Identifying and validating biomarkers for a cell therapy product for Huntington's disease

Huntington’s disease (HD) is a neurodegenerative disorder in which striatal medium spiny neurons (MSNs) are selectively vulnerable and die early in the course of the disease. No treatments or disease-modifying therapies exist for HD, making this a significant unmet need. We have adopted a regenerative medicine approach to repair the neural circuitry and to alleviate the cognitive and motor symptoms of the disease. To achieve this, we developed an embryonic stem cell-derived MSN (hESC-MSN) cell therapy product, called RHD-001, which can be transplanted into the brain, where it replaces the function of striatal MSNs lost to the disease process. An important aspect of this is that it re-connects cortical and subcortical basal ganglia circuitry. Over the last 10 years, we have characterised RHD-001 extensively (including demonstrating survival in the brain, safety, the ability to functionally synapse with the host brain, the positive electrophysiological benefit of the graft, gene expression profiling of the product, and alleviation of motor and cognitive deficits). Recently, we undertook a gap analysis with the Cell and Gene Therapy Catapult, which concluded that we have compelling body of evidence to support taking RHD-001 forward for regulatory consideration, but identified two gaps: (i) validation of a biomarker panel for quality control of RHD-001 prior to transplantation and (ii) a long-term GLP toxicity study. It is essential to have the biomarker panel in place for use in the GLP toxicity study, so this application is to develop and validate the biomarker panel. Development and validation of the biomarker panel will be based on 8 existing batches of RHD-001 product. Each batch has already been transplanted into rodent brains with long-term survival times, and subsequently designated as either an ‘optimal’ or ‘suboptimal’ batch of cells, based on MSN marker expression in the grafts and the propensity to integrate into the host brain. Single-cell RNAseq analysis of these batches will allow identification of biomarkers that can differentiate between optimal and suboptimal batches. These biomarkers will form the basis of the quality control (QC) panel, which will include on-target genes, off-target genes and genes associated with undifferentiated stem cells. We will then prospectively validate the suitability of this QC panel to predict optimal batches (after testing the suitability of antibodies and primers to be used for our flow cytometry/qRT-PCR assays). To address this, we will differentiate 10 new batches of optimal or suboptimal cells, with suboptimal batches created through subtle, unfavourable manipulations of the differentiation protocol prior to cryopreservation. Aliquots of the new batches will be screened using the QC panel following which these batches will be transplanted into the rodent brain. 12 weeks post-transplantation, grafts will be graded as optimal/suboptimal so that we can assess the extent to which their status was predicted by the QC panel. The establishment of this validated QC panel is a critical step in our progression to the clinic and will enable the GLP toxicity study, which is the final hurdle for the preclinical package in preparation for the MHRA.