Human-derived NLS enhance the gene transfer efficiency of chitosan.

Diogo B. Bitoque,Sónia Simão,Ana V. Oliveira,Raquel L. Sequeira,Ana M. Rosa da Costa,Gabriela A. Silva,Joana Morais,Tiago M. Fortunato,Sofia M. Calado

doi:10.1042/bsr20201026

Abstract

Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA.In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5.These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.

Highlights

Gene therapy entails the transfer of therapeutic genetic material into cells where the production of the encoded protein will occur to treat or prevent a disease, by replacement of a missing or defective gene [1,2,3,4]
Since the mixing method can influence the properties of polyplexes [37], we evaluated if adding the total amount of nuclear localization signals (NLS) peptides to the chitosan solution or adding it in equal amounts to either the chitosan or DNA solutions had an impact on polyplex formation
No increase in transfection efficiency was observed for CSedac5 polyplexes compared with polyplexes without NLS peptides. These results indicate that transfection efficiency is improved by polyplexes with insulin growth factor binding proteins (IGFBP)-3 peptides but not with polyplexes with IGFBP-5 peptides, suggesting, as hypothesized above

Summary

Introduction

Gene therapy entails the transfer of therapeutic genetic material into cells where the production of the encoded protein will occur to treat or prevent a disease, by replacement of a missing or defective gene [1,2,3,4]. We have previously explored the gene delivery properties of chitosan and hyaluronic acid [10,11,12,13,14] and confirmed the cause of the low transfection efficiency to be the inability of the polyplex/DNA load to enter the nucleus. To overcome these obstacles, we have explored chemical modification of the vectors [10,12] as a strategy to increase the gene transfer efficiency. We evaluate the efficiency of transfection of our chitosan-based vectors after the incorporation of nuclear localization signals (NLS)

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Conclusion