Increasing Enzyme Mannose-6-Phosphate Levels but Not Miglustat Coadministration Enhances the Efficacy of Enzyme Replacement Therapy in Pompe Mice.

Pompe disease is a rare glycogen storage disorder caused by a deficiency in the lysosomal enzyme acid -glucosidase, which leads to muscle weakness, cardiac and respiratory failure, and early mortality. Alglucosidase alfa, a recombinant human acid -glucosidase, was the first approved treatment of Pompe disease, but its uptake into skeletal muscle via the cation-independent mannose-6-phosphate (M6P) receptor (CIMPR) is limited. Avalglucosidase alfa has received marketing authorization in several countries for infantile-onset and/or late-onset Pompe disease.

Secreted acid sphingomyelinase as a potential gene therapy for limb girdle muscular dystrophy 2B.

Efficient sarcolemmal repair is required for muscle cell survival, with deficits in this process leading to muscle degeneration. Lack of the sarcolemmal protein dysferlin impairs sarcolemmal repair by reducing secretion of the enzyme acid sphingomyelinase (ASM), and causes limb girdle muscular dystrophy 2B (LGMD2B). The large size of the dysferlin gene poses a challenge for LGMD2B gene therapy efforts aimed at restoring dysferlin expression in skeletal muscle fibers.

Priapism in a Fabry disease mouse model is associated with upregulated penile nNOS and eNOS expression.

Fabry disease is a glycosphingolipidosis caused by deficient activity of α-galactosidase A; it is one of a few diseases that are associated with priapism, an abnormal prolonged erection of the penis. The goal of this study was to investigate the pathogenesis of Fabry disease-associated priapism in a mouse model of the disease. We found that Fabry mice develop late-onset priapism.

Reversibility of neuropathology in Tay-Sachs-related diseases.

The GM2 gangliosidoses are progressive neurodegenerative disorders due to defects in the lysosomal β-N-acetylhexosaminidase system. Accumulation of β-hexosaminidases A and B substrates is presumed to cause this fatal condition. An authentic mouse model of Sandhoff disease (SD) with pathological characteristics resembling those noted in infantile GM2 gangliosidosis has been described.

Gene transfer corrects acute GM2 gangliosidosis--potential therapeutic contribution of perivascular enzyme flow.

The GM2 gangliosidoses are fatal lysosomal storage diseases principally affecting the brain. Absence of β-hexosaminidase A and B activities in the Sandhoff mouse causes neurological dysfunction and recapitulates the acute Tay-Sachs (TSD) and Sandhoff diseases (SD) in infants. Intracranial coinjection of recombinant adeno-associated viral vectors (rAAV), serotype 2/1, expressing human β-hexosaminidase α (HEXA) and β (HEXB) subunits into 1-month-old Sandhoff mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord.

Adeno-associated virus-mediated expression of β-hexosaminidase prevents neuronal loss in the Sandhoff mouse brain.

Sandhoff disease, a GM2 gangliosidosis caused by a deficiency in β-hexosaminidase, is characterized by progressive neurodegeneration. Although loss of neurons in association with lysosomal storage of glycosphingolipids occurs in patients with this disease, the molecular pathways that lead to the accompanying neurological defects are unclear. Using an authentic murine model of GM2 gangliosidosis, we examined the pattern of neuronal loss in the central nervous system and investigated the effects of gene transfer using recombinant adeno-associated viral vectors expressing β-hexosaminidase subunits (rAAV2/1-Hex).

Distribution of acid sphingomyelinase in rodent and non-human primate brain after intracerebroventricular infusion.

One treatment approach for lysosomal storage diseases (LSDs) is the systemic infusion of recombinant enzyme. Although this enzyme replacement is therapeutic for the viscera, many LSDs have central nervous system (CNS) components that are not adequately treated by systemic enzyme infusion. Direct intracerebroventricular (ICV) infusion of a high concentration of recombinant human acid sphingomyelinase (rhASM) into the CNS over a prolonged time frame (hours) has shown therapeutic efficacy in a mouse model of Niemann-Pick A (NP/A) disease.

Systemic administration of AAV8-α-galactosidase A induces humoral tolerance in nonhuman primates despite low hepatic expression.

In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal.

Adeno-associated virus-mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E(-/-) mice.

Background: The secretory form of acid sphingomyelinase (ASM) is postulated to play a key role in the retention and aggregation of lipoproteins in the subendothelial space of the arterial wall by converting sphingomyelin in lipoproteins into ceramide. The present study aimed to determine whether the level of circulating ASM activity affects lesion development in mouse model of atherosclerosis.

Methods: Apolipoprotein E deficient (ApoE(-/-) ) mice were injected intravenously with a recombinant adeno-associated virus (AAV8-ASM) that constitutively expressed high levels of human ASM in liver and plasma.

Preexisting immunity and low expression in primates highlight translational challenges for liver-directed AAV8-mediated gene therapy.

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models.

Evaluation of systemic follistatin as an adjuvant to stimulate muscle repair and improve motor function in Pompe mice.

Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice.

Glycoengineered acid alpha-glucosidase with improved efficacy at correcting the metabolic aberrations and motor function deficits in a mouse model of Pompe disease.

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart.

Pulmonary delivery of recombinant acid sphingomyelinase improves clearance of lysosomal sphingomyelin from the lungs of a murine model of Niemann-Pick disease.

Systemic administration of recombinant acid sphingomyelinase (rhASM) into ASM deficient mice (ASMKO) results in hydrolysis of the abnormal storage of sphingomyelin in lysosomes of the liver, spleen and lung. However, the efficiency with which the substrate is cleared from the lung, particularly the alveolar macrophages, appears to be lower than from the other visceral tissues. To determine if delivery of rhASM into the air spaces of the lung could enhance clearance of pulmonary sphingomyelin, enzyme was administered to ASMKO mice by intranasal instillation.

Systemic Insulin-like growth factor-1 reverses hypoalgesia and improves mobility in a mouse model of diabetic peripheral neuropathy.

Peripheral neuropathy is a particularly debilitating complication of both type 1 and type 2 diabetes characterized by sensory and motor neuron damage and decreased circulating levels of insulin-like growth factor 1 (IGF-1). Quite often, an early hyperalgesia is followed by hypoalgesia and muscle weakness. Hypoalgesia can lead to significant morbidity for which there is no current treatment.

Ability of adeno-associated virus serotype 8-mediated hepatic expression of acid alpha-glucosidase to correct the biochemical and motor function deficits of presymptomatic and symptomatic Pompe mice.

The availability of a murine model of Pompe disease has enabled an evaluation of the relative merits of various therapeutic paradigms, including gene therapy. We report here that administration of a recombinant adeno-associated virus serotype 8 (AAV8) vector (AAV8/DC190-GAA) encoding human acid alpha-glucosidase (GAA) into presymptomatic Pompe mice resulted in nearly complete correction of the lysosomal storage of glycogen in all the affected muscles. A relatively high dose of AAV8/DC190-GAA was necessary to attain a threshold level of GAA for inducing immunotolerance to the expressed enzyme and for correction of muscle function, coordination, and strength.

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.

Correction of the Biochemical and Functional Deficits in Fabry Mice Following AAV8-mediated Hepatic Expression of α-galactosidase A.

The advent of novel adeno-associated virus (AAV) serotype vectors with higher transduction activity has encouraged a re-evaluation of the merits of this delivery platform for a variety of diseases. We report here that administration of a recombinant AAV8-based serotype vector encoding human α-galactosidase A into Fabry mice facilitated more rapid and significantly higher levels of production of the enzyme than an AAV2 vector. This translated into improved clearance of globotriaosylceramide, the glycosphingolipid that accumulates in the lysosomes of affected Fabry cells, and to correction of the peripheral neuropathy shown associated with this disease.

Correction of the biochemical and functional deficits in fabry mice following AAV8-mediated hepatic expression of alpha-galactosidase A.

The advent of novel adeno-associated virus (AAV) serotype vectors with higher transduction activity has encouraged a re-evaluation of the merits of this delivery platform for a variety of diseases. We report here that administration of a recombinant AAV8-based serotype vector encoding human alpha-galactosidase A into Fabry mice facilitated more rapid and significantly higher levels of production of the enzyme than an AAV2 vector. This translated into improved clearance of globotriaosylceramide, the glycosphingolipid that accumulates in the lysosomes of affected Fabry cells, and to correction of the peripheral neuropathy shown associated with this disease.

Effective gene therapy in an authentic model of Tay-Sachs-related diseases.

Tay-Sachs disease is a prototypic neurodegenerative disease. Lysosomal storage of GM2 ganglioside in Tay-Sachs and the related disorder, Sandhoff disease, is caused by deficiency of beta-hexosaminidase A, a heterodimeric protein. Tay-Sachs-related diseases (GM2 gangliosidoses) are incurable, but gene therapy has the potential for widespread correction of the underlying lysosomal defect by means of the secretion-recapture cellular pathway for enzymatic complementation.

AAV8-mediated hepatic expression of acid sphingomyelinase corrects the metabolic defect in the visceral organs of a mouse model of Niemann-Pick disease.

Acid sphingomyelinase deficiency is a lysosomal storage disorder in which the defective lysosomal hydrolase fails to degrade sphingomyelin. The resulting accumulation of substrate in the lysosomes of histiocytic cells leads to hepatosplenomegaly and severe pulmonary inflammation. Administration of a recombinant AAV1 vector encoding human acid sphingomyelinase to acid sphingomyelinase knockout (ASMKO) mice effectively reduced the accumulated substrate in all of the affected visceral organs.

AAV2 vector harboring a liver-restricted promoter facilitates sustained expression of therapeutic levels of alpha-galactosidase A and the induction of immune tolerance in Fabry mice.

The successful application of gene therapy for the treatment of genetic diseases such as Fabry is reliant on the development of vectors that are safe and that facilitate sustained expression of therapeutic levels of the transgene product. Here, we report that intravenous administration of a recombinant AAV2 vector encoding human alpha-galactosidase A under the transcriptional control of a liver-restricted enhancer/promoter (AAV2/DC190-alphagal) generated significantly higher levels of expression in BALB/c and Fabry mice than could be realized using the ubiquitous CMV promoter (AAV2/CMVHI-alphagal). Moreover, AAV2/DC190-alphagal-mediated hepatic expression of alpha-galactosidase A was sustained for 12 months in BALB/c mice and was associated with a significantly reduced immune response to the expressed enzyme.

Partial correction of the alpha-galactosidase A deficiency and reduction of glycolipid storage in Fabry mice using synthetic vectors.

Background: Fabry disease is a recessive, X-linked disorder caused by a deficiency of the lysosomal enzyme alpha-galactosidase A, leading to an accumulation of the glycosphingolipid globotriaosylceramide (GL-3) in most tissues of the body. The goal of this study was to determine if systemic delivery of a nonviral vector could correct the enzyme deficiency and reduce the levels of GL-3 in different tissues of a transgenic knockout mouse model of the disease.

Methods: Cationic lipid was complexed with a CpG-depleted plasmid DNA vector and then injected intravenously into Fabry mice.

Demonstration of feasibility of in vivo gene therapy for Gaucher disease using a chemically induced mouse model.

Progress towards developing gene therapy for Gaucher disease has been hindered by the lack of an animal model. Here we describe a mouse model of Gaucher disease which has a chemically induced deficiency of glucocerebrosidase and that accumulates elevated levels of glucosylceramide (GL-1) in the lysosomes of Kupffer cells. Administration of mannose-terminated glucocerebrosidase (Cerezyme) resulted in the reduction of GL-1 levels in the livers of these animals.

Correction of the nonlinear dose response improves the viability of adenoviral vectors for gene therapy of Fabry disease.

Systemic administration of recombinant adenoviral vectors for gene therapy of chronic diseases such as Fabry disease can be limited by dose-dependent toxicity. Because administration of a high dose of Ad2/CMVHI-alpha gal encoding human alpha-galactosidase A results in expression of supraphysiological levels of the enzyme, we sought to determine whether lower doses would suffice to correct the enzyme deficiency and lysosomal storage abnormality observed in Fabry mice. Reducing the dose of Ad2/CMVHI-alpha gal by 10-fold (from 10(11) to 10(10) particles/mouse) resulted in a greater than 200-fold loss in transgene expression.

Adenovirus-transduced lung as a portal for delivering alpha-galactosidase A into systemic circulation for Fabry disease.

Gene therapy efforts have focused primarily on the use of either the liver or skeletal muscle as depot organs for the production of a variety of therapeutic proteins that act systemically. Here we examined the lung to determine whether it could function as yet another portal for the secretion of proteins into the circulation. Fabry disease is caused by a deficiency of the lysosomal hydrolase alpha-galactosidase A, resulting in the abnormal deposition of the glycosphingolipid globotriaosylceramide (GL-3) in vascular lysosomes.