NCE Year 3

Project Description and Rationale:
Amyotrophic Lateral Sclerosis (ALS) is the most common adult motor neuron disease, affecting 30,000 people in the US and the typical age of onset is in the mid-50s or slightly younger. ALS is a degenerative neural disease in which the damage and death of neurons results in progressive loss of the body’s functions until death, which is usually in 3-5 years of diagnosis. Current ALS treatments are primarily supportive, and providing excellent clinical care is essential for patients with ALS; however, there is an urgent need for treatments that significantly change the disease course. The only FDA approved, disease-specific medication for treatment of ALS is Rilutek (riluzole); which demonstrated only a modest effect on survival (up to 3 mo.) in clinical trials.
The ALS Disease Team/Early Translational project is focused on developing an ALS therapy based on human embryonic stem cell (ESC) derived neural stem cells (NSC) and/or astrocyte precursor cells transplanted into the ventral horn of the spinal cord. Several lines of evidence strongly support the approach of transplanting cells that exhibit the capacity to migrate, proliferate and mature into normal astrocytes and provide a neuroprotection for motor neurons to reduce/prevent neural damage and disease progression.

Year 3 (6 month) Progress Summary:
NSCs generated with clonal enrichment from ESI-017 ESCs have a similar capacity to stably expand in vitro; and to survive, migrate and differentiate into neuronal and astrocytic cells in vivo without generating teratomas or other unwanted tissue formations when implanted into nude or SOD1 ALS rats. UCSF4 NSCs were originally selected as the developmental candidate, however, we changed to ESI-017 NSCs because: 1) UC Davis found ESI-017 NSC generation relatively easy but were unable to produce UCSF4 NSCs even with several method modifications; and 2) histology suggests that ESI-017 NSCs produce mature astrocytes earlier than UCSF4 NSCs.
ESI-017 NSCs generated at UC Davis under research conditions with predominantly GMP compatible reagents were implanted into athymic rats in order to compare the in vivo fate to NSCs generated at UCSD. Animals were perfused 2 months and 6 months post implant. Histology showed graft survival and differentiation of implanted ESI-017 NSCs generated at UCSD and UC Davis are similar, and further confirm successful transfer of NSC production methods the UC Davis.

The aggressive disease presentation of the current SOD1 rat ALS model results in a very short 2-month treatment window which exceeds the length of time for the migration, expansion, differentiation and maturation of sufficient astrocytes to provide a neural protective effect; and the variability of α-motor neuron counts precludes the use of this animal model demonstration of efficacy or proof-of-concept. We discussed these issues during a “pre-pre-IND” call with CBER/OCTGT/FDA on September 9, 2014. Prior to the call, Mercedes Serabian, Chief, Pharmacology/Toxicology Branch provided informal general comments which included examples of POC study endpoints were provided (e.g. motor neuron counts, levels of glutamate transport, electrophysiology/neurophysiology, etc.). During the call, discussion regarding glutamate excitotoxicity in ALS, and demonstrating that our heNSCs (or glial progeny) have the capacity to preserve/replace lost glutamate transporter activity in a model of ALS was “on the table” as potentially acceptable demonstration of POC, and we should have another pre-pre-IND call when we have such data.

In order to generate POC data for further discussion with the FDA we preformed additional histology on existing spinal cord tissue and initiated a collaboration with Don Cleveland’s and Brian Kaspar’s labs to perform in vitro co-culture experiments using ESI-017 NSCs generated at UCSD.

Histology: additional histological evaluation of spinal cord tissue from ESI-017 implanted nude and SOD1 rats demonstrated GLAST expression in grafted human astrocytes, suggesting active glutamate buffering activity.

In vitro co-culture experiments: ESI-017 NSCs generated at UCSD were shipped to Brian Kaspar’s lab where they were expanded and differentiated into astrocytes using their published methods. Human astrocytes were co-cultured with GFP positive motor neurons (MN) and at various time during culture, images were recorded and processed for survival cell counts and neurite length measurements. After 5 days of co-culture, astrocytes generated from ESI-017 NSCs provided motor neuron support similar to that provided by the normal control astrocytes whereas astrocytes derived from NSCs isolated post mortem from spinal cord tissue of patients with either familial ALS (FALS) or sporadic ALS (SALS) were toxic to motor neurons in co-culture. This co-culture experiment was repeated with similar findings (data analysis is in progress).
Collaboration with the Kaspar lab will continue beyond the end of this award.