Increasing survival of motor neuron 2, centromeric (SMN2) exon 7 inclusion to express more full-length SMN protein in motor neurons is a promising approach to treat spinal muscular atrophy (SMA), a genetic neurodegenerative disease. Previously, we identified a potent 29-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) that blocks an SMN2 intronic splicing silencer element and efficiently promotes exon 7 inclusion in transgenic mouse peripheral tissues after systemic administration. Here we address its efficacy in the spinal cord-a prerequisite for disease treatment-and its ability to rescue a mild SMA mouse model that develops tail and ear necrosis, resembling the distal tissue necrosis reported in some SMA infants. Using a microosmotic pump, we directly infused the ASO into a lateral cerebral ventricle in adult mice expressing a human SMN2 transgene; the ASO gave a robust and long-lasting increase in SMN2 exon 7 inclusion measured at both the mRNA and protein levels in spinal cord motor neurons. A single embryonic or neonatal intracerebroventricular ASO injection strikingly rescued the tail and ear necrosis in SMA mice. We conclude that this MOE ASO is a promising drug candidate for SMA therapy, and, more generally, that ASOs can be used to efficiently redirect alternative splicing of target genes in the CNS.[Keywords: Spinal muscular atrophy; SMN2; antisense oligonucleotide; splicing correction; spinal cord; mouse models] Supplemental material is available at http://www.genesdev.org.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the SMN1 gene that result in a deficiency of SMN protein. One approach to treat SMA is to use antisense oligonucleotides (ASOs) to redirect the splicing of a paralogous gene, SMN2, to boost production of functional SMN. Injection of a 2′-O-2-methoxyethyl–modified ASO (ASO-10-27) into the cerebral lateral ventricles of mice with a severe form of SMA resulted in splice-mediated increases in SMN protein and in the number of motor neurons in the spinal cord, which led to improvements in muscle physiology, motor function and survival. Intrathecal infusion of ASO-10-27 into cynomolgus monkeys delivered putative therapeutic levels of the oligonucleotide to all regions of the spinal cord. These data demonstrate that central nervous system–directed ASO therapy is efficacious and that intrathecal infusion may represent a practical route for delivering this therapeutic in the clinic.

Emerging genetic and clinical evidence suggests a link between Gaucher disease and the synucleinopathies Parkinson disease and dementia with Lewy bodies. Here, we provide evidence that a mouse model of Gaucher disease ( Gba1 D409V/D409V ) exhibits characteristics of synucleinopathies, including progressive accumulation of proteinase K-resistant α-synuclein/ubiquitin aggregates in hippocampal neurons and a coincident memory deficit. Analysis of homozygous ( Gba1 D409V/D409V ) and heterozygous ( Gba1 D409V/+ and Gba1 +/− ) Gaucher mice indicated that these pathologies are a result of the combination of a loss of glucocerebrosidase activity and a toxic gain-of-function resulting from expression of the mutant enzyme. Importantly, adeno-associated virus-mediated expression of exogenous glucocerebrosidase injected into the hippocampus of Gba1 D409V/D409V mice ameliorated both the histopathological and memory aberrations. The data support the contention that mutations in GBA1 can cause Parkinson disease-like α-synuclein pathology, and that rescuing brain glucocerebrosidase activity might represent a therapeutic strategy for GBA1 -associated synucleinopathies.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a deficiency of survival motor neuron (SMN) due to mutations in the SMN1 gene. In this study, an adeno-associated virus (AAV) vector expressing human SMN (AAV8-hSMN) was injected at birth into the CNS of mice modeling SMA. Western blot analysis showed that these injections resulted in widespread expression of SMN throughout the spinal cord, and this translated into robust improvement in skeletal muscle physiology, including increased myofiber size and improved neuromuscular junction architecture. Treated mice also displayed substantial improvements on behavioral tests of muscle strength, coordination, and locomotion, indicating that the neuromuscular junction was functional. Treatment with AAV8-hSMN increased the median life span of mice with SMA-like disease to 50 days compared with 15 days for untreated controls. Moreover, injecting mice with SMA-like disease with a human SMN-expressing self-complementary AAV vector -a vector that leads to earlier onset of gene expression compared with standard AAV vectors -led to improved efficacy of gene therapy, including a substantial extension in median survival to 157 days. These data indicate that CNS-directed, AAV-mediated SMN augmentation is highly efficacious in addressing both neuronal and muscular pathologies in a severe mouse model of SMA.

Mutations of GBA1, the gene encoding glucocerebrosidase, represent a common genetic risk factor for developing the synucleinopathies Parkinson disease (PD) and dementia with Lewy bodies. PD patients with or without GBA1 mutations also exhibit lower enzymatic levels of glucocerebrosidase in the central nervous system (CNS), suggesting a possible link between the enzyme and the development of the disease. Previously, we have shown that early treatment with glucocerebrosidase can modulate α-synuclein aggregation in a presymptomatic mouse model of Gaucher-related synucleinopathy (Gba1 D409V/D409V) and ameliorate the associated cognitive deficit. To probe this link further, we have now evaluated the efficacy of augmenting glucocerebrosidase activity in the CNS of symptomatic Gba1 D409V/D409V mice and in a transgenic mouse model overexpressing A53T α-synuclein. Adeno-associated virus-mediated expression of glucocerebrosidase in the CNS of symptomatic Gba1 D409V/D409V mice completely corrected the aberrant accumulation of the toxic lipid glucosylsphingosine and reduced the levels of ubiquitin, tau, and proteinase K-resistant α-synuclein aggregates. Importantly, hippocampal expression of glucocerebrosidase in Gba1 D409V/D409V mice (starting at 4 or 12 mo of age) also reversed their cognitive impairment when examined using a novel object recognition test. Correspondingly, overexpression of glucocerebrosidase in the CNS of A53T α-synuclein mice reduced the levels of soluble α-synuclein, suggesting that increasing the glycosidase activity can modulate α-synuclein processing and may modulate the progression of α-synucleinopathies. Hence, increasing glucocerebrosidase activity in the CNS represents a potential therapeutic strategy for GBA1-related and non-GBA1-associated synucleinopathies, including PD.lysosomal storage diseases | mouse models | MAPT | memory defect M utations in the gene for glucocerebrosidase (GBA1) present the highest genetic risk factor for developing synucleinopathies such as Parkinson disease (PD) and dementia with Lewy bodies (DLB) (1-5). The central nervous system (CNS) of Gaucher patients and carriers who present with parkinsonism and dementia harbor deposits of α-synuclein-positive Lewy bodies (LBs) and Lewy neurites (LNs) in hippocampal neurons and their processes resembling those noted in patients with classical PD and DLB (6, 7). Aspects of these characteristics have also been noted in the CNS of several mouse models of neuropathic and nonneuropathic Gaucher disease (8-10). Consequently, a causal relationship has been suggested between the loss of glucocerebrosidase activity or the lysosomal accumulation of undegraded metabolites and the development of PD and DLB. A more direct link between glucocerebrosidase activity and α-synuclein metabolism has been highlighted by studies of Gaucher cells and mice indicating that a reduction in glucocerebrosidase activity by pharmacological or genetic interventions resulted in increased levels of α-synuclein aggregates (9-12). Moreover, a decrease in glucoce...

Inherited metabolic disorders that affect the central nervous system typically result in pathology throughout the brain; thus, gene therapy strategies need to achieve widespread delivery. We previously found that although intraventricular injection of the neonatal mouse brain with adeno-associated virus serotype 2 (AAV2) results in dispersed gene delivery, many brain structures were poorly transduced. This limitation may be overcome by using different AAV serotypes because the capsid proteins use different cellular receptors for entry, which may allow enhanced global targeting of the brain. We tested this with AAV1 and AAV5 vectors. AAV5 showed very limited brain transduction after neonatal injection, even though it has different transduction patterns than AAV2 in adult brain injections. In contrast, AAV1 vectors, which have not been tested in the brain, showed robust widespread transduction. Complementary patterns of transduction between AAV1 and AAV2 were established and maintained in the adult brain after neonatal injection. In the majority of structures, AAV1 transduced many more cells than AAV2. Both vectors transduced mostly neurons, indicating that differential expression of receptors on the surfaces of neurons occurs in the developing brain. The number of cells positive for a vector-encoded secreted enzyme (␤-glucuronidase) was notably greater and more widespread in AAV1-injected brains. A comprehensive analysis of AAV1-treated brains from ␤-glucuronidase-deficient mice (mucopolysaccharidosis type VII) showed complete reversal of pathology in all areas of the brain for at least 1 year, demonstrating that the combination of this serotype and experimental strategy is therapeutically effective for treating global neurometabolic disorders.Lysosomal storage diseases are inherited metabolic disorders characterized by mutations in genes encoding acid hydrolases (23). The loss of enzyme activity results in cell dysfunction due to accumulation of undegraded substrates in the lysosomal compartment. Most of these diseases have global storage lesions in the central nervous system (CNS) that result in progressive neurodegeneration and mental retardation. The collective frequency of all of the lysosomal storage diseases is approximately 1 in 5,000 live births, which represents about 20% of single-gene disorders that affect the brain (22, 31).Treatment strategies for the global pathology in the brain must include widespread gene delivery so that cellular "enzyme pumps" can become established throughout the CNS. These pumps would then distribute the enzyme to nontransduced areas by multiple mechanisms, such as secretion and uptake by distal cells (37), axonal transport (26), and migrating progenitor cells (26,35). We recently showed that widespread gene delivery can be achieved by injecting an adeno-associated virus type 2 (AAV2) vector directly into the cerebral lateral ventricles at birth, and allowing the cerebrospinal fluid to deliver the virus throughout the CNS (25). However, many areas of the brain did not show s...

Mutations in the CLN2 gene, which encodes a lysosomal serine protease, tripeptidyl-peptidase I (TPP I), result in an autosomal recessive neurodegenerative disease of children, classical late-infantile neuronal ceroid lipofuscinosis (cLINCL). cLINCL is inevitably fatal, and there currently exists no cure or effective treatment. In this report, we provide the characterization of the first CLN2-targeted mouse model for cLINCL. CLN2-targeted mice were fertile and apparently healthy at birth despite an absence of detectable TPP I activity. At ϳ7 weeks of age, neurological deficiencies became evident with the onset of a tremor that became progressively more severe and was eventually accompanied by ataxia. Lifespan of the affected mice was greatly reduced (median survival, 138 d), and extensive neuronal pathology was observed including a prominent accumulation of cytoplasmic storage material within the lysosomal-endosomal compartment, a loss of cerebellar Purkinje cells, and widespread axonal degeneration. The CLN2-targeted mouse therefore recapitulates much of the pathology and clinical features of cLINCL and represents an animal model that should provide clues to the normal cellular function of TPP I and the pathogenic processes that underlie neuronal death in its absence. In addition, the CLN2-targeted mouse also represents a valuable model for the evaluation of different therapeutic strategies.

Lentiviral vectors have proven to be promising tools for transduction of central nervous system (CNS) cells in vivo and in vitro. In this study, CNS transduction patterns of lentiviral vectors pseudotyped with envelope glycoproteins from Ebola virus, murine leukemia virus (MuLV), lymphocytic choriomeningitis virus (LCMV), or the rabies-related Mokola virus were compared to a vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Mokola-, LCMV-, and VSV-G-pseudotyped vectors transduced similar populations, including striatum, thalamus, and white matter. Mokola-pseudotyped vectors were the most efficient of the three. MuLV-pseudotyped lentivirus efficiently transduced striatum and hippocampal dentate gyrus. In contrast, no transduction resulted from injection of Ebola-pseudotyped virus in the CNS. The same pattern was observed in vitro with primary cultured oligodendrocytes. LCMV, MuLV, and Ebola pseudotypes were the most stable. These results demonstrate that targeted transduction in the CNS can be achieved using specific envelope glycoproteins to pseudotype lentiviral vectors, and support the use of Mokola-pseudotyped and MuLV-pseudotyped lentiviral vectors as efficient and stable alternatives to VSV-G-pseudotyped vectors for experiments in the mouse CNS.