Regenerative Medicine - From Bench to Clinic

Tuesday, 27th July 2021

In June 2021, BlueRock Therapeutics announced that the first patient had been dosed in a phase I trial of a new cell therapy for the treatment of Parkinson’s disease¹, developed in conjunction with Memorial Sloan Kettering Cancer Center. The therapy, known as DA01, consists of dopaminergic neurons derived from pluripotent stem cells, which are surgically transplanted into the brain of patients with advanced Parkinson’s disease. Specifically, the cells will be transplanted into the putamen, a brain area that undergoes significant neurodegeneration during Parkinson’s disease. The primary aim of the trial is to determine the safety and tolerability of the treatment at 1 year post-surgery, and will also assess the survival of the transplanted cells and effects on movement.

DA01 represents a new treatment modality in the field of regenerative medicine, which involves the repair or replacement of damaged or diseased tissue to restore normal tissue function. This type of stem cell-derived cell therapy has wide ranging potential, including for other neurodegenerative diseases, diabetes, cardiovascular disease, retinopathy and spinal injury. While this is not the first clinical study of stem cell-derived dopaminergic neurons for Parkinson’s disease, it could represent a significant advance in the rapidly growing field of cell therapy and regenerative medicine, as the researchers behind the therapy have devised a scalable and reproducible method for the manufacture of an off-the-shelf cell therapy product, DA01^2,3.

Protocol for Differentiation of hPSCs to Dopaminergic Neurons

The protocol for the differentiation of human pluripotent stem cells (hPSCs) into mature midbrain dopaminergic neurons uses a cocktail of small molecules and proteins. The main ingredients for neuronal induction are the small molecules SB 431542, LDN 193189, CHIR 99021 (CHIR), and Y-27632, along with Sonic Hedgehog (Shh) protein. Boosting CHIR concentration from day 4 of the protocol, led to improved midbrain specification and reduced contamination with unwanted cell types. Differentiation of midbrain dopaminergic neurons was achieved by switching to media containing the growth factors BDNF, GDNF and TGF-ß3, supplemented with Ascorbic Acid, Dibutyryl cAMP, and CHIR. Substitution of CHIR for DAPT occurred in the latter stages of differentiation (see protocol snapshot). The resulting cells were subjected to extensive molecular, biochemical and electrophysiological analysis and were found to reproducibly exhibit the hallmarks of midbrain dopaminergic (mDA) neurons. Batches were cryopreserved, thawed and when analyzed found to retain functional properties in vivo and in vitro. When transplanted into the striatum in a hemiparkinsonian rat model, differentiated mDA neurons exhibited long-term survival and animals showed functional recovery, as indicated by reduced amphetamine-induced rotational behavior.

Transition from the Lab to the Clinic

The generation of mDA neurons at clinical scale, however, is more than a robust differentiation protocol. When transitioning from preclinical to clinical studies patient safety is of the utmost importance so requires defined differentiation conditions, and traceability and consistency of raw materials. In order to generate mDA neurons that are safe for cell therapy of Parkinson’s disease, manufacturers are adopting a risk-based approach to manufacturing at all stages of the process, i.e. adopting and following the guidelines for current good manufacturing practice (cGMP) as far as possible.

To address the need to move to GMP, the team behind the original research adapted the initial protocols for generating dopaminergic neurons for the development of the DA01 product. Standard operating procedures (SOPs) were developed to minimize batch-to-batch variability for the final cell product. One specific adaptation was that the original culture conditions using mouse fibroblast feeders and knockout serum replacement (KSR) based media, were switched to a feeder-free system to eliminate materials of animal origin. In addition to efficacy studies in animal models, stringent safety testing of the resulting DA01 neurons, including biodistribution, toxicology and tumorigenicity, was performed, as would be expected for any conventional therapy.

The manufacture of DA01 starts with well characterized hPSCs manufactured under cGMP conditions. The protocol uses small molecules and proteins as raw materials (aka ancillary reagents) in the differentiation process, and these were performance-tested by the researchers. However, using reagents intended for standard research (i.e. research-use-only or RUO products) as ancillary reagents, may not always meet the safety and performance standards required to for a commercial a cell therapy product, even though they are not intended to be present in the final product

Ancillary Material Grade Small Molecules

Small molecules offer several advantages over bioactive proteins in manipulation of stem cells for the manufacture of cell therapies. Small molecules are synthetically manufactured, so theoretically offer low batch-to-batch variability and high purity. It is important to work with chemical manufacturers that fully implement a quality system (e.g., ISO9001:2005 or GMP) to ensure that batch consistency and high purity are delivered. This will translate to consistent and reproducible activity. Additionally, small molecules are cell permeant and have rapid and reversible effects (see Five Reasons to use Small Molecules in Stem Cell Research for more information).

The recently launched Ancillary Material Grade (AM Grade) Small Molecules range from Bio-Techne offer an enhanced quality alternative to RUO reagents and are suited for use in the manufacture of cell therapy products. These small molecules undergo enhanced QC testing and a more detailed Quality Assurance (QA) review, including traceability of starting materials, and are subjected to an animal-free manufacturing process. The final products are also bioburden and endotoxin tested (see Figure 1). These additional quality checks assure the suitability of these products for use as ancillary reagents in the manufacture of cell therapies for regenerative medicine. Although RUO products are suitable for the early stages of cell therapy protocol development, switching to enhanced quality materials early for the manufacturing and testing phases avoids making costly changes later on. The closer a cell therapy approaches to the clinic, the more expensive and difficult it becomes to alter reagents and components.

Ancillary Material Grade Quality Attributes

Figure 1: A comparison of standard catalog (RUO) with Ancillary Material Grade products. Each block represents a stage in the manufacturing process, control measure or guidelines followed to assure the quality of the final product; the size of the bar represents the relative time expended.

Key small molecules for cell therapies, such as CHIR and Y-27632, are available as GMP small molecules. Bio-Techne GMP proteins are also available and offer further manufacturing controls. GMP standards are designed to provide guidelines around product identity, purity and quality, and compliant ancillary reagents will ensure end-product compliance. AM Grade Small Molecules offer an alternative when a GMP version is not achievable and provide a practical solution to ensure the best quality product is used in the cell therapy manufacture.