The adeno-associated virus 2 genome and Rep 68/78 proteins interact with cellular sites of DNA damage.

Nuclear DNA viruses simultaneously access cellular factors that aid their life cycle while evading inhibitory factors by localizing to distinct nuclear sites. Adeno-associated viruses (AAVs), which are Dependoviruses in the family Parvovirinae, are non-enveloped icosahedral viruses, which have been developed as recombinant AAV vectors to express transgenes. AAV2 expression and replication occur in nuclear viral replication centers (VRCs), which relies on cellular replication machinery as well as coinfection by helper viruses such as adenoviruses or herpesviruses, or exogenous DNA damage to host cells. AAV2 infection induces a complex cellular DNA damage response (DDR), in response to either viral DNA or viral proteins expressed in the host nucleus during infection, where VRCs co-localized with DDR proteins. We have previously developed a modified iteration of a viral chromosome conformation capture (V3C-seq) assay to show that the autonomous parvovirus minute virus of mice localizes to cellular sites of DNA damage to establish and amplify its replication. Similar V3C-seq assays to map AAV2 show that the AAV2 genome co-localized with cellular sites of DNA damage under both non-replicating and replicating conditions. The AAV2 non-structural protein Rep 68/78, also localized to cellular DDR sites during both non-replicating and replicating infections, and also when ectopically expressed. Ectopically expressed Rep could be efficiently re-localized to DDR sites induced by micro-irradiation. Recombinant AAV2 gene therapy vector genomes derived from AAV2 localized to sites of cellular DNA damage to a lesser degree, suggesting that the inverted terminal repeat origins of replication were insufficient for targeting.

AAV9-DOK7 gene therapy reduces disease severity in Smn2B/- SMA model mice.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disease caused by deletions or mutations in the survival motor neuron (SMN1) gene. An important hallmark of disease progression is the pathology of neuromuscular junctions (NMJs). Affected NMJs in the SMA context exhibit delayed maturation, impaired synaptic transmission, and loss of contact between motor neurons and skeletal muscle. Protection and maintenance of NMJs remains a focal point of therapeutic strategies to treat SMA, and the recent implication of the NMJ-organizer Agrin in SMA pathology suggests additional NMJ organizing molecules may contribute. DOK7 is an NMJ organizer that functions downstream of Agrin. The potential of DOK7 as a putative therapeutic target was demonstrated by adeno-associated virus (AAV)-mediated gene therapy delivery of DOK7 in Amyotrophic Lateral Sclerosis (ALS) and Emery Dreyefuss Muscular Dystrophy (EDMD). To assess the potential of DOK7 as a disease modifier of SMA, we administered AAV-DOK7 to an intermediate mouse model of SMA. AAV9-DOK7 treatment conferred improvements in NMJ architecture and reduced muscle fiber atrophy. Additionally, these improvements resulted in a subtle reduction in phenotypic severity, evidenced by improved grip strength and an extension in survival. These findings reveal DOK7 is a novel modifier of SMA.

Functional characterization of SMN evolution in mouse models of SMA.

Spinal Muscular Atrophy (SMA) is a monogenic neurodegenerative disorder and the leading genetic cause of infantile mortality. While several functions have been ascribed to the SMN (survival motor neuron) protein, their specific contribution to the disease has yet to be fully elucidated. We hypothesized that some, but not all, SMN homologues would rescue the SMA phenotype in mouse models, thereby identifying disease-relevant domains. Using AAV9 to deliver Smn homologs to SMA mice, we identified a conservation threshold that marks the boundary at which homologs can rescue the SMA phenotype. Smn from Danio rerio and Xenopus laevis significantly prevent disease, whereas Smn from Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe was significantly less efficacious. This phenotypic rescue correlated with correction of RNA processing defects induced by SMN deficiency and neuromuscular junction pathology. Based upon the sequence conservation in the rescuing homologs, a minimal SMN construct was designed consisting of exons 2, 3, and 6, which showed a partial rescue of the SMA phenotype. While a significant extension in survival was observed, the absence of a complete rescue suggests that while the core conserved region is essential, additional sequences contribute to the overall ability of the SMN protein to rescue disease pathology.

AAV9-mediated delivery of miR-23a reduces disease severity in Smn2B/-SMA model mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/- SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA.

A Direct Comparison of IV and ICV Delivery Methods for Gene Replacement Therapy in a Mouse Model of SMARD1.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is an infantile autosomal recessive disease caused by the loss of the ubiquitously expressed IGHMBP2 gene. SMARD1 causes degeneration of alpha-motor neurons, resulting in distal muscle weakness, diaphragm paralysis, and respiratory malfunction. We have reported that delivery of a low dose of AAV9-IGHMBP2 to the CNS results in a significant rescue of the SMARD1 mouse model (nmd). To examine how a delivery route can impact efficacy, a direct comparison of intravenous (IV) and intracerebroventricular (ICV) delivery of AAV9-IGHMBP2 was performed. Using a low-dose, both IV and ICV delivery routes led to a significant extension in survival and increased body weight. Conversely, only ICV-treated animals demonstrated improvements in the hindlimb muscle, neuromuscular junction, and motor function. The hindlimb phenotype of IV-treated mice resembled the untreated nmd mice. We investigated whether the increased survival of IV-treated nmd mice was the result of a positive impact on the cardiac function. Our results revealed that cardiac function and pathology were similarly improved in IV- and ICV-treated mice. We concluded that while IV delivery of a low dose does not improve the hindlimb phenotype and motor function, partial restoration of cardiac performance is sufficient to significantly extend survival.

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCFSlmb degron.

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1 Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

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.

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.