Subcutaneous administration of TC007 reduces disease severity in an animal model of SMA

Background Spinal Muscular Atrophy (SMA) is the leading genetic cause of infantile death. It is caused by the loss of functional Survival Motor Neuron 1 (SMN1). There is a nearly identical copy gene, SMN2, but it is unable to rescue from disease due to an alternative splicing event that excises a necessary exon (exon 7) from the majority of SMN2-derived transcripts. While SMNΔ7 protein has severely reduced functionality, the exon 7 sequences may not be specifically required for all activities. Therefore, aminoglycoside antibiotics previously shown to suppress stop codon recognition and promote translation read-through have been examined to increase the length of the SMNΔ7 C-terminus. Results Here we demonstrate that subcutaneous-administration of a read-through inducing compound (TC007) to an intermediate SMA model (Smn-/-; SMN2+/+; SMNΔ7) had beneficial effects on muscle fiber size and gross motor function. Conclusion Delivery of the read-through inducing compound TC007 reduces the disease-associated phenotype in SMA mice, however, does not significantly extend survival

Huntington's disease patient-derived astrocytes display electrophysiological impairments and reduced neuronal support

In Huntington’s disease (HD), while the ubiquitously expressed mutant Huntingtin (mtHTT) protein primarily compromises striatal and cortical neurons, glia also undergo disease-contributing alterations. Existing HD models using human induced pluripotent stem cells (iPSCs) have not extensively characterized the role of mtHTT in patient-derived astrocytes. Here physiologically mature astrocytes are generated from HD patient iPSCs. These human astrocytes exhibit hallmark HD phenotypes that occur in mouse models, including impaired inward rectifying K+ currents, lengthened spontaneous Ca2+ waves and reduced cell membrane capacitance. HD astrocytes in co-culture provided reduced support for the maturation of iPSC-derived neurons. In addition, neurons exposed to chronic glutamate stimulation are not protected by HD astrocytes. This iPSC-based HD model demonstrates the critical effects of mtHTT on human astrocytes, which not only broadens the understanding of disease susceptibility beyond cortical and striatal neurons but also increases potential drug targets

Molecular genetics of spinal muscular atrophy : insights into various routes of therapeutic intervention

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Vita."May 2009"Thesis (Ph. D.) University of Missouri-Columbia 2009.[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT REQUEST OF AUTHOR.] Spinal Muscular Atrophy is an autosomal recessive neuromuscular disorder that is the leading genetic cause of infant mortality. In humans, a nearly identical copy gene is present called SMN2 that is retained in all SMA patients and encodes an identical protein compared to SMN1. However, SMN1 and SMN2 differ by a silent C to T transition at the 5' end of exon 7. This single nucleotide difference results in the production of an alternatively spliced isoform, called SMN[delta]7, which encodes an unstable and non-functional protein. Therefore, the absence of the short peptide encoded by SMN exon 7 is critical in the disease development process. A cytoplasmic localization signal has been previously identified within SMN exon 7 and can readily transport SMN and heterologous proteins to the cytoplasm. While this peptide is important for SMN protein function, here it is shown heterologous sequences can seemingly compensate for the SMN exon 7 peptide, regarding: SMN protein localization, protein stability, supporting neurite outgrowth, and snRNP assembly. Consistent with this, aminoglycosides that suppress efficient recognition of stop codons, known as "read-through" (Rt), resulted in significantly increased levels of SMN protein in SMA patient fibroblasts. It is also shown in this work these aminoglycosides increase lifespan and gross-motor function in SMA model mice. Collectively, these experiments help to provide insight into therapeutics designed to induce readthrough of the SMN[delta]7 stop codon, as well as other routes of therapeutic intervention to be used in combination with a read-through event.Includes bibliographical references

Analysis of a read-through promoting compound in a severe mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is the leading genetic cause of infantile death and caused by the loss of functional Survival Motor Neuron 1(SMN1). The remaining copy gene, SMN2, is unable to rescue from disease because the primary gene product lacks the final coding exon, exon 7, due to an alternative splicing event. While SMNΔ7 is a rapidly degraded protein, exon 7 is not specifically required in a sequence-specific manner to confer increased functionality to this truncated protein. Based upon this molecular observation, aminoglycosides have been examined to artificially elongate the C-terminus of SMNΔ7 by “read-through” of the stop codon. An SMNΔ7 read-through event benefits intermediate mouse models of SMA. Here we demonstrate that delivery of a read-through inducing compound directly to the CNS can partially lessen the severity of a severe model of SMA (Smn−/−; SMN2+/+), albeit not to the extent seen in the less severe model. This further demonstrates the utility of read-through inducing compounds in SMA

Optimization of trans-Splicing for Huntington's Disease RNA Therapy

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the Huntingtin (HTT) gene. We have previously demonstrated that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically reduce and repair mutant HTT (mtHTT). Here, the targeted tethering efficacy of the pre-mRNA trans-splicing modules (PTM) in HTT was optimized. Various PTMs that targeted the 3′ end of HTT intron 1 or the intron 1 branch point were shown trans-splice into an HTT mini-gene, as well as the endogenous HTT pre-mRNA. PTMs that specifically target the endogenous intron 1 branch point increased the trans-splicing efficacy from 1–5 to 10–15%. Furthermore, lentiviral expression of PTMs in a human HD patient iPSC-derived neural culture significantly reversed two previously established polyQ-length dependent phenotypes. These results suggest that pre-mRNA repair of mtHTT could hold therapeutic benefit and it demonstrates an alternative platform to correct the mRNA product produced by the mtHTT allele in the context of HD

Stimulation of GABA-induced Ca2+ influx enhances maturation of human induced pluripotent stem cell-derived neurons.

Optimal use of patient-derived, induced pluripotent stem cells for modeling neuronal diseases is crucially dependent upon the proper physiological maturation of derived neurons. As a strategy to develop defined differentiation protocols that optimize electrophysiological function, we investigated the role of Ca(2+) channel regulation by astrocyte conditioned medium in neuronal maturation, using whole-cell patch clamp and Ca(2+) imaging. Standard control medium supported basic differentiation of induced pluripotent stem cell-derived neurons, as assayed by the ability to fire simple, single, induced action potentials. In contrast, treatment with astrocyte conditioned medium elicited complex and spontaneous neuronal activity, often with rhythmic and biphasic characteristics. Such augmented spontaneous activity correlated with astrocyte conditioned medium-evoked hyperpolarization and was dependent upon regulated function of L-, N- and R-type Ca(2+) channels. The requirement for astrocyte conditioned medium could be substituted by simply supplementing control differentiation medium with high Ca(2+) or γ-amino butyric acid (GABA). Importantly, even in the absence of GABA signalling, opening Ca(2+) channels directly using Bay K8644 was able to hyperpolarise neurons and enhance excitability, producing fully functional neurons. These data provide mechanistic insight into how secreted astrocyte factors control differentiation and, importantly, suggest that pharmacological modulation of Ca(2+) channel function leads to the development of a defined protocol for improved maturation of induced pluripotent stem cell-derived neurons

Delivery of a read-through inducing compound, TC007, lessens the severity of a SMA animal model

Spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality and is caused by the loss of a functional SMN1 gene. In humans, there exists a nearly-identical copy gene known as SMN2 that encodes an identical protein as SMN1, but differs by a silent C to T transition within exon 7. This single nucleotide difference produces an alternatively spliced isoform, SMNΔ7, which encodes a rapidly degraded protein. The absence of the short peptide encoded by SMN exon 7 is critical in the disease development process; however, heterologous sequences can partially compensate for the SMN exon 7 peptide in several cellular assays. Consistent with this, aminoglycosides, compounds that can suppress efficient recognition of stop codons, resulted in significantly increased levels of SMN protein in SMA patient fibroblasts. We now examine the potential therapeutic capabilities of a novel aminoglycoside, TC007. In an intermediate SMA model (Smn−/−; SMN2+/+; SMNΔ7), when delivered directly to the central nervous system (CNS), TC007 induces SMN in both the brain and spinal cord, significantly increases lifespan (∼30%) and increases ventral horn cell number, consistent with its ability to increase SMN levels in induced pluripotent stem cell-derived human SMA motor neuron cultures. Collectively, these experiments are the first in vivo examination of therapeutics for SMA designed to induce read-through of the SMNΔ7 stop codon to show increased benefit by direct administration to the CNS