Stem Cell-Derived Astrocyte Precursor Transplants in Amyotrophic Lateral Sclerosis

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 Food and Drug Administration approved, disease-specific medication for treatment of ALS is Rilutek (riluzole); which demonstrated only a modest effect on survival (up to 3 months) 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 healthy astrocytes which can provide a neuroprotective effect for motor neurons and reduce or prevent neural damage and disease progression in ALS. Strong evidence has been generated from extensive studies in culture dishes and in animal models to support the concept that providing normal astrocytes in the proximity of α-motor neurons can protect them from neural damage. Project Plan and Progress: Multiple ESC lines were acquired in 2 rounds based on early and later availability. The first round of ESCs included ESCs from City of Hope (GMP H9) and the University of California, San Francisco (UCSF4). The second round included ESCs from the University of California, San Francisco [UCSFB6 (aka UCSF4.2) and UCSFB7 (aka UCSF4.3)] and from BioTime (ESI-017). These ESC lines were tested for their ability to survive and expand under conditions required for producing a cellular therapy (FDA GMP-like and GTP compliant conditions). From these ESC lines, NSCs were generated, expanded and characterized to determine their ability to produce stable and consistent populations of NSCs under conditions required for producing a cellular therapy. For the first round of cell lines, both UCSF4 and H9 were successfully induced to produce NSCs, which were mechanically enriched, expanded and implanted into immunodeficient rats and a rat model of ALS (SOD1G93A). For this small-scale in vivo screen, implanted UCSF4 and H9 NSCs survived, migrated and differentiated into neurons and astrocytic cells in 3-5 weeks, without producing tumors or other unwanted structures. NSCs from both UCSF4 and H9 performed similarly in culture and in vivo, thus the decision to use UCSF4 in the larger-scale in vivo studies for safety (implant into immunodeficient rats) and efficacy/proof of concept (SOD1G93A ALS model rats) was weighted by the difficulties obtaining H9 for future studies for a therapeutic product. These larger-scale studies began August 2013 (earlier than projected), with expected completion in February 2014. For the second round of ESC lines (UCSFB6, UCSFB7 and BioTime ESI-017), UCSFB6 and UCSFB7 ESCs expanded well, while ESI-017 expansion was less robust. Because UCSFB6 and UCSFB7 ESCs are from the same blastomere, we decided to continue to NSC production with only UCSFB7, keeping UCSFB6 in reserve as a back-up. UCSFB7 ESCs were successfully induced to produce NSCs, which were mechanically enriched, expanded and implanted into immunodeficient rats and a rat model of ALS (SOD1G93A). The results from these studies are pending (some animals are still in-life), but early histology suggests the cell survival is similar to UCSF4 and H9. A second round of large-scale in vivo studies is planned to start January 2014 to evaluate this NSC line. By September 2014, the “best” NSC line will be selected as a therapeutic candidate for definitive pre-clinical studies and entry into clinical trials. ESC production under GMP-like condition has been completed at the UC Davis GMP facility. UC Davis generated the first batch of NSCs, which were not sufficiently homogeneous for successful expansion beyond approximately passage 10. This prompted UCSD to investigate multiple enrichment strategies, which were tested on multiple cell lines to ensure method reproducibility. A mechanical enrichment method reproducibly resulted in more homogeneous NSC cultures, capable of expansion for 20 – 30 passages, or more. The NSC generation and enrichment methods are currently being transferred to UC Davis and the UCSD scientist who developed the methods will work side-by-side with the UC Davis GMP production team to ensure successful method transfer to the GMP facility. UCSF4 NSCs are also in use in a CIRM supported early translation study for spinal cord injury.

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 Food and Drug Administration approved, disease-specific medication for treatment of ALS is Rilutek (riluzole); which demonstrated only a modest effect on survival (up to 3 months) 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 healthy astrocytes which can provide a neuroprotective effect for motor neurons and reduce or prevent neural damage and disease progression in ALS. Year 2 Progress Summary: The longer-term, larger-scale in vivo safety and efficacy studies using the lines that showed the most promise during previous screening studies (UCSF4 and ESI-017 NSCs) have been completed. The safety studies were performed in immunodeficient rats to evaluate the survival, migration, differentiation, function and tumorigenicity of implanted NSCs at 3 weeks, 2 months and 6 months post implant. The efficacy studies were conducted in a transgenic SOD1G93A ALS rat model to evaluate safety and cell fate in the background of disease, as well as, to evaluate disease-modifying activity (e.g. neural protection/proof-of-concept) of the implanted NSCs. NOTE: A labeling error occurred during expansion and banking of the ESCs at UC Davis, and the cell line labeled as UCSFB7 (aka UCSF4.3) was determined by DNA fingerprinting to actually be ESI-017. Previous NSC generation, characterization and in vivo screening data was reported for cell line UCSFB7 (aka UCSF4.3), which was actually for ESI-017. Both UCSF4 and ESI-017 NSCs were deemed acceptable in 2 out of 3 of the minimal acceptance criteria: 1) Long-term survival in nude and SOD1G93A rats 2) No formation of tumors or other unwanted structures when implanted into nude or SOD rats. The third criterion: at least 10% greater α-motor neuron counts in cell-injected animals as compared to medium injected controls (or cell-injected side compared to the non-injected, or contralateral side) was not met due to a) variability of α-motor neuron counts and b) the aggressive nature of the current SOD1G93A rat ALS model and resulting 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 in all implanted animals. UCSF4 NSCs were originally selected as the developmental candidate, however, there are compelling reasons to reconsider ESI-017 NSCs: 1) UC Davis has found ESI-017 NSCs relatively easy to generate and is having difficulty generating UCSF4 NSCs; and 2) recent hisotological evaluations suggest that ESI-017 NSCs produce mature astrocytes earlier in vivo than UCSF4 NSCs. We are working with UC Davis on generation of UCSF4 NSCs and are quantifying astrocyte maturation histology (e.g. GFAP) to make a well-supported developmental candidate selection. In parallel, mRNA sequencing has been performed 1) on cells produced in the course of this project to identify potential markers predictive of in vivo fate, 2) on naïve SOD1G93A rats to explore markers of disease onset and progression that could potentially be used as surrogate markers of disease modulation in place of motor neuron counts, and 3) on NSCs implanted into nude and SOD1G93A rats to identify potential markers of long-term post-transplant NSC cell fate and host response.

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.