Transplantation of Gene-Corrected Motor Neurons as a Therapeutic Strategy for Spinal Muscular Atrophy
Authors: Kathrin Meyer, Carlos J Miranda, Brian K Kaspar
Journal: Molecular Therapy (2013)

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 intrinsic DNA damage-repair system during replication, leading to permanent correction of the mutation.1Parekh-Olmedo H Kmiec EB Progress and prospects: targeted gene alteration (TGA).Gene Ther. 2007; 14: 1675-1680Crossref PubMed Scopus (41) Google Scholar TGA technology has become increasingly relevant to translational 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 transplantation in a mouse model of spinal muscular atrophy (SMA).2Corti S Nizzardo M Simone C Falcone M Nardini M Ronchi D et al.Genetic correction of human induced pluripotent stem cells from patients with spinal muscular atrophy.Sci Transl Med. 2012; 4: 165ra162Crossref PubMed Scopus (168) Google Scholar Unlike other strategies commonly used to alter genomic sequences, such as zinc-finger nucleases or transcription activator–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, gene editing with oligonucleotides has clinical potential for approaches in which a selection of corrected cells is possible.3Perez-Pinera P Ousterout DG Gersbach CA Advances in targeted genome editing.Curr Opin Chem Biol. 2012; 16: 268-277Crossref PubMed Scopus (119) Google Scholar iPS cell technology permits such selection and enables the differentiation of the newly corrected stem cells into almost any cell type, which naturally led to the idea of using a patient'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 leading to the degeneration of motor neurons in children and death within the first two years of life. This disorder manifests through autosomal 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 nonintegrating-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 successfully corrected, leading to increased protein production. The corrected iPS cells were then differentiated into spinal motor neurons, the cell type that is most prominently affected in this disease. Confirming a previous study,4Ebert AD Rose Jr, FF Mattis VB Lorson CL Thomson JA Svensen CN Induced pluripotent stem cells from a spinal muscular atrophy patient.Nature. 2009; 457: 277-280Crossref PubMed Scopus (1181) Google Scholar SMA motor neurons showed reduced size and decreased survival. Importantly, this phenotype was reversed in motor neurons generated from the oligonucleotide-corrected iPS lines. Corti et al. performed gene expression profiling and splicing analyses to evaluate differences in the transcriptome of the generated motor 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, underscoring the important role of the SMN protein in the assembly of components of the spliceosome,5Gabanella F Butchbach ME Saieva L Carissimi C Burghes AHM Pellizzoni L Ribonucleoprotein assembly defects correlate with spinal muscular atrophy severity and preferentially affect a subset of spliceosomal snRNPs.PLoS ONE. 2007; 2: e921Crossref PubMed Scopus (248) Google Scholar 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 engraftment potential of the human iPS cell–derived motor neurons in the standard mouse model of SMA.6Le TT Pham LT Butchbach ME Zhang HL Monani UR Coovert DD et al.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. 2005; 14: 845-857Crossref PubMed Scopus (486) Google Scholar Transplanted motor neurons differentiated from human embryonic stem (ES) cells have been shown to engraft and integrate into the spinal cord of SMNΔ7 mice as well as in models of spinal cord injury and amyotrophic lateral sclerosis.7Rossi SL Nistor G Wyatt T Yin HZ Poole AJ Weiss JH et al.Histological and functional benefit following transplantation of motor neuron progenitors to the injured rat spinal cord.PLoS ONE. 2010; 5: e11852Crossref PubMed Scopus (79) Google Scholar,8Wyatt TJ Rossi SL Siegenthaler MM Frame J Robles R Nistor G et al.Human motor neuron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss.Stem Cells Int. 2011; 2011: 207230Crossref PubMed Scopus (39) Google Scholar The human ES cell–derived motor neurons secreted various growth factors, thereby generating a beneficial neurotro­phic effect upon the remaining endogenous cells. The new study confirms similar behavior of the iPS cell–derived motor neurons. Transplantation moderately increased survival of the SMNΔ7 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 neuroprotective factors benefiting endogenous motor neurons. Indeed, the observed increase 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 SMNΔ7 mice.9Corti S Nizzardo M Nardini M Donadoni C Salani S Ronchi D et al.Embryonic stem cell–derived neural stem cells improve spinal muscular atrophy phenotype in mice.Brain. 2010; 133(Pt 2): 465-481Crossref Scopus (94) Google Scholar,10Corti S Nizzardo M Nardini M Donadoni C Salani S Ronchi D et al.Neural stem cell transplantation can ameliorate the phenotype of a mouse model of spinal muscular atrophy.J Clin Invest. 2008; 118: 3316-3330Crossref PubMed Scopus (116) Google Scholar Interestingly, transplantation of motor neurons derived from SMA iPS cells also increased growth, strength, and survival of the mice, although to a smaller extent 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 transplanted might be explained by either a lower level of engraftment of the cells or reduced function due to the SMA mutations, and further studies are necessary to distinguish these effects. The use of iPS cell–derived motor neurons has potential for therapeutic approaches 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. demonstrated remarkable axon outgrowth over long distances and improved cell survival.11Lu P Wang Y Graham L McHale K Gao M Wu D et al.Long-distance growth and connectivity of neural stem cells after severe spinal cord injury.Cell. 2012; 150: 1264-1273Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar In addition, new protocols have been developed to improve the differentiation efficiency of motor neurons.12Hester ME Murtha MJ Song S Rao M Miranda CJ Meyer K et al.Rapid and efficient generation of functional motor neurons from human pluripotent stem cells using gene delivered transcription factor codes.Mol Ther. 2011; 19: 1905-1912Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar These improvements have the potential to increase engraftment and muscle innervation by the transplanted motor neurons. For type I SMA, the production 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 motor 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 virus encoding normal SMN shortly after birth led to an essentially complete rescue of the disease phenotype, whereas treatment at later time points of the disease showed less benefit.13Foust KD Wang X McGovern VL Braun L Bevan AK Haidet AM et al.Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN.Nat Biotechnol. 2010; 28: 271-274Crossref PubMed Scopus (591) Google Scholar In summary, the field of motor neuron transplantation raises hope for treating neurodegenerative disorders such as SMA. However, much additional research is necessary to identify the appropriate time window for transplantation, the optimal type and number of cells, and the combination with the right growth factors and parameters for optimal success. Therefore, studies to elucidate environmental influences and development of methods to improve motor neuron survival and proper connections are imperative. The ultimate goal for motor neuron replacement remains the integration, growth, functional maturation, and innervation of muscle. Significant challenges must still be overcome, but with recent advances in the field we are moving closer toward this goal.