Target For Cancer Gene Therapy Research Articles

Tunable metal-organic frameworks assist in catalyzing DNAzymes with amplification platforms for biomedical applications.

Various binding modes of tunable metal organic frameworks (MOFs) and functional DNAzymes (Dzs) synergistically catalyze the emergence of abundant functional nanoplatforms. Given their serial variability in formation, structural designability, and functional controllability, Dzs@MOFs tend to be excellent building blocks for the precise "intelligent" manufacture of functional materials. To present a clear outline of this new field, this review systematically summarizes the progress of Dz integration into MOFs (MOFs@Dzs) through different methods, including various surface infiltration, pore encapsulation, covalent binding, and biomimetic mineralization methods. Atomic-level and time-resolved catalytic mechanisms for biosensing and imaging are made possible by the complex interplay of the distinct molecular structure of Dzs@MOF, conformational flexibility, and dynamic regulation of metal ions. Exploiting the precision of DNAzymes, MOFs@Dzs constructed a combined nanotherapy platform to guide intracellular drug synthesis, photodynamic therapy, catalytic therapy, and immunotherapy to enhance gene therapy in different ways, solving the problems of intracellular delivery inefficiency and insufficient supply of cofactors. MOFs@Dzs nanostructures have become excellent candidates for biosensing, bioimaging, amplification delivery, and targeted cancer gene therapy while emphasizing major advancements and seminal endeavors in the fields of biosensing (nucleic acid, protein, enzyme activity, small molecules, and cancer cells), biological imaging, and targeted cancer gene delivery and gene therapy. Overall, based on the results demonstrated to date, we discuss the challenges that the emerging MOFs@Dzs might encounter in practical future applications and briefly look forward to their bright prospects in other fields.

Biocompatible Iron Oxide Nanoparticles for Targeted Cancer Gene Therapy: A Review.

In recent years, gene therapy has made remarkable achievements in tumor treatment. In a successfully cancer gene therapy, a smart gene delivery system is necessary for both protecting the therapeutic genes in circulation and enabling high gene expression in tumor sites. Magnetic iron oxide nanoparticles (IONPs) have demonstrated their bright promise for highly efficient gene delivery target to tumor tissues, partly due to their good biocompatibility, magnetic responsiveness, and extensive functional surface modification. In this review, the latest progress in targeting cancer gene therapy is introduced, and the unique properties of IONPs contributing to the efficient delivery of therapeutic genes are summarized with detailed examples. Furthermore, the diagnosis potentials and synergistic tumor treatment capacity of IONPs are highlighted. In addition, aiming at potential risks during the gene delivery process, several strategies to improve the efficiency or reduce the potential risks of using IONPs for cancer gene therapy are introduced and addressed. The strategies and applications summarized in this review provide a general understanding for the potential applications of IONPs in cancer gene therapy.

Large-scale screening of i-motif binding compounds in the silkworm, Bombyx mori

DNA secondary structure i-motif involves in gene transcription and considered as a novel target for cancer gene therapy. I-motif-binding compounds can either stabilize or destroy the structure, resulting in change in target gene transcription. In this study, a large-scale screening of binding compounds was conducted using the i-motif structure of BmPOUM2, a transcription factor in silkworm, Bombyx mori. Surface plasmon resonance imaging (SPRi) high-throughput binding screening of 3642 compounds found 60 compounds with an binding affinity Kd of 10−7-10−6 M. SPRi and circular dichroism (CD) double screening demonstrated that the BmPOUM2 i-motif structure bound the compounds IF1, IF3, IF4, IF6 and IF7 with Kd of 10−7 M, and the compounds IF2 and tetrakis (4-N-methylpyridyl) porphine (TMPyP4) with a Kd of 10−8 M. Interestingly, IF2, IF3, IF4, IF6 and IF7 promoted the binding of the i-motif-binding protein BmILF with the i-motif structure, whereas TMPyP4 inhibited the binding. This study provided a list of compounds that have potential applications in functional analysis of i-motif structure and in pesticide and drug development through gene transcription regulation by i-motif structure.

Mesenchymal stem cells engineered by modified polyethylenimine polymer for targeted cancer gene therapy, in vitro and in vivo.

Cell-based delivery system is a promising strategy to protect therapeutic agents from the immune system and provide targeted delivery. Mesenchymal stem cells (MSCs) have recently been introduced as an encouraging vehicle in cell-based gene therapy due to their unique features including tumor-tropic property and migratory ability. However, gene transfer into MSCs is limited due to low efficiency and cytotoxicity of carriers. In this study, we designed a novel delivery system based on polyethylenimine (PEI25 ) to improve these features of carrier and transfect plasmid encoding TRAIL to MSCs. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a death ligand of TNF family with selective effect on cancerous cells. Then, death induction and migration ability of TRAIL-expressing MSCs was studied in melanoma cells. The effect of engineered-MSCs as an antitumor vehicle was also investigated in mice bearing melanoma cells. Our findings indicated that heterocyclic amine derivative of PEI25 showed significant improvement in MSCs viability determined by MTT assay and gene expression using fluorescent microscopy, flow cytometry, and Western blot analysis. We observed that engineered-MSCs could migrate toward and induce cell death in B16F0 cells in vitro. The single administration of TRAIL-expressing MSCs could delay tumor appearance and efficiently reduce tumor weights. Hematoxylin and eosin staining of tumor sections revealed extensive neoplastic cells necrosis. Furthermore, engineered-MSCs could migrate and localize to tumors sites within 5 days. Our results indicated that MSCs engineered by modified-PEI/TRAIL complexes could be considered as a promising cellular vehicle for targeted tumor suppression.

Homotypic Targeting Delivery of siRNA with Artificial Cancer Cells.

The camouflage with cell membrane bestows nanoparticles with cell-like functions, such as specific recognition, long blood circulation, and immune escaping. For cancer therapy, the nanoparticles camouflaged with cancer cell membrane (CCM) from homologous cells show homotypic targeting delivery of small molecule compounds, photosensitizers, or enzymes to the tumors. However, effective gene therapy encounters difficulties by this approach due to the properties of nucleic acids. Herein, a cancer cell-like gene delivery system is developed using an excellent polymer poly(β-amino ester) (PBAE) to condense small interfering RNA (siRNA) (targeting to Plk1 gene) into nanoparticles (PBAE/siPlk1) as the core, which is further camouflaged with CCM. These novel biomimetic nanoparticles CCM/PBAE/siPlk1 (CCMPP) demonstrate highly specific targeting to homotypic cancer cells, effective downregulation of PLK1 level, and inducing apoptosis of cancer cells. Based on the homotypic binding adhesion molecules on the CCM, the cellular internalization and homotypic-targeting accumulation to the tumors are clearly improved. CCMPP induces highly efficient apoptosis of cancer cells both in vitro and in vivo and results in significant tumor inhibition. The artificial cancer cells with homotypic properties can serve as a biomimetic delivery system for cancer-targeted gene therapy.

Cancer-targeted PEDF-DNA therapy for metastatic colorectal cancer.

Colorectal cancer (CRC) is a major cause of cancer-related mortality worldwide. Moreover, metastasis is one of the main causes of death in CRC patients. Nanotechnology-based gene therapy has shown significant therapeutic benefits in recent clinical trials for cancer treatment. Recent studies have shown that pigment epithelium-derived factor (PEDF) protein can inhibit tumor growth and metastasis by anti-angiogenesis and pro-apoptosis. In this study, we prepared a PEDF-DNA-loaded liposome for cancer-targeted gene therapy for metastatic CRC using an iRGD peptide. Our results showed that cancer-targeted PEDF-DNA liposomes (R-LP/PEDF) exhibited enhanced inhibitory effects on invasion, migration, and pro-apoptosis of CRC cells in vitro. In addition, it reduced metastasis tumor nodules in lung and prolonged the survival time in a mouse model of metastatic CRC.

Cancer Targeted Gene Therapy for Inhibition of Melanoma Lung Metastasis with eIF3i shRNA Loaded Liposomes.

Eukaryotic translation initiation factors 3i (eIF3i) is a proto-oncogene that is overexpressed in various tumors, reducing its expression by eIF3i shRNA is a promising strategy to inhibit tumor growth or metastasis. Tumor cell is the target of eIF3i shRNA so that tumor-site accumulation could be important for fulfilling its therapeutic effect. Thus, the iRGD modified liposome (R-LP) was rationally synthesized to enhance the antitumor effect by active targeted delivery of eIF3i shRNA to B16F10 melanoma cells. R-LP encapsulating eIF3i shRNA gene (R-LP/sheIF3i) were prepared by a film dispersion method. The transfection experiment proves that R-LP could effectively transfect B16F10 cells. R-LP/sheIF3i notably restrained the migration, invasion, and adhesion of melanoma cells in vitro. In a mouse model of lung metastasis, R-LP/sheIF3i administered by intravenous injection suppressed pulmonary metastasis of melanoma by dramatically downregulated eIF3i expression and subsequently inhibiting tumor neovascularization and tumor cells proliferation in vivo. Our results provide a basis for tumor cells targeting strategies to reduce the expression of eIF3i by RNAi in the treatment of tumor metastasis.

Abstract 4784: Combination of sorafenib with liver cancer-targeted gene therapy exerts synergistic efficacy against hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. Sorafenib, a multikinase inhibitor, is the first-line target therapy for patients with advanced HCC, but the response rate is low. Thus, development of more effective therapeutics for HCC is needed. Previously we have established a HCC-targeted BikDD gene therapy vector driven by a liver cancer-specific α-fetoprotein promoter/enhancer and transcriptional amplification module. Systemic administration of liposome-loaded HCC-targeted BikDD gene therapy vector effectively suppressed tumor growth and prolonged survival in multiple orthotopic HCC mouse models with no toxicity. Here, we further investigated whether combination of sorafenib with gene therapy could enhance anti-HCC activity. The results showed that combination of BikDD gene therapy with HCC targeted therapeutic agent sorafenib synergistically killed a panel of liver cancer cells but not normal cells in vitro by WST-1 assays and analysis of combination index according to the Chou and Talalay method. Moreover, a low dose of sorafenib combined with BikDD gene therapy augmented tumor inhibition and better mice survival compared with separate treatment alone in xenograft and syngeneic orthotopic HCC mouse models. These results suggest that sorafenib combined with HCC-targeted gene therapy may improve the therapeutic efficacy and may be a potential alternative therapeutics for HCC. Citation Format: Huei-Yue Dai, Long-Yuan Li. Combination of sorafenib with liver cancer-targeted gene therapy exerts synergistic efficacy against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4784.

Role of TMPRSS4 Modulation in Breast Cancer Cell Proliferation.

Background:TMPRSS4 is a novel Type II transmembrane serine protease found at the surface of the cells and is involved in the development and cancer progression. However, TMPRSS4 functions in breast cancer remain poor understand. The present study investigated the function of TMPRSS4 in the breast cancer cells and the potential mechanistic action underling. Materials and Methods:The lentiviral vectors causing TMPRSS4 down-regulation and over-expression were established and transfected in MDA-MB-468 and MCF-7 cells, respectively. By using the CCK-8 assay, cell proliferation was analyzed. Moreover, western blot was used to detect the expression of certain proteins related to cell apoptosis (Bax and Bcl2) signaling pathway and telomere maintenance (POT1, TPP1, and UBE2D3). Cell cycle and cell apoptosis were also analyzed by using the Flow cytometry analysis. TMPRSS4 expression was detected at the mRNA level and protein level by performing qPCR and western blot technique, respectively. Results:TMPRSS4 expression is inhibited in stable transfected MDA-MB-468-shTMPRSS4 cells compared to the control MDA-MB-468-NC and its expression is up-regulated in stable transfected MCF-7-TMPTSS4 compared to its control MCF-7-NC. Moreover, TMPRSS4 silencing in breast cancer reduces cells proliferation by promoting cell cycle arrest in G2/M phase, cell apoptosis, and telomere maintenance impairment while the TMPRSS4 overexpression increases cells proliferation through cell apoptosis reduction and telomere maintenance reinforcement associated with insignificant change in cell cycle progression. Conclusion:TMPRSS4 plays important roles in cancer progression and may be considered as a good therapeutic target for cancer gene therapy especially breast cancer.

Delivery siRNA with a novel gene vector for glioma therapy by targeting Gli1.

BackgroundGene therapy has recently shown considerable clinical benefit in cancer therapy during the past few years, and the application of this choice in cancer treatments is increasing continually. Gli1 is an ideal candidate target for cancer gene therapy and is important for tumorigenesis.MethodsIn this study, we developed a novel gene delivery system with a self-assembly method by using a 1,2-dioleoyl-3-trimethylammonium-propane and methoxy poly (ethylene glycol)-poly(lactide) copolymer (DMP), with zeta potential of 32.7 mV and measuring 35.6 nm. The effect of this delivery system was tested in vitro and in vivo.ResultsDMP showed good performance in delivering siRNA to glioma cells in vitro with high transfection performance (98%). Moreover, DMP–Gli1si shows a satisfactory anti-glioma effect via induction of cell apoptosis and cell growth inhibition in vitro. Furthermore, for subcutaneous tumor-bearing mice, treatment with the DMP–Gli1si complex significantly inhibited tumor growth by inhibiting Gli1 protein expression, promoting apoptosis, and reducing proliferation.ConclusionThe complex of Gli1 siRNA and DMP may potentially play an important role as a new drug in the clinical treatment of gliomas.

Anti-apoptosis Effect of Decoy Receptor 3 in Cholangiocarcinoma Cell Line TFK-1.

Background:Decoy receptor 3 (DcR3) is a protein with anti-apoptotic effect that belongs to the tumor necrosis factor receptor superfamily. DcR3 is highly expressed in a variety of malignant tumors including cholangiocarcinoma and its expression was found to be related to the clinical stage, the invasion, and the metastasis of the tumor. This in vitro study aimed to investigate the effect of downregulated expression of DcR3 on cell viability, cell apoptosis, and cell cycle in cholangiocarcinoma cell line TFK-1.Methods:Three different cell lines were cultured: human cholangiocarcinoma TFK-1, human biliary epithelial carcinoma HuCCT-1, and human cholangiocarcinoma RBE. The cholangiocarcinoma cell line with the highest expression of DcR3 was selected for further investigation. The expression of DcR3 was silenced/knocked down by transfection with DcR3-siRNA in the selected cell line. Various biological phenotype parameters such as cell viability, apoptosis, and cell cycle were observed.Results:The mRNA and protein levels of DcR3 were measured in the three cell lines, and TFK-1 was selected. After the treatment with DcR3-siRNA for 48 h, DcR3 mRNA and protein expression in the treatment group were 38.45% (P < 0.01) and 48.03% (P < 0.05) of that of the control, respectively. It was found that the cell viability decreased to 61.87% of the control group (P < 0.01) after the downregulation of DcR3 in cholangiocarcinoma cell line TFK-1 by transfection with DcR3-siRNA, while the percentage of apoptotic cells was 2.98 times as compared with the control group (P < 0.05). Compared with the control group the ratio of G0/G1 increased, and the ratio of G2/M decreased in the treatment group. However, the differences were not statistically significant.Conclusions:The effect of DcR3 on the growth and apoptosis of cholangiocarcinoma has been demonstrated. DcR3 is not only a predictive marker for malignant tumor but it is also likely to be a potential target for cancer gene therapy. Further studies should focus on exploring the binding ligand of DcR3, the signaling pathway involved, and the molecular mechanism for the regulation of DcR3 expression in cholangiocarcinoma.

Abstract 3134: Hierarchical gene master regulators of papillary and anaplastic thyroid cancer phenotype

Abstract The incidence of thyroid cancer (TCa) has doubled in the last decade. Papillary thyroid cancer (PTC), that comprises of 80% of all TCas, is treatable with great outcomes. However, undifferentiated anaplastic thyroid cancer (ATC), with very poor prognosis, is considered a clinical challenge and currently a losing battle. Moreover, the molecular mechanisms responsible for various forms of TCa are largely unknown. We believe that the cells of histopathologically distinct regions of a heterogeneous thyroid tumor are governed by different sets of master gene regulators. The smart manipulation of such master genes will be able to selectively destroy cancer cells but not normal tissue, opening a novel and much more effective avenue in the thyroid cancer targeted gene therapy. Our analysis established the gene hierarchical governance in each region based on their Gene Commanding Height (GCH). GCH is a measure that combines the gene expression coordination with other genes and the expression stability among biological replicas provided by the internal homeostatic mechanisms. Here, we provide experimental evidence that standard papillary (BCPAP) and anaplastic (8505C) human thyroid cancer cell lines have different master regulators. We identified the master regulators of BCPAP and 8505C, and determined their GCH. Transfection of master gene regulators of a particular cell line has significantly larger effects on the cell line they command than on other cells. We found that the stable transfection with TMEM194A, a nuclear envelope protein, regulated twice more genes in BCPAP than in 8505C cells. The analysis using human thyroid cancer cells reaffirmed our hypothesis of the existence of hierarchical master gene regulators and that the phenotypic changes can be manipulated with the introduction of these genes. We further validate these concepts using human thyroid biopsy samples. We found substantial differences in the GCH scores of cancer versus normal tissue of a surgically removed 32.0mm papillary carcinoma from the left lobe of a 33y old male. Because of such differences between the cancer region and the normal tissue, manipulation of cancer regulators is expected to affect the cancer cells in a greater degree than the normal cells. These results suggest that we have defined a master gene regulator hierarchy in thyroid cancer and extrapolation of this analysis to compare anaplastic and papillary thyroid cancer will lead to novel gene therapeutic modalities. Our long-term goal is to identify master regulators of cancer nodules for each patient and develop personalized cancer therapy targeting these master regulators. Citation Format: Neha Yashpal Tuli, Craig Berzofsky, Rachana Maniyar, Sanjukta Chakraborty, Ghada Ben Rahoma, Sarnath Singh, Jan Geliebter, Raj K. Tiwari, Sanda Iacobas, Dumitru A. Iacobas. Hierarchical gene master regulators of papillary and anaplastic thyroid cancer phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3134. doi:10.1158/1538-7445.AM2017-3134

Knockdown of SKA1 gene inhibits cell proliferation and metastasis in human adenoid cystic carcinoma.

The spindle and kinetochore-associated complex subunit 1(SKA1) is a newly discovered gene, which has been associated with mitosis and tumorigenesis. However, its role insalivary adenoid cystic carcinoma (SACC) is still unknown, and the invasive and metastatic mechanism in SACC is still unclear. To explore the molecular mechanism of SKA1 in the process of malignant proliferation and metastasis in adenoid cystic cancer (ACC) cells, we employed lentivirus-mediated short hairpin RNA to knockdown SKA1 in SACC-83 cells. The results demonstrated that the lentivirus-mediated shRNA-targeting SKA1 lead to a significant down-regulation of SKA1 expression. Knockdown of SKA1 inhibited cell proliferation, cell invasion, migration and the cell cycle arrest. Furthermore, knockdown of SKA1 reduced the Ndc80, CDK4, Cyclin D1, Cyclin E1, Cyclin B1 and matrix metalloproteinase-9 (MMP-9) protein expression, but increased the p27 protein expression. These findings indicated that SKA1 might be a promising target for cancer gene therapy in human ACC.

Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy

BackgroundBacterial toxins have evolved to an effective therapeutic option for cancer therapy. The Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with selective cytotoxicity. The transmembrane tight junction proteins claudin-3 and -4 are known high affinity CPE receptors. Their expression is highly upregulated in human cancers, including breast, ovarian and colon carcinoma. CPE binding to claudins triggers membrane pore complex formation, which leads to rapid cell death. Previous studies demonstrated the anti-tumoral effect of treatment with recombinant CPE-protein. Our approach aimed at evaluation of a selective and targeted cancer gene therapy of claudin-3- and/or claudin-4- expressing colon carcinoma in vitro and in vivo by using translation optimized CPE expressing vector.MethodsIn this study the recombinant CPE and a translation optimized CPE expressing vector (optCPE) was used for targeted gene therapy of claudin-3 and/or -4 overexpressing colon cancer cell lines. All experiments were performed in the human SW480, SW620, HCT116, CaCo-2 and HT-29 colon cancer and the isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines. Claudin expression analysis was done at protein and mRNA level, which was confirmed by immunohistochemistry. The CPE induced cytotoxicity was analyzed by the MTT cytotoxicity assay. In addition patient derived colon carcinoma xenografts (PDX) were characterized and used for the intratumoral in vivo gene transfer of the optCPE expressing vector in PDX bearing nude mice.ResultsClaudin-3 and -4 overexpressing colon carcinoma lines showed high sensitivity towards both recCPE application and optCPE gene transfer. The positive correlation between CPE cytotoxicity and level of claudin expression was demonstrated. Transfection of optCPE led to targeted, rapid cytotoxic effects such as membrane disruption and necrosis in claudin overexpressing cells. The intratumoral optCPE in vivo gene transfer led to tumor growth inhibition in colon carcinoma PDX bearing mice in association with massive necrosis due to the intratumoral optCPE expression.ConclusionsThis novel approach demonstrates that optCPE gene transfer represents a promising and efficient therapeutic option for a targeted suicide gene therapy of claudin-3 and/or claudin-4 overexpressing colon carcinomas, leading to rapid and effective tumor cell killing in vitro and in vivo.

Glucocorticoid receptor: A potent cellular &amp; gene therapy target for cancer and cancer stem cells

Glucocorticoid receptor (GR) is expressed in the cytoplasm of almost all cells, if not all of cancer and non-cancer cells. Moreover, unlike many other factors implicated with cancer, it is neither overexpressed nor is it expressed on cell-membrane surface to qualify logically as a viable target for treating cancer. GR is importantly linked with alternate pathway of energy metabolism in cancer cells and our research indicated that cancer cells possibly tend to avoid activation of GR which would have otherwise instigate that energy demanding alternate pathway called gluconeogenesis. We discovered a way to induce cancer cell-selective GR-transactivation which leads to among many things, gluconeogenesis, reversal of epithelial-to-mesenchymal transition (EMT), drug- sensitization in drug-resistant cancer cells etc. Thus, we proved, although it warrants further studies, that GR in cancer cells behave differently and hence it can be a viable target for the treatment of cancer.

Anti-EGFR immunonanoparticles containing IL12 and salmosin genes for targeted cancer gene therapy

Tumor-directed gene delivery is of major interest in the field of cancer gene therapy. Varied functionalizations of non-viral vectors have been suggested to enhance tumor targetability. In the present study, we prepared two different types of anti-EGF receptor (EGFR) immunonanoparticles containing pDNA, neutrally charged liposomes and cationic lipoplexes, for tumor-directed transfection of cancer therapeutic genes. Even though both anti-EGFR immunonanoparticles had a high binding affinity to the EGFR-positive cancer cells, the anti-EGFR immunolipoplex formulation exhibited approximately 100-fold higher transfection to the target cells than anti-EGFR immunoliposomes. The lipoplex formulation also showed a higher transfection to SK-OV-3 tumor xenografts in mice. Thus, IL12 and/or salmosin genes were loaded in the anti-EGFR immunolipoplexes and intravenously administered to mice carrying SK-OV-3 tumors. Co-transfection of IL12 and salmosin genes using anti-EGFR immunolipoplexes significantly reduced tumor growth and pulmonary metastasis. Furthermore, combinatorial treatment with doxorubicin synergistically inhibited tumor growth. These results suggest that anti-EGFR immunolipoplexes containing pDNA encoding therapeutic genes could be utilized as a gene-transfer modality for cancer gene therapy.

Folate-conjugated polyspermine for lung cancer–targeted gene therapy

Biodegradable polyamines have long been studied as potential recombinant viral gene vectors. Spermine (SPE) is an endogenous tetra-amine with excellent biocompatibility yet poor gene condensation capacity. We have previously synthesized a polyspermine based on SPE and poly(ethylene glycol) (PEG) diacrylate (SPE-alt-PEG) for enhanced transfection performance, but the synthesized SPE-alt-PEG still lacked specificity towards cancer cells. In this study, folic acid (FA) was incorporated into SPE-alt-PEG to fabricate a targeted gene delivery vector (FA-SPE-PEG) via an acylation reaction. FA-SPE-PEG exhibited mild cytotoxicity in both cancer cells and normal cells. FA-SPE-PEG possessed higher transfection efficiency than PEI 25K and Lipofectamine® 2000 in two tested cancer cell lines at functional weight ratios, and its superiority over untargeted SPE-alt-PEG was prominent in cells with overexpressed folate receptors (FRs). Moreover, in vivo delivery of green fluorescent protein (GFP) with FA-SPE-PEG resulted in highest fluorescent signal intensity of all investigated groups. FA-SPE-PEG showed remarkably enhanced specificity towards cancer cells both in vivo and in vitro due to the interaction between FA and FRs. Taken together, FA-SPE-PEG was demonstrated to be a prospective targeted gene delivery vector with high transfection capacity and excellent biocompatibility.

Modification of the hTERT promoter by heat shock elements enhances the efficiency and specificity of cancer targeted gene therapy

Purpose: One of the current challenges facing cancer gene therapy is the tumour-specific targeting of therapeutic genes. Effective targeting in gene therapy requires accurate spatial and temporal control of gene expression. To develop a sufficient and accurate tumour-targeting method for cancer gene therapy, we have investigated the use of hyperthermia to control the expression of a transgene under the control of the human telomerase reverse transcriptase (hTERT) promoter and eight heat shock elements (8HSEs). Materials and methods: Luciferase reporters were constructed by inserting eight HSEs and the hTERT promoter (8HSEs-hTERTp) upstream of the pGL4.20 vector luciferase gene. The luciferase activity of the hTERT promoter and 8HSEs-hTERT promoter were then compared in the presence and absence of heat. The differences in luciferase activity were analysed using dual luciferase assays in SW480 (high hTERT expression), MKN28 and MRC-5 cells (low hTERT expression). The luciferase activity of the Hsp70B promoter was also compared to the 8HSEs-hTERT promoter in the above listed cell lines. Lentiviral vector and heat-induced expression of EGFP expression under the control of the 8HSEs-hTERT promoter in cultured cells and mouse tumour xenografts was measured by reverse transcription polymerase (RT-PCR), Western blot and immunofluorescence assays. Results: hTERT promoter activity was higher in SW480 cells than in MKN28 or MRC-5 cells. At 43 °C, the luciferase activity of the 8HSEs-hTERT promoter was significantly increased in SW480 cells, but not in MKN28 or MRC-5 cells. Importantly, the differences in luciferase activity were much more obvious in both high (SW480) and low (MKN28 and MRC-5) hTERT expressing cells when the activity of the 8HSEs-hTERT promoter was compared to the Hsp70B promoter. Moreover, under the control of 8HSEs-hTERT promoter in vitro and in vivo, EGFP expression was obviously increased by heat treatment in SW480 cells but not in MKN28 or MRC-5 cells, nor was expression increased under normal temperature conditions. Conclusions: The hTERT promoter is a potentially powerful tumour-specific promoter and gene therapy tool for cancer treatment. Incorporating heat-inducible therapeutic elements (8HSEs) into the hTERT promoter may enhance the efficiency and specificity of cancer targeting gene therapy under hyperthermic clinical conditions.

Targeted Inhibition of the miR-199a/214 Cluster by CRISPR Interference Augments the Tumor Tropism of Human Induced Pluripotent Stem Cell-Derived Neural Stem Cells under Hypoxic Condition.

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However, the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p, miR-199a-3p, and miR-214 in the microRNA gene cluster. Meanwhile, the expression levels of their targets related to regulation of hypoxia-stimulated cell migration, such as HIF1A, MET, and MAPK1, were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy.

Folic acid conjugated amine functionalized cerium oxide nanoparticle for cancer targeted gene therapy

Event Abstract Back to Event Folic acid conjugated amine functionalized cerium oxide nanoparticle for cancer targeted gene therapy Kishor Sarkar1, Harini V. Krishnan1, G Vinoth Kumar2, K Suresh Babu2, Vinayak Sant1 and Shilpa Sant1, 3, 4 1 University of Pittsburgh, Department of Pharmaceutical Sciences, United States 2 Pondicherry University, Centre for Nano Science and Technology, India 3 University of Pittsburgh, Department of Bioengineering, United States 4 University of Pittsburgh, McGowan Institute for Regenerative Medicine, United States Introduction: Gene therapy has become alternative therapeutic approach to treat genetic diseases such as cancer[1]. Nonviral vectors have gained tremendous attraction over the viral vector due to its low toxicity, non-immunogenicity and low production cost[2]-[4]. Recently, rare earth metal oxide especially cerium oxide nanoparticle (CNP) has widely been used in biomedical fields such as drug delivery, bio-imaging, nanomedicine and tissue engineering application because of its inherent antioxidant activity, free radical scavenging activity and radioprotection to normal cell[5],[6]. It is also reported that CNP increased the ROS (reactive oxygen species) in cancer cell[7]. Therefore, we hypothesized that therapeutic gene (like siRNA, p53 etc.) delivery by means of CNP may give the dual therapeutic effect in conjugation with increased ROS level in cancer cell for the treatment of cancer. Here we report the functionalization of CNP by low molecular weight polyethyleneimine (LPEI, 2 kDa) followed by conjugation of folic acid (FA) for targeted gene delivery. Methods: LPEI was conjugated with CNP (LPEI-conj-CNP) through carboxylation followed by amidation reaction. Then, FA was reacted with LPEI-conj-CNP by carbodiimide to get FA-LPEI-conj- CNP. CNP/pDNA (enhanced green fluorescent protein, EGFP encoding pDNA) complexes were prepared at various weight ratios and characterized by agarose gel electrophoresis assay and dynamic light scattering (DLS). In vitro transfection efficiency, toxicity, ROS and underlying mechanistic pathway were also evaluated in HeLa cell. Results: Poor water dispersibility of CNP at physiological pH is a major drawback for biological field especially gene/ drug delivery application. CNP forms stable dispersion in acidic pH only. Despite the positive zeta potential of CNP, it did not bind any DNA at any weight ratio due to poor dispersibility. But, the DNA complexation capability of CNP significantly increased after conjugation of LPEI and LPEI-conj-CNP complexed all pDNA at a very low weight ratio of 0.5:1. Due to presence of lots of primary amine groups in LPEI, the water dispersibility of CNP increased drastically and resulted better DNA complexation through the cationic primary amine groups. The transfection efficiency of CNP increased after conjugation of LPEI but the efficiency increased significantly after FA conjugation with LPEI-conj-CNP. FA-LPEI-conj-CNP/pDNA complex at weight ratio of 15:1 showed highest transfection efficiency which also surpassed the efficiency offered by PEI (25 kDa) (Figure 1). It was also observed that FA-LPEI-conj-CNP internalized into cell via folate receptor as well as caveolae mediated pathway and resulted enhanced transfection efficiency. While LPEI-conj-CNP followed clathrin mediated pathway followed by lysosomal compartment entrapment and as a result showed low transfection efficiency. It was found that green fluorescence (fluorescent dichlorofluorescein, DCF obtained through oxidation of nonfluorescent dichlorofluorescein diacetate, DCFA by ROS) increased drastically by FA-LPEI-conj-CNP compared to all CNPs and H2O2 treated cell indicating the increase in ROS (Figure 2). Conclusion: The transfection efficiency as well as ROS of CNP increased after functionalization of CNP especially after FA conjugation. Therefore, FA-LPEI-conj-CNP may be useful candidate in nonviral gene therapy for cancer treatment by using its dual property including gene delivery and ROS enhancement. This work was financially supported by Start-up funds from University of Pittsburgh School of Pharmacy