Bortezomib-mediated reduction in anti-AAV IgG titres is insufficient to facilitate reinfection with AAV2/8-DC190-alphaGal.

<p><b>a.</b> At twenty-four weeks following the administration of AAV2/8-EV (including 20 weeks of bortezomib treatment between weeks 4 and 24), mice were re-infected with 5×10¹¹ AAV2/8-DC190-alphaGal/mouse vector particles. Sera were collected at the indicated times, and the titres of anti-AAV IgG were determined. <b>b.</b> A subset of the sera samples was assayed for the total amount of IgG using ELISA. <b>c.</b> The level of α-galactosidase A in serum samples from mice infected with AAV2/8-DC190-alphaGal. Expression of α-galactosidase A was observed in mice that had not been previously infected with AAV2/8-EV. <b>d.</b> Immunohistochemistry was performed on liver sections from mice that were administered AAV2/8-DC190-alphaGal. The figures show representative data from two studies with similar outcomes.</p

Bortezomib treatment significantly reduces anti-AAV IgG titre. a.

<p>Mice were infected with AAV2/8-EV and treated with bortezomib, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034684#s2" target="_blank">Materials and Methods</a> section. Sera were collected at the indicated times, and the levels of anti-AAV IgG were determined by titration. <b>b.</b> A subset of the same sera samples was assayed for the total amount of IgG using ELISA. Representative data from two separate experiments with similar outcomes are shown.</p

Bortezomib treatment differentially affects T cells.

<p><b>a.</b> Mononuclear cells were prepared from spleens and analysed for the T cell markers CD4, CD8, and CD44 using flow cytometry. Plots depict CD44 expression gated on the CD4⁺ (left panel) and CD8⁺ (right panel) populations. <b>b.</b> A summary of the data from <b>a</b> is shown in a quantitative format. <b>c.</b> Mononuclear cells isolated from spleen were reactivated with AAV2/8-EV for 72 h <i>in vitro</i>. Supernatants were collected and assayed for IL-2 (left panel) or interferon-γ (right panel) levels using ELISA. The figures show representative data from two studies with similar outcomes.</p

Muscle glycogen synthase protein is elevated in GAA^−/− mice.

<p>Tissue lysates were prepared for the indicated tissues from each mouse strain. Lysates from 5 mice were pooled and 100 µg of protein from the pooled lysate was analyzed by Western blot analysis. All of the blots were probed with an anti-GAPDH antibody (∼37 kDa in all panels) to verify equal protein loading of all the test samples. (A) An anti-muscle glycogen synthase antibody was used to probe the blots containing samples from the heart, triceps, and quad. An anti-liver glycogen synthase antibody was used to probe the blot containing samples from the liver. (B) An anti-phospho-glycogen synthase (Ser641) antibody was used to probe the blots for phosphorylated GS. (C) Glycogen synthase transcript levels are not elevated in GAA^−/− mice compared to wild type animals. RNA was isolated from triceps and heart of C57Bl/6 and GAA^−/− mice and probed with qPCR primers as described under “Experimental Procedures.” Values shown are means ± SEM.</p

rhGAA treatment normalizes glycogen synthase protein and activity levels in heart of GAA^−/− mice.

<p>Groups (n = 5) of 3 to 4-month old mice of the indicated strains were dosed with rhGAA (100 mg/kg) weekly for 4 weeks by tail vein injection. Muscle homogenates were prepared, pooled and analyzed by: (A) Western blot using an anti-GS antibody and (B) densitometry analysis of the western blots (with GS normalized to GAPDH)., Samples were also processed for measurements of (C) glycogen synthase activity (D) tissue glycogen levels. Values are means ± SEM. Values are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing groups. ***<i>P</i><0.001.</p

Glycogen is not reduced in GAA^−/− mice lacking S6K1 and S6K2.

<p>Glycogen was quantified in tissue lysates collected from groups (n = 10) of 3–4 month old mice of the indicated strains. Values shown are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing all groups to C57Bl/6. ***<i>P</i><0.001.</p

Glycogenin levels are dysregulated in GAA^−/− mice and normalized by rhGAA treatment.

<p>Homogenates from triceps were prepared for the indicated strains. Lysates from 5 mice for each strain were pooled and 100 µg of protein analyzed by Western blot. A monoclonal antibody to glycogenin was used to probe the blots. (<i>A</i>) lysates not treated with amyloglucosidase. (<i>B)</i>, treated with amyloglucosidase. (<i>C</i>) GAA^−/− mice were dosed with rhGAA as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056181#pone-0056181-g004" target="_blank">Figure 4</a>. Lysates from the strains indicated in this panel were not treated with amyloglucosidase.</p

Percentage of the total administered dose of hASM detected in the (A) brain, (B) spinal cord and (C) serum of rats following 6 h ICV infusion.

<p>Animals were infused with 0.1, 1.0 or 10 mg/kg of hASM or aCSF. For both the brain and spinal cord, less than 0.2% of the total dose could be detected in each region. Cumulatively, the level of hASM detected in the brain and spinal cord was less than 1% of the total dose administered. No significant differences were observed between regions or different dose groups. Significant (p<.01) levels of hASM were detected in serum for each dose.</p

G6P (A) and hexokinase (B) levels are elevated in GAA^−/− mice compared to wild type (C57/Bl6) and reduced by rhGAA treatment.

<p>GAA^−/− mice were dosed with rhGAA as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056181#pone-0056181-g004" target="_blank">Figure 4</a>. G6P and hexokinase was quantified in heart and triceps homogenates from the strains indicated. Values are means ± SEM. Data was analyzed by one-way ANOVA followed by Newman-Keuls comparing groups. ***<i>P</i><0.001.</p

hASM levels in (A) rat brain, (B) spinal cord and (C) serum following a 6 h ICV infusion of hASM at different doses (0.1, 1.0 and 10 mg/kg) or aCSF.

<p>Significant (p<.01) levels of hASM were detected in all regions of the brain (i.e., cortex, preoptic region, striatum, hippocampus, thalamus, hypothalamus, midbrain, cerebellum and brainstem) in rats infused with 1 and 10 mg/kg of the enzyme. Rats infused with the 0.1 mg/kg dose displayed significant hASM levels in the cortex, preoptic region, striatum and hippocampus. No significant differences in hASM levels between regions were observed regardless of the dose tested. In each region of the spinal cord, significant (p<.01) levels of hASM were also detected in rats infused at either 1 or 10 mg/kg vs. rats infused with aCSF (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016313#pone-0016313-g002" target="_blank">Figure 2b</a>). Detected hASM levels in rats infused with 0.1 mg/kg were not significantly above background. Significant (p<.01) levels of hASM were detected in serum for all hASM infused rats regardless of the dose of enzyme used.</p