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Enhanced genedelivery with LAH4-L1 peptide modified gene deliverysystems
by Nan Liu
Institution: | Universität Freiburg |
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Department: | |
Degree: | |
Year: | 2016 |
Keywords: | |
Posted: | 2/5/2017 12:00:00 AM |
Record ID: | 2106548 |
Full text PDF: | https://www.freidok.uni-freiburg.de/data/11100 |
It has been demonstrated that successful gene transfer requires efficient cellular uptake, sufficient endosomal escape and nucleus entry. Many gene delivery systems show only high levels of cellular uptake but result in poor transfection or transduction efficiency in certain cell types. These gene delivery systems typically only have one strategy for endosomal escape, like proton sponge effect, phospholipase activity or membrane destabilization effect. This can be insufficient in certain cell lines, especially in hard-to-transfect cell lines. Additional approaches should be developed to overcome this major bottleneck for successful gene transfer. In this study, we hypothesized that two different endosomal escape strategies could be combined together for efficient gene delivery via electrostatic interaction. LAH4-L1 peptide was combined with AAV2 virus to form AAV2/LAH4-L1 complexes. Besides, LAH4-L1 peptide was combined with PAMAM dendrimer-mediated transfection to form PAMAM/DNA/LAH4-L1 complexes. Thus, the AAV2/LAH4-L1 complexes possess two endosomal escape strategies: phospholipase activity and membrane destabilization effect. And PAMAM/DNA/LAH4-L1 complexes have not only proton sponge effect but also membrane destabilization effect. In the study of AAV2/LAH4-L1 complexes, J774A.1 cells, a murine macrophage cell line, was employed as the cell model for hard-to-transfect cells. The transduction results demonstrated that LAH4-L1 peptide could markedly improve AAV2-mediated transduction in macrophages with high cell viability. The visualization of intracellular trafficking indicated that LAH4-L1 could enhance the endosomal escape of AAV2 in macrophages. Besides, AAV2/LAH4-L1 complexes induced long-term gene expression in macrophages. More important, compared to uncoated AAV2, AAV2/LAH4-L1 complexes showed enhanced storage stability and possess the ability of antiserum, which extends its clinical application in vivo. Taken together, the study of AAV2/LAH4-L1 complexes presents an effective strategy for gene delivery into macrophages. In the study of PAMAM/DNA/LAH4-L1 (PSL) complexes, transfection studies revealed that PSL complexes achieved excellent efficiency in several cell lines including a hard-to-transfect cell line at low N/P ratios with high cell viability, thus overcoming the efficiency-toxicity dilemma of PAMAM-mediated transfection. Additionally, the PSL complexes showed high anti-serum ability and exhibited outstanding transfection efficiency even in medium containing 50% fetal calf serum (higher than 90% in HeLa cells). Moreover, high levels of long-term gene expression (over 30% in HeLa cells) could be observed by using the sleeping beauty transposon system as genetic payloads of PSL complexes. Furthermore, PSL complexes not only exhibited high cellular uptake but also showed enhanced endosomal escape and unpacking DNA compared to PAMAM/DNA complexes. These results demonstrate the simple association of cationic polymer, LAH4-L1 peptide and sleeping beauty transposon system by molecular self-assembly as a… Advisors/Committee Members: Süss, Regine (advisor).
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