Suppression of pullulanase-induced cytotoxic T cell response with a dual promoter in GSD IIIa mice
Jeong-A Lim, … , Priya S. Kishnani, Baodong Sun
Jeong-A Lim, … , Priya S. Kishnani, Baodong Sun
Published October 20, 2022
Citation Information: JCI Insight. 2022;7(23):e152970. https://doi.org/10.1172/jci.insight.152970.
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Research Article Genetics Therapeutics

Suppression of pullulanase-induced cytotoxic T cell response with a dual promoter in GSD IIIa mice

Abstract

Glycogen debranching enzyme deficiency in glycogen storage disease type III (GSD III) results in excessive glycogen accumulation in multiple tissues, primarily the liver, heart, and skeletal muscle. We recently reported that an adeno-associated virus vector expressing a bacterial debranching enzyme (pullulanase) driven by the ubiquitous CMV enhancer/chicken β-actin (CB) promoter cleared glycogen in major affected tissues of infant GSD IIIa mice. In this study, we developed a potentially novel dual promoter consisting of a liver-specific promoter (LSP) and the CB promoter for gene therapy in adult GSD IIIa mice. Ten-week treatment with an adeno-associated virus vector containing the LSP-CB dual promoter in adult GSD IIIa mice significantly increased pullulanase expression and reduced glycogen contents in the liver, heart, and skeletal muscle, accompanied by the reversal of liver fibrosis, improved muscle function, and a significant decrease in plasma biomarkers alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Compared with the CB promoter, the dual promoter effectively decreased pullulanase-induced cytotoxic T lymphocyte responses and enabled persistent therapeutic gene expression in adult GSD IIIa mice. Future studies are needed to determine the long-term durability of dual promoter–mediated expression of pullulanase in adult GSD IIIa mice and in large animal models.

Authors

Jeong-A Lim, Priya S. Kishnani, Baodong Sun

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Figure 6

AAV-Dual-Pull recovered liver abnormalities and improved muscle function after 10 weeks of treatment.

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AAV-Dual-Pull recovered liver abnormalities and improved muscle function...
(A–C) Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase (CK) activities were measured to evaluate liver and muscle damage. Data are shown as the mean ± SD; n = 5 for each group. Ordinary 1-way ANOVA for ALT (A) and CK (C); Kruskal-Wallis test for AST (B); *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus UT. (D) Trichrome staining of liver sections was performed for the detection of liver fibrosis. Substantial fibrotic tissues (blue) were observed in the livers of UT, chicken β-actin–treated (CB-treated), and cotreated mice (top). Separated blue-stained areas from the trichrome images are shown in black and white by ImageJ using the threshold color option. At least 3 mice in each group were examined, and representative images are shown (bottom). Scale bar: 50 μm. (E) Quantitative analysis of the black-and-white images in D using ImageJ. The optical density of each image was measured for the quantitation of liver fibrosis. Three different areas of each mouse from n = 3 mice in each group were examined. Data are shown as the mean ± SD. Ordinary 1-way ANOVA; **P < 0.01, ***P < 0.001, ****P < 0.0001 versus UT. (F) The ratio of liver to body weight was measured to determine liver size (hepatomegaly). Data are shown as the mean ± SD; n = 5 for each group. Ordinary 1-way ANOVA; **P < 0.01, ***P < 0.001, ****P < 0.0001 versus UT. (G) Treadmill test was used to evaluate exercise intolerance for the UT and AAV-treated GSD IIIa mice. The graph represents the maximum running distance. Data are shown as the mean ± SD; n = 5 for each group. Ordinary 1-way ANOVA, ****P < 0.0001 versus UT. UT, untreated.