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

"Of Mice and Measures": A Project to Improve How We Advance Duchenne Muscular Dystrophy Therapies to the Clinic.

A new line of dystrophic mdx mice on the DBA/2J (D2) background has emerged as a candidate to study the efficacy of therapeutic approaches for Duchenne muscular dystrophy (DMD). These mice harbor genetic polymorphisms that appear to increase the severity of the dystropathology, with disease modifiers that also occur in DMD patients, making them attractive for efficacy studies and drug development. This workshop aimed at collecting and consolidating available data on the pathological features and the natural history of these new D2/mdx mice, for comparison with classic mdx mice and controls, and to identify gaps in information and their potential value.

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.

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.

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).

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.

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.

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.

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.

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.

Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy.

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.

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.

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.

CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy.

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.

Intracerebroventricular infusion of acid sphingomyelinase corrects CNS manifestations in a mouse model of Niemann-Pick A disease.

Niemann-Pick A (NPA) disease is a lysosomal storage disorder (LSD) caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously, we showed that the storage pathology in the ASM knockout (ASMKO) mouse brain could be corrected by intracerebral injections of cell, gene and protein based therapies. However, except for instances where distal areas were targeted with viral vectors, correction of lysosomal storage pathology was typically limited to a region within a few millimeters from the injection site.

Development of a single vector system that enhances trans-splicing of SMN2 transcripts.

RNA modalities are developing as a powerful means to re-direct pathogenic pre-mRNA splicing events. Improving the efficiency of these molecules in vivo is critical as they move towards clinical applications. Spinal muscular atrophy (SMA) is caused by loss of SMN1.

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.

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.

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.

Intraparenchymal injections of acid sphingomyelinase results in regional correction of lysosomal storage pathology in the Niemann-Pick A mouse.

Niemann-Pick A disease (NPD-A) is caused by a deficiency of acid sphingomyelinase (ASM) leading to the intracellular accumulation of sphingomyelin and cholesterol in lysosomes. We evaluated the effects of direct intraparenchymal brain injections of purified recombinant human ASM (hASM) at correcting the storage pathology in a mouse model of NPD-A (ASMKO). Different doses (0.

Timing of therapeutic intervention determines functional and survival outcomes in a mouse model of late infantile batten disease.

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice.

Combination brain and systemic injections of AAV provide maximal functional and survival benefits in the Niemann-Pick mouse.

Niemann-Pick disease (NPD) is caused by the loss of acid sphingomyelinase (ASM) activity, which results in widespread accumulation of undegraded lipids in cells of the viscera and CNS. In this study, we tested the effect of combination brain and systemic injections of recombinant adeno-associated viral vectors encoding human ASM (hASM) in a mouse model of NPD. Animals treated by combination therapy exhibited high levels of hASM in the viscera and brain, which resulted in near-complete correction of storage throughout the body.

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.

Gene transfer of human acid sphingomyelinase corrects neuropathology and motor deficits in a mouse model of Niemann-Pick type A disease.

Niemann-Pick type A disease is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity. Previously we showed that storage pathology in the ASM knockout (ASMKO) mouse brain can be corrected by adeno-associated virus serotype 2 (AAV2)-mediated gene transfer. The present experiment compared the relative therapeutic efficacy of different recombinant AAV serotype vectors (1, 2, 5, 7, and 8) using histological, biochemical, and behavioral endpoints.

Identification of novel biomarkers for Niemann-Pick disease using gene expression analysis of acid sphingomyelinase knockout mice.

Although several therapies are available or being developed for lysosomal storage disorders (LSDs), assessment of therapeutic efficacy is challenged by the lack of markers to assess disease progression and severity. This is particularly true for rare diseases such as LSDs, since natural history data from human populations are often lacking. Herein we describe the use of gene expression analysis in the acid sphingomyelinase-deficient mouse model (ASMKO) of Types A and B Niemann-Pick disease (NPD) to identify novel serum biomarkers.