502 www.moleculartherapy.org vol. 21 no. 3 march 2013

© The American Society of Gene & Cell Therapycommentaries

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: Brian.Kaspar@NationwideChildrens.org

gene editing with oligonucleotides has clinical potential for approaches in which a selection of corrected cells is possible.3 iPS cell technology permits such selection and enables the differentiation of the newly cor-rected stem cells into almost any cell type, which naturally led to the idea of using a pa-tient’s own cells as an autologous source for transplantation. In the new study, Corti et al. generated iPS cell lines from two patients with type I SMA, a devastating disease lead-ing to the degeneration of motor neurons in children and death within the first two years of life. This disorder manifests through au-tosomal recessive inheritance and is caused by reduced levels of survival motor neuron (SMN) protein, which is encoded by either of two nearly identical, paralogous genes called SMN1 and SMN2. SMA patients lack a functional copy of SMN1 and therefore rely solely on SMN2 for the production of the protein. Unfortunately, SMN2 contains a point mutation in exon 7 that alters the splicing and leads to reduced production of the full-length protein.

Whereas the majority of iPS protocols rely on viral vectors, Corti et al. used a non-integrating-plasmid transfection method to generate their iPS lines. The two SMA iPS lines were then transfected with the point mutation–correcting oligonucleotide, and subclones were screened for permanent correction of the genotype. In about 4% of the subclones, one SMN2 allele was success-fully corrected, leading to increased protein production. The corrected iPS cells were then differentiated into spinal motor neu-rons, the cell type that is most prominently affected in this disease. Confirming a pre-vious study,4 SMA motor neurons showed reduced size and decreased survival. Importantly, this phenotype was reversed in motor neurons generated from the

Targeted gene alteration strategies (TGAs) make use of single-stranded

DNA oligonucleotides to correct disease-causing point mutations. The introduced oligonucleotides bind to their genomic target sequence and induce the cell’s in-trinsic DNA damage-repair system during replication, leading to permanent correc-tion of the mutation.1 TGA technology has become increasingly relevant to transla-tional medicine since the development of cell reprogramming methods to generate induced pluripotent stem (iPS) cells from fully differentiated somatic cells. Hundreds of iPS cell lines have been generated from patients suffering from a wide spectrum of diseases. In the December issue of Science Translational Medicine, Corti et al. reported the successful combination of iPS and TGA technology with therapeutic cell transplan-tation in a mouse model of spinal muscular atrophy (SMA).2

Unlike other strategies commonly used to alter genomic sequences, such as zinc-finger nucleases or transcription activa-tor–like effector nucleases, oligonucleotides are easy to design and manufacture, and they also exhibit less nonspecific binding to off-target sequences. Therefore, despite the low overall efficiency of this method,

oligonucleotide-corrected iPS lines. Corti et al. performed gene expression profiling and splicing analyses to evaluate differences in the transcriptome of the generated mo-tor neurons. They demonstrated that the expression profile of the corrected SMA motor neurons was more similar to that observed in cells generated from a healthy relative. In addition, changes in splicing of a variety of transcripts were observed, un-derscoring the important role of the SMN protein in the assembly of components of the spliceosome,5 which may lead to the identification of additional therapeutic targets. However, the iPS cell–derived motor neurons were immature when the gene expression profiling was performed. Therefore, genes involved in neuronal development and differentiation might be overrepresented.

Corti et al. next evaluated the engraft-ment potential of the human iPS cell–de-rived motor neurons in the standard mouse model of SMA.6 Transplanted motor neu-rons differentiated from human embry-onic stem (ES) cells have been shown to engraft and integrate into the spinal cord of SMND7 mice as well as in models of spinal cord injury and amyotrophic lateral scle-rosis.7,8 The human ES cell–derived motor neurons secreted various growth factors, thereby generating a beneficial neurotro-phic effect upon the remaining endogenous cells. The new study confirms similar be-havior of the iPS cell–derived motor neu-rons. Transplantation moderately increased survival of the SMND7 mice from ~14 days to ~24 days. Although a few human motor neurons seemed to grow axons and form neuromuscular junctions, the major effect was probably due to the secretion of neu-roprotective factors benefiting endogenous motor neurons. Indeed, the observed in-crease in survival was similar to that seen in an earlier study by the same group in which they transplanted embryonic mouse neural stem cells into the spinal cords of SMND7 mice.9,10 Interestingly, transplantation of motor neurons derived from SMA iPS cells also increased growth, strength, and sur-vival of the mice, although to a smaller ex-tent than that observed with the corrected SMA motor neurons or the non-SMA control cells. The slightly reduced benefit observed when SMA motor neurons were

Transplantation of Gene-Corrected Motor Neurons as a Therapeutic Strategy for Spinal Muscular AtrophyKathrin Meyer1, Carlos J Miranda1 and Brian K Kaspar1,2,3

doi:10.1038/mt.2013.23

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© 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: Brian.Kaspar@NationwideChildrens.org

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