Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A Novel Gene Therapy Approach for GSD III Using an AAV Vector Encoding a Bacterial Glycogen Debranching Enzyme

doi: 10.1016/j.omtm.2020.05.034

Figure Lengend Snippet: Pullulanase Reduced Glycogen Accumulation in the Liver and Muscles and Reversed Liver Fibrosis in GSD IIIa Mice (A) Glycogen contents were measured in the liver, heart, and skeletal muscle. Consistent with the enzyme activity results, there is no difference in liver glycogen content between the UT- and CB-treated GSD IIIa mice at 3 months of age, but glycogen level was reduced significantly in the CB+LSP-treated liver at 5 months of age. Glycogen content was profoundly decreased in the heart and skeletal muscle of both CB- and CB+LSP-treated mice. The graph represents the mean ± SD n = 4 for WT, n = 5 for UT, n = 8 for CB, and n = 7 for CB+LSP; Student’s t test. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001. (B) Periodic acid-Schiff (PAS) staining was used for detection of glycogen accumulation in tissues. Untreated GSD IIIa mice showed intense PAS-positive glycogen accumulation (purple) in the liver, heart, and skeletal muscle. CB treatment had no effect on liver glycogen accumulation; in contrast, CB+LSP treatment markedly reduced glycogen accumulation in the liver. Glycogen buildup was profoundly cleared in the heart and skeletal muscle by both CB and CB+LSP treatments. At least 3 mice were examined in each group, and representative images are shown. Scale bar, 50 μm. (C) Trichrome staining was used for detection of liver fibrosis. The blue staining (arrows) indicates the presence of fibrotic tissues in the liver of UT- and CB-treated GSD IIIa mice. CB+LSP treatment successfully reversed liver fibrosis. At least 3 mice were examined in each group, and representative images are shown. Scale bar, 50 μm.

Article Snippet: A 2.2 kb DNA containing the codon-optimized Pullulanase cDNA ( ; Bacillus subtilis strain 168, amyX , GenBank: NP_390871.2 ) linked with a hemagglutinin (HA)-tag sequence (5′-TACCCATACGATGTTCCAGATTACGCT-3′) at the 3′ end was synthesized and cloned into the pcDNA3.1(+) vector using KpnI and EcoR I sites (pcDNA3.1-Pull) at GenScript (Piscataway, NJ, USA).

Techniques: Activity Assay, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A Novel Gene Therapy Approach for GSD III Using an AAV Vector Encoding a Bacterial Glycogen Debranching Enzyme

doi: 10.1016/j.omtm.2020.05.034

Figure Lengend Snippet: Pullulanase Normalized Disease Symptoms and Improved Muscle Function (A) The ratio of liver to body weight was measured to determine hepatomegaly. The ratio did not change by the CB treatment. However, the liver size was significantly reduced by the CB+LSP treatment. The dotted line represents the level of liver to body weight ratio in WT mice (4.5% ± 0.2%, n = 6). The graph represents the mean ± SD n = 7 for UT (3 months) and CB+LSP, n = 8 for CB, and n = 5 for UT (5 months). Each dot/triangle represents an individual mouse. Student’s t test. ∗p < 0.05. (B) The activity of alanine aminotransferase (ALT) was measured in the plasma from UT- and AAV-treated GSD IIIa mice to evaluate liver function. The ALT level was slightly reduced (p = 0.09) in the CB-treated mice compared to UT. The ALT level was remarkably increased in UT mice from age 3 to 5 months. CB+LSP treatment significantly reduced plasma ALT activity compared with UT mice. The graph represents the mean ± SD n = 7 for UT (3 months) and CB+LSP, n = 8 for CB, and n = 5 for UT (5 months). Each dot/triangle represents an individual mouse. The dotted line represents the ALT level in WT mice (98.1 ± 33.24 U/L, n = 4). Student’s t test. ∗p < 0.05 and ∗∗∗∗p < 0.0001. (C) The concentrations of urinary Glc4 were assessed in the UT and AAV-CB-Pull (CB)-treated GSD IIIa mice. The CB-treated mice showed a significant decrease in the level of urinary Glc4 compared to the UT mice. The urinary creatinine level was used for normalization. The graph represents the mean ± SD, n = 6 for UT and n = 8 for CB. Each dot represents an individual mouse. The dotted line represents the WT level (4.4 ± 1.7 mmol/mol of creatinine, n = 7). Student’s t test. ∗p < 0.05. (D) Treadmill test was used for evaluating exercise intolerance in WT, UT-, and AAV-treated GSD IIIa mice. The graph represents the maximum running distance. The running distance gradually declined in the UT mice with time (UT, dots). However, the running distance was steady in the AAV-treated mice (AAV, squares). Wire hang test was used to assess limb muscle strength. The time of latency to fall was measured and the maximum time from three independent trials was used for comparison. The hanging time was also progressively decreased in UT mice with age (UT, dots), but AAV treatments significantly improved the hanging time (AAV, squares). Rota-rod test was used to measure muscle strength, motor coordination, and balance. The mice were tested during three sessions using the accelerating Rota-rod protocol (4.0–40.0 rpm), and the latency to fall was recorded. AAV-treated mice also showed significant improvement in Rota-rod performance compared to the UT mice. The data represents the mean ± SD, n = 5 for WT and n = 7 for both UT- and AAV-treated GSD IIIa mice. Student’s t test. ††p < 0.01, †††p < 0.001, and †††p < 0.0001, WT compared to UT. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001, AAV compared to UT.

Article Snippet: A 2.2 kb DNA containing the codon-optimized Pullulanase cDNA ( ; Bacillus subtilis strain 168, amyX , GenBank: NP_390871.2 ) linked with a hemagglutinin (HA)-tag sequence (5′-TACCCATACGATGTTCCAGATTACGCT-3′) at the 3′ end was synthesized and cloned into the pcDNA3.1(+) vector using KpnI and EcoR I sites (pcDNA3.1-Pull) at GenScript (Piscataway, NJ, USA).

Techniques: Activity Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: A Novel Gene Therapy Approach for GSD III Using an AAV Vector Encoding a Bacterial Glycogen Debranching Enzyme

doi: 10.1016/j.omtm.2020.05.034

Figure Lengend Snippet: AAV-Mediated Gene Delivery of Pullulanase in the Liver and Muscles of GSD IIIa Mice (A) The diagram shows the constructs of AAV vector containing the 2.2 kb codon-optimized Pullulanase cDNA under the control of the ubiquitous CMV enhancer/chicken β-actin promoter (CB) and the liver-specific promoter (LSP). ITR, inverted terminal repeats; poly(A), human growth hormone polyadenylation signal sequence. (B) Experimental flow chart of AAV treatments in GSD IIIa mice. Fifteen mice were injected with AAV9-CB-Pull at 2 weeks of age at a dose of 1 × 10 13 vg/kg. Eight mice were sacrificed after 10 weeks of treatment (3 months old, gray arrow) for sample collection; the remaining seven mice received a second AAV injection with AAV8-LSP-Pull at the same dose. Tissues and plasma were analyzed after another 10 weeks (5 months old, black arrow). Behavioral tests were performed at ages 2, 2.5, 3, 4, and 5 months old (open arrows). (C) AAV genome copy numbers were determined by real-time PCR using gene-specific primers for Pullulanase (see ). The copy number was high in the heart and low in the liver and skeletal muscle of the AAV9-CB-Pull-treated mice at 3 months of age (CB, gray). 10 weeks after subsequent AAV8-LSP-Pull treatment (CB+LSP, black), the AAV copy number profoundly increased in the liver. The graph represents the mean ± SD n = 5 for each group. vg/genome, vector genome copies per nucleus (genome). (D) Western blot analysis using an anti-HA antibody confirmed the expression of Pullulanase in tissues of the AAV-treated GSD IIIa mice. Pullulanase was undetectable in any tissues of the untreated (UT) mice. Pullulanase was not detectable in the liver of the CB-treated mice but was highly expressed in the liver of CB+LSP-treated mice. The heart showed a high level and skeletal muscle showed a low level (but detectable) of Pullulanase in both CB- and CB+LSP-treated mice. β-Actin was used as a loading control. (E) Pullulanase activity was evaluated in the liver, heart, and skeletal muscle using the Pullulanase activity kit (Megazyme). Consistent with the western blot results, the enzyme activity was not detectable in the CB-treated liver but drastically increased in the CB+LSP-treated liver. The enzyme activity was significantly elevated in the heart and skeletal muscle of both CB- and CB+LSP-treated mice compared to the UT mice. The graph represents the mean ± SD n = 5 for UT, n = 8 for CB, and n = 7 for CB+LSP; Student’s t test. *p < 0.05, ***p < 0.001, and ****p < 0.0001.

Article Snippet: A 2.2 kb DNA containing the codon-optimized Pullulanase cDNA ( ; Bacillus subtilis strain 168, amyX , GenBank: NP_390871.2 ) linked with a hemagglutinin (HA)-tag sequence (5′-TACCCATACGATGTTCCAGATTACGCT-3′) at the 3′ end was synthesized and cloned into the pcDNA3.1(+) vector using KpnI and EcoR I sites (pcDNA3.1-Pull) at GenScript (Piscataway, NJ, USA).

Techniques: Construct, Plasmid Preparation, Sequencing, Injection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Activity Assay