Study on efficient delivery of recombinant adenoviruses and its application to stem cell-based gene therapy

DoctorRecombinant adenoviruses (rAds) have been widely used for gene delivery in vitro and in vivo, especially in clinical cancer gene therapy due to broad tropisms, efficient transduction in dividing and non-dividing cells, high level of transgene production, high immunogenicity, and low risk of insertional mutagenesis. However, their therapeutic efficiency is limited by several obstacles regarding low transduction efficiency in several cell types lacking coxsackievirus and adenovirus receptor (CAR) such as mesenchymal stem cells (MSCs), various cancer cells, and dendritic cells (DCs), and preexisting neutralizing antibody (nAb). Thus, development of novel strategy for an efficient delivery of rAd into rAd-resistant cell types and for overcoming preexisting nAb has been required. Here, I studied about cell-permeable peptide (CPP)- and/or ferric ion (Fe3+)-based delivery of rAd into CAR-negative cells, and evaluated its applicability to MSC-based gene therapy of hard-to-cure diseases. In the first part of this study, it was demonstrated that a novel CPP (HP4) derived from herring protamine appeared to enter C6Bu1 rat glioma cells more rapidly than other known CPPs such as Tat, Antp, and Hph-1. Moreover, HP4 significantly enhanced in vitro transduction of rAd into MSCs, various cancer cells, and DCs, which are relatively resistant to rAd infection. Enhancement of rAd delivery into C6Bu1 and MSCs by HP4 is 20- and 7-fold higher than that by Tat, respectively. However, their transduction efficiency was still limited even at high concentration. Because oligomerization of CPPs was known as a strategy to enhance intracellular DNA delivery by CPP/DNA complexes, the effect of branched oligomerization of CPP on rAd transduction into MSCs was investigated. Branched oligomerization of CPPs significantly enhanced the transduction of MSCs by rAd in a CPP type-independent manner. In particular, tetrameric CPPs (tCPPs) increased transduction efficiency at 3000-5000-fold lower concentrations than monomeric CPPs. Furthermore, while only about 60% of MSCs were maximally transduced at 500 ？？M of monomeric CPPs, greater than 95% of MSCs were transduced with 0.1 ？？M of tCPPs. tCPPs also significantly increased the formation and net surface charge of CPP/rAd complexes as well as the binding of rAd to cell membranes at a greater degree than monomeric CPPs, followed by rapid internalization into MSCs. Alternatively, Fe3+ was found to enhance the transduction efficiency of rAd-resistant cell types such as MSCs, HeLa, and CT26 cells by forming coprecipitates with rAd (FePO4/rAd), of which efficiency was higher than that of tCPP. Interestingly, combination of Fe3+ and tCPP synergistically increased the transgene expression level by up to 20-fold compared to Fe3+ alone. FePO4/rAd, but not tCPP/rAd, significantly evaded nAb, which was further enhanced by cotreatment with tCPP even in the presence of a high nAb titer. Cocomplexes of tCPP/FePO4/rAd may efficiently coat the rAd surface, enabling more complete encapsulation and protecting rAd from nAb. The synergistic effect between Fe3+ and tCPP on the rAd transduction efficiency was likely due to augmentation of the net surface charge and rAd binding to the cellular membrane. The in vivo transgene expression of rAd was substantially enhanced by cotreatment with Fe3+ and tCPP in the presence (3- to 6-fold) or absence (2-fold increase) of nAb, meaning that the combined use of Fe3+ and tCPP may be a promising approach for in vivo rAd-mediated gene delivery as well as ex-vivo transduction of target cells.To investigate whether rAd-mediated therapeutic gene delivery using tCPP or Fe3+ could improve the therapeutic potential of MSCs, two different tissue injury models were used. In a critical-size calvarial defect model, the inclusion of tCPPs in ex vivo transduction of rAd expressing bone morphogenetic protein 2 into MSCs promoted highly mineralized bone formation. In addition, MSCs which were transduced with rAd expressing brain-derived neurotrophic factor in the presence of tCPPs improved functional recovery in a spinal cord injury model. These results demonstrated the potential for tCPPs and Fe3+ to provide innovative tool for MSC-based gene therapy as well as for basic research of stem cells