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.

Species-specific differences in the processing of acid α-glucosidase are due to the amino acid identity at position 201.

Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R(200) and A(204) inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAA(H201L) but not rhGAA(WT) to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAA(H201L) but not rhGAA(WT) was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues.

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, TGFß, 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.

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.

Role of viral hemagglutinin glycosylation in anti-influenza activities of recombinant surfactant protein D.

BACKGROUND: Surfactant protein D (SP-D) plays an important role in innate defense against influenza A viruses (IAVs) and other pathogens. METHODS: We tested antiviral activities of recombinant human SP-D against a panel of IAV strains that vary in glycosylation sites on their hemagglutinin (HA). For these experiments a recombinant version of human SP-D of the Met11, Ala160 genotype was used after it was characterized biochemically and structurally. RESULTS: Oligosaccharides at amino acid 165 on the HA in the H3N2 subtype and 104 in the H1N1 subtype are absent in collectin-resistant strains developed in vitro and are important for mediating antiviral activity of SP-D; however, other glycans on the HA of these viral subtypes also are involved in inhibition by SP-D. H3N2 strains obtained shortly after introduction into the human population were largely resistant to SP-D, despite having the glycan at 165. H3N2 strains have become steadily more sensitive to SP-D over time in the human population, in association with addition of other glycans to the head region of the HA. In contrast, H1N1 strains were most sensitive in the 1970s-1980s and more recent strains have become less sensitive, despite retaining the glycan at 104. Two H5N1 strains were also resistant to inhibition by SP-D. By comparing sites of glycan attachment on sensitive vs. resistant strains, specific glycan sites on the head domain of the HA are implicated as important for inhibition by SP-D. Molecular modeling of the glycan attachment sites on HA and the carbohydrate recognition domain of SPD are consistent with these observations. CONCLUSION: Inhibition by SP-D correlates with presence of several glycan attachment sites on the HA. Pandemic and avian strains appear to lack susceptibility to SP-D and this could be a contributory factor to their virulence.

Lysosomal acid alpha-glucosidase consists of four different peptides processed from a single chain precursor.

Pompe's disease is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). GAA is synthesized as a 110-kDa precursor containing N-linked carbohydrates modified with mannose 6-phosphate groups. Following trafficking to the lysosome, presumably via the mannose 6-phosphate receptor, the 110-kDa precursor undergoes a series of complex proteolytic and N-glycan processing events, yielding major species of 76 and 70 kDa. During a detailed characterization of human placental and recombinant human GAA, we found that the peptides released during proteolytic processing remained tightly associated with the major species. The 76-kDa form (amino acids (aa) 122-782) of GAA is associated with peptides of 3.9 kDa (aa 78-113) and 19.4 kDa (aa 792-952). The 70-kDa form (aa 204-782) contains the 3.9- and 19.4-kDa peptide species as well as a 10.3-kDa species (aa 122-199). A similar set of proteolytic fragments has been identified in hamster GAA, suggesting that the multicomponent character is a general phenomenon. Rabbit anti-peptide antibodies have been generated against sequences in the proteolytic fragments and used to demonstrate the time course of uptake and processing of the recombinant GAA precursor in Pompe's disease fibroblasts. The results indicate that the observed fragments are produced intracellularly in the lysosome and not as a result of nonspecific proteolysis during purification. These data demonstrate that the mature forms of GAA characterized by polypeptides of 76 or 70 kDa are in fact larger molecular mass multicomponent enzyme complexes.

cDNA cloning, DNA binding, and evolution of mammalian transcription factor IIIA.

cDNA for rat transcription factor IIIA (TFIIIA) was cloned by degenerate PCR and rapid amplification of cDNA ends. This cDNA coded for a protein with nine Cys(2)His(2) zinc fingers and a non-finger C-terminal tail; 63% amino acid (aa) sequence identity was observed with the Xenopus TFIIIA zinc finger region. Recombinant rat protein containing only the nine fingers afforded DNase I protection of the identical nucleotides protected by Xenopus laevis native TFIIIA on the Xenopus 5S RNA gene internal control region. A putative mouse TFIIIA clone was identified in an expressed sequence tag database by sequence similarity to rat TFIIIA. Recombinant nine-finger protein from this clone afforded DNase I protection of the Xenopus 5S rRNA gene like the native frog protein as did a recombinant nine-finger form of a putative human TFIIIA clone. These DNA binding results demonstrate that these clones code for the respective mammalian TFIIIAs. Rodent and human TFIIIAs share about 87% aa sequence identity in their zinc finger regions and have evolved to about the same extent as X. laevis and Xenopus borealis TFIIIAs. A monoclonal antibody against human p53 tumor suppressor bound to rat and mouse TFIIIA but not to human TFIIIA in Western blots. The N-terminal regions of rodent and human TFIIIA do not contain the oocyte-specific initiating Met and accompanying conserved residues found in fish and amphibian TFIIIAs. In their non-finger C-terminal tails, mammalian and amphibian TFIIIAs share a conserved transcription activation domain as well as conserved nuclear localization and nuclear export signals.