Kristin M. Taylor

Inhibition of glycogen biosynthesis via mTORC1 suppression as an adjunct therapy for Pompe disease.

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1-2years of age to a more slowly progressive course that causes significant morbidity and early mortality in children and adults. Recombinant human GAA (rhGAA) improves clinical outcomes with variable results. Adjunct therapy that increases the effectiveness of rhGAA may benefit some Pompe patients. Co-administration of the mTORC1 inhibitor rapamycin with rhGAA in a GAA knockout mouse reduced muscle glycogen content more than rhGAA or rapamycin alone. These results suggest mTORC1 inhibition may benefit GSDs that involve glycogen accumulation in muscle.

Antisense Oligonucleotide-mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease.

Pompe disease is an autosomal recessive disorder caused by a deficiency of acid α-glucosidase (GAA; EC 3.2.1.20) and the resultant progressive lysosomal accumulation of glycogen in skeletal and cardiac muscles. Enzyme replacement therapy using recombinant human GAA (rhGAA) has proven beneficial in addressing several aspects of the disease such as cardiomyopathy and aberrant motor function. However, residual muscle weakness, hearing loss, and the risks of arrhythmias and osteopenia persist despite enzyme therapy. Here, we evaluated the relative merits of substrate reduction therapy (by inhibiting glycogen synthesis) as a potential adjuvant strategy. A phosphorodiamidate morpholino oligonucleotide (PMO) designed to invoke exon skipping and premature stop codon usage in the transcript for muscle specific glycogen synthase (Gys1) was identified and conjugated to a cell penetrating peptide (GS-PPMO) to facilitate PMO delivery to muscle. GS-PPMO systemic administration to Pompe mice led to a dose-dependent decrease in glycogen synthase transcripts in the quadriceps, and the diaphragm but not the liver. An mRNA response in the heart was seen only at the higher dose tested. Associated with these decreases in transcript levels were correspondingly lower tissue levels of muscle specific glycogen synthase and activity. Importantly, these reductions resulted in significant decreases in the aberrant accumulation of lysosomal glycogen in the quadriceps, diaphragm, and heart of Pompe mice. Treatment was without any overt toxicity, supporting the notion that substrate reduction by GS-PPMO-mediated inhibition of muscle specific glycogen synthase represents a viable therapeutic strategy for Pompe disease after further development.

Dysregulation of Multiple Facets of Glycogen Metabolism in a Murine Model of Pompe Disease

Pompe disease, also known as glycogen storage disease (GSD) type II, is caused by deficiency of lysosomal acid α-glucosidase (GAA). The resulting glycogen accumulation causes a spectrum of disease severity ranging from a rapidly progressive course that is typically fatal by 1 to 2 years of age to a slower progressive course that causes significant morbidity and early mortality in children and adults. The aim of this study is to better understand the biochemical consequences of glycogen accumulation in the Pompe mouse. We evaluated glycogen metabolism in heart, triceps, quadriceps, and liver from wild type and several strains of GAA−/− mice. Unexpectedly, we observed that lysosomal glycogen storage correlated with a robust increase in factors that normally promote glycogen biosynthesis. The GAA−/− mouse strains were found to have elevated glycogen synthase (GS), glycogenin, hexokinase, and glucose-6-phosphate (G-6-P, the allosteric activator of GS). Treating GAA−/− mice with recombinant human GAA (rhGAA) led to a dramatic reduction in the levels of glycogen, GS, glycogenin, and G-6-P. Lysosomal glycogen storage also correlated with a dysregulation of phosphorylase, which normally breaks down cytoplasmic glycogen. Analysis of phosphorylase activity confirmed a previous report that, although phosphorylase protein levels are identical in muscle lysates from wild type and GAA−/− mice, phosphorylase activity is suppressed in the GAA−/− mice in the absence of AMP. This reduction in phosphorylase activity likely exacerbates lysosomal glycogen accumulation. If the dysregulation in glycogen metabolism observed in the mouse model of Pompe disease also occurs in Pompe patients, it may contribute to the observed broad spectrum of disease severity.

Induction of immune tolerance to a therapeutic protein by intrathymic gene delivery.

The efficacy of recombinant enzyme therapy for genetic diseases is limited in some patients by the generation of a humoral immune response to the therapeutic protein. Inducing immune tolerance to the protein prior to treatment has the potential to increase therapeutic efficacy. Using an AAV8 vector encoding human acid α-glucosidase (hGAA), we have evaluated direct intrathymic injection for inducing tolerance. We have also compared the final tolerogenic states achieved by intrathymic and intravenous injection. Intrathymic vector delivery induced tolerance equivalent to that generated by intravenous delivery, but at a 25-fold lower dose, the thymic hGAA expression level was 10,000-fold lower than the liver expression necessary for systemic tolerance induction. Splenic regulatory T cells (Tregs) were apparent after delivery by both routes, but with different phenotypes. Intrathymic delivery resulted in Tregs with higher FoxP3, TGFβ, and IL-10 mRNA levels. These differences may account for the differences noted in splenic T cells, where only intravenous delivery appeared to inhibit their activation. Our results imply that different mechanisms may be operating to generate immune tolerance by intrathymic and intravenous delivery of an AAV vector, and suggest that the intrathymic route may hold promise for decreasing the humoral immune response to therapeutic proteins in genetic disease indications.

Adeno‐associated virus‐mediated expression of acid sphingomyelinase decreases atherosclerotic lesion formation in apolipoprotein E−/− mice

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.

S6 Kinase 2 Deficiency Improves Glucose Disposal in Mice Fed a High FatDiet

The mammalian target of rapamycin complex 1 (mTORC1) regulates insulin-mediated glucose metabolism, cell proliferation, the oxidative branch of the pentose phosphate pathway, de novo lipogenesis, and autophagy. Ribosomal S6 kinase 1 (S6K1) and 2 (S6K2) are downstream effectors of mTORC1. To characterize the role of S6K2 in insulin-mediated metabolism, the response of S6K2 deficient mice (S6K2-/-) to a glucose challenge was compared to that of wild-type (C57BL/6) and diabetes resistant strains (BALB/c and A/J) after 35 weeks on a high fat diet (HFD). Although S6K2-/- mice fed a HFD gained as much weight as the wild-type C57BL/6 control mice, unlike the wild-type mice they remained glucose tolerant, insulin sensitive, and had lower basal blood glucose levels. Moreover, unlike S6K1 deficient mice, S6K2-/- mice have increased basal plasma insulin levels and increased β-cell mass compared to C57BL/6, BALB/c, and A/J mice. Administration of insulin to S6K2-/- and C57BL/6 mice fed a Standard Diet (SD) resulted in phosphorylation of Ser307 on skeletal muscle Insulin Receptor Substrate 1 (IRS-1); however, when both strains were fed a HFD, phosphorylation of IRS-1 Ser307 was maintained in S6K2-/- mice but inhibited in C57BL/6 mice. Taken together, these results suggest that S6K2 inhibition may represent a strategy for treating type 2 diabetes.

531. Balloon Catheter-Mediated Hepatic Vein Delivery of a Viral Vector Mitigates Neutralization by Anti-Viral Antibodies and Results in Efficient Transduction of Rabbit Liver

The liver, and in particular, hepatocytes, represent an attractive target for both viral and non-viral gene transfer vectors. However, there are certain delivery issues associated with both of these vector systems that make their use in humans problematic. In particular, for viral vectors, the existence of anti-viral antibodies in the general population represents a potential barrier to their effective use in the clinic. Building upon our earlier success using balloon catheters to deliver plasmid DNA by way of the hepatic venous circulation to rabbit liver, we have used this same model to ask if we could use a similar approach to deliver a model viral vector. Specifically, we have compared a local, balloon catheter-mediated hepatic vein delivery protocol to systemic delivery of a CMV-driven adenoviral vector expressing b-galactosidase in terms of liver transduction efficiency, toxicity, and cell types transduced. We have also made this comparison in the presence of defined anti-AdV antibody titers in the recipient rabbits. In the naive animal, local balloon catheter-based delivery conferred an advantage in overall liver transduction when compared to systemic delivery of an identical dose of virus. In rabbits bearing anti-AdV antibody titers equivalent to those found in pooled human serum, this difference in expression between local and systemic delivery was even more striking. Importantly, in the presence of passively-administered anti-AdV antibodies, balloon-catheter mediated delivery resulted in expression levels that were comparable to those obtained by systemic delivery of an equivalent dose in a naive animal. Since in general, systemic delivery of AdV in naive animal models results in expression levels of secreted proteins regarded as therapeutic, the present results predict that retrograde delivery by a hepatic vein route using a balloon catheter should result in therapeutic levels of expression, even in the presence of average human levels of anti-AdV antibodies. Further support for this hypothesis is the finding that this local hepatic vein delivery approach resulted in the majority of expression originating from hepatocytes even in the passively immunized animals. In contrast, systemic delivery of AdV in passively immunized animals resulted in the majority of expression originating from non-hepatocytes. This latter finding implies that this local approach should help minimize the transduction of antigen-presenting cells, thereby reducing any immune consequences of viral transduction. Taken together, these data suggest that coupled with additional features such as a hepatocyte-specific expression cassette, balloon catheter viral-mediated transduction of the liver may represent a practical clinical approach for treating indications for which hepatocytes can be used as a depot for therapeutic protein production.