Year 2

Considerable progress was made on transitioning cells and cell production methods from research-scale to translational/clinical scale, including initial cell production in a GMP facility with GMP compatible production methods. Additionally, extensive characterization of the amyotrophic lateral sclerosis (ALS) disease animal model was completed and cells were evaluated for potential efficacy in this ALS disease animal model. These activities are key for continued progress in cellular therapy development, which is a multi-stage process that requires increasing focus on the details of the methods, stringent requirements for reagents/materials, greater scale, and more thorough product characterization during the transition to an approved cellular therapy.

Specifically, we made significant progress in three major areas:

First, we found evidence for efficacy using neural stem cells made at Life Technologies. In brief, during Year 1, the rat ALS disease model was shown to be a more aggressive disease model with an earlier disease onset and more rapid progression to end-stage and death than the model that had been used in previous studies. During Year 2, this more aggressive ALS disease model was further characterized with the identification of a reliable marker of disease onset, and demonstration that alpha motor neuron sparing by implanted cells could be detected and measured even, despite the aggressive nature of disease progression in this rat model.

We found that H9 NSCs produced by Life Technologies, when implanted into the rat ALS disease model, survived, migrated extensively into the area where alpha motor neurons are located, differentiated into cells that appear to be astrocytes, and provided a protective effect for the alpha motor neurons. This protective effect was determined by comparing the survival of alpha motor neurons on the side of the rat spinal cord where NSCs were implanted with the side of the spinal cord that did not have cells implanted. The side of the spinal cord where the NSCs were implanted showed approximately 10% more surviving alpha motor neurons than the matching side of the spinal cord that did not have cells implanted.

Second, cells from the various production methods were subjected to gene sequencing as part of the development of molecular characterization methods. This sequencing information was critical to identify whether cells produced by various methods were typical for the cell type, or exhibited qualities that indicated they were not optimal cell populations. These methods will be used to identify optimal markers for characterizing cell populations as part of current cell production development and for future quality control assays.

Third, during Year 2, Life Technologies further developed their pilot-scale embryonic stem cell (ESC) growth and differentiation methods to be more easily adaptable to cell production under Good Manufacturing Practices (GMP). This involved increasing the scale of cell production, and where possible, substituting reagent grade reagents and materials with reagents and materials that would be required or preferred for producing a cell therapy for use in humans (produced GMP, non-animal origin, well characterized). These conditions are not ideal for many ESC lines, and in Year 1, only one (H9) of the 4 starting ESC lines was successfully adapted to these culture conditions, however, 3 additional ESC lines were acquired to increase the number of potential clinical ESC candidate cell lines. One of these ESC lines (UCSFB7 from the University of California, San Francisco) was successfully adapted to the pilot ESC culture conditions, and resulted in the production of NSCs, and with AP production in progress. Because the research version of ESC line H9 has been used to successfully produce NSCs at Life Technologies, agreements are in progress for City of Hope for NSC cell production using the H9 ESCs, that have been banked under GMP conditions at City of Hope. In addition, pilot-scale cell production was initiated earlier than originally planned at the University of California, Davis GMP facility. The plan is to produce NSCs and APs under conditions that UC Davis has found to be successful in the past, and transition these methods to GMP compliance. To date, UC Davis has produced ESCs from 3 ESC lines [UCSF4, UCSF4.2 (a.k.a. UCSFB6) and UCSF4.3 (a.k.a. UCSFB7] and has produced NSCs from ESC line UCSF4. The UCSF4 NSCs are scheduled to be shipped to UCSD for testing in the ALS disease animal model in early June, 2012, and NSC production from ESC lines UCSF4.2 and UCSF4.3 is expected to begin in late June 2012.