Introduction
Although we previously demonstrated that AAV8-hSPINK1 safely exhibited significant alleviation of chronic pancreatitis (CP) progression1, the limited pancreas specificity of AAV8 leads to off-target expression of the transgenes. Additionally, the CMV promoter, as a ubiquitous promoter, lacks tissue preference, hindering its usage in AAV-mediated gene therapy for pancreatic diseases.
Aims & Methods
We aimed to construct a novel pancreas acini-specific promoter to restrict the ectopic expression of transgenes and further enhancing the precision of gene therapy for pancreatic diseases. By integrating human scRNA-seq database, the TiGER database, and our mouse RNA-seq data from Part II, we identified highly conserved genes that are richly expressed in pancreatic acini across species. Sequences surrounding the transcriptional start site (TSS, designated as +1) were selected as candidate compacted pancreas-specific promoters. We first validated transcriptional activity of the candidate promoters using dual-luciferase reporter assay. Subsequently, we packaged compacted promoters into AAV to drive ZsGreen gene expression and injected into mice. Pancreas and liver were collected and immunofluorescence, qRT-PCR, and Western blot were performed to characterised the efficiency and tissue selectivity of the candidate promoters. Finally, the synergistic effects of combining short enhancer fragments from human or rat amylase genes with the candidate promoters were assessed to enhance transcriptional activity.
Results
We selected the promoter sequences originated from hPRSS1 (408 bp), rCela1(-500/+8) (508 bp), rCela1(-205/+8) (213 bp), hSPINK1 (120 bp) as candidates. In vitro dual luciferase activity assays results indicated that the relative luciferase activities of hPRSS1 promoter and rCela1(-500/+8) promoter were 2.77±1.64 and 3.91±0.93, respectively (compared with CMV [100]), which were significantly higher than that of the empty vector group (0.199±0.20) (all P < 0.001). However, the rCela1(-205/+8) and hSPINK1 promoters exhibited no significant promoter activity. Compared to the CMV promoter, the ZsGreen expression levels driven by the hPRSS1 and rCela1(-500/+8) promoter were lower, whereas the pancreas-to-liver ZsGreen mRNA ratio demonstrated that both compacted promoters had pronounced pancreatic transduction preference, which elevated pancreatic-specific expression by 77.81- and 63.02-fold, respectively (all P < 0.001). We finally introduction of human amylase AMY2A (34 bp) and rat Amy2a3 (40 bp) enhancer into hPRSS1 and rCela1(-500/+8) promoters. The Amy2(rat)-hPRSS1 promoter markedly increased the pancreatic transduction efficiency by 8-fold, and was selected as the optimal compacted enhancer-promoter sequence.
Conclusion
The compact pancreatic acini-specific enhancer-promoter, Amy2(rat)-hPRSS1, dramatically improved the pancreas gene transfer efficiency in vivo and eliminating liver tropism, serving a promising tool for pancreas-specific gene therapy targeting exocrine pancreatic diseases, including acute pancreatitis, chronic pancreatitis, and precancerous lesions of pancreatic cancer.
References
1. Wang YC, Mao XT, Sun C, et al. Pancreas-directed AAV8-hSPINK1 gene therapy safely and effectively protects against pancreatitis in mice. Gut 2024;73:1142-1155.
Disclosure
All authors declare no conflicts of interest.
