Systemic delivery of a glucosylceramide synthase inhibitor reduces CNS substrates and increases lifespan in a mouse model of type 2 Gaucher disease.

Neuropathic Gaucher disease (nGD), also known as type 2 or type 3 Gaucher disease, is caused by a deficiency of the enzyme glucocerebrosidase (GC). This deficiency impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to their accumulation in the brains of patients and mouse models of the disease. These accumulated substrates have been thought to cause the severe neuropathology and early death observed in patients with nGD and mouse models. Substrate accumulation is evident at birth in both nGD mouse models and humans affected with the most severe type of the disease. Current treatment of non-nGD relies on the intravenous delivery of recombinant human glucocerebrosidase to replace the missing enzyme or the administration of glucosylceramide synthase inhibitors to attenuate GluCer production. However, the currently approved drugs that use these mechanisms do not cross the blood brain barrier, and thus are not expected to provide a benefit for the neurological complications in nGD patients. Here we report the successful reduction of substrate accumulation and CNS pathology together with a significant increase in lifespan after systemic administration of a novel glucosylceramide synthase inhibitor to a mouse model of nGD. To our knowledge this is the first compound shown to cross the blood brain barrier and reduce substrates in this animal model while significantly enhancing its lifespan. These results reinforce the concept that systemically administered glucosylceramide synthase inhibitors could hold enhanced therapeutic promise for patients afflicted with neuropathic lysosomal storage diseases.

Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease.

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.

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. To evaluate whether such an approach would translate to a larger brain, rhASM was infused into the lateral ventricles of both rats and Rhesus macaques, and the resulting distribution of enzyme characterized qualitatively and quantitatively. In both species, ICV infusion of rhASM resulted in parenchymal distribution of enzyme at levels that were therapeutic in the NP/A mouse model. Enzyme distribution was global in nature and exhibited a relatively steep gradient from the cerebrospinal fluid compartment to the inner parenchyma. Additional optimization of an ICV delivery approach may provide a therapeutic option for LSDs with neurologic involvement.

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. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.

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. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.

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. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.

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. Administration of AAV8/DC190-GAA into older Pompe mice with overt disease manifestations was also effective at correcting the lysosomal storage abnormality. However, these older mice exhibited only marginal improvements in motor function and no improvement in muscle strength. Examination of histologic sections showed evidence of skeletal muscle degeneration and fibrosis in aged Pompe mice whose symptoms were abated or rescued by early but not late treatment with AAV8/DC190-GAA. These results suggest that AAV8-mediated hepatic expression of GAA was effective at addressing the biochemical and functional deficits in Pompe mice. However, early therapeutic intervention is required to maintain significant muscle function and should be an important consideration in the management and treatment of Pompe 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. The higher levels of alpha-galactosidase A expression also allowed for a more rapid induction of immunotolerance to the enzyme. Recombinant AAV8 vectors that facilitated hepatic-restricted expression of high levels of alpha-galactosidase A conferred immunotolerance to the expressed enzyme as early as 30 days post-treatment. Animals expressing lower levels of the hydrolase, such as those treated with an AAV2-based vector or with lower doses of the AAV8-based vector, were also able to develop immunotolerance, but only after a more extended time period. Adoptive transfer of T cells isolated from the spleens of immunotolerized mice suppressed the formation of antibodies in naïve recipient animals, suggesting the possible role of regulatory T cells in effecting this state.

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. The higher levels of α-galactosidase A expression also allowed for a more rapid induction of immunotolerance to the enzyme. Recombinant AAV8 vectors that facilitated hepatic-restricted expression of high levels of α-galactosidase A conferred immunotolerance to the expressed enzyme as early as 30 days post-treatment. Animals expressing lower levels of the hydrolase, such as those treated with an AAV2-based vector or with lower doses of the AAV8-based vector, were also able to develop immunotolerance, but only after a more extended time period. Adoptive transfer of T cells isolated from the spleens of immunotolerized mice suppressed the formation of antibodies in naïve recipient animals, suggesting the possible role of regulatory T cells in effecting this state.

Inhibition of pulmonary fibrosis by the chemokine IP-10/CXCL10.

Pulmonary fibrosis is an enigmatic and devastating disease with few treatment options, now thought to result from abnormal wound healing in the lung in response to injury. We have previously noted a role for the chemokine interferon gamma-inducible protein of 10 kD (IP-10)/CXC chemokine ligand 10 in the regulation of cutaneous wound healing, and consequently investigated whether IP-10 regulates pulmonary fibrosis. We found that IP-10 is highly expressed in a mouse model of pulmonary fibrosis induced by bleomycin. IP-10-deficient mice exhibited increased pulmonary fibrosis after administration of bleomycin, suggesting that IP-10 limits the development of fibrosis in this model. Substantial fibroblast chemoattractant and proliferative activities were generated in the lung after bleomycin exposure. IP-10 significantly inhibited fibroblast responses to the chemotactic, but not the proliferative activity generated, suggesting that IP-10 may attenuate fibroblast accumulation in bleomycin-induced pulmonary fibrosis by limiting fibroblast migration. Consistent with this inhibitory activity of IP-10 on fibroblast migration, fibroblast accumulation in the lung after bleomycin exposure was dramatically increased in IP-10-deficient mice compared with wild-type mice. Conversely, transgenic mice overexpressing IP-10 were protected from mortality after bleomycin exposure, and demonstrated decreased fibroblast accumulation in the lung after challenge compared with wild-type mice. Our findings suggest that interruption of fibroblast recruitment may represent a novel therapeutic strategy for pulmonary fibrosis, which could have applicability to a wide range of fibrotic illnesses.

Leukotriene B4 receptor BLT1 mediates early effector T cell recruitment.

Leukotriene B4 (LTB4) was originally described as a potent lipid myeloid cell chemoattractant, rapidly generated from innate immune cells, that activates leukocytes through the G protein-coupled receptor BLT1. We report here that BLT1 is expressed on effector CD4+ T cells generated in vitro as well as in vivo when effector T cells migrate out of the lymphoid compartment and are recruited into peripheral tissues. BLT1 mediated LTB4-induced T helper type 1 (T(H)1) and T(H)2 cell chemotaxis and firm adhesion to endothelial cells under flow, as well as early CD4+ and CD8+ T cell recruitment into the airway in an asthma model. Our findings show that the LTB4-BLT1 pathway is involved in linking early immune system activation and early effector T cell recruitment.