Efficient Gene Delivery into Dendritic Cells by Fiber-Mutant Adenovirus Vectors

Abstract

Dendritic cells (DC) are professional antigen-presenting cells with a key function in the immune system as initiators of T-cell responses against microbial pathogens and tumors. Therefore, the immunization using DC loaded with tumorassociated antigens is potential to represent a powerful method of inducing anti-tumor immunity. Recent studies have demonstrated the usefulness of DC genetically modiˆed by adenovirus vectors (Ad) to immunotherapy, while su‹cient gene transduction into DC is required for high doses of Ad. Entry of Ad into target cells occurs by serial two steps: the binding of Ad-ˆber knob to Coxsackie-adenovirus receptor (CAR) on the cell surface, and the subsequent interaction between Arg-Gly-Asp (RGD) motif located in Ad-penton base and a v -integrins. The reverse transcription-polymerase chain reaction analysis revealed that the relative resistance of DC to Ad-mediated gene transfer was due to the absence of CAR expression, and that DC expressed adequate a v -integrins. Therefore, we investigated whetherˆber-mutant Ad (FM-Ad) containing the RGD sequence in theˆber knob can e‹ciently transduce and express high levels of the foreign gene into DC. The gene delivery by FM-Ad was more e‹cient than that by conventional Ad in both murine DC lines and normal human DC. Furthermore, both antigen presentations via major histocompatibility complex class I molecules and in vivo antigen-speciˆc cytotoxic T lymphocyte induction by DC transduced with antigen gene by FM-Ad were superior to those by DC applied with conventional Ad. We propose that a v -integrin-targeted FM-Ad is a very powerful tool to implement DC-based vaccination strategies.

Dendritic cells transduced with gp100 gene by RGD fiber-mutant adenovirus vectors are highly efficacious in generating anti-B16BL6 melanoma immunity in mice

“…These data clearly demonstrate that AdRGD is a very potent vector system for ex vivo genetic modification of DCs, as we showed in our previous studies using reporter genes. 15,16,18 Allogenic and syngeneic T-cell proliferation-stimulating ability of AdRGD-gp100/mBM-DCs and Ad-gp100/ mBM-DCs…”

“…From the perspective of the exploitation of DC-based immunotherapy, we previously reported a method using AdRGD that enhances the ex vivo gene transduction efficiency of murine and human DCs. 15,16,18 In recent years, the cloning of various MAAs interacting with the melanocyte differentiation and melanogenesis pathways has opened new possibilities for the development of active immunotherapy designed to cause the rejection of malignant melanoma. [19][20][21][22][23] gp100, an MAA, is consistently expressed in both murine and human melanomas, 27,40 and has been shown to be highly immunogenic and an important target for active-specific immunotherapy in humans.…”

“…[52][53][54] AdRGD-LacZ and AdRGD-Null, which were constructed previously, 15,16,18 were used as control vectors in the present study. All recombinant viruses were propagated in 293 cells, purified by two rounds of CsCl density centrifugation, dialyzed, and stored at À801C.…”

“…[10][11][12] However, the low or lack of coxsackie-adenovirus receptor (primary Ad-receptor) expression on the DC surface makes sufficient gene transduction to DCs by conventional Ad difficult. [13][14][15][16] Likewise, the cytopathic effect of high-dose Ad on DCs remains an impediment to this strategy. We recently reported that RGD fiber-mutant Ad (AdRGD), which targets a v -integrin during attachment to cells by insertion of the RGD peptide into the HI loop of the fiber knob, 17 exhibited highly efficient foreign gene transduction in murine and human DCs compared to conventional Ad.…”

“…We recently reported that RGD fiber-mutant Ad (AdRGD), which targets a v -integrin during attachment to cells by insertion of the RGD peptide into the HI loop of the fiber knob, 17 exhibited highly efficient foreign gene transduction in murine and human DCs compared to conventional Ad. 15,16,18 Moreover, a murine DC line (DC2.4 cells) transduced with ovalbumin (OVA) gene by AdRGD could elicit a more effective OVA-specific immune response in vaccinated mice than a cell line transduced by conventional Ad. 18 Although these results suggest that efficient gene transduction by AdRGD would improve the efficacy of DC-based gene immunotherapy, the use of primary DCs and the targeting of natural TAA rather than a continuously proliferating DC line and highly immunogenic model antigen is desirable in order to establish the predominancy of AdRGD in DCbased immunotherapy.…”