neural stem cells as a therapeutic approach for amyotrophic lateral sclerosis
TRANSCRIPT
Molecular Therapy vol. 21 no. 3 march 2013 503
© The American Society of Gene & Cell Therapy commentaries
transplanted might be explained by either a lower level of engraftment of the cells or re-duced function due to the SMA mutations, and further studies are necessary to distin-guish these effects.
The use of iPS cell–derived motor neurons has potential for therapeutic ap-proaches in SMA. Using a patient’s own cells reduces the risk of immune reactions, and such cells have the intrinsic potential to replace lost motor neurons and build new neuromuscular junctions. Improvements in transplantation of human fetal neural stem cells were recently achieved in a rat model of severe spinal cord injury. By embedding the cells into a fibrin matrix containing a growth-factor cocktail, Lu et al. demon-strated remarkable axon outgrowth over long distances and improved cell survival.11 In addition, new protocols have been devel-oped to improve the differentiation efficien-cy of motor neurons.12 These improvements have the potential to increase engraftment and muscle innervation by the transplanted motor neurons. For type I SMA, the pro-duction of iPS cell–derived cells remains a particular challenge because of the short window of opportunity to successfully treat these patients, due to the early loss of mo-tor neurons in this disorder. By the time SMA is diagnosed and skin biopsy samples for the reprogramming can be recovered, the patient would probably have lost many endogenous motor neurons. Therefore, later time points for transplantation of the motor neurons should be tested. Finally, motor neuron transplantations are also well suited to support gene therapy approaches for SMA. Delivery of adeno-associated vi-rus encoding normal SMN shortly after birth led to an essentially complete rescue of the disease phenotype, whereas treat-ment at later time points of the disease showed less benefit.13
In summary, the field of motor neu-ron transplantation raises hope for treating neurodegenerative disorders such as SMA. However, much additional research is nec-essary to identify the appropriate time win-dow for transplantation, the optimal type and number of cells, and the combination with the right growth factors and param-eters for optimal success. Therefore, studies to elucidate environmental influences and development of methods to improve mo-tor neuron survival and proper connections are imperative. The ultimate goal for motor
neuron replacement remains the integra-tion, growth, functional maturation, and innervation of muscle. Significant challeng-es must still be overcome, but with recent advances in the field we are moving closer toward this goal.
REFERENCES1. Parekh-Olmedo, H and Kmiec, EB (2007). Progress
and prospects: targeted gene alteration (TGA). Gene Ther 14: 1675–1680.
2. Corti, S, Nizzardo, M, Simone, C, Falcone, M, Nardini, M, Ronchi, D et al. (2012). Genetic correction of hu-man induced pluripotent stem cells from patients with spinal muscular atrophy. Sci Transl Med 4: 165ra162.
3. Perez-Pinera, P, Ousterout, DG and Gersbach, CA (2012). Advances in targeted genome editing. Curr Opin Chem Biol 16: 268–277.
4. Ebert, AD, Rose, FF Jr, Mattis, VB, Lorson, CL, Thom-son, JA and Svensen, CN (2009). Induced pluripotent stem cells from a spinal muscular atrophy patient. Nature 457: 277–280.
5. Gabanella, F, Butchbach, ME, Saieva, L, Carissimi, C, Burghes, AHM and Pellizzoni, L (2007). Ribonucleo-protein assembly defects correlate with spinal muscu-lar atrophy severity and preferentially affect a subset of spliceosomal snRNPs. PLoS ONE 2: e921.
6. Le, TT, Pham, LT, Butchbach, ME, Zhang, HL, Monani, UR, Coovert, DD et al. (2005). SMNDelta7, the major product of the centromeric survival motor neuron (SMN2) gene, extends survival in mice with spinal
muscular atrophy and associates with full-length SMN. Hum Mol Genet 14: 845–857.
7. Rossi, SL, Nistor, G, Wyatt, T, Yin, HZ, Poole, AJ, Weiss, JH et al. (2010). Histological and functional benefit following transplantation of motor neuron progenitors to the injured rat spinal cord. PLoS ONE 5: e11852.
8. Wyatt, TJ, Rossi, SL, Siegenthaler, MM, Frame, J, Robles, R, Nistor, G et al. (2011). Human motor neu-ron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss. Stem Cells Int 2011: 207230.
9. Corti, S, Nizzardo, M, Nardini, M, Donadoni, C, Salani, S, Ronchi, D et al. (2010). Embryonic stem cell–derived neural stem cells improve spinal muscular atrophy phenotype in mice. Brain 133(Pt 2): 465–481.
10. Corti, S, Nizzardo, M, Nardini, M, Donadoni, C, Salani, S, Ronchi, D et al. (2008). Neural stem cell transplantation can ameliorate the phenotype of a mouse model of spinal muscular atrophy. J Clin Invest 118: 3316–3330.
11. Lu, P, Wang, Y, Graham, L, McHale, K, Gao, M, Wu, D et al. (2012). Long-distance growth and connectivity of neural stem cells after severe spinal cord injury. Cell 150: 1264–1273.
12. Hester, ME, Murtha, MJ, Song, S, Rao, M, Miranda, CJ, Meyer, K et al. (2011). Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcrip-tion factor codes. Mol Ther 19: 1905–1912.
13. Foust, KD, Wang, X, McGovern, VL, Braun, L, Bevan, AK, Haidet, AM et al. (2010). Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol 28: 271–274.
1Center for Gene Therapy, The Research Insti-tute at Nationwide Children’s Hospital, Colum-bus, Ohio, USA; 2Biomedical Sciences Graduate Program, College of Medicine, The Ohio State University, Columbus, Ohio, USA; 3Department of Pediatrics and Neuroscience, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio, USACorrespondence: Brian K Kaspar, The Research Institute at Nationwide Children’s Hospital, The Ohio State University, 700 Children’s Drive, WA3022, Columbus, Ohio 43205, USA. E-mail: [email protected]
Neural Stem Cells as a Therapeutic Approach for Amyotrophic Lateral SclerosisLaura Ferraiuolo1, Ashley Frakes1,2 and Brian K Kaspar1,2,3
doi:10.1038/mt.2013.24
Proliferating neural stem cells (NSCs) were first identified in the late 1960s
in the adult rat brain1 as multipotent self-renewing stem cells, able to differentiate into neurons, astrocytes, and oligodendrocytes. NSC transplantation is being evaluated to treat traumatic brain or spinal cord injury
and neurodegenerative diseases. Amyo-trophic lateral sclerosis (ALS) is a fatal neuro degenerative disorder characterized by progressive loss of upper and lower motor neurons and chronic inflamma-tion leading to paralysis and death due to respiratory failure. In the December issue of Science Translational Medicine, Teng et al.2 reported successful use of transplanted, undifferen tiated multipotent NSCs to pro-long survival in a mouse model of ALS. They found that the experimental treat-ment is both safe and potentially beneficial to preserve neurons that have been spared by the disease at the time of treatment. The results further indicate the benefits of early intervention and suggest that targeting the spinal cord at different sites will protect both motor and respiratory function. The authors conclude that a combination of therapeutic
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approaches, from gene therapy to cell trans-plantation, targeting different pathogenic mechanisms of the disorder, is likely to lead to successful therapy.
ALS is largely sporadic in origin, with only about 10% of total cases having a family history of the disease. The genetic causes are known for about 50% of familial cases; the only risk factor clearly associated with the disease is aging.3 Mutations in the superox-ide dismutase 1 (SOD1) gene are one of the most common causes of familial ALS, and transgenic animals carrying the mutant gene recapitulate the pathophysiology of ALS. Although the main feature of the disease is motor-neuron death, in recent years it has become clear that the nonneuronal cells sur-rounding the vulnerable neurons play a cru-cial role in disease progression and represent a promising therapeutic target.4 However, drug therapies aiming at targeting single cell types and/or single pathogenic mechanisms have failed to provide a successful therapeu-tic outcome. The development of a cell-based therapy able to target multiple components of the affected system could be beneficial by both supporting dying neurons and improv-ing the milieu of the spinal cord by secreting neurotrophic and anti-inflammatory factors. Early studies in rodent models of ALS have shown that transplantation of human NSCs can transiently improve motor performance as well as moderately increase survival with-out major side effects.5,6
Teng et al. made use of a highly standard-ized protocol and common experimental material at four institutions, involving 11 independent, double-blinded studies, to evaluate the therapeutic effects of mouse and human NSC transplantation into the mutant SOD1 mouse model. In a first series of experiments, investigators transplanted nestin-positive, multipotent mouse NSCs into one site in the lumbar spinal cord of two groups of ALS mice: early-to-mid-symptomatic stages and late end stage. The single injection resulted in robust and extensive integration of donor cells in the lumbar cord, 99% of which were non-neuronal cells. Additionally, coordinated hind-limb function evaluated by the rotarod test was improved in the early-to-mid-symptomatic group and survival was 10 days longer than in the noninjected controls. The authors determined the mechanisms by which transplanted mouse and human NSCs were acting to delay host-neuron
degeneration by observing that NSCs se-creted increased levels of the trophic factors nerve-growth factor, brain-derived neuro-trophic factor, and glial cell line–derived growth factor (GDNF), all of which enhance motor-neuron survival.
Next, the authors sought to evaluate the therapeutic effects of human NSCs (hNSCs) and the effects of intervening at earlier time points. The injections of hNSCs were per-formed bilaterally at one site (L2) or multiple sites (C6, T10, L1, and L3) in the spinal cord of mutant SOD1 mice at 8–10 weeks of age. Although clinical signs are not yet evident at this age, histopathology reveals clear signs of neurodegeneration. After NSC injection at a single site (L2), motor performance im-proved and there was a mild increase in sur-vival; the multisite approach showed more encouraging results. The authors report a remarkable survival increase of 200 days or up to a full year in 40% and 20%, respectively, of animals injected with hNSCs at all four sites of the spinal cord during the presymptomatic stage of disease. Interestingly, the remaining 40% of mice injected with hNSCs showed a very mild increase in survival. These find-ings may be caused by variation in the level of hNSC engraftment achieved in segments of the spinal cord regulating vital functions such as respiration. Despite the survival variability, meta-analysis of these studies produced an estimated hazard ratio of 0.33, meaning that the risk of death of treated animals was 67% lower than in controls.
Interestingly, this large study confirmed that the beneficial effects of this therapeutic approach derived mainly from the ability of undifferentiated NSCs to secrete growth factors that protect spared motor neurons. Further studies are needed to identify the factors associated with transplants that re-sult in significant increases in survival. It will also be useful to assess survival of the trans-planted cells over time and to determine whether multiple injections at different stag-es could further improve survival or if the transplanted cells are negatively affected by the local disease environment. Recent stud-ies suggest that ALS might originate focally and then spread through the spinal cord in both sporadic and familial cases.7 More-over, this evidence has led to the hypothesis that misfolded SOD1 might be acting as a prion-like protein, propagating the disease from cell to cell.8 In this case, the beneficial effects of wild-type transplanted cells would
be transient, and they could also acquire toxic properties. Interestingly, Teng et al. found no improvement when NSCs were injected in late-symptomatic animals. As the authors suggest, this result could be due to the already extensive loss of motor neu-rons, and it would be interesting to investi-gate whether the cells implanted at later time points present a secretome and functional characteristics that are similar to that of the NSCs implanted at earlier stages or whether the more aggressive environment of the spi-nal cord at later stages of the disease inhibits survival and function of the transplants.
As Teng and colleagues suggest, NSCs have the potential to be a valuable thera-peutic vehicle because they are site-specific and become integrated into the parenchyma. However, engineering the NSCs to increase their migratory power within the spinal cord or to enhance their secretion of neu-rotrophic and anti-inflammatory factors might increase their efficacy. Human NSCs and mesenchymal stem cells engineered to secrete high levels of GDNF have already been shown to have beneficial effects in animal models of ALS.9 However, unilateral injection of engineered hNSCs in the spinal cord of ALS rats protected dying motor neu-rons but not their projection to muscle, with minimal impact on motor performance and survival.10 By contrast, injection of mesen-chymal stem cells secreting high levels of GDNF in the muscles of ALS rats protected the neuromuscular junction and signifi-cantly increased survival.11 This suggests that NSC injections in the spinal cord might lead to a better outcome if combined with other therapies aimed at preserving the neuro-muscular junction.
Two clinical trials are currently testing the safety and the effects of intra–spinal cord delivery of hNSCs in ALS patients. The first was initiated in 2010, and phase I is now complete.12 Twelve patients received either 5 unilateral or 5 bilateral (10 total) injec-tions into the lumbar spinal cord at doses of 100,000 cells per injection. This first trial was designed with the aim of evaluating safety and tolerability of the procedure in ALS pa-tients, and thus far there is no indication that the cells are toxic or injurious to the spinal cord. Although this phase of the trial was not aimed at evaluating efficacy, the authors report mild but encouraging improvements in terms of disease progression. However, as suggested by the study by Teng et al.,2 the aim
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of the next phase of this clinical trial will be targeting the cervical spinal cord to preserve respiratory function and increase patient survival. The other trial, which began in July 2012, is currently recruiting participants with the goal of evaluating safety as well as clinical outcomes (http://clinicaltrials.gov/ct2/show/NCT01640067?term=neural+stem+cells+and+als&rank=1).
In summary, stem cell transplantation for severe diseases such as ALS shows pre-clinical signs of modest improvements. The field continues to make advances in delivery of cells to the spinal cord as well as in under-standing the mechanisms for the beneficial effects seen in the rodent models, highlight-ing the importance of continued research and development in this area.
REFERENCES1. Altman, J and Das, GD (1965). Autoradiographic and
histological evidence of postnatal hippocampal neuro-genesis in rats. J Comp Neurol 124: 319–335.
2. Teng, YD, Benn, SC, Kalkanis, SN, Shefner, JM, Onario, RC, Cheng, B et al. (2012). Multimodal actions of neu-ral stem cells in a mouse model of ALS: a meta-analysis. Sci Transl Med 4: 165ra164.
3. Al-Chalabi, A, Jones, A, Troakes, C, King, A, Al-Sarraj, S and van den Berg, LH (2012). The genetics and
neuropathology of amyotrophic lateral sclerosis. Acta Neuropathol 124: 339–352.
4. Ilieva, H, Polymenidou, M and Cleveland, DW (2009). Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J Cell Biol 187: 761–772.
5. Xu, L, Yan, J, Chen, D, Welsh, AM, Hazel, T, Johe, K et al. (2006). Human neural stem cell grafts ameliorate motor neuron disease in SOD-1 transgenic rats. Transplanta-tion 82: 865–875.
6. Yan, J, Xu, L, Welsh, AM, Chen, D, Hazel, T, Johe, K et al. (2006). Combined immunosuppressive agents or CD4 antibodies prolong survival of human neural stem cell grafts and improve disease outcomes in amyotrophic lateral sclerosis transgenic mice. Stem Cells 24: 1976–1985.
7. Ravits, JM and La Spada, AR (2009). ALS motor pheno-type heterogeneity, focality, and spread: deconstructing motor neuron degeneration. Neurology 73: 805–811.
8. Grad, LI, Guest, WC, Yanai, A, Pokrishevsky, E, O’Neill, MA, Gibbs, E et al. (2011). Intermolecular transmission of superoxide dismutase 1 misfolding in living cells. Proc Natl Acad Sci USA 108: 16398–16403.
9. Kaspar, BK (2008). Mesenchymal stem cells as trojan horses for GDNF delivery in ALS. Mol Ther 16: 1905–1906.
10. Suzuki, M, McHugh, J, Tork, C, Shelley, B, Klein, SM, Aebischer, P et al. (2007). GDNF secreting human neu-ral progenitor cells protect dying motor neurons, but not their projection to muscle, in a rat model of familial ALS. PLoS One 2: e689.
11. Suzuki, M, McHugh, J, Tork, C, Shelley, B, Hayes, A, Bellantuono, I et al. (2008). 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 16: 2002–2010.
12. Glass, JD, Boulis, NM, Johe, K, Rutkove, SB, Federici, T, Polak, M et al. (2012). Lumbar intraspinal injection of neural stem cells in patients with amyotrophic lateral sclerosis: results of a phase I trial in 12 patients. Stem Cells 30: 1144–1151.
1Gene Therapy Center, University of Massachusetts Medical School, Worcester, Massachusetts, USA; 2Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USACorrespondence: Guangping Gao, Gene Therapy Center, 368 Plantation Street, AS6, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA. E-mail: [email protected]
Gene Therapy for Canavan’s Disease Takes a Step ForwardSeemin S Ahmed1,2 and Guangping Gao1,2
doi:10.1038/mt.2013.25
Canavan’s disease (CD) is a rare but dev-astating pediatric leukodystrophy that
causes progressive spongy neurodegen-eration and is invariably fatal in congenital form.1 The disease is associated with >54 loss-of-function mutations2–4 in the enzyme aspartoacylase (ASPA), leads to accumula-tion of the substrate N-acetyl aspartic acid
(NAA) in the brain, and is diagnosed via the presence of NAA aciduria.1 CD is char-acterized by dysmyelination, intramyelinic edema (leading to hydrocephalus), and extensive vacuolation of the central nervous system (CNS) white matter.5 Currently there is no established therapy that affects pro-gression of the disease, and survival is based primarily on improved general medical care. A previous gene therapy attempt us-ing liposome-encapsulated plasmid DNA6 had shown encouraging although transient decreases in local NAA concentrations in the treated brains, which prompted a gene therapy clinical protocol using recombi-nant AAV serotype 2 (rAAV2) in the hope of better dissemination of the vector and more sustainable NAA reductions.7 In a re-cent issue of Science Translational Medicine,
Leone et al.8 report long-term follow-up of 13 of the 28 patients enrolled in this trial, who received intracranial injections of first-generation rAAV vectors-based on serotype 2 nearly a decade ago.
The study evaluates the long-term safety, dosing parameters, and efficacy of the treat-ment. The findings suggest that widespread ASPA gene transfer throughout the entire CNS might be necessary for alleviating the extensive neuropathology and maximizing outcomes in patients with CD. Additionally, the findings underline the importance of early intervention, because improvements in younger patients appeared to be more pro-nounced than those in older patients. The study suggests that rAAV-mediated gene therapy is the most promising therapeutic modality for CD to date; it seems likely that gene therapy for CD and other inherited neurological diseases will advance rapidly in the near future as less invasive delivery methods and more efficient vectors for pan-CNS transductions are developed.9–11
The authors of the study had to deal with several challenges in addition to the rarity of the disease. Enrollment of age-matched children who showed similar trends in dis-ease progression was difficult because of the sheer variety of mutations that cause CD. Patients with similar phenotypes and com-plete lack of ASPA activity were enrolled and grouped into cohorts based on age. Although the levels of NAA were not fully normalized by the gene therapy, there was an encouraging trend toward reductions in NAA in the treated brains. In-depth assess-ment of individual brain regions showed a statistically significant improvement over untreated subjects in one of the four regions assessed. Unfortunately, the varied rates of disease progression and the small number of patients confounded interpretation of the results. Indeed, only when the authors had removed the oldest cohorts from some anal-yses were they able to identify statistically significant changes. Nevertheless, the trial is a step forward in rAAV gene therapy at-tempts, showing encouraging but marginal improvements in the most characteristic fea-ture of CD: NAA accumulation in the brain.
The authors studied atrophy of brain mass by serial magnetic resonance imaging followed by digital image processing to es-timate enlargement of ventricles, changes in morphology of anatomical landmarks, or decrease of white matter mass by