Approach For Cancer Gene Therapy Research Articles

Abstract LB_B02: Novel PAMs from somatic mutations produce numerous pancreatic cancer-specific CRISPR-Cas9 targets for selective cell elimination: A novel gene therapy approach for cancer

Abstract Somatic mutations accumulate as we age and are mostly found within the noncoding regions which make up of 98% of the human genome. CRISPR-Cas9, on the other hand, can be designed to induce double strand breaks (DSBs) at specific locations in the genome, and DSBs are known to be cytotoxic. CRISPR-Cas9 requires the presence of a 3-5 nucleotide protospacer adjacent motif (PAM) to serve as a binding signal for Cas9, in which Cas9 would not bind nor cut the target in its absence. We hypothesized that we could identify a large number of somatic 5’-NGG-3’ PAMs that arise from single base substitutions (SBSs) in pancreatic cancers (PCs; e.g. NCG -> NGG), and these novel PAMs could serve as cancer-specific CRISPR-Cas9 targets for selective cell elimination. First, we performed whole genome sequencing (WGS) of three PC cell lines and their corresponding normal cell lines followed by tumor-normal (T-N) subtraction to identify somatic SBSs. PAMfinder, a software designed for somatic PAM discovery, was executed to identify somatic PAMs in each case. On average, 4548 somatic SBSs were found per PC case, in which 417 of them produced somatic PAMs (9.2% of total SBSs). PAMs with >95% variant allele frequency (VAF) and that produced sgRNAs with minimal off-target activity (based on CRISPOR) were further validated through Sanger sequencing, which resulted in an average of 32/33 confirmed targets per cell line. Mutational signature analyses also revealed that clock-like signatures were predominant, suggesting that aging itself could give rise to novel PAMs. We subsequently tested the specificity and selectivity of our patient-specific sgRNAs derived from two PC cases and showed that on-target mutations were only detected in the target cell lines and not in the corresponding normal or other PC cell lines lacking the targets. This result was further substantiated by comprehensive WGS analyses in a negative control cell line which demonstrated no off-target activity. Then, two PC cell lines were co-cultured and transduced with patient-specific sgRNAs identified from one of the PC cell lines, and showed >75% selective cell killing via flow cytometry. Finally, to examine the prevalence of somatic PAMs in different tumor types, we obtained 591 T-N subtracted variant call files from ICGC and performed tumor purity correction and PAMfinder analyses for each sample. We found that in two separate PC projects (APGI-AU and PACA-CA), the number of novel PAMs were 703 (N=44) and 863 (N=130), respectively, on average; in the lung cancer (LUCA-KR) and esophageal cancer (OCCAMS-GB) projects, an average of 3319 (N=29) and 4739 (N=388) novel PAMs were discovered, respectively. Overall, our results showed that adult solid tumors harbor hundreds, if not thousands, of somatic PAMs, and these PAMs could serve as novel CRISPR-Cas9 targets to selectively kill cancer cells. Citation Format: Selina Shiqing K Teh, Kirsten Bowland, Alexis Bennett, Eitan Halper-Stromberg, Alyza Skaist, Aparna Pallavajjala, Jacqueline Tang, Fidel Cai, Hong Liang, Sarah Wheelan, Robert B Scharpf, Nicholas J Roberts, James R Eshleman. Novel PAMs from somatic mutations produce numerous pancreatic cancer-specific CRISPR-Cas9 targets for selective cell elimination: A novel gene therapy approach for cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B02.

RNA Interference against ATP as a Gene Therapy Approach for Prostate Cancer.

Chemotherapeutic agents targeting energy metabolism have not achieved satisfactory results in different types of tumors. Herein, we developed an RNA interference (RNAi) method against adenosine triphosphate (ATP) by constructing an interfering plasmid-expressing ATP-binding RNA aptamer, which notably inhibited the growth of prostate cancer cells through diminishing the availability of cytoplasmic ATP and impairing the homeostasis of energy metabolism, and both glycolysis and oxidative phosphorylation were suppressed after RNAi treatment. Further identifying the mechanism underlying the effects of ATP aptamer, we surprisingly found that it markedly reduced the activity of membrane ionic channels and membrane potential which led to the dysfunction of mitochondria, such as the decrease of mitochondrial number, reduction in the respiration rate, and decline of mitochondrial membrane potential and ATP production. Meanwhile, the shortage of ATP impeded the formation of lamellipodia that are essential for the movement of cells, consequently resulting in a significant reduction of cell migration. Both the downregulation of the phosphorylation of AMP-activated protein kinase (AMPK) and endoplasmic reticulum kinase (ERK) and diminishing of lamellipodium formation led to cell apoptosis as well as the inhibition of angiogenesis and invasion. In conclusion, as the first RNAi modality targeting the blocking of ATP consumption, the present method can disturb the respiratory chain and ATP pool, which provides a novel regime for tumor therapies..

Interleukin-24-mediated antitumor effects against human glioblastoma via upregulation of P38 MAPK and endogenous TRAIL-induced apoptosis and LC3-II activation-dependent autophagy

BackgroundMelanoma differentiation-associated gene 7 (Mda-7) encodes IL-24, which can induce apoptosis in cancer cells. A novel gene therapy approach to treat deadly brain tumors, recombinant mda-7 adenovirus (Ad/mda-7) efficiently kills glioma cells. In this study, we investigated the factors affecting cell survival and apoptosis and autophagy mechanisms that destroy glioma cells by Ad/IL-24.MethodsHuman glioblastoma U87 cell line was exposed to a multiplicity of infections of Ad/IL-24. Antitumor activities of Ad/IL-24 were assessed by cell proliferation (MTT) and lactate dehydrogenase (LDH) release analysis. Using flow cytometry, cell cycle arrest and apoptosis were investigated. Using the ELISA method, the tumor necrosis factor (TNF-α) level was determined as an apoptosis-promoting factor and Survivin level as an anti-apoptotic factor. The expression levels of TNF-related apoptosis inducing ligand(TRAIL) and P38 MAPK genes were assessed by the Reverse transcription-quantitative polymerase chain reaction(RT‑qPCR) method. The expression levels of caspase-3 and protein light chain 3-II (LC3-II) proteins were analyzed by flow cytometry as intervening factors in the processes of apoptosis and autophagy in the cell death signaling pathway, respectively.ResultsThe present findings demonstrated that transduction of IL-24 inhibited cell proliferation and induced cell cycle arrest and cell apoptosis in glioblastoma. Compared with cells of the control groups, Ad/IL24-infected U87 cells exhibited significantly increased elevated caspase-3, and TNF-α levels, while the survivin expression was decreased. TRAIL was shown to be upregulated in tumor cells after Ad/IL-24 infection and studies of the apoptotic cascade regulators indicate that Ad/IL-24 could further enhance the activation of apoptosis through the TNF family of death receptors. In the current study, we demonstrate that P38 MAPK is significantly activated by IL-24 expression. In addition, the overexpression of mda-7/IL-24 in GBM cells induced autophagy, which was triggered by the upregulation of LC3-II.ConclusionsOur study demonstrates the antitumor effect of IL-24 on glioblastoma and may be a promising therapeutic approach for GBM cancer gene therapy.

Enhanced RNA knockdown efficiency with engineered fusion guide RNAs that function with both CRISPR-CasRx and hammerhead ribozyme

BackgroundCRISPR-Cas13 is a newly emerging RNA knockdown technology that is comparable to RNAi. Among all members of Cas13, CasRx degrades RNA in human cells with high precision and effectiveness. However, it remains unclear whether the efficiency of this technology can be further improved and applied to gene therapy.ResultsIn this study, we fuse CasRx crRNA with an antisense ribozyme to construct a synthetic fusion guide RNA that can interact with both CasRx protein and ribozyme and tested the ability of this approach in RNA knockdown and cancer gene therapy. We show that the CasRx-crRNA-ribozyme system (CCRS) is more efficient for RNA knockdown of mRNAs and non-coding RNAs than conventional methods, including CasRx, shRNA, and ribozyme. In particular, CCRS is more effective than wild-type CasRx when targeting multiple transcripts simultaneously. We next use bladder cancer as a model to evaluate the anticancer effects of CCRS targeting multiple genes in vitro and in vivo. CCRS shows a higher anticancer effect than conventional methods, consistent with the gene knockdown results.ConclusionsThus, our study demonstrates that CCRS expands the design ideas and RNA knockdown capabilities of Cas13 technology and has the potential to be used in disease treatment.

Cell-Penetrating Peptide and siRNA-Mediated Therapeutic Effects on Endometriosis and Cancer In Vitro Models.

Gene therapy is a powerful tool for the development of new treatment strategies for various conditions, by aiming to transport biologically active nucleic acids into diseased cells. To achieve that goal, we used highly potential delivery vectors, cell-penetrating peptides (CPPs), as oligonucleotide carriers for the development of a therapeutic approach for endometriosis and cancer. Despite marked differences, both of these conditions still exhibit similarities, like excessive, uncoordinated, and autonomous cellular proliferation and invasion, accompanied by overlapping gene expression patterns. Thus, in the current study, we investigated the therapeutic effects of CPP and siRNA nanoparticles using in vitro models of benign endometriosis and malignant glioblastoma. We demonstrated that CPPs PepFect6 and NickFect70 are highly effective in transfecting cell lines, primary cell cultures, and three-dimensional spheroids. CPP nanoparticles are capable of inducing siRNA-specific knockdown of therapeutic genes, ribonucleotide reductase subunit M2 (RRM2), and vascular endothelial growth factor (VEGF), which results in the reduction of in vitro cellular proliferation, invasion, and migration. In addition, we proved that it is possible to achieve synergistic suppression of endometriosis cellular proliferation and invasion by combining gene therapy and hormonal treatment approaches by co-administering CPP/siRNA nanoparticles together with the endometriosis-drug danazol. We suggest a novel target, RRM2, for endometriosis therapy and as a proof-of-concept, we propose a CPP-mediated gene therapy approach for endometriosis and cancer.

Synthesis of 9-(6-Deoxy-α-L-Talofuranosyl)-6-Methylpurine and 9-(6-Deoxy-β-D-Allofuranosyl)-6-Methylpurine Nucleosides.

6-Methylpurine (MeP) is a cytotoxic adenine analog that does not exhibit selectivity when administered systemically and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli purine nucleoside phosphorylase (PNP). 9-(6-Deoxy-β-D-allofuranosyl)-6-methylpurine [methyl(allo)-MePR, 18] and 9-(6-deoxy-α-L-talofuranosyl)-6-methylpurine [methyl(talo)-MePR, 21] were synthesized as potential prodrugs for MeP in the E. coli PNP/prodrug cancer gene therapy approach. The detailed syntheses of [methyl(allo)-MePR] and [methyl(talo)-MePR] are described. The glycosyl donors, 1,2-di-O-acetyl-3,5-di-O-benzyl-α-D-allofuranose (12) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-α-L-talofuranose (16) were prepared from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranose (4) in nine and eleven steps, respectively. Vorbrüggen coupling of the latter glycosyl donors with 6-methylpurine (3), followed by deprotection of the sugar hydroxyl groups, gave the title compounds in good overall yields. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of 6-methylpurine Basic Protocol 2: Preparation of the D-allofuranose derivative (12) Basic Protocol 3: Preparation of 6-deoxy-α-L-talofuranoside Basic Protocol 4: Preparation of methyl(allo)-MePR (18) Basic Protocol 5: Preparation of methyl(talo)-MePR (21).

Inhibition of miR‐155 in MCF‐7 breast cancer cell line by gold nanoparticles functionalized with antagomir and AS1411 aptamer

MicroRNAs are key factors for many biological functions. These regulatory molecules affect various gene networks and involve the subsequent signaling pathways. Therefore, disrupting the expression of these molecules is associated with multiple anomalies in the cells and body. One of the most important related abnormalities is the incidence of cancer. Thus, targeting microRNAs (miRNAs) is an effective approach for cancer gene therapy. Various factors are used for this purpose, including the antagomir nucleotide structure. There are some obstacles in the delivery of nucleotide therapeutics to the target cells, however, the use of nanoparticles could partly overcome these defeciencies. On the other hand, targeted delivery of antagomirs using aptamers, reduces nonspecific effects on nontarget cells. Considering the above, in this study, we designed and fabricated a nanocarrier composed of gold nanoparticles (GNPs), antagomir-155, and nucleolin specific aptamer for breast cancer study and therapy. Here, GNPs were synthesized using citrate reduction and were modified by polyA sequences, AS1411 aptamer, and antagomir-155. Attachment of molecules were confirmed using gel electrophoresis, atomic force microscopy imaging and electrochemical test. The specific entry of modified nanoparticles was investigated by fluorescence microscopy. The efficacy of modified nanoparticles was evaluated using a quantitative polymerase chain reaction (q-PCR) for miR-155 and its target gene. Efficient and specific delivery of AuNP-Apt-anti-miR-155 to target cells was confirmed in comparison with the control cell. The q-PCR analysis showed not only a significant decrease in mir-155 levels but also an elevated TP53INP1 mRNA, direct target of miR-155. The proposed structure inhibits proliferation and stimulates apoptosis by increasing the expression of TP53INP1. Our results suggest that AuNP-Apt-anti-miR-155 could be a promising nano constructorfor breast cancer treatment.

P19Arf sensitizes B16 melanoma cells to interferon-β delivered via mesenchymal stem cells in vitro.

The immune stimulatory and anti-neoplastic functions of type I interferon have long been applied for the treatment of melanoma. However, the systemic application of high levels of this recombinant protein is often met with toxicity. An approach that provides localized, yet transient, production of type I interferon may overcome this limitation. We propose that the use of mesenchymal stem cells (MSCs) as delivery vehicles for the production of interferon-β (IFNβ) may be beneficial when applied together with our cancer gene therapy approach. In our previous studies, we have shown that adenovirus-mediated gene therapy with IFNβ was especially effective in combination with p19Arf gene transfer, resulting in immunogenic cell death. Here we showed that MSCs derived from mouse adipose tissue were susceptible to transduction with adenovirus, expressed the transgene reliably, and yet were not especially sensitive to IFNβ production. MSCs used to produce IFNβ inhibited B16 mouse melanoma cells in a co-culture assay. Moreover, the presence of p19Arf in the B16 cells sensitizes them to the IFNβ produced by the MSCs. These data represent a critical demonstration of the use of MSCs as carriers of adenovirus encoding IFNβ and applied as an anti-cancer strategy in combination with p19Arf gene therapy.

Non-viral Delivery of Nucleic Acids: Insight Into Mechanisms of Overcoming Intracellular Barriers.

Delivery of genes, including plasmid DNAs, short interfering RNAs (siRNAs), and messenger RNAs (mRNAs), using artificial non-viral nanotherapeutics is a promising approach in cancer gene therapy. However, multiple physiological barriers upon systemic administration remain a key challenge in clinical translation of anti-cancer gene therapeutics. Besides extracellular barriers including sequestration of gene delivery nanoparticles from the bloodstream by resident organ-specific macrophages, and their poor extravasation and tissue penetration in tumors, overcoming intracellular barriers is also necessary for successful delivery of nucleic acids. Whereas for RNA delivery the endosomal barrier holds a key importance, transfer of DNA cargo additionally requires translocation into the nucleus. Better understanding of crossing membrane barriers by nucleic acid nanoformulations is essential to the improvement of current non-viral carriers. This review aims to summarize relevant literature on intracellular trafficking of non-viral nanoparticles and determine key factors toward surmounting intracellular barriers. Moreover, recent data allowed us to propose new interpretations of current hypotheses of endosomal escape mechanisms of nucleic acid nanoformulations.

Biodegradable Black Phosphorus Nanosheets Mediate Specific Delivery of hTERT siRNA for Synergistic Cancer Therapy.

Human telomerase reverse transcriptase (hTERT) has been found to be closely related to tumor transformation, growth, and metastasis. Thus, the delivery of hTERT small interfering RNA (siRNA) is an important approach for cancer gene therapy. However, the single anticancer effect of gene silencing is often limited by poor specificity or low efficiency in siRNA delivery and release. In this work, we present small and thin black phosphorus (BP) nanosheets as a biodegradable delivery system for hTERT siRNA. The BP nanosheets prepared with poly(ethylene glycol) (PEG) and polyethylenimine (PEI) modification (PPBP), exhibited high siRNA loading capacity and robust cell uptake. The PPBP nanosheets also exhibited potent photodynamic therapy/photothermal therapy (PDT/PTT) activities when exposed to different wavelengths of laser irradiation. More importantly, PPBP nanosheets underwent a gradual degradation when presented in a mixture of low pH and reactive oxygen species (ROS)-rich environment. The degradation of PPBP was strengthened especially after local and minimal invasive PDT treatment, because of excessive ROS production. Further delivery and release of siRNA to the cytoplasm for gene silencing was achieved by PEI-aided escape from the acidic lysosome. Thus, PPBP-siRNA efficiently inhibited tumor growth and metastasis by specific delivery of hTERT siRNA and a synergistic combination of targeted gene therapy, PTT and PDT.

Abstract B31: Death of endothelial cells induced by paracrine exposure to factors derived from murine melanoma cells upon interferon-beta gene therapy

Abstract Tumor vasculature plays a central role in tumor progression, making it a therapeutic target. Inhibition of angiogenesis has the potential to slow down tumor progression and inhibit metastasis. Interferon-beta (IFN) is a cytokine that shows antitumor effects, including immune activation. In addition, IFN inhibits angiogenesis by reducing the production of proangiogenic factors and by inducing death of endothelial cells. Gene therapy is showing significant results in cancer treatment including when recombinant adenoviral vectors are used for the delivery of therapeutic genes. In our previous studies, we developed a specialized adenoviral vector for the delivery of IFN to p53-positive cells. We propose that the impact of our cancer gene therapy approach may include the inhibition of angiogenesis. Here we explore the response of endothelial cells exposed to IFN through distinct routes of delivery. The efficient transduction of tEnd cells, a murine endothelial cell line, was confirmed by detection of GFP encoded by the control vector (Ad-GFP). In addition, tEnd cells readily produced IFN, as measured by ELISA, after treatment with Ad-IFN. Unexpectedly, IFN gene transfer did not alter angiogenic characteristics, such as migration or tube formation as evidenced in wound healing and tubulogenesis assays, respectively. Our results also indicate that the gene transfer of IFN does not impact cell viability since treatment did not provoke the accumulation of hypodiploid cells, was not associated with AnnexinV staining, and did not impact clonogenesis or MTT staining. These data indicate that expression of IFN by the transduced endothelial cells may not have any effect, a surprise given IFN's well-known ability to inhibit tumor angiogenesis. We next sought to investigate the impact of IFN treatment in a model that mimics certain aspects of the tumor microenvironment. In the first set of assays, B16 (mouse melanoma, p53wt) tumor cells were transduced, washed to remove excess virus particles, and only after were they cocultivated with tEnd cells, permiting the direct contact of adenoviral transduced tumor cells and non-treated endothelial cells. In order to differentiate the two cell lines, we used tEnd cells marked with the dTomato fluorescent protein (which we call tEndTO). Strikingly, the number of tEndTO cells was drastically reduced in the presence of B16 cells treated with Ad-IFN, suggesting that treated tumor cells promote a paracrine effect that inhibits tEnd cells. This point was further explored by exposing tEnd cells to conditioned medium derived from B16 cells that had been transduced with the recombinant adenoviral vectors before analysis of the viability of the endothelial cells. We observed significantly reduced colony formation and MTT staining accompanied by greatly increased accumulation of hypodiploid cells as well as AnnexinV staining when the tEnd cells were cultivated in conditioned medium derived from Ad-IFN transduced tumor cells, but not Ad-GFP. These data suggest that the exposure of tEnd to the secretome of Ad-IFN tranduced B16 cells results in the death of the endothelial cells in a paracrine fashion. In the context of gene therapy for melanoma, the use of modified adenoviral vectors carrying IFN as the therapeutic gene may be of great interest. Our results show a remarkable paracrine effect on endothelial cells when they are exposed to factors produced by melanoma cells transduced with Ad-IFN. Currently, we are continuing our evaluation of the use of these vectors to inhibit melanoma angiogenesis to cause retardation of tumor growth, though special attention will be given to the differential response associated with the route of IFN delivery and the possibility that additional factors are required to bring about endothelial cell death. Citation Format: Igor de Luna Vieira, Rodrigo Esaki Tamura, Bryan Eric Strauss. Death of endothelial cells induced by paracrine exposure to factors derived from murine melanoma cells upon interferon-beta gene therapy [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B31.

Quantum Dots-siRNA Nanoplexes for Gene Silencing in Central Nervous System Tumor Cells.

RNA interfering (RNAi) using short interfering RNA (siRNA) is becoming a promising approach for cancer gene therapy. However, owing to the lack of safe and efficient carriers, the application of RNAi for clinical use is still very limited. In this study, we have developed cadmium sulphoselenide/Zinc sulfide quantum dots (CdSSe/ZnS QDs)-based nanocarriers for in vitro gene delivery. These CdSSe/ZnS QDs are functionalized with polyethyleneimine (PEI) to form stable nanoplex (QD-PEI) and subsequently they are used for siRNA loading which specially targets human telomerase reverse transcriptase (TERT). High gene transfection efficiency (>80%) was achieved on two glioblastoma cell lines, U87 and U251. The gene expression level (49.99 ± 10.23% for U87, 43.28 ± 9.66% for U251) and protein expression level (51.58 ± 7.88% for U87, 50.69 ± 7.59% for U251) of TERT is observed to decrease substantially after transfecting the tumor cells for 48 h. More importantly, the silencing of TERT gene expression significantly suppressed the proliferation of glioblastoma cells. No obvious cytotoxicity from these QD-PEI nanoplexes were observed over at 10 times of the transfected doses. Based on these results, we envision that QDs engineered here can be used as a safe and efficient gene nanocarrier for siRNA delivery and a promising tool for future cancer gene therapy applications.

SC06.03 Intraoperative Therapies in Malignant Pleural Mesothelioma

SC06.03 Intraoperative Therapies in Malignant Pleural Mesothelioma

Sonoporation by microbubbles as gene therapy approach for liver cancer

Purpose: We use an innovative method, known as sonoporation, to induce the expression of silenced gene in liver cancer cells (HepG2), such as (but nit restricted to) TRAIL in a specific manner. Aim of the project is the re-activation of silenced apoptotic pathway in liver cancer models, using diagnostic microbubble synovial as plasmidic gene delivery. Material and methods: HepG2 ATCC were used to assess all the experiments. Microbubble (Sonovue®) were used at standard condition according to manufacturer's instructions. pEGFP-TRAIL plasmid (Plasmid #10953 addgene) and the respective control were selected and propagated in LB broth in order to obtain the necessary amount. Plasmid were purified with Invitrogen PureLink (thermo-fisher scientific Cod. K210017) kit. Transfection was mediated by Ultrasound device (Sonitron 2000, Artison corporation®) compared with standard protocol for lipofectamine 2000 (Invitrogen). GFP (Green Fluorescent Protein) was acquired via FACS excalibur DB analysis. Results: HepG2 cells were used to achieve the TRAIL-GFP recombinant protein transfection. Cells were collected and re-suspended in PBS1X and Cell Cycle Buffer. FACS analysis was performed and results were analysed with Cell-Quest and ModIFit software. Among the several condition, cytotoxic parameters were acquired (5 MHz, 100% Duty Cycle, and 3 W/cm2, 60 s) with over than 80% cells in Pre-G1 phase; meanwhile lower parameters were not enough for gene delivery (1 MHz, 30% Duty Cycle, and 1 W/cm2 60 s). Best parameters were collected between 3 MHz, 100% Duty Cycle, and 1 W/cm2, 30 s). Data showed a dose dependent effect in terms of output energy. 30 – 50% transfection efficacy was acquired and TRAIL re-expression induced apoptotic effect. Conclusion: Results showed the possibility to restore the expression of pro-apoptotic gene TRAIL in a liver cancer model HepG2. The future goal, in our vision, is the translation in animal model of our system, in order to evaluate the in-vivo effect of plasmidic gene therapy in hepatocarcinoma cells.

Role of nanotechnology and gene delivery systems in TRAIL-based therapies.

Since its identification as a member of the tumour necrosis factor (TNF) family, TRAIL (TNF-related apoptosis-inducing ligand) has emerged as a new avenue in apoptosis-inducing cancer therapies. Its ability to circumvent the chemoresistance of conventional therapeutics and to interact with cancer stem cells (CSCs) self-renewal pathways, amplified its potential as a cancer apoptotic agent. Many recombinant preparations of this death ligand and monoclonal antibodies targeting its death receptors have been tested in monotherapy and combinational clinical trials. Gene therapy is a new approach for cancer treatment which implies viral or non-viral functional transgene induction of apoptosis in cancer cells or repair of the underlying genetic abnormality on a molecular level. The role of this approach in overcoming the traditional barriers of radiation and chemotherapeutics systemic toxicity, risk of recurrence, and metastasis made it a promising platform for cancer treatment. The recent first Food Drug Administration (FDA) approved oncolytic herpes virus for melanoma treatment brings forth the potency of the cancer gene therapy approach in the future. Many gene delivery systems have been studied for intratumoural TRAIL gene delivery alone or in combination with chemotherapeutic agents to produce synergistic cancer cytotoxicity. However, there still remain many obstacles to be conquered for this different gene delivery systems. Nanomedicine on the other hand offers a new frontier for clinical trials and biomedical research. The FDA approved nanodrugs motivates horizon exploration for other nanoscale designed particles’ implications in gene delivery. In this review we aim to highlight the molecular role of TRAIL in apoptosis and interaction with cancer stem cells (CSCs) self-renewal pathways. Finally, we also aim to discuss the different roles of gene delivery systems, mesenchymal cells, and nanotechnology designs in TRAIL gene delivery.

Bifidobacterial recombinant thymidine kinase-ganciclovir gene therapy system induces FasL and TNFR2 mediated antitumor apoptosis in solid tumors.

BackgroundDirectly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms.MethodsBifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression.ResultsThe results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy.ConclusionsBF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim without inducing necroptosis and autophagy. Furthermore, BF-rTK + GCV showed to repress the inflammation of tumor through downregulating TNF-α expression. Survival analysis results of multiple cancer models confirmed that BF-rTK + GCV system has a wide field of application in solid tumor gene therapy.

Live imaging of transgene expression in Cloudman S91 melanoma cells after polyplex-mediated gene delivery

Utilizing nanoparticles made of cationic polymers as gene carriers is a promising approach in cancer gene therapy. One of the major requirements for successful gene delivery is DNA translocation into the nuclei of cancer cells. Nuclear envelope breakdown during mitosis has been considered as the most favorable opportunity for DNA translocation to the nucleus. Here, we aimed to study the influence of mitosis on polyplex-mediated gene delivery using time-lapse microscopy as a safe and accurate tool. Studying of reporter gene expression on a single cell level enabled to confirm the significance of cell division for gene delivery to Cloudman S91 melanoma cells, in spite of minor mitosis-independent transfection, and to discover some important details of polyplex delivery process. We have found that cell division can result in only one post-mitotic transfected cell of the two that could indicate non-uniform distribution of a very small number of intact plasmid DNA between daughter cells. According to our data, the shorter the time interval from polyplex addition to cell division, the longer time is required for the start of reporter gene expression after completed cytokinesis that presumably is a result of gradual polyplex dissociation in cell. Most probably, the development of new gene delivery carriers which would combine the strong ability to protect DNA and ability to release it during mitosis can provide an increase in intact DNA molecule number per cell, uniform DNA distribution between two post-mitotic cells, and fast reporter gene expression resulting in superior transfection of proliferating cells.

170. TransfeX and TransIT-LT1 Mediated Gene Delivery to Cervical and Oral Squamous Cell Carcinoma Cells

While TransIT-LT1 is not quite as effective, TransfeX facilitates unusually high levels of transgene expression at levels comparable to that achieved by adenoviral transduction (our unpublished data). Transfection of HeLa cells with TransfeX exhibited the highest transfection activity obtained in our laboratory, and was almost 5 times higher than that achieved with TransIT-LT1. In HSC-3 cells, transfection efficiency of TransfeX was 29 times higher than that of TransIT-LT1. Very high levels of luciferase activity were obtained in H357 cells with TransfeX. TransfeX is likely to be beneficial in various cancer gene therapy approaches and other gene therapies involving non-viral vectors.

3D multicellular models reflect the efficiency of MSC-directed enzyme/prodrug treatment.

Mesenchymal stromal cells (MSC) exhibit beneficial properties to serve as cellular vehicles for enzyme/prodrug cancer gene therapy approaches. We have previously shown that engineered human adipose tissue-derived MSC in combination with non-toxic prodrug mediated substantial cytotoxic and antitumor effect. The aim of this study was to develop advanced 3D cultivation method to serve for modelling of the therapeutic outcome in vitro. We have used engineered MSC expressing fusion transgene cytosine deaminase::uracilphosphoribosyltransferase (CD-MSC) in combination with prodrug 5-fluorocytosine (5FC). This therapeutic regimen designated CD-MSC/5FC was combined with the human melanoma cells A375 or EGFP-A375 in both standard monolayer culture and 3-dimensional (3D) multicellular nodules. The extent of cytotoxicity was evaluated by standard viability assay MTS, ATP-based luminescence assay, fluorimetric test, measurement of Caspase-3/7 activation and DNA laddering. The data have shown that the extent of cytotoxic bystander effect mediated by CD-MSC/5FC is significantly lower in 3D culture conditions. However, these data better recapitulate the therapeutic efficiency as observed previously in vivo. We suggest here to use the 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models.

Lentivirus-Mediated siRNA Targeting ER-α Inhibits Tumorigenesis and Induces Apoptosis in Hepatocarcinoma Cells.

Background and Objectives. Estrogen receptor-α (ER-α) plays important roles in hepatocarcinogenesis. Recent studies have shown that ER-α could lead to cell cycle progression or inhibition of apoptosis. To better understand the role of ER-α, RNA interference (RNAi) was used to inhibit ER-α expression in the human hepatocellular carcinoma (HCC) cells. Methods. Lentivirus-mediated ER-α small interfering RNA (siRNA) was transfected into HCC cells Hep3B. ER-α expression was monitored by real-time polymerase chain reaction (PCR) and western blot. Cell proliferation, apoptosis, and invasion were examined by methyl thiazol tetrazolium (MTT), flow cytometry (FCM), and invasion assay, respectively. Results. ER-α siRNA efficiently downregulated the expression of ER-α in Hep3B cells at both mRNA and protein levels in a time-dependent manner. ER-α siRNA also inhibited cell proliferation and reduced cell invasion (compared with other groups, P < 0.05, resp.). Furthermore, knockdown of ER-α slowed down the cell population at S phase and increased the rate of apoptosis (P < 0.05, resp.). Conclusion. ER-α knockdown suppressed the growth of HCC cells. Thus, ER-α may play a very important role in carcinogenesis of HCC and its knockdown may offer a new potential gene therapy approach for human liver cancer in the future.