An efficient targeted radiotherapy/gene therapy strategy utilising human telomerase promoters and radioastatine and harnessing radiation-mediated bystander effects

BACKGROUND: Targeted radiotherapy achieves malignant cell-specific concentration of radiation dosage by tumour-affinic molecules conjugated to radioactive atoms. Combining gene therapy with targeted radiotherapy is attractive because the associated cross-fire irradiation of the latter induces biological bystander effects upon neighbouring cells overcoming low gene transfer efficiency. METHODS: We sought to maximise the tumour specificity and efficacy of noradrenaline transporter (NAT) gene transfer combined with treatment using the radiopharmaceutical meta-[(131)I]iodobenzylguanidine ([(131)I]MIBG). Cell-kill was achieved by treatment with the beta-decay particle emitter [(131)I]MIBG or the alpha-particle emitter [(211)At]MABG. We utilised our novel transfected mosaic spheroid model (TMS) to determine whether this treatment strategy could result in sterilisation of spheroids containing only a small proportion of NAT-expressing cells. RESULTS: The concentrations of [(131)I]MIBG and [(211)At]MABG required to reduce to 0.1% the survival of clonogens derived from the TMS composed of 100% of NAT gene-transfected cells were 1.5 and 0.004 MBq/ml (RSV promoter), 8.5 and 0.0075 MBq/ml (hTR promoter), and 9.0 and 0.008 MBq/ml (hTERT promoter), respectively. The concentrations of radiopharmaceutical required to reduce to 0.1% the survival of clonogens derived from 5% RSV/NAT and 5% hTERT/NAT TMS were 14 and 23 MBq/ml, respectively, for treatment with [(131)I]MIBG and 0.018 and 0.028 MBq/ml, respectively, for treatment with [(211)At]MABG. CONCLUSIONS: These results indicate that the telomerase promoters have the capacity to drive the expression of the NAT. The potency of [(211)At]MABG is approximately three orders of magnitude greater than that of [(131)I]MIBG. Spheroids composed of only 5% of cells expressing NAT under the control of the RSV or hTERT promoter were sterilised by radiopharmaceutical treatment. This observation is indicative of bystander cell-kill.     

Modeling the time dependent distribution of a new 153Sm complex for targeted radiotherapy purpose

BackgroundFor radioimmunotherapy purposes, a chemical complex with high absorption in cancer tumor is required. New chemicals are to be examined for their concentration in tumor and healthy organs. These are labeled with β-emitting radioisotopes to irradiate the tumor while deposited inside it.AimTo study the capability of recently developed chemical complex in targeting cancer tumor and investigate the distribution of ¹⁵³Sm-TPTTC in rat organs as function of time.Materials and methodsThe chemical complex – [Tris(1,10-phenanthroline)Samarium(III)] trithiocyanate was prepared and labeled with ¹⁵³Sm radioisotope. The labeled complex was injected to a population of tumor bearing mice. In 2, 4, 24, 48, 96 h after injection the animals were sacrificed and the concentration of Samarium complex was measured in various organs such as blood, heart, intestine, colon, liver, spleen, kidney, sternum and bone.ResultsThe concentration of the radiopharmaceutical in various organs was measured at different times. The temporal behavior of biodistribution of ¹⁵³Sm-TPTTC was modeled and drawn as function of time.ConclusionIt is shown that ¹⁵³Sm-TPTTC is concentrated in tumor tissue and liver much more than in other organs. The variation of pharmaceutical concentration in all organs is described with summation of eight exponential terms and it approximates our experimental data with precision better than 2%.     

microRNAs: a new emerging class of players for disease diagnostics and gene therapy

microRNAs (miRNAs) are a new class of non-protein-coding small RNAs, which regulate the expression of more than 30% protein-coding genes. The unique expression profiles of different miRNAs in different types of cancers and at different stages in one cancer type suggest that miRNAs can function as novel biomarkers for disease diagnostics and may present a new strategy for miRNA gene therapy. Anti-miRNAs and antisense oligonucleotides (ASO) have been employed to inhibit specific miRNA expression in vitro and in vivo for investigational and clinical purposes. Although miRNA-based diagnostics and gene therapy are still in their infancy, their huge potentials will meet our need for future disease diagnostics and gene therapy. High efficient delivery of miRNAs into targeted sites, designing accurate anti-miRNA/ASOs, and related biosafety issues are three major challenges in this field.     

Nuclear estrogen receptor targeted photodynamic therapy: selective uptake and killing of MCF-7 breast cancer cells by a C17alpha-alkynylestradiol-porphyrin conjugate

We hypothesized that over-expression of estrogen receptor (ER) in hormone-sensitive breast cancer could be harnessed synergistically with the tumor-migrating effect of porphyrins to selectively deliver estrogen-porphyrin conjugates into breast tumor cells, and preferentially kill the tumor cells upon exposure to red light. In the present work we synthesized four (4) conjugates of C17-alpha-alkynylestradiol and chlorin e6-dimethyl ester with varying tether lengths, and showed that all these conjugates specifically bound to recombinant ER alpha. In a cellular uptake assay with ER-positive MCF-7 and ER-negative MDA-MB 231 human breast cancer cell-lines, we observed that one such conjugate (E17-POR, XIV) was selectively taken up in a dose-dependent and saturable manner by MCF-7 cells, but not by MDA-MB 231 cells. Furthermore, MCF-7 cells, but not MDA-MB 231 cells, were selectively and efficiently killed by exposure to red light after incubation with E17-POR. Therefore, the combination approach, including drug and process modalities has the potential to be applied clinically for hormone-sensitive cancers in organs where ER is significantly expressed. This could potentially be carried out either as monotherapy involving a photo-induced selective destruction of tumor cells and/or adjuvant therapy in post-surgical treatment for the destruction of residual cancer cells in tissues surrounding the tumor.     

Combined effects of radiotherapy and endostatin gene therapy in melanoma tumor model

PEgr–Endostatin–EGFP plasmid was constructed to investigate its expression properties induced by ionizing irradiation and the effect of pEgr–Endostatin–EGFP gene-radiotherapy on melanoma tumor-bearing mice. The pEgr–Endostatin–EGFP plasmid was transfected into B16 cell line with liposome. The expression property of endostatin was investigated by RT-PCR and that of EGFP was detected by flow cytometry. Tumor-bearing mice were treated by the plasmid injection and 2 Gy X-irradiation of three fractions. Tumor growth was observed for 18 days after treatment. Change of tumor capillary formation was measured with histochemistry assay at the end of the experiment. The expression of GFP in B16 melanoma cells was detected after X-irradiation with 0.05–20 Gy. Time-course studies showed that the expression of GFP in B16 cells reached its peak at 8 h after irradiation with 2 Gy. The injection of pEgr–Endostatin–EGFP recombinant plasmid into the implanted B16 melanoma in C57BL/6J mice followed by local X-irradiation could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density. The inhibitory effect of pEgr–Endostatin–EGFP gene-radiotherapy on the growth of B16 melanoma is correlated with the marked decrease of intratumoral vascularization. The present data point to the potential of an anti-angiogenic approach in gene-radiotherapy of cancer.     

Introduction to the background, principles, and state of the art in suicide gene therapy

Gene therapy is defined as a technology that aims to modify the genetic component of cells to gain therapeutic benefits. Suicide gene therapy (or gene-directed enzyme prodrug therapy [GDEPT]) is a two-step treatment for cancer (especially, solid tumors). In the first step, a gene for a foreign enzyme is delivered to the tumor by a vector. Following the expression of the foreign enzyme, a prodrug is administered during the second step, which is selectively activated in the tumor. This article discusses the principles and the theoretical background of GDEPT. A special emphasis is put on enzyme/prodrug systems developed for GDEPT, the design of prodrugs and the kinetic of their activation, the types and the mechanisms of bystander effect and its immunological implications. The possible strategies to improve GDEPT are also discussed.     

Proteostasis regulation at the endoplasmic reticulum: a new perturbation site for targeted cancer therapy

To deal with the constant challenge of protein misfolding in the endoplasmic reticulum (ER), eukaryotic cells have evolved an ER protein quality control (ERQC) mechanism that is integrated with an adaptive stress response. The ERQC pathway is comprised of factors residing in the ER lumen that function in the identification and retention of aberrantly folded proteins, factors in the ER membrane for retrotranslocation of misfolded polypeptides, and enzymes in the cytosol that degrade retrotranslocated proteins. The integrated stress response (termed ER stress or unfolded protein response, UPR) contains several signaling branches elicited from the ER membrane, which fine-tune the rate of protein synthesis and entry into the ER to match the ER folding capacity. The fitness of the cell, particularly those bearing a high secretory burden, is critically dependent on functional integrity of the ER, which in turn relies on these stress-attenuating mechanisms to maintain protein homeostasis, or proteostasis. Aberrant proteostasis can trigger cellular apoptosis, making these adaptive stress response systems attractive targets for perturbation in treatment of cell malignancies. Here, we review our current understanding of how the cell preserves ER proteostasis and discuss how we may harness the mechanistic information on this process to develop new cancer therapeutics.     

骨髓基质细胞的特征及其在细胞和基因治疗中的应用

骨髓基质细胞是一类独特的间质干细胞,可分化为多种非造血系的组织。骨髓基质细胞具有贴壁生长的特性,因而易于在体外分离和扩增;另外骨髓基质细胞可在体内外表达多种治疗性的外湖目的基因。因此,骨髓基质细胞被认为是一种理想的治疗性细胞的基因治疗中的靶细胞。本文对骨髓基质细胞的研究进展及其在细胞和基因治疗中的应用作一综述。     

Targeted gene delivery to CD117‐expressing cells in vivo with lentiviral vectors co‐displaying stem cell factor and a fusogenic molecule

The development of a lentiviral system to deliver genes to specific cell types could improve the safety and the efficacy of gene delivery. Previously, we have developed an efficient method to target lentivectors to specific cells via an antibody–antigen interaction in vitro and in vivo. We report herein a targeted lentivector that harnesses the natural ligand–receptor recognition mechanism for targeted modification of c‐KIT receptor‐expressing cells. For targeting, we incorporate membrane‐bound human stem cell factor (hSCF), and for fusion, a Sindbis virus‐derived fusogenic molecule (FM) onto the lentiviral surface. These engineered vectors can recognize cells expressing surface CD117, resulting in efficient targeted transduction of cells in an SCF‐receptor dependent manner in vitro, and in vivo in xenografted mouse models. This study expands the ability of targeting lentivectors beyond antibody targets to include cell‐specific surface receptors. Development of a high titer lentivector to receptor‐specific cells is an attractive approach to restrict gene expression and could potentially ensure therapeutic effects in the desired cells while limiting side effects caused by gene expression in non‐target cells. Biotechnol. Bioeng. 2009; 104: 206–215 © 2009 Wiley Periodicals, Inc.     

Medicinal chemistry of plasmid DNA with peptide nucleic acids: A new strategy for gene therapy

Summary In this chapter, we describe an approach using a peptide nucleic acid (PNA) clamp to directly and irreversibly modify plasmid DNA, without affecting either its supercoiled conformation or its ability to be efficiently transcribed. This strategy enables investigators to functionalize their gene of interest by direct coupling of ligands (fluorophores, peptide, proteins, sugars or oligonucleotides) to plasmid DNA. This approach provides versatile tools to study the mechanisms of gene delivery and to circumvent some of the main obstacles of synthetic gene delivery systems, such as specific targeting and efficient delivery. The proof-of-principal of PNA-dependent gene chemistry (PDGC) was demonstrated with a fluorescently labeled PNA that allowed generation of a highly fluorescent preparation of plasmid DNA that was functionally and conformationally intact. Fluorescent-PNA/DNA was used to identify critical parameters involved in naked DNA and non-viral gene delivery technology. The greatest potential of PDGC lies in the ability to attach specific ligands (e.g., peptides, proteins) to the plasmid DNA in order to overcome cellular barriers of non-viral gene delivery systems. In this regard, specific examples of ligands coupled to DNA are described and their effect on increasing the efficacy of gene therapy is presented     

Exome sequencing and targeted gene panels: a simulated comparison of diagnostic yield using data from 158 patients with rare diseases

Next-generation sequencing (NGS) has altered clinical genetic testing by widening the access to molecular diagnosis of genetically determined rare diseases. However, physicians may face difficulties selecting the best diagnostic approach. Our goal is to estimate the rate of possible molecular diagnoses missed by different targeted gene panels using data from a cohort of patients with rare genetic diseases diagnosed with exome sequencing (ES). For this purpose, we simulated a comparison between different targeted gene panels and ES: the list of genes harboring clinically relevant variants from 158 patients was used to estimate the theoretical rate of diagnoses missed by NGS panels from 53 different NGS panels from eight different laboratories. Panels presented a mean rate of missed diagnoses of 64% (range 14%-100%) compared to ES, representing an average predicted sensitivity of 36%. Metabolic abnormalities represented the group with highest mean of missed diagnoses (86%), while seizure represented the group with lowest mean (46%). Focused gene panels are restricted in covering select sets of genes implicated in specific diseases and they may miss molecular diagnoses of rare diseases compared to ES. However, their role in genetic diagnosis remains important especially for well-known genetic diseases with established genetic locus heterogeneity.     

A multiscale cell‐based model of tumor growth for chemotherapy assessment and tumor‐targeted therapy through a 3D computational approach

ObjectivesComputational modeling of biological systems is a powerful tool to clarify diverse processes contributing to cancer. The aim is to clarify the complex biochemical and mechanical interactions between cells, the relevance of intracellular signaling pathways in tumor progression and related events to the cancer treatments, which are largely ignored in previous studies.Materials and MethodsA three‐dimensional multiscale cell‐based model is developed, covering multiple time and spatial scales, including intracellular, cellular, and extracellular processes. The model generates a realistic representation of the processes involved from an implementation of the signaling transduction network.ResultsConsidering a benign tumor development, results are in good agreement with the experimental ones, which identify three different phases in tumor growth. Simulating tumor vascular growth, results predict a highly vascularized tumor morphology in a lobulated form, a consequence of cells'' motile behavior. A novel systematic study of chemotherapy intervention, in combination with targeted therapy, is presented to address the capability of the model to evaluate typical clinical protocols. The model also performs a dose comparison study in order to optimize treatment efficacy and surveys the effect of chemotherapy initiation delays and different regimens.ConclusionsResults not only provide detailed insights into tumor progression, but also support suggestions for clinical implementation. This is a major step toward the goal of predicting the effects of not only traditional chemotherapy but also tumor‐targeted therapies.