Abstract
BackgroundHuman adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. Many target cells, however, express low levels of Ad5 native receptor, the Coxsackie-Adenovirus Receptor (CAR), and thus are resistant to Ad5 infection. The Protein Transduction Domain of the HIV Tat protein, namely PTDtat, has been shown to mediate protein transduction in a wide range of cells. We hypothesize that re-targeting Ad5 vector via the PTDtat motif would improve the efficacy of Ad5-mediated gene delivery.ResultsIn this study, we genetically incorporated the PTDtat motif into the knob domain of Ad5 fiber, and rescued the resultant viral vector, Ad5.PTDtat. Our data showed the modification did not interfere with Ad5 binding to its native receptor CAR, suggesting Ad5 infection via the CAR pathway is retained. In addition, we found that Ad5.PTDtat exhibited enhanced gene transfer efficacy in all of the cell lines that we have tested, which included both low-CAR and high-CAR decorated cells. Competitive inhibition assays suggested the enhanced infectivity of Ad5.PTDtat was mediated by binding of the positively charged PTDtat peptide to the negatively charged epitopes on the cells' surface. Furthermore, we investigated in vivo gene delivery efficacy of Ad5.PTDtat using subcutaneous tumor models established with U118MG glioma cells, and found that Ad5.PTDtat exhibited enhanced gene transfer efficacy compared to unmodified Ad5 vector as analyzed by a non-invasive fluorescence imaging technique.ConclusionGenetic incorporation of the PTDtat motif into Ad5 fiber allowed Ad5 vectors to infect cells via an alternative PTDtat targeting motif while retaining the native CAR-mediated infection pathway. The enhanced infectivity was demonstrated in both cultured cells and in in vivo tumor models. Taken together, our study identifies a novel tropism expanded Ad5 vector that may be useful for clinical gene therapy applications.
Highlights
Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases
It is known that infection of Ad5 is initiated by attachment of its capsid fiber protein to the cell surface coxsackievirus adenovirus receptor (CAR), which is followed by interaction of its penton base with αv integrins that triggers the internalization of the viruses [47]
The modified Ad5 (Ad5.PTDtat) and the unmodified control (Ad5) were both replication deficient as their E1 region, which is essential for Ad5 replication, was replaced with a CMV promoter-driven green fluorescence protein (GFP) reporter gene
Summary
Human adenovirus serotype 5 (Ad5) has been widely explored as a gene delivery vector for a variety of diseases. It is known that infection of Ad5 is initiated by attachment of its capsid fiber protein to the cell surface coxsackievirus adenovirus receptor (CAR), which is followed by interaction of its penton base with αv integrins that triggers the internalization of the viruses [47]. Many target cells, such as malignant tumor cells, are found to express very low level of CAR, and are resistant to Ad5 infection. Genetic incorporation of RGD peptide and/or a polylysine epitope into the knob domain allowed Ad5 to infect cells through alternative receptors (cell surface integrins for RGD and negatively charged epitopes such as heparan sulfate proteoglycans for polylysine), greatly improving the gene delivery efficacy Ad5 vectors in many target cells [12,13,14,15]
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