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Translational/Regenerative Therapy Approach
Disease Team CIRM Grant
Progenitor Cells Secreting GDNF for the Treatment of ALS
Amyotrophic lateral sclerosis (ALS or Lou Gehrig’s disease) is a devastating and lethal disease resulting in the degeneration of neurons located in the brain and spinal cord responsible for controlling muscle function. Progression from early muscle twitches to complete paralysis and death usually happens within four years. There is currently no cure for ALS and only one approved therapeutic agent, Riluzole, which has been shown to minimally slow the progress of the disease.
The CIRM disease team project aims to use a powerful combined neural progenitor cell and growth factor approach to treat patients with amyotrophic lateral sclerosis. Human neural progenitor cells found early in brain development can be isolated and expanded in culture to large banks of billions of cells. When transplanted into animal models of ALS, they have been shown to mature into support cells for dying motor neurons called astrocytes. In other studies, growth factors such as glial cell line-derived growth factor (or GDNF) have been shown to protect motor neurons from damage in a number of different animal models, including ALS. However, delivering GDNF to the spinal cord has been almost impossible, as it does not cross from the blood to the tissue of the spinal cord. The idea behind the current proposal is to modify human neural progenitor cells to produce GDNF and then transplant these cells into patients. There they act as "Trojan horses," arriving at sick motor neurons and delivering the drug exactly where it is needed. A number of advances in human neural progenitor cell biology, along with new surgical approaches, have allowed us to create this disease team approach.
Human neural progenitor cells (hNPCs) can be expanded in vitro as spheres, genetically modified, and survive following transplantation into the nervous system of animal models.
A) Representative phase image of human neural progenitors (hNPC) grown as spheres in EGF-supplemented media. B-C) Representative immunofluorescence for hNPC markers nestin (B), GFAP and Tuj1(C). D)Representative image of hNPC genetically modified to produce the powerful growth factor GDNF (green). E) In vivo image of hNPCs (green) following transplantation into the rat spinal cord.
A) Representative picture of ChAT (green-motor neurons) and SC121 (red-human specific) markers in animals transplanted with CNS010-hNPCs secreting GDNF (10X). Note the increased number of motor neurons ipsilateral to the graft site (left side of the spinal cord). B) immunofluorescence for ChAT and SC121 showing the close proximity of hNPCs to motor neurons (20X). C) Immunohistochemistry for GDNF; arrows indicate GDNF labeled motor neurons, dashed line represent the separation between grey and white matter.
The focus of the proposal is to perform essential preclinical studies in relevant preclinical animal models to establish optimal doses and safe procedures for translating this progenitor cell and growth factor therapy into human patients. The Phase 1/2a clinical study will inject the cells into one side of the lumbar spinal cord (that supplies the legs with neural impulses) of 12 ALS patients from the state of California. The progression in the treated leg versus the non-treated leg will be compared to see if the cells slow down progression of the disease. This is the first time a combined progenitor cell and growth factor treatment has been explored for patients with ALS.
CIRM Disease Team II: DR2A-05320
Genevieve Gowing, PhD; Brandon Shelley; Pablo Avalos, MD; Jessica Latter; Maximus Chen; Amanda Hurley; Renee Paradis; Kevin Staggenborg; Leslie Garcia; Jessica Zelaya; and Jacalyn McHugh, MS
Nichols NL, Gowing G, Satriotomo I, Nashold LJ, Dale EA, Suzuki M, Avalos P, Mulcrone PL, McHugh J, Svendsen CN, Mitchell GS. Intermittent hypoxia and stem cell implants preserve breathing capacity in a rodent model of ALS. Am J Respir Crit Care Med. 2012 Dec 6. [Epub ahead of print] PMID:23220913.
Suzuki M, McHugh J, Tork C, Shelley B, Klein SM, Aebischer P, Svendsen CN. GDNF secreting human neural progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS.PLoS One.2007;2(8):e689.
Klein SM, Behrstock S, McHugh J, Hoffmann K, Wallace K, Suzuki M, Aebischer P, Svendsen CN. GDNF delivery using human neural progenitor cells in a rat model of ALS. Hum Gene Ther. 2005;16(4):509-21.
Upper Motor Neuron Degeneration in ALS
One aim of this research is to gain a better understanding of the origin of ALS. Conflicting reports exist in support of either a distal (muscle) or upper/lower motor neuron (cortical/spinal) origin of disease. By analyzing tissue from cortical motor neurons, spinal motor neurons,ventral root motor axons and muscle tissue at various stages of disease (pre-/non-symptomatic, early-symptomatic, symptomatic and endpoint), we are investigating where degeneration first occurs in the SOD1G93A rat model of ALS.This is important for developing appropriate future therapies targeting the early stages of disease in order to prevent or slow disease progression and extend survival of those affected by the disease.
In a second aim, in an effort to protect motor neurons in the brain of our rat model of ALS, we will transplant neural progenitor cells (NPCs) modified to secrete growth factors into the motor cortex.
Gretchen Miller, PhD
Labeling of upper motor
Combining Muscle-Derived GDNF Delivery and Spinal Stem Cell Transplantation to Promote Motor Neuron Survival and Function in an ALS Rat Model
Therapeutic strategies for effective treatment in ALS will likely require the combination of several therapeutic approaches. Using various approaches, the first goal of this study is to confirm the overt neuroprotective effect of glial cell line-derived neurotrophic factor (GDNF) on motor neurons survival and function in the SOD1G93A rat model of ALS. Secondly, we propose combining the viral delivery of the growth factor glial cell line-derived neurotrophic factor (GDNF) to the muscles of SOD1G93A rats with the spinal transplantation of human neural progenitors/stem cells (hNPCs) secreting GDNF. Our hope is to generate a combination of treatments with significant beneficial additive effects that can be translated to clinical trial. Moreover, the identification of a successful combination will lead to further studies by our group to identify strategies that would further enhance the therapeutic effect of our approach.
Genevieve Gowing, PhD; Gretchen Miller, PhD; Jessica Latter; Maximus Chen; Renee Paradis; Kevin Staggenborg; Leslie Garcia
Suzuki M, McHugh J, Tork C, Shelley B, Hayes A, Bellantuono I, Aebischer P, Svendsen CN. Direct muscle delivery of GDNF with human mesenchymal stem cells improves motor neuron survival and function in a rat model of familial ALS. Mol Ther. 2008;16(12):2002-10.
Disease modeling using induced pluripotent stem (iPS) cells:
iPS colonies labeled with markers of pluripotency (left) alkaline phosphatase and
Genetic psychomotor retardation
Mutations in the X-linked monocarboxylatetransporter 8 (MCT8) gene are the cause for the
Allan-Herndon-Dudley syndrome (AHDS). AHDS is characterized by a severe form of psychomotor retardation that is accompanied by abnormal thyroid hormone (TH) levels in the blood serum. MCT8 plays a role in the transport of thyroid hormones (THs) across cell membranes into the nervous system. Although the endocrinological phenotype of AHDS is well characterized, the mechanisms underlying MCT8 deficiencies are poorly understood. Moreover, there is currently very little known about the pathophysiological deficits of AHDS patients. The recent emergence of technologies for the generation of induced pluripotent stem (iPS) cells from mature human cells provides a new, unprecedented platform for studying genetic disorders in humans. We have generated and characterized iPS cells derived from AHDS patients and controls. Using established protocols, these iPS cells are differentiated into various neural lineages. Following differentiation, these cells are being examined for both a neurological and an endocrinological phenotype. Further, these experiments will serve as a platform to design molecule screens for the treatment of AHDS.
Gad Vatine, PhD; Soshana Svendsen, PhD; Dhruv Sareen, PhD
HD iPS cells were differentiated
Huntington’s disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. In collaboration with the RMI iPSC Core and the HD Consortium, we are generating and characterizing iPSC lines from HD patient cells. We have reported CAG-expansion-associated phenotypes, such as vulnerability to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. We are currently continuing our studies on the HD iPSCs to elucidate and validate HD-related phenotypes to model disease in vitro and in vivo to gain further mechanistic insights into HD and explore novel drug targets for this devastating disorder.
HD Foundation Fellowship and NIH U24 grant.
Virginia Mattis, PhD; Colton Tom
The Huntington’s Disease iPS Consortium.* Induced pluripotent stem cells from patients with Huntington’s disease show CAG repeat-associated phenotypes. Cell Stem Cell. 2012;11:264-278. (*Svendsen CN corresponding author)
Parkinson’s disease (PD) is a neurodegenerative disorder primarily characterized by a loss of dopamine neurons, but which also leads to many other pathological changes. While mainly sporadic, there are familial forms of PD that involve missense mutations, duplication, or triplication of the a-synuclein (a-SYN) gene. Based on such genetic linkage, our group previously reported that over-expression of mutant a-SYN using lentiviral infection in dopaminergic (DA) neurons differentiated from human embryonic stem cells (hESCs) can recapitulate some aspects of the PD. Interestingly, we observed that the mutant form of a-SYN localizes more in the nucleus than the wildtype form of a-SYN does. This observation led us to address whether nuclear localized a-SYN is specifically toxic to DA neurons or not. To this end, we generated lentiviral vector of a-SYN with/without nuclear localization signal (NLS). After infection in DA neurons differentiated from hESCs in vitro, we compared the temporal effects of a-SYN with NLS to one without NLS in DA neurons by counting co-labeled cells of both tyrosine hydroxylase (TH) and a-SYN. There was no statistically significant difference between them, which suggests that nuclear localized a-SYN is unlikely to be specifically toxic to DA neurons. This study helps us understand the pathologic mechanism of familial PD.
Howon Kim, PhD; Soshana Svendsen, PhD
Schneider BL, Seehus CR, Capowski EE, Aebischer P, Zhang SC, Svendsen CN. Over-expression of alpha-synuclein in human neural progenitors leads to specific changes in fate and differentiation. Hum Mol Genet. 2007;16(6):651-66.
Spinal Muscular Ahy
Spinal muscular atrophy (SMA) is one of the most common lethal genetic diseases in children. One in thirty-five people carry a mutation in a gene called survival of motor neurons 1 (SMN1), which is responsible for this disease. If two carriers have children together they have a one in four chance of having a child with SMA. Children with Type I SMA seem fine until around six months of age, at which time they begin to show lack of muscular development and slowly develop a “floppy” syndrome over the next six months. Following this period, SMA children become less able to move and are eventually paralyzed by the disease by three years of age or earlier. We know that this mutation causes the death of motor neurons—which are important for making muscle cells work. Interestingly, there is a second gene which can lessen the severity of the disease process (SMN2). Children with more copies of this modifying gene have less severe symptoms and can live for longer periods of time (designated Type II, III, and IV, and living longer periods respectively). There is no therapy for SMA at the current time. One of the roadblocks is that there are no human models for this disorder, as it is very difficult to make the motor neurons in the laboratory that die in the disease. The researchers in the current proposal have recently created pluripotent stem cells from a patient with Type I SMA (the most severe) and shown that motor neurons grown out from the pluripotent stem cells also die in the culture dish just as they do in children. This is an important model for SMA. The proposed research takes this model of SMA and extends it to Type II and Type III children in order to have a wider range of disease severity in the culture dish (Type IV is very rare and difficult to obtain samples). The research then develops new technologies to produce very large numbers of motor neurons and perform large-scale analysis of their survival profiles. Finally, it will explore whether novel compounds can slow down the degeneration of motor neurons in this model, which should lead to the discovery of new drugs that may then be used to treat the disease.
Dhruv Sareen, PhD; Loren Ornelas
Gene Targeting and Editing
Gene targeting is a genetic manipulation technique that provides the method of marking cellular markers and studying loss or gain of function via homologous recombination. In human pluripotent stem cells (PSCs) and induced PSCs (iPSCs), the homologous recombination is relatively hard to achieve compared to mouse PSCs, due to slower rate of proliferation and little later stage of PSCs. To increase efficiency of homologous recombination, we are trying to apply helper-dependent adenovirus for higher transfer of ectopic genes into cells, and TALEN for induction of double strand breakage. These techniques are being used to mark the final differentiated cells from each disease-modeled iPSC.
Howon Kim, PhD; Seigo Hatada, PhD
Sareen D, Ebert AD, Heins BM, McGivern JV, Ornelas L, Svendsen CN. Inhibition of apoptosis blocks human motor neuron cell death in a stem cell model of spinal muscular atrophy. PLOS One. 2012;7(6):e39113.
Ebert AD, Yu J, Rose FF Jr, Mattis VB, Lorson CL, Thomson JA, Svendsen CN. Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature. 2008;457(7227):277-80.