Developing a good manufacturing practice (GMP) qualified source of hESC-derived DA neurons

Isabelle Rivière, PhD
Direction, Cell Therapy and Cell Engineering Facility
Memorial Sloan Kettering

Mark Tomishima, PhD
Director, Sloan Kettering Institute Stem Cell Facility
Associate Member, Center for Stem Cell Biology
Memorial Sloan Kettering

Any new therapy has to meet several requirements to be considered by the US Food and Drug Administration (FDA). These regulations are designed to protect patients, doctors, and hospitals. They require researchers to have the facilities and reagents as well as the protocols to produce a reliable and safe product.

This project focuses on the identification of cells and reagents that will allow for the production of clinical-grade DA neurons under good manufacturing practice (GMP) conditions. Under the leadership of Isabelle Rivière and Mark Tomishima, whose expertise encompasses translational research and stem cell biology, respectively, the group will identify the right pluripotent stem cell — one that can fulfill the stringent requirements for cellular therapies by the FDA — and source media and other reagents that are GMP compliant. In addition, they will establish production standards for a large-scale bank of frozen (cryopreserved) DA neurons to be used in the transplantation studies.

The team will also provide materials to advance studies performed by the other groups of the consortium.

Transplantation and behavioral studies in murine hosts

Viviane Tabar, MD
Member, Center for Stem Cell Biology
Member, Center for Cell Engineering
Memorial Sloan Kettering

Experiments can be carried out in vitro in a cell culture dish or in vivo in living organisms. These two approaches are neither redundant nor competing, but rather act in concert to better our understanding of the physiology and biology of hESC-derived DA neurons and Parkinson’s disease. Neurodegenerative diseases are often characterized by changes in behavior and changes in functions of daily life — both virtually impossible to model in a laboratory cell culture dish.

Under the guidance of Viviane Tabar, the team will develop standard operating procedures that will allow us to test the impact of transplanted, hESC-derived DA neurons on the health and behavior of parkinsonian animals. Not only will we gain insights into the safety and function of these cells, we will also learn about potential variability of the preparations. Furthermore, these models will be used to evaluate additional projects — “Cell purification and enrichment of A9 type DA neurons” and “Use of engineered cell surface polysialic acid (PSA) to promote in vivo integration and fiber outgrowth of grafted DA neuron.”

Transplantation in MPTP-lesioned rhesus monkeys

Jeffrey H. Kordower, PhD
Rush University

Dustin Wakeman
Rush University

Studies in rodents have allowed us to gain valuable insights into the pathology and biology of almost all human biology and disease biology. However, while making a fine model system, mice are exactly that: a model. Sometimes a positive effect in mice isn’t recapitulated later in human trials, or unpredicted side effects and off-target effects emerge only in clinical trials. Ultimately safety and function are best evaluated in primates before therapies find their way into the clinic.

Jeffrey H. Kordower brings years of experience in cellular therapies for neurodegenerative diseases and behavioral studies to the consortium. Dustin Wakeman is experienced in novel techniques for maintaining fetal hNSCs and the standardization of stereotaxic injection of hNSC in a nonhuman primate model of PD. He is an investigator on several projects focusing on preclinical testing of dopamine neurons derived from pluripotent cells, both human embryonic stem cells (HESC) and patient-derived induced pluripotent stem cells (iPSC), in the MPTP primate model of PD.

The Kordower laboratory will evaluate the DA neuron product produced under GMP conditions, perform large-animal studies and behavioral experiments, and process tissues for later analysis.

Cell purification and enrichment of A9 type DA neurons

Lorenz Studer, MD
Director, Center for Stem Cell Biology
Member, Center for Cell Engineering
Memorial Sloan Kettering

Fetal tissue transplantation studies for Parkinson’s disease have had mixed outcomes in double-blinded clinical trials. One possible explanation for this is the heterogeneity of the transplant (graft). A mixed population may be desired in some cases, but the more knowledge about the graft composition we have, the more reliable the product should be.

For this project, Lorenz Studer’s team uses directed differentiation of hESCs, which allows for the routine production of large quantities of DA neurons. Although the cultures are already largely positive for DA markers, further purification especially of A9 DA neurons may be desirable. A9 DA neurons are the specific cell type lost in Parkinson’s disease. Using monoclonal antibodies against cell surface markers specifically expressed on A9 DA neurons, this project is targeted at the specific isolation of transplantable A9 DA neurons.

Use of engineered cell surface polysialic acid (PSA) to promote in vivo integration and fiber outgrowth of grafted DA neuron

Urs Rutishauser, PhD
Member, Center for Stem Cell Biology
Memorial Sloan Kettering

Abderrahman El Maarouf, PhD
Research Fellow
Memorial Sloan Kettering

Billions of signals are transmitted between different parts of the brain. These signals coordinate basic functions of human life and are the basis of our cognitive function. If cells are transplanted into this complex network, it is essential that they form the right connections.

Neurons — the cells able to make these connections — send out extensions, termed axons, to establish this grid. This process of axon outgrowth and increased neuronal plasticity is facilitated polysialic acid (PSA), which is added to proteins on the surface of neurons.

In this project we interrogate the ability of polysialyltransferase (PST) to promote the outgrowth of cellular processes that may be essential for repair of the injured adult nervous system.

Establishing hESC-derived DA neuron source with inducible safety switches

Michel Sadelain, MD, PhD
Director, Center for Cell Engineering
Executive Committee, Center for Stem Cell Biology
Memorial Sloan Kettering

Embryonic stem cells (ESCs) are very powerful cells, capable of generating every cell type of the human body. Our understanding of how that is achieved is growing daily. Herein lies the challenge.

We need to ensure that the cells transplanted are only of the dopaminergic cell type and free of pluripotent stem cells. While our protocols are highly efficient, and additional purification steps are taken, we are testing an additional safety strategy. By introducing a “safety switch” into the transplanted cells we could, in theory, specifically eliminate the grafted cells using a small drug-like compound.

For this project we are testing various genomic locations in the ESC line for the introduction of one of two suicide genes with different mechanisms of action. If these experiments are promising, the final product will contain such safety switches.

Clinical Trial Development Team

Claire Henchcliffe, MD
Weill Cornell Medical College

Flint Beal, MD
Weil Cornell Medical College

While scientists in the lab are busy finding the right conditions to make the best possible transplant product, our team is active in designing the clinical trial.

This task is not trivial. Prior to the first transplant, we need to identify the most suitable patients, carefully weighing the risks and benefits for this group.

We also need to consider the best strategy to evaluate the outcome of the trial. How can we measure improvement? How can we ensure safety for the patient and the clinician? How long will it take to see an improvement and when will we know how long the therapy will last?

All these questions and more are discussed by our team of experts, encompassing clinicians, physician-scientists, ethicists, trial experts, and statisticians.