Carlos J. Miranda

BACKGROUND: Spinal muscular atrophy type 1 (SMA1) is a progressive, monogenic motor neuron disease with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. We studied functional replacement of the mutated gene encoding survival motor neuron 1 (SMN1) in this disease. METHODS: vg per kilogram). The primary outcome was safety. The secondary outcome was the time until death or the need for permanent ventilatory assistance. In exploratory analyses, we compared scores on the CHOP INTEND (Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders) scale of motor function (ranging from 0 to 64, with higher scores indicating better function) in the two cohorts and motor milestones in the high-dose cohort with scores in studies of the natural history of the disease (historical cohorts). RESULTS: As of the data cutoff on August 7, 2017, all 15 patients were alive and event-free at 20 months of age, as compared with a rate of survival of 8% in a historical cohort. In the high-dose cohort, a rapid increase from baseline in the score on the CHOP INTEND scale followed gene delivery, with an increase of 9.8 points at 1 month and 15.4 points at 3 months, as compared with a decline in this score in a historical cohort. Of the 12 patients who had received the high dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently. Elevated serum aminotransferase levels occurred in 4 patients and were attenuated by prednisolone. CONCLUSIONS: In patients with SMA1, a single intravenous infusion of adeno-associated viral vector containing DNA coding for SMN resulted in longer survival, superior achievement of motor milestones, and better motor function than in historical cohorts. Further studies are necessary to confirm the safety and efficacy of this gene therapy. (Funded by AveXis and others; ClinicalTrials.gov number, NCT02122952 .).

Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.

Stem cell-derived motor neurons (MNs) are increasingly utilized for modeling disease in vitro and for developing cellular replacement strategies for spinal cord injury and diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Human embryonic stem cell (hESC) differentiation into MNs, which involves retinoic acid (RA) and activation of the sonic hedgehog (SHH) pathway is inefficient and requires up to 60 days to develop MNs with electrophysiological properties. This prolonged differentiation process has hampered the use of hESCs, in particular for high-throughput screening. We evaluated the MN gene expression profile of RA/SHH-differentiated hESCs to identify rate-limiting factors involved in MN development. Based on this analysis, we developed an adenoviral gene delivery system encoding for MN inducing transcription factors: neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM/homeobox protein 3 (Lhx3). Strikingly, delivery of these factors induced functional MNs with mature electrophysiological properties, 11-days after gene delivery, with >60–70% efficiency from hESCs and human induced pluripotent stem cells (hiPSCs). This directed programming approach significantly reduces the time required to generate electrophysiologically-active MNs by approximately 30 days in comparison to conventional differentiation techniques. Our results further exemplify the potential to use transcriptional coding for rapid and efficient production of defined cell types from hESCs and hiPSCs. Stem cell-derived motor neurons (MNs) are increasingly utilized for modeling disease in vitro and for developing cellular replacement strategies for spinal cord injury and diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Human embryonic stem cell (hESC) differentiation into MNs, which involves retinoic acid (RA) and activation of the sonic hedgehog (SHH) pathway is inefficient and requires up to 60 days to develop MNs with electrophysiological properties. This prolonged differentiation process has hampered the use of hESCs, in particular for high-throughput screening. We evaluated the MN gene expression profile of RA/SHH-differentiated hESCs to identify rate-limiting factors involved in MN development. Based on this analysis, we developed an adenoviral gene delivery system encoding for MN inducing transcription factors: neurogenin 2 (Ngn2), islet-1 (Isl-1), and LIM/homeobox protein 3 (Lhx3). Strikingly, delivery of these factors induced functional MNs with mature electrophysiological properties, 11-days after gene delivery, with >60–70% efficiency from hESCs and human induced pluripotent stem cells (hiPSCs). This directed programming approach significantly reduces the time required to generate electrophysiologically-active MNs by approximately 30 days in comparison to conventional differentiation techniques. Our results further exemplify the potential to use transcriptional coding for rapid and efficient production of defined cell types from hESCs and hiPSCs.