Orally administered branaplam does not impact neurogenesis in juvenile mice, rats, and dogs.

Branaplam is a therapeutic agent currently in clinical development for the treatment of infants with type 1 spinal muscular atrophy (SMA). Since preclinical studies showed that branaplam had cell-cycle arrest effects, we sought to determine whether branaplam may affect postnatal cerebellar development and brain neurogenesis. Here, we describe a novel approach for developmental neurotoxicity testing (DNT) of a central nervous system (CNS) active drug. The effects of orally administered branaplam were evaluated in the SMA neonatal mouse model (SMNΔ7), and in juvenile Wistar Hannover rats and Beagle dogs. Histopathological examination and complementary immunohistochemical studies focused on areas of neurogenesis in the cerebellum (mice, rats, and dogs), and the subventricular zone of the striatum and dentate gyrus (rats and dogs) using antibodies directed against Ki67, phosphorylated histone H3, cleaved caspase-3, and glial fibrillary acidic protein. Additionally, image-analysis based quantification of calbindin-D28k and Ki67 was performed in rats and dogs. The patterns of cell proliferation and apoptosis, and neural migration and innervation in the cerebellum and other brain regions of active adult neurogenesis did not differ between branaplam- and control-treated animals. Quantitative image analysis did not reveal any changes in calbindin-D28k and Ki67 expression in rats and dogs. The data show that orally administered branaplam has no impact on neurogenesis in juvenile animals. Application of selected immunohistochemical stainings in combination with quantitative image analysis on a few critical areas of postnatal CNS development offer a reliable approach to assess DNT of CNS-active drug candidates in juvenile animal toxicity studies.

Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy.

Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood-brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141-150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.

Rescue of gene-expression changes in an induced mouse model of spinal muscular atrophy by an antisense oligonucleotide that promotes inclusion of SMN2 exon 7.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by disruption of the survival motor neuron 1 (SMN1) gene, partly compensated for by the paralogous gene SMN2. Exon 7 inclusion is critical for full-length SMN protein production and occurs at a much lower frequency for SMN2 than for SMN1. Antisense oligonucleotide (ASO)-mediated blockade of an intron 7 splicing silencer was previously shown to promote inclusion of SMN2 exon 7 in SMA mouse models and mediate phenotypic rescue. However, downstream molecular consequences of this ASO therapy have not been defined. Here we characterize the gene-expression changes that occur in an induced model of SMA and show substantial rescue of those changes in central nervous system tissue upon intracerebroventricular administration of an ASO that promotes inclusion of exon 7, with earlier administration promoting greater rescue. This study offers a robust reference set of preclinical pharmacodynamic gene expression effects for comparison of other investigational therapies for SMA.

Current advances in drug development in spinal muscular atrophy.

PURPOSE OF REVIEW: Spinal muscular atrophy (SMA) is a pediatric neuromuscular condition characterized by progressive proximal muscle weakness. It is one of the most common genetic causes of infant mortality across different races and is caused by mutation of the survival of motor neuron 1 (SMN1) gene on chromosome 5q13. RECENT FINDINGS: To date, there have been many therapeutics developments for SMA targeting various potential pathways such as increasing SMN gene expression, enhancing SMN2 exon 7 inclusion, neuroprotection, cell replacement, and gene therapy. SUMMARY: Although SMA remains an incurable disease to date, recent advances in the field of basic and translational research have enhanced our understanding of the pathogenesis of the disease and opened new possibilities for therapeutic intervention. This article reviews and highlights past and current translational research on SMA therapeutics.

Treatment of spinal muscular atrophy cells with drugs that upregulate SMN expression reveals inter- and intra-patient variability.

Spinal muscular atrophy (SMA) is a genetic neuromuscular disorder caused by mutations in the SMN1 gene. The homologous copy (SMN2) is always present in SMA patients. SMN1 gene transcripts are usually full-length (FL), but exon 7 is spliced out in a high proportion of SMN2 transcripts (delta7) (Δ7). Advances in drug therapy for SMA have shown that an increase in SMN mRNA and protein levels can be achieved in vitro. We performed a systematic analysis of SMN expression in primary fibroblasts and EBV-transformed lymphoblasts from seven SMA patients with varying clinical severity and different SMN1 genotypes to determine expression differences in two accessible tissues (skin and blood). The basal expression of SMN mRNA FL and Δ7 in fibroblasts and lymphoblasts was analyzed by quantitative real-time PCR. The FL-SMN and FL/Δ7 SMN ratios were higher in control cells than in patients. Furthermore, we investigated the response of these cell lines to hydroxyurea, valproate and phenylbutyrate, drugs previously reported to upregulate SMN2. The response to treatments with these compounds was heterogeneous. We found both intra-patient and inter-patient variability even within haploidentical siblings, suggesting that tissue and individual factors may affect the response to these compounds. To optimize the stratification of patients in clinical trials, in vitro studies should be performed before enrolment so as to define each patient as a responder or non-responder to the compound under investigation.