Amyotrophic lateral sclerosis (ALS), a lethal disease lacking effective treatments, is characterized by the loss of upper and lower motor neurons. 5-10% of ALS is familial, but the majority of ALS cases are sporadic with unknown causes. The lifetime risk is approximately 1 in 2000. This corresponds to ~30,000 affected individuals in the United States and ~5000 in the Collaborative Funding Partner country. There is currently only one FDA-approved compound, Rilutek, that extends lifespan by a maximum of three months. Although the causes of ALS are unknown and the presentation of the disease highly variable, common to all forms of ALS is the significant loss of motor neurons leading to muscle weakness, paralysis, respiratory failure and ultimately death. It is likely that many pathways are affected in the disease and focusing on a single pathway may have limited impact on survival. In addition, as ALS is diagnosed at a time that significant cell loss has occurred, an attempt to spare further cell loss would have significant impact on survival.
Several findings support the approach of glial (cells surrounding the motor neurons) transplants. Despite the relative selectivity of motor neuron cell death in ALS, published studies demonstrate that glial transporters critical for the appropriate balance of glutamate surrounding the motor neurons are affected both in animal models and in tissue from sporadic and familial ALS. The significance of non-neuronal cells in the disease process has been well characterized using SOD1 mouse models representing many of the key aspects of the human disease. In addition, transplantation using glial-restricted precursors (GRPs) that differentiate into astrocytes in SOD1 mutant rats has been shown to increase survival. Motor neurons have a process, the axon, up to a meter in length which connects the cell body to its target, the muscle. The ability to appropriately rewire and ensure functional connections after motor neuron replacement remains a daunting task with no evidence to date that this will be possible in humans. Therefore, we will focus on the development of an ALS therapy based on hES-derived astrocyte precursor cell transplants to prevent the progression of ALS.
Our proposed project will develop clinical grade stem-cell derived astrocyte precursor transplants for therapy in a prospective Phase I clinical trial. We will: 1) generate astrocyte precursors from three different sources of human embryonic stem cell (hESC) lines; 2) identify the hESC line and glial progenitor combination that has the best characteristics of minimal toxicity, best efficiency in generating astrocytes, and reducing disease phenotypes in vivo in a rat model of ALS; 3) manufacture the appropriate cells in a GMP facility required by the FDA; 4) work with our established clinical team to design a Phase I safety trial; and 5) submit an application for an invesitgational new drug (IND) within the next four years.
Statement of Benefit to California:
Amyotrophic lateral sclerosis (ALS; also known as Lou Gehrig's Disease) is a common and devastating adult motor neuron disease that afflicts many Californians. In the absence of a cure, or an effective treatment, the cost of caring for patients with ALS is substantial, and the consequences on friends and family members similarly takes a devastating toll. Our goal is to develop a safe and effective cell transplant therapy for ALS by starting with human embryonic stem cells. If successful, this advance will hopefully diminish the cost of caring for the many Californians with ALS, extend their useful lives, and improve their quality of life. In addition, the development of this type of therapeutic approach in California will serve as an important proof of principle and stimulate the formation of businesses that seek to develop these types of therapies in California with consequent economic benefit.
Year 1Project 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.