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
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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 7

Both the LSP and LSP-CB dual promoter decreased pullulanase-induced CTL response in GSD IIIa mice.

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Both the LSP and LSP-CB dual promoter decreased pullulanase-induced CTL ...
(A) Paraffin-embedded liver sections from GSD IIIa mice 2 weeks after AAV injection were stained with an anti-CD4+ or anti-CD8α antibody to detect cytotoxic T cell responses. Infiltrations of CD4+ or CD8α+ lymphocytes (brown stained, arrows) were abundant in the CB-treated livers, moderately present in the cotreated livers. CD4+ or CD8α+ cells (arrowheads) were occasionally seen in the dual-treated livers and almost absent in the LSP-treated livers. Images are representative of at least 3 mice in each group. Scale bar: 50 μm. (B) Mouse IFN-γ ELISpot was performed to detect cell-mediated immune responses against pullulanase. Splenocytes isolated 2 weeks after AAV injection were stimulated by the final pullulanase peptides pool (Supplemental Figure 2). The levels of pullulanase-induced IFN-γ secretion were determined by the number of spot-forming units (SFU) per million splenocytes and are represented in the graph. The number of SFU was the highest in the CB-treated mice, significantly decreased in the dual- and cotreated mice, and the lowest in the LSP-treated mice. Data are shown as the mean ± SD; n = 3, triplicates. Ordinary 1-way ANOVA; *P < 0.05, **P < 0.01.