-ready conditions. In parallel a quinolinic acid model of HD has been set up in a macaque (m) model, with demonstration that the lesions can be detected non-invasively by PET and MRI, and that they correlate with behavioural deficits. The initial objective was to mimic the clinical conditions in which HD patients would receive CTP
(i.e. allografting, macaque to macaque) to validate non-invasive tools for exploring safety and efficacy. To this end we have successfully derived and quality assessed miPSCs and have established and optimized a battery of assays to analyze the immunogenic potential of transplanted cells. This was necessary to progress to transplantation experiments in a macaque and also important to assess the potential immunogenicity of various types of mCTPs in vivo and post-mortem. Allotransplantation will be important to validate the methodologies chosen by the consortium, and the feasibility of in vivo non-invasive assessment of cell survival, differentiation and adverse events, including rejection. In preparation for transplants of human- GMP CTPs into macaque we have addressed the issue of peripheral immunosuppression to prevent humanto- primate xenograft rejection and have validated equipment and processes required for the combined expertise of two partners in two different countries to performing imaging and cognitive testing in
xenotransplanted macaques. 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 now complete and available to the consortium, and is being prepared for publication. On the basis of this, the most useful tasks from CAPIT-HD have been selected for inclusion in the preliminary revised battery (CAPIT-HD2) along with novel objective assessment tools of motor, cognitive, behavioural and functional outcomes, all created and validated by the consortium. Task selection is complete and the draft CAPIT-HD2 battery has been
determined. Ethical permission to test the battery has been obtained in all centres and beta testing of the new battery is currently ongoing. A review of existing post-operative rehabilitation literature has been performed and reported. In addition a novel dual task has been developed to assess striatal function and both animal and clinical work is ongoing to determine the timing and nature of interventions to enhance the function of grafts postoperatively. In order to manage the complexity of the regulatory environments for the production of cell product for use in man we prepared an audited report to confirm that all of the necessary Ethical and Regulatory approvals relating to the Repair-HD project have in fact been obtained, and regulatory oversight committee and ethical advisory committees were established and convened. In order to understand and to solve the key ethical and regulatory challenges associated with the design of such a trial, we held a workshop of experts in cell transplantation, surgical trials, and ethics, and have prepared a report addressing the ethical and regulatory challenges.

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.
xi. 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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