Novel Nonviral Gene Delivery Strategies For Cancer Therapy

Successful gene therapy requires targeted gene delivery with the assistance of vectors.Nonviral gene vectors,including cationic lipids and cationic polymers,are particularly attractive due to their excellent safety,biocompatibility,and the potential for large-scale manufacture.However,there are still many issues within nonviral gene delivery,leading to compromised clinical application for cancer therapy.First,nonviral gene delivery needs to overcome multiple extra-and intra-cellular barriers,which restricts the gene transfer efficacy of nonviral vectors,leading to limited therapeutic efficacy.Second,potential problems can be associated with the application of therapeutic genes.Take TRAIL gene delivery as an example,locally expressed TRAIL protein not only induces apoptosis of tumor cells,but also stimulates cytoprotective autophagy of tumor cells,which attenuated TRAIL-induced apoptosis and decreased the anticancer activity of TRAIL.Moreover,targeting tumor cells in desmoplastic tumors achieves unsatisfactory therapeutic outcome,suggesting that modification of tumor microenvironment is also important for effective cancer therapy.To solve these problems,we proposed specific strategies to improve the therapeutic efficacy of nonviral gene delivery and further accelerate its clinical translation.In the first part,we explored the strategy to improve the transfection efficacy of nonviral gene delivery with specific small molecules.Although the detailed mechanisms of transfection by nonviral vectors are still not completely understood,nuclear envelope,which completely encapsulates chromatin,was reported to be one of the key obstacles for plasmid DNA(pDNA)delivery.Cationic polymers,such as poly(ethylene imine)(PEI),are widely used for pDNA delivery.However,the nanoparticles of PEI/DNA are too large(?100 nm)to enter the nuclei through nuclear pores after their entry into the cells and the nuclear envelope breakdown in mitosis is believed to facilitate the nuclear entry of polyplexes.To jump the nuclear envelope barrier,we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle to increase the proportion of mitotic cells with disappeared nuclear envelope during transfection,facilitating the nuclear uptake of polyplexes and the subsequent gene expression.Compared with PEI,the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammoniumbromide](B-PDEAEA)which responses to cellular stimuli and releases free pDNA in cytoplasm is less sensitive to RO-3306 increased transfection efficacy,indicating the distinction in nuclear entry for different vectors.Next,B-PDEAEA was further used to explore how small molecules can enhance the transfection efficacy of polymers with responsiveness.B-PDEAEA responses to the high level of intracellular reactive oxygen species(ROS)in cancer cells to quickly release free DNA in cytoplasm,resulting in significantly improved transfection efficacy.Although cancer cells constantly generate higher levels of intracellular ROS than normal cells,the ROS levels may still not be enough to trigger the vigorous response,especially when concerning the heterogeneity in cancer cell lines.Here,we report SAHA(vorinostat),which is a clinical histone deacetylase inhibitor and anticancer drug,induces the ROS accumulation in cancer cells,and facilitates the charge reversal process of B-PDEAEA and the cellular dissociation of the delivered gene from the vectors.As a result,SAHA remarkably increases the gene transfection efficacy in an ROS-dependent manner.Importantly,SAHA synergizes with B-PDEAEA mediated therapeutic gene TNF-related apoptosis-inducing ligand(TRAIL)delivery in inducing apoptosis of cancer cells.In the second part,we focused on improving the therapeutic efficacy of TRAIL gene delivery.TRAIL-based gene therapy not only induces apoptosis selectively in cancer cells while sparing normal cells,but also could overcome the drawbacks of recombinant TRAIL protein,such as short half-life,inefficient delivery to target sites and reduced sensitivity.However,as an apoptosis inducer,TRAIL may also promote autophagy in tumor cells,which attenuated TRAIL-induced apoptosis and decreased the anticancer activity of TRAIL.Autophagy inhibition could synergize with TRAIL and augment TRAIL-induced tumor cell apoptosis.Therefore,we designed the first autophagy-inhibiting cationic polymer by incorporating the autophagy inhibitor hydroxychloroquine into the gene carrier,to deliver pDNA encoding for cancer gene therapy.The copolymerization of methacryloyl chloroquine(MACQ)with 2-(Dimethylamino)ethyl methacrylate(DMAEMA)not only improves transfection efficacy through hydrophobic modification,but also endows the copolymer with autophagy-blocking capability,which further sensitizes cancer cells to TRAIL induced apoptosis,providing new insights into the development of nonviral vectors.In the third part,we investigated the approach for desmoplastic tumor therapy with nonviral gene delivery and showed-the importance of tumor microenvironment modulation.The tumour microenvironment is crucial for tumour initiation,progression,and metastasis,indicating the necessity of microenvironment modification for efficient cancer therapy.Relaxin(RLN)is a potent antifibrotic peptide,which is an endogenous hormone and can be used to decrease the fibrosis for tumor microenvironment modulation.Nevertheless,the short circulation half-life and the potential off-target effect of RLN peptide restricts its direct use for therapy.Targeted delivery of RLN plasmid(pRLN)with lipid nanoparticles into the tumor tissue,however,generated local expression of RLN,which improved the infiltration of immune cells to induce anticancer effect by modulating the microenvironment.We also discovered that macrophages constituted the main source of the receptor of RLN for desmoplastic tumors and demonstrated the important involvmement of macrophages to deplete fibrosis.