A Thermoreversible 3D Manufacturing Platform for Scalable Production of Dopaminergic Neurons for Parkinson's Disease Therapy

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PROJECT SUMMARY Parkinson's Disease (PD) affects more than 1 million adults in the U.S, and cell replacement therapy (CRT) using human embryonic stem cell (hESC) derived midbrain dopaminergic (DA) neurons has shown great promise in animal studies in alleviating symptoms of PD. However, current manufacturing strategies for cell therapies are constrained by the industry standard format – monolayer, 2-dimensional (2D), cell culture – that additionally suffer from the lot-to-lot variability of animal-derived materials such as Matrigel as well as harsh enzymatic cell retrieval methods. Axent Biosciences Inc. is developing a proprietary 3-dimensional (3D) hydrogel based culture method to offer a critical extra dimension for cells to expand during production and thereby significantly increase the cell quantity produced per culture volume. Furthermore, our proprietary hydrogel technology is fully synthetic and avoids animal derived products, and its temperature responsive phase change enables gentle, high viability cell harvesting by cooling. We have shown that our 3D culture approach enables highly scalable expansion of hESCs, followed by 3D differentiation to DA neurons. Notably, we have achieved up to 25 times the quantity of potent DA neurons produced per culture volume with our 3D culture format compared to standard 2D formats. Moving forward, our overall objective is to scale up our 3D cell manufacturing process to produce high-purity, functional hESC-derived DA neurons and validate their in vitro and in vivo functionality to develop our Chemistry, Manufacturing, and Controls (CMC) towards filing an Investigational New Drug (IND) application. In Aim 1, we will scale up production of hESC-derived DA neurons to a pilot bioreactor capable of suppling sufficient numbers of cells for a Phase I clinical trial. In Aim 2, we will develop a purification strategy to remove residual contaminating cell types. In Aim 3, we will validate in vivo functionality of DA neurons produced in 3D bioreactor process and will benchmark against DA neurons generated in a conventional 2D production format. Successful completion of these aims will yield the basis for a cGMP-compliant manufacturing platform capable of producing higher quantities of functional DA neurons per culture volume compared to current 2D culture formats and thereby overcome a significant bottleneck for the development of PD cell therapies. Furthermore, our manufacturing process is generalizable and can be tuned to manufacture numerous cell types from hESCs. Finally, the purification process will minimize the risk of contaminating cell types in the final formulation, thereby increasing the safety profile of our candidate therapy in preparation for an FDA INTERACT meeting and subsequent filing of an IND application. To build toward this goal further in a Phase II SBIR proposal, we will include IND-enabling preclinical studies – particularly a 9- month animal study to validate safety – to progress translation of our DA neuron therapy for PD to the clinic.