Erkan Y. Osman

Bifunctional RNAs Targeting the Intronic Splicing Silencer N1 Increase SMN Levels and Reduce Disease Severity in an Animal Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients. Although the SMN2 coding sequence has the potential to produce full-length SMN, nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein. SMN2, however, is an excellent therapeutic target. Previously, we developed antisense-based oligonucleotides (bifunctional RNAs) that specifically recruit SR/SR-like splicing factors and target a negative regulator of SMN2 exon-7 inclusion within intron-6. As a means to optimize the antisense sequence of the bifunctional RNAs, we chose to target a potent intronic repressor downstream of SMN2 exon 7, called intronic splicing silencer N1 (ISS-N1). We developed RNAs that specifically target ISS-N1 and concurrently recruit the modular SR proteins SF2/ASF or hTra2β1. RNAs were directly injected in the brains of SMA mice. Bifunctional RNA injections were able to elicit robust induction of SMN protein in the brain and spinal column of neonatal SMA mice. Importantly, hTra2β1-ISS-N1 and SF2/ASF-ISS-N1 bifunctional RNAs significantly extended lifespan and increased weight in the SMNΔ7 mice. This technology has direct implications for SMA therapy and provides similar therapeutic strategies for other diseases caused by aberrant splicing.

Morpholino antisense oligonucleotides targeting intronic repressor Element1 improve phenotype in SMA mouse models

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of Survival Motor Neuron-1 (SMN1). In all SMA patients, a nearly identical copy gene called SMN2 is present, which produces low levels of functional protein owing to an alternative splicing event. To prevent exon-skipping, we have targeted an intronic repressor, Element1 (E1), located upstream of SMN2 exon 7 using Morpholino-based antisense oligonucleotides (E1(MO)-ASOs). A single intracerebroventricular injection in the relatively severe mouse model of SMA (SMNΔ7 mouse model) elicited a robust induction of SMN protein, and mean life span was extended from an average survival of 13 to 54 days following a single dose, consistent with large weight gains and a correction of the neuronal pathology. Additionally, E1(MO)-ASO treatment in an intermediate SMA mouse (SMN(RT) mouse model) significantly extended life span by ∼700% and weight gain was comparable with the unaffected animals. While a number of experimental therapeutics have targeted the ISS-N1 element of SMN2 pre-mRNA, the development of E1 ASOs provides a new molecular target for SMA therapeutics that dramatically extends survival in two important pre-clinical models of disease.

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Enhancement of SMN protein levels in a mouse model of spinal muscular atrophy using novel drug-like compounds

Spinal muscular atrophy (SMA) is a neurodegenerative disease that causes progressive muscle weakness, which primarily targets proximal muscles. About 95% of SMA cases are caused by the loss of both copies of the SMN1 gene. SMN2 is a nearly identical copy of SMN1, which expresses much less functional SMN protein. SMN2 is unable to fully compensate for the loss of SMN1 in motor neurons but does provide an excellent target for therapeutic intervention. Increased expression of functional full‐length SMN protein from the endogenous SMN2 gene should lessen disease severity. We have developed and implemented a new high‐throughput screening assay to identify small molecules that increase the expression of full‐length SMN from a SMN2 reporter gene. Here, we characterize two novel compounds that increased SMN protein levels in both reporter cells and SMA fibroblasts and show that one increases lifespan, motor function, and SMN protein levels in a severe mouse model of SMA.

Defining the therapeutic window in a severe animal model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the loss of a single gene, Survival Motor Neuron-1 (SMN1). Administration of a self-complementary Adeno-Associated Virus vector expressing full-length SMN cDNA (scAAV-SMN) has proven an effective means to rescue the SMA phenotype in SMA mice, either by intravenous (IV) or intracerebroventricular (ICV) administration at very early time points. We have recently shown that ICV delivery of scAAV9-SMN is more effective than a similar dose of vector administered via an IV injection, thereby providing an important mechanism to examine a timeline for rescuing the disease and determining the therapeutic window in a severe model of SMA. In this report, we utilized a relatively severe mouse model of SMA, SMNΔ7. Animals were injected with scAAV9-SMN vector via ICV injection on a single day, from P2 through P8. At each delivery point from P2 through P8, scAAV9-SMN decreased disease severity. A near complete rescue was obtained following P2 injection while a P8 injection produced a ∼ 40% extension in survival. Analysis of the underlying neuromuscular junction (NMJ) pathology revealed that late-stage delivery of the vector failed to provide protection from NMJ defects despite robust SMN expression in the central nervous system. While our study demonstrates that a maximal benefit is obtained when treatment is delivered during pre-symptomatic stages, significant therapeutic benefit can still be achieved after the onset of disease symptoms.

Development and characterization of an SMN2-based intermediate mouse model of Spinal Muscular Atrophy

Spinal Muscular Atrophy (SMA) is due to the loss of the survival motor neuron gene 1 (SMN1), resulting in motor neuron (MN) degeneration, muscle atrophy and loss of motor function. While SMN2 encodes a protein identical to SMN1, a single nucleotide difference in exon 7 causes most of the SMN2-derived transcripts to be alternatively spliced resulting in a truncated and unstable protein (SMNΔ7). SMA patients retain at least one SMN2 copy, making it an important target for therapeutics. Many of the existing SMA models are very severe, with animals typically living less than 2 weeks. Here, we present a novel intermediate mouse model of SMA based upon the human genomic SMN2 gene. Genetically, this model is similar to the well-characterized SMNΔ7 model; however, we have manipulated the SMNΔ7 transgene to encode a modestly more functional protein referred to as SMN read-through (SMN(RT)). By introducing the SMN(RT) transgene onto the background of a severe mouse model of SMA (SMN2(+/+);Smn(-/-)), disease severity was significantly decreased based upon a battery of phenotypic parameters, including MN pathology and a significant extension in survival. Importantly, there is not a full phenotypic correction, allowing for the examination of a broad range of therapeutics, including SMN2-dependent and SMN-independent pathways. This novel animal model serves as an important biological and therapeutic model for less severe forms of SMA and provides an in vivo validation of the SMN(RT) protein.

Plastin-3 extends survival and reduces severity in mouse models of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a leading genetic cause of infantile death and is caused by the loss of survival motor neuron-1 (SMN1). Importantly, a nearly identical gene is present called SMN2; however, the majority of SMN2-derived transcripts are alternatively spliced and encode a truncated, dysfunctional protein. Recently, several compounds designed to increase SMN protein have entered clinical trials, including antisense oligonucleotides (ASOs), traditional small molecules, and gene therapy. Expanding beyond SMN-centric therapeutics is important, as it is likely that the breadth of the patient spectrum and the inherent complexity of the disease will be difficult to address with a single therapeutic strategy. Several SMN-independent pathways that could impinge upon the SMA phenotype have been examined with varied success. To identify disease-modifying pathways that could serve as stand-alone therapeutic targets or could be used in combination with an SMN-inducing compound, we investigated adeno-associated virus-mediated (AAV-mediated) gene therapy using plastin-3 (PLS3). Here, we report that AAV9-PLS3 extends survival in an intermediate model of SMA mice as well as in a pharmacologically induced model of SMA using a splice-switching ASO that increases SMN production. PLS3 coadministration improves the phenotype beyond the ASO, demonstrating the potential utility of combinatorial therapeutics in SMA that target SMN-independent and SMN-dependent pathways.

Optimization of Morpholino Antisense Oligonucleotides Targeting the Intronic Repressor Element1 in Spinal Muscular Atrophy

Loss of Survival Motor Neuron-1 (SMN1) causes Spinal Muscular Atrophy, a devastating neurodegenerative disease. SMN2 is a nearly identical copy gene; however SMN2 cannot prevent disease development in the absence of SMN1 since the majority of SMN2-derived transcripts are alternatively spliced, encoding a truncated, unstable protein lacking exon 7. Nevertheless, SMN2 retains the ability to produce low levels of functional protein. Previously we have described a splice-switching Morpholino antisense oligonucleotide (ASO) sequence that targets a potent intronic repressor, Element1 (E1), located upstream of SMN2 exon 7. In this study, we have assessed a novel panel of Morpholino ASOs with the goal of optimizing E1 ASO activity. Screening for efficacy in the SMNΔ7 mouse model, a single ASO variant was more active in vivo compared with the original E1(MO)-ASO. Sequence variant eleven (E1(MOv11)) consistently showed greater efficacy by increasing the lifespan of severe Spinal Muscular Atrophy mice after a single intracerebroventricular injection in the central nervous system, exhibited a strong dose-response across an order of magnitude, and demonstrated excellent target engagement by partially reversing the pathogenic SMN2 splicing event. We conclude that Morpholino modified ASOs are effective in modifying SMN2 splicing and have the potential for future Spinal Muscular Atrophy clinical applications.

Comparison of independent screens on differentially vulnerable motor neurons reveals alpha-synuclein as a common modifier in motor neuron diseases

The term “motor neuron disease” encompasses a spectrum of disorders in which motor neurons are the primary pathological target. However, in both patients and animal models of these diseases, not all motor neurons are equally vulnerable, in that while some motor neurons are lost very early in disease, others remain comparatively intact, even at late stages. This creates a valuable system to investigate the factors that regulate motor neuron vulnerability. In this study, we aim to use this experimental paradigm to identify potential transcriptional modifiers. We have compared the transcriptome of motor neurons from healthy wild-type mice, which are differentially vulnerable in the childhood motor neuron disease Spinal Muscular Atrophy (SMA), and have identified 910 transcriptional changes. We have compared this data set with published microarray data sets on other differentially vulnerable motor neurons. These neurons were differentially vulnerable in the adult onset motor neuron disease Amyotrophic Lateral Sclerosis (ALS), but the screen was performed on the equivalent population of neurons from neurologically normal human, rat and mouse. This cross species comparison has generated a refined list of differentially expressed genes, including CELF5, Col5a2, PGEMN1, SNCA, Stmn1 and HOXa5, alongside a further enrichment for synaptic and axonal transcripts. As an in vivo validation, we demonstrate that the manipulation of a significant number of these transcripts can modify the neurodegenerative phenotype observed in a Drosophila line carrying an ALS causing mutation. Finally, we demonstrate that vector-mediated expression of alpha-synuclein (SNCA), a transcript decreased in selectively vulnerable motor neurons in all four screens, can extend life span, increase weight and decrease neuromuscular junction pathology in a mouse model of SMA. In summary, we have combined multiple data sets to identify transcripts, which are strong candidates for being phenotypic modifiers, and demonstrated SNCA is a modifier of pathology in motor neuron disease.

Analysis of Azithromycin Monohydrate as a Single or a Combinatorial Therapy in a Mouse Model of Severe Spinal Muscular Atrophy

BACKGROUND Spinal muscular atrophy (SMA) is a neurodegenerative autosomal recessive disorder characterized by the loss of α-motor neurons. A variety of molecular pathways are being investigated to elevate SMN protein expression in SMA models and in the clinic. One of these approaches involves stabilizing the SMNΔ7 protein by inducing translational read-through. Previous studies have demonstrated that functionality and stability are partially restored to the otherwise unstable SMNΔ7 by the addition of non-specific C-terminal peptide sequences, or by inducing a similar molecular event through the use of read-through inducing compounds such as aminoglycosides. OBJECTIVE The objective was to determine the efficacy of the macrolide Azithromycin (AZM), an FDA approved read-through-inducing compound, in the well-established severe mouse model of SMA. METHODS Initially, dosing regimen following ICV administrations of AZM at different post-natal days and concentrations was determined by their impact on SMN levels in disease-relevant tissues. Selected dose was then tested for phenotypic parameters changes as compared to the appropriate controls and in conjugation to another therapy. RESULTS AZM increases SMN protein in disease relevant tissues, however, this did not translate into similar improvements in the SMA phenotype in a severe mouse model of SMA. Co-administration of AZM and a previously developed antisense oligonucleotide that increases SMN2 splicing, resulted in an improvement in the SMA phenotype beyond either AZM or ASO alone, including a highly significant extension in survival with improvement in body weight and movement. CONCLUSIONS It is important to explore various approaches for SMA therapeutics, hence compounds that specifically induce SMNΔ7 read-through, without having prohibitive toxicity, may provide an alternative platform for a combinatorial treatment. Here we established that AZM activity at a low dose can increase SMN protein in disease-relevant animal model and can impact disease severity.