Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model.

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 2'-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 micro-osmotic 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.

SMN regulates axonal local translation via miR-183/mTOR pathway.

Reduced expression of SMN protein causes spinal muscular atrophy (SMA), a neurodegenerative disorder leading to motor neuron dysfunction and loss. However, the molecular mechanisms by which SMN regulates neuronal dysfunction are not fully understood. Here, we report that reduced SMN protein level alters miRNA expression and distribution in neurons. In particular, miR-183 levels are increased in neurites of SMN-deficient neurons. We demonstrate that miR-183 regulates translation of mTor via direct binding to its 3' UTR. Interestingly, local axonal translation of mTor is reduced in SMN-deficient neurons, and this can be recovered by miR-183 inhibition. Finally, inhibition of miR-183 expression in the spinal cord of an SMA mouse model prolongs survival and improves motor function of Smn-mutant mice. Together, these observations suggest that axonal miRNAs and the mTOR pathway are previously unidentified molecular mechanisms contributing to SMA pathology.

Augmenting CNS glucocerebrosidase activity as a therapeutic strategy for parkinsonism and other Gaucher-related synucleinopathies.

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.

Systemic administration of a recombinant AAV1 vector encoding IGF-1 improves disease manifestations in SMA mice.

Spinal muscular atrophy is a progressive motor neuron disease caused by a deficiency of survival motor neuron. In this study, we evaluated the efficacy of intravenous administration of a recombinant adeno-associated virus (AAV1) vector encoding human insulin-like growth factor-1 (IGF-1) in a severe mouse model of spinal muscular atrophy. Measurable quantities of human IGF-1 transcripts and protein were detected in the liver (up to 3 months postinjection) and in the serum indicating that IGF-1 was secreted from the liver into systemic circulation. Spinal muscular atrophy mice administered AAV1-IGF-1 on postnatal day 1 exhibited a lower extent of motor neuron degeneration, cardiac and muscle atrophy as well as a greater extent of innervation at the neuromuscular junctions compared to untreated controls at day 8 posttreatment. Importantly, treatment with AAV1-IGF-1 prolonged the animals' lifespan, increased their body weights and improved their motor coordination. Quantitative polymerase chain reaction and western blot analyses showed that AAV1-mediated expression of IGF-1 led to an increase in survival motor neuron transcript and protein levels in the spinal cord, brain, muscles, and heart. These data indicate that systemically delivered AAV1-IGF-1 can correct several of the biochemical and behavioral deficits in spinal muscular atrophy mice through increasing tissue levels of survival motor neuron.

CNS expression of glucocerebrosidase corrects alpha-synuclein pathology and memory in a mouse model of Gaucher-related synucleinopathy.

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.

Gene transfer to the CNS is efficacious in immune-primed mice harboring physiologically relevant titers of anti-AAV antibodies.

Central nervous system (CNS)-directed gene therapy with recombinant adeno-associated virus (AAV) vectors has been used effectively to slow disease course in mouse models of several neurodegenerative diseases. However, these vectors were typically tested in mice without prior exposure to the virus, an immunological scenario unlikely to be duplicated in human patients. Here, we examined the impact of pre-existing immunity on AAV-mediated gene delivery to the CNS of normal and diseased mice. Antibody levels in brain tissue were determined to be 0.6% of the levels found in systemic circulation. As expected, transgene expression in brains of mice with relatively high serum antibody titers was reduced by 59-95%. However, transduction activity was unaffected in mice that harbored more clinically relevant antibody levels. Moreover, we also showed that markers of neuroinflammation (GFAP, Iba1, and CD3) and histopathology (hematoxylin and eosin (H&E)) were not enhanced in immune-primed mice (regardless of pre-existing antibody levels). Importantly, we also demonstrated in a mouse model of Niemann Pick Type A (NPA) disease that pre-existing immunity did not preclude either gene transfer to the CNS or alleviation of disease-associated neuropathology. These findings support the continued development of AAV-based therapies for the treatment of neurological disorders.

Merits of combination cortical, subcortical, and cerebellar injections for the treatment of Niemann-Pick disease type A.

Niemann-Pick disease Type A (NPA) is a neuronopathic lysosomal storage disease (LSD) caused by the loss of acid sphingomyelinase (ASM). The goals of the current study are to ascertain the levels of human ASM that are efficacious in ASM knockout (ASMKO) mice, and determine whether these levels can be attained in non-human primates (NHPs) using a multiple parenchymal injection strategy. Intracranial injections of different doses of AAV1-hASM in ASMKO mice demonstrated that only a small amount of enzyme (<0.5 mg hASM/g tissue) was sufficient to increase survival, and that increasing the amount of hASM did not enhance this survival benefit until a new threshold level of >10 mg hASM/g tissue was reached. In monkeys, injection of 12 tracts of AAV1-hASM resulted in efficacious levels of enzyme in broad regions of the brain that was aided, in part, by axonal transport of adeno-associated virus (AAV) and movement through the perivascular space. This study demonstrates that a combination cortical, subcortical, and cerebellar injection protocol could provide therapeutic levels of hASM to regions of the NHP brain that are highly affected in NPA patients. The information from this study might help design new AAV-mediated enzyme replacement protocols for NPA and other neuronopathic LSDs in future clinical trials.

IGF-1 delivery to CNS attenuates motor neuron cell death but does not improve motor function in type III SMA mice.

The efficacy of administering a recombinant adeno-associated virus (AAV) vector encoding human IGF-1 (AAV2/1-hIGF-1) into the deep cerebellar nucleus (DCN) of a type III SMA mouse model was evaluated. High levels of IGF-1 transcripts and protein were detected in the spinal cord at 2 months post-injection demonstrating that axonal connections between the cerebellum and spinal cord were able to act as conduits for the viral vector and protein to the spinal cord. Mice treated with AAV2/1-hIGF-1 and analyzed 8 months later showed changes in endogenous Bax and Bcl-xl levels in spinal cord motor neurons that were consistent with IGF-1-mediated anti-apoptotic effects on motor neurons. However, although AAV2/1-hIGF-1 treatment reduced the extent of motor neuron cell death, the majority of rescued motor neurons were non-functional, as they lacked axons that innervated the muscles. Furthermore, treated SMA mice exhibited abnormal muscle fibers, aberrant neuromuscular junction structure, and impaired performance on motor function tests. These data indicate that although CNS-directed expression of IGF-1 could reduce motor neuron cell death, this did not translate to improvements in motor function in an adult mouse model of type III SMA.

A cell-penetrating peptide enhances delivery and efficacy of phosphorodiamidate morpholino oligomers in mdx mice.

Antisense RNA technology is a strategy for the treatment of Duchenne muscular dystrophy (DMD), a progressive and universally fatal X-linked neuromuscular disease caused by frameshift mutations in the gene encoding dystrophin. Phosphorodiamidate morpholino oligomers (PMOs) are an antisense RNA platform that is used clinically in patients with DMD to facilitate exon skipping and production of an internally truncated, yet functional, dystrophin protein. Peptide-conjugated PMOs (PPMOs) are a next-generation platform in which a cell-penetrating peptide is conjugated to the PMO backbone, with the goal of increasing cellular uptake. RC-1001 is a PPMO that contains a proprietary cell-penetrating peptide and targets the Dmd mutation in mdx mice. It was evaluated in mdx mice for exon 23 skipping, dystrophin production, and functional efficacy. Single-dose RC-1001 dose dependently increased exon skipping and dystrophin protein levels in striated muscle and is associated with improvements in muscle function. Dystrophin protein levels were durable for 60 days. Three doses, each given 1 month apart, increased exon skipping to 99% in quadriceps and 43% in heart, with dystrophin protein levels at 39% and 9% of wild type, respectively. These findings support clinical development of PPMO therapies for the treatment of DMD.

Intraventricular enzyme replacement improves disease phenotypes in a mouse model of late infantile neuronal ceroid lipofuscinosis.

Late infantile neuronal ceroid lipofuscinosis (LINCL) is an autosomal recessive neurodegenerative disease caused by mutations in CLN2, which encodes the lysosomal protease tripeptidyl peptidase 1 (TPP1). LINCL is characterized clinically by progressive motor and cognitive decline, and premature death. Enzyme-replacement therapy (ERT) is currently available for lysosomal storage diseases affecting peripheral tissues, but has not been used in patients with central nervous system (CNS) involvement. Enzyme delivery through the cerebrospinal fluid is a potential alternative route to the CNS, but has not been studied for LINCL. In this study, we identified relevant neuropathological and behavioral hallmarks of disease in a mouse model of LINCL and correlated those findings with tissues from LINCL patients. Subsequently, we tested if intraventricular delivery of TPP1 to the LINCL mouse was efficacious. We found that infusion of recombinant human TPP1 through an intraventricular cannula led to enzyme distribution in several regions of the brain of treated mice. In vitro activity assays confirm increased TPP1 activity throughout the rostral-caudal extent of the brain. Importantly, treated mice showed attenuated neuropathology, and decreased resting tremor relative to vehicle-treated mice. This data demonstrates that intraventricular enzyme delivery to the CNS is feasible and may be of therapeutic value.

Delivery of AAV-IGF-1 to the CNS extends survival in ALS mice through modification of aberrant glial cell activity.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor system. Recent work in rodent models of ALS has shown that insulin-like growth factor-1 (IGF-1) slows disease progression when delivered at disease onset. However, IGF-1's mechanism of action along the neuromuscular axis remains unclear. In this study, symptomatic ALS mice received IGF-1 through stereotaxic injection of an IGF-1-expressing viral vector to the deep cerebellar nuclei (DCN), a region of the cerebellum with extensive brain stem and spinal cord connections. We found that delivery of IGF-1 to the central nervous system (CNS) reduced ALS neuropathology, improved muscle strength, and significantly extended life span in ALS mice. To explore the mechanism of action of IGF-1, we used a newly developed in vitro model of ALS. We demonstrate that IGF-1 is potently neuroprotective and attenuates glial cell-mediated release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). Our results show that delivering IGF-1 to the CNS is sufficient to delay disease progression in a mouse model of familial ALS and demonstrate for the first time that IGF-1 attenuates the pathological activity of non-neuronal cells that contribute to disease progression. Our findings highlight an innovative approach for delivering IGF-1 to the CNS.

Temporal neuropathologic and behavioral phenotype of 6neo/6neo Pompe disease mice.

Pompe disease (glycogen storage disease II) is caused by mutations in the acid alpha-glucosidase gene. The most common form is rapidly progressive with glycogen storage, particularly in muscle, which leads to profound weakness, cardiac failure, and death by the age of 2 years. Although usually considered a muscle disease, glycogen storage also occurs in the CNS. We evaluated the progression of neuropathologic and behavioral abnormalities in a Pompe disease mouse model (6neo/6neo) that displays many features of the human disease. Homozygous mutant mice store excess glycogen within large neurons of hindbrain, spinal cord, and sensory ganglia by the age of 1 month; accumulations then spread progressively within many CNS cell types. "Silver degeneration" and Fluoro-Jade C stains revealed severe degeneration in axon terminals of primary sensory neurons at 3 to 9 months. These abnormalities were accompanied by progressive behavioral impairment on rotorod, wire hanging, and foot fault tests. The extensive neuropathologic alterations in this model suggest that therapy of skeletal and cardiac muscle disorders by systemic enzyme replacement therapy may not be sufficient to reverse functional deficits due to CNS glycogen storage, particularly early-onset, rapidly progressive disease. A better understanding of the basis for clinical manifestations is needed to correlate CNS pathology with Pompe disease manifestations.

Intracranial delivery of CLN2 reduces brain pathology in a mouse model of classical late infantile neuronal ceroid lipofuscinosis.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a lysosomal storage disorder caused by mutations in CLN2, which encodes lysosomal tripeptidyl peptidase I (TPP1). Lack of TPP1 results in accumulation of autofluorescent storage material and curvilinear bodies in cells throughout the CNS, leading to progressive neurodegeneration and death typically in childhood. In this study, we injected adeno-associated virus (AAV) vectors containing the human CLN2 cDNA into the brains of CLN2(-/-) mice to determine therapeutic efficacy. AAV2CUhCLN2 or AAV5CUhCLN2 were stereotaxically injected into the motor cortex, thalamus, and cerebellum of both hemispheres at 6 weeks of age, and mice were then killed at 13 weeks after injection. Mice treated with AAV2CUhCLN2 and AAV5CUhCLN2 contained TPP1 activity at each injection tract that was equivalent to 0.5- and 2-fold that of CLN2(+/+) control mice, respectively. Lysosome-associated membrane protein 1 immunostaining and confocal microscopy showed intracellular targeting of TPP1 to the lysosomal compartment. Compared with control animals, there was a marked reduction of autofluorescent storage in the AAV2CUhCLN2 and AAV5CUhCLN2 injected brain regions, as well as adjacent regions, including the striatum and hippocampus. Analysis by electron microscopy confirmed a significant decrease in pathological curvilinear bodies in cells. This study demonstrates that AAV-mediated TPP1 enzyme replacement corrects the hallmark cellular pathologies of cLINCL in the mouse model and raises the possibility of using AAV gene therapy to treat cLINCL patients.

Distribution of a lysosomal enzyme in the adult brain by axonal transport and by cells of the rostral migratory stream.

A portion of the lysosomal enzymes produced by cells is secreted, diffuses through extracellular spaces, and can be taken up by distal cells via mannose-6-phosphate receptor-mediated endocytosis. This provides the basis for treating lysosomal storage diseases, many of which affect the CNS. Normal enzyme secreted from a cluster of genetically corrected cells has been shown to reverse storage lesions in a zone of surrounding brain tissue in mouse disease models. However, low levels of enzyme activity and reduction of storage lesions also have been observed at sites in the brain that may not be explained by a contiguous gradient of secreted enzyme diffusing away from the genetically corrected cells. No direct evidence for alternative mechanisms of enzyme transport has been shown, and little is understood about the intracellular movement of lysosomal enzymes in neurons. We investigated whether axonal transport could occur, by expressing an eukaryotic lysosomal enzyme that can be visualized in tissue sections (beta-glucuronidase) in brain structures that have defined axonal connections to other structures. This resulted in the transfer of enzyme to, and a reversal of storage lesions in, neurons that project to the gene expression site, but not in nearby structures that would have been corrected if the effect had been mediated by diffusion. In addition, transduction of cells in the subventricular zone resulted in the uptake of beta-glucuronidase by cells entering the rostral migratory stream. Gene transfer to specific neuronal circuits or cells in migratory pathways may facilitate delivery to the global brain lesions found in these disorders.

A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration.

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 approximately 7 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.

Transduction of the choroid plexus and ependyma in neonatal mouse brain by vesicular stomatitis virus glycoprotein-pseudotyped lentivirus and adeno-associated virus type 5 vectors.

Evaluation of gene transfer into the developing mouse brain has shown that when adeno-associated virus serotype 1 (AAV1) or AAV2 vectors are injected into the cerebral lateral ventricles at birth, widespread parenchymal transduction occurs. Lentiviral vectors have not been tested by this route. In this study, we found that injection of lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) resulted in targeted transduction of the ependymal cells lining the ventricular system and the choroid plexus along the entire rostrocaudal axis of the brain, whereas a Mokola pseudotype transduced only a few cells after injection into the neonatal ventricle. In contrast, when lentiviral vectors pseudotyped with either VSV-G or Mokola glycoprotein are injected into the adult mouse brain, they transduce similar patterns of cells. An Ebola-Zaire-pseudotyped vector did not transduce any neonatal CNS cells, as was also the case for adult parenchymal injections. Long-term gene expression (12 months) occurred with a constitutively active mammalian promoter and a self-inactivating long terminal repeat (LTR), whereas the cytomegalovirus promoter in a vector with an intact LTR was expressed only in short-term experiments. We found that an AAV5 vector also targeted the ependymal and choroid plexus cells throughout the ventricular system. This vector exhibited limited penetration from the ventricle to other structures, which was significantly different from the previously reported patterns of transduction after intraventricular injection of AAV1 and AAV2 vectors.

Genetically modified NT2N human neuronal cells mediate long-term gene expression as CNS grafts in vivo and improve functional cognitive outcome following experimental traumatic brain injury.

Human Ntera-2 (NT2) cells can be differentiated in vitro into well-characterized populations of NT2N neurons that engraft and mature when transplanted into the adult CNS of rodents and humans. They have shown promise as treatments for neurologic disease, trauma, and ischemic stroke. Although these features suggest that NT2N neurons would be an excellent platform for ex vivo gene therapy in the CNS, stable gene expression has been surprisingly difficult to achieve in these cells. In this report we demonstrate stable, efficient, and nontoxic gene transfer into undifferentiated NT2 cells using a pseudotyped lentiviral vector encoding the human elongation factor 1-alpha promoter and the reporter gene eGFP. Expression of eGFP was maintained when the NT2 cells were differentiated into NT2N neurons after treatment with retinoic acid. When transplanted into the striatum of adult nude mice, transduced NT2N neurons survived, engrafted, and continued to express the reporter gene for long-term time points in vivo. Furthermore, transplantation of NT2N neurons genetically modified to express nerve growth factor significantly attenuated cognitive dysfunction following traumatic brain injury in mice. These results demonstrate that defined populations of genetically modified human NT2N neurons are a practical and effective platform for stable ex vivo gene delivery into the CNS.

Gene delivery to the mouse brain with adeno-associated virus.

The efficient transduction of postmitotic cells by adeno-associated virus (AAV) makes it an excellent vector to deliver marker, functional, or therapeutic genes to the mammalian brain. An attractive feature of AAV is that all the viral-coding sequences are removed when engineering the recombinant genome, thereby limiting the extent of cell toxicity and immune response that are often associated with viral gene transcription. Of the seven described AAV serotypes, AAV serotype-2 (AAV2) is the most studied gene-transfer vehicle for in the mammalian brain. A feature of AAV2 transduction in the brain is that the vector remains confined to the injection site and predominately infects neurons rather than glia (2-8). The limited diffusion of AAV2 vectors is beneficial for controlled gene delivery. For instance, targeting therapeutic genes only to brain structures showing pathology would eliminate complications associated with vector diffusion and subsequent expression in healthy structures, and is an important consideration when designing treatment strategies for localized neurodegenerative diseases. The same is true for other experimental paradigms, such as investigating the function of genes in specific brain structures or using marker genes in tract-tracing experiments. Although AAV2 vectors were shown to remain predominately at the injection site, one study demonstrated that the vector itself may undergo axonal transport in inter-regional systems (9).

Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by mutations in survival motor neuron 1 (SMN1). Previously, we showed that central nervous system (CNS) delivery of an adeno-associated viral (AAV) vector encoding SMN1 produced significant improvements in survival in a mouse model of SMA. Here, we performed a dose-response study in SMA mice to determine the levels of SMN in the spinal cord necessary for efficacy, and measured the efficiency of motor neuron transduction in the spinal cord after intrathecal delivery in pigs and nonhuman primates (NHPs). CNS injections of 5e10, 1e10, and 1e9 genome copies (gc) of self-complementary AAV9 (scAAV9)-hSMN1 into SMA mice extended their survival from 17 to 153, 70, and 18 days, respectively. Spinal cords treated with 5e10, 1e10, and 1e9 gc showed that 70-170%, 30-100%, and 10-20% of wild-type levels of SMN were attained, respectively. Furthermore, detectable SMN expression in a minimum of 30% motor neurons correlated with efficacy. A comprehensive analysis showed that intrathecal delivery of 2.5e13 gc of scAAV9-GFP transduced 25-75% of the spinal cord motor neurons in NHPs. Thus, the extent of gene expression in motor neurons necessary to confer efficacy in SMA mice could be obtained in large-animal models, justifying the continual development of gene therapy for SMA.

Prospects for the gene therapy of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by a deficiency of functional SMN protein because of mutations in SMN1. A decrease in SMN activity results in motor neuron cell loss in the spinal cord, leading to a weakness of the proximal muscles responsible for crawling, walking, head/neck control and swallowing as well as the involuntary muscles that control breathing and coughing. Thus, patients present with pulmonary manifestations, paralysis and a shortened lifespan. Gene therapy is emerging as a promising therapeutic strategy for SMA given that the molecular basis for this monogenic disorder is well established. Recent advances and findings from preclinical studies in animal models provide optimism that gene therapy might be an effective therapeutic strategy for treating SMA.