Possibility for Transcriptional Targeting of Cancer-Associated Fibroblasts-Limitations and Opportunities.

Cancer-associated fibroblasts (CAF) are attractive therapeutic targets in the tumor microenvironment. The possibility of using CAFs as a source of therapeutic molecules is a challenging approach in gene therapy. This requires transcriptional targeting of transgene expression by cis-regulatory elements (CRE). Little is known about which CREs can provide selective transgene expression in CAFs. We hypothesized that the promoters of FAP, CXCL12, IGFBP2, CTGF, JAG1, SNAI1, and SPARC genes, the expression of whose is increased in CAFs, could be used for transcriptional targeting. Analysis of the transcription of the corresponding genes revealed that unique transcription in model CAFs was characteristic for the CXCL12 and FAP genes. However, none of the promoters in luciferase reporter constructs show selective activity in these fibroblasts. The CTGF, IGFBP2, JAG1, and SPARC promoters can provide higher transgene expression in fibroblasts than in cancer cells, but the nonspecific viral promoters CMV, SV40, and the recently studied universal PCNA promoter have the same features. The patterns of changes in activity of various promoters relative to each other observed for human cell lines were similar to the patterns of activity for the same promoters both in vivo and in vitro in mouse models. Our results reveal restrictions and features for CAF transcriptional targeting.

MicroRNA-Regulated Gene Delivery Systems for Research and Therapeutic Purposes.

Targeted gene delivery relies on the ability to limit the expression of a transgene within a defined cell/tissue population. MicroRNAs represent a class of highly powerful and effective regulators of gene expression that act by binding to a specific sequence present in the corresponding messenger RNA. Involved in almost every aspect of cellular function, many miRNAs have been discovered with expression patterns specific to developmental stage, lineage, cell-type, or disease stage. Exploiting the binding sites of these miRNAs allows for construction of targeted gene delivery platforms with a diverse range of applications. Here, we summarize studies that have utilized miRNA-regulated systems to achieve targeted gene delivery for both research and therapeutic purposes. Additionally, we identify criteria that are important for the effectiveness of a particular miRNA for such applications and we also discuss factors that have to be taken into consideration when designing miRNA-regulated expression cassettes.

Regulated expression of murine CD40L by a lentiviral vector transcriptionally targeted through its endogenous promoter.

BACKGROUND: Targeted lentiviral vectors may contribute to circumventing genotoxicity associated with uncontrolled transcription of therapeutic genes. Some vectors replacing strong viral sequences for gene promoters such as ß-globin, CD4, CD19 or Igκ were able to drive tissue-specific expression of the transgene. Gene therapy, however, faces even greater hurdles when the therapeutic transgene is subject to strict regulatory mechanisms. This is the case of the CD40LG gene, which encodes for the CD154 (also known as CD40L) molecule, transiently expressed upon activation on CD4(+) T cells. Mutations in this gene cause the X-linked hyper IgM syndrome (HIGM1) in humans because the interaction of CD40L with its ligand CD40 triggers signals that are critical for the immunobiology of B lymphocytes. METHODS: We developed a lentiviral vector containing the murine Cd40lg cDNA under the control of its endogenous promoter. RESULTS: The CD4(+) BW5147 T cells transduced with the pCd40lg-Cd40lg lentiviral vector express CD40L only upon stimulation. The intensity of the expression correlates with the number of vector integrations per cell and detected molecules rapidly decay after removing the stimulating agent. The tissue-specific, activation-dependent and reversible expression of CD40L fully mimics the physiological induction and disappearance of the molecule from the surface of murine T lymphocytes. The functional activity of the regulated lentiviral vector is demonstrated by the ability of transduced BW5147 cells to promote the proliferation of purified B cell splenocytes. CONCLUSIONS: We have developed a fine-regulated lentiviral vector that can be a model for expressing molecules subject to stringent regulatory mechanisms.

Increasing cancer-specific gene expression by targeting overexpressed α5ß1 integrin and upregulated transcriptional activity of NF-κB.

We developed a modular multifunctional nonviral gene delivery system by targeting the overexpressed cancer surface receptor α5ß1 integrin and the upregulated transcriptional activity of the cancer resistance mediating transcription factor NF-κB, thereby introducing a new form of transcriptional targeting. NF-κB regulated therapy can improve specificity of gene expression in cancer tissue and also may offset NF-κB mediated cancer resistance. We delivered a luciferase gene under the control of an NF-κB responsive element (pNF-κB-Luc) encapsulated in a PR_b peptide functionalized stealth liposome that specifically targets the α5ß1 integrin and achieved increased gene expression in DLD-1 colorectal cancer cells compared to BJ-fibroblast healthy cells in vitro. The multitargeted system was also able to differentiate between cancer cells and healthy cells better than either of the individually targeted systems. In addition, we constructed a novel cancer therapeutic plasmid by cloning a highly potent diphtheria toxin fragment A (DTA) expressing gene under the control of an NF-κB responsive element (pNF-κB-DTA). A dose-dependent reduction of cellular protein expression and increased cytotoxicity in cancer cells was seen when transfected with PR_b functionalized stealth liposomes encapsulating the condensed pNF-κB-DTA plasmid. Our therapeutic delivery system specifically eradicated close to 70% of a variety of cancer cells while minimally affecting healthy cells in vitro. Furthermore, the modular nature of the nonviral design allows targeting novel pairs of extracellular receptors and upregulated transcription factors for applications beyond cancer gene therapy.

Transcriptional Targeting Approaches in Cardiac Gene Transfer Using AAV Vectors.

Cardiac-targeted transgene delivery offers new treatment opportunities for cardiovascular diseases, which massively contribute to global mortality. Restricted gene transfer to cardiac tissue might protect extracardiac organs from potential side-effects. This could be mediated by using cis-regulatory elements, including promoters and enhancers that act on the transcriptional level. Here, we discuss examples of tissue-specific promoters for targeted transcription in myocytes, cardiomyocytes, and chamber-specific cardiomyocytes. Some promotors are induced at pathological states, suggesting a potential use as "induction-by-disease switches" in gene therapy. Recent developments have resulted in the identification of novel enhancer-elements that could further pave the way for future refinement of transcriptional targeting, for example, into the cardiac conduction system.

Short regulatory DNA sequences to target brain endothelial cells for gene therapy.

Gene vectors targeting CNS endothelial cells allow to manipulate the blood-brain barrier and to correct genetic defects in the CNS. Because vectors based on the adeno-associated virus (AAV) have a limited capacity, it is essential that the DNA sequence controlling gene expression is short. In addition, it must be specific for endothelial cells to avoid off-target effects. To develop improved regulatory sequences with selectivity for brain endothelial cells, we tested the transcriptional activity of truncated promoters of eleven (brain) endothelial-specific genes in combination with short regulatory elements, i.e., the woodchuck post-transcriptional regulatory element (W), the CMV enhancer element (C), and a fragment of the first intron of the Tie2 gene (S), by transfecting brain endothelial cells of three species. Four combinations of regulatory elements and short promoters (Cdh5, Ocln, Slc2a1, and Slco1c1) progressed through this in-vitro pipeline displaying suitable activity. When tested in mice, the regulatory sequences C-Ocln-W and C-Slc2a1-S-W enabled a stronger and more specific gene expression in brain endothelial cells than the frequently used CAG promoter. In summary, the new regulatory elements efficiently control gene expression in brain endothelial cells and may help to specifically target the blood-brain barrier with gene therapy vectors.

Towards Clinical Implementation of Adeno-Associated Virus (AAV) Vectors for Cancer Gene Therapy: Current Status and Future Perspectives.

Adeno-associated virus (AAV) vectors have gained tremendous attention as in vivo delivery systems in gene therapy for inherited monogenetic diseases. First market approvals, excellent safety data, availability of large-scale production protocols, and the possibility to tailor the vector towards optimized and cell-type specific gene transfer offers to move from (ultra) rare to common diseases. Cancer, a major health burden for which novel therapeutic options are urgently needed, represents such a target. We here provide an up-to-date overview of the strategies which are currently developed for the use of AAV vectors in cancer gene therapy and discuss the perspectives for the future translation of these pre-clinical approaches into the clinic.

Polyethyleneimine-polyethylene glycol copolymer targeted by anti-HER2 nanobody for specific delivery of transcriptionally targeted tBid containing construct.

The present research seeks to investigate the process of mixing targeted gene delivery and transcriptional targeting. We have conjugated Polyethylenimine polymers (PEI) and molecules of poly (ethylene glycol). The next step was covalent attachment of anti-HER2 variables domains of camelid heavy chains antibodies (VHHs) or nanobodies (Nbs) to the distal terminals of NHS-PEG3500 in PEI-PEG nanoparticles. The whole procedure yielded PEI-PEG-Nb immunoconjugates. Having determined the properties of polyplexes, steps were taken to investigate the most efficient ratio of PEI polymers to pDNA molecules (N/P) so that the greatest rate of transfection may be obtained. This immune targeted nano biopolymer could condense the gene constructs that coded a transcriptionally targeted truncated -Bid (tBid) killer gene which was controlled by the breast cancer-specific MUC1 promoter. The favourable physicochemical properties matching both the size and zeta potential were observed in engineered polyplexes. Elevated transfection efficiency in HER2 positive cell lines using Nb-modified polyplexes were shown by the results of flow cytometry as compared against non-modified particles. 1.6 and 4.8 fold higher transfection efficiencies were observed in in vitro gene expression researches which used PEI-PEG-Nb/pGL4.50 compared to the situation when native PEI polymers were utilized in both BT-474 and SK-BR-3, respectively. A 2.22 and 3.62 fold rise in the level of tBid gene expression in BT-474 and SK-BR-3 cell lines relative to unmodified PEI treated cells was the result of transfection with PEI-PEG-Nb/pMUC1-tBid, respectively. In those HER2-positive cells which were transfected by targeted polyplexes, higher levels of cell death were observed. This fact points not only to the effective targeted delivery, but it is also indicative of transcriptional targeting efficiency of tBid killer gene when its expression is controlled by MUC1 promoter.

Targeted Diphtheria Toxin-Based Therapy: A Review Article.

Cancer remains one of the leading causes of death worldwide. Conventional therapeutic strategies usually offer limited specificity, resulting in severe side effects and toxicity to normal tissues. Targeted cancer therapy, on the other hand, can improve the therapeutic potential of anti-cancer agents and decrease unwanted side effects. Targeted applications of cytolethal bacterial toxins have been found to be especially useful for the specific eradication of cancer cells. Targeting is either mediated by peptides or by protein-targeting moieties, such as antibodies, antibody fragments, cell-penetrating peptides (CPPs), growth factors, or cytokines. Together with a toxin domain, these molecules are more commonly referred to as immunotoxins. Targeting can also be achieved through gene delivery and cell-specific expression of a toxin. Of the available cytolethal toxins, diphtheria toxin (DT) is one of the most frequently used for these strategies. Of the many DT-based therapeutic strategies investigated to date, two immunotoxins, OntakTM and TagraxofuspTM, have gained FDA approval for clinical application. Despite some success with immunotoxins, suicide-gene therapy strategies, whereby controlled tumor-specific expression of DT is used for the eradication of malignant cells, are gaining prominence. The first part of this review focuses on DT-based immunotoxins, and it then discusses recent developments in tumor-specific expression of DT.

Evaluation of cellular and transcriptional targeting of breast cancer stem cells via anti-HER2 nanobody conjugated PAMAM dendrimers.

According to the cancer stem cell (CSC) theory, a small subset of cells with stem cell-like characteristics is responsible for tumor initiation, progression, and recurrence. CD44+/CD24- phenotype is assumed to be one of the main characteristics of the breast CSCs. We developed an MDA-MB-231 cell line overexpressing cell surface HER2 antigen for the evaluation of targeting efficiency of anti-HER2 nanobody (Nb)-conjugated polyamidoamine (PAMAM) polyplexes. Apoptosis-inducing tBid gene under control of CXCR1 promoter was delivered by this nanoparticle. Cellular uptake study showed higher uptake of Nb-targeted PAMAM carriers compared to non-targeted nanoparticles after 6 h of incubation. Gene expression analysis showed a significant rise in the expression of tBid in both MDA-MB-231/HER2+ and MDA-MB-231 compared to the two other cell lines. The same effect was observed after transfection with Nb-conjugated polyplexes within MDA-MB-231/HER2+ cell line compared to non-conjugated PAMAM polyplexes. We confirmed the killing efficiency of the gene construct in both MDA-MB-231/HER2+ and MDA-MB-231 cell lines by caspase 3 activity assay. These findings suggest that imposing pre-entry and post-entry restrictions on tBid killer gene might be a promising approach to specifically target the breast CSCs.

MicroRNA199a-Based Post-transcriptional Detargeting of Gene Vectors for Hepatocellular Carcinoma.

A gene therapeutic platform needs to be both efficient and safe. The criterion of safety is particularly important for diseases like hepatocellular carcinoma (HCC), which develop in a background of an already compromised liver. Gene vectors can be constructed either by targeting HCC or by detargeting liver and/or other major organs. miRNA-based negative detargeting has gained considerable attention in recent times due to its effectiveness and the ease with which it can be adapted into current gene delivery vectors. In this study, we provide a proof-of-concept using miRNA199a as a negative targeting agent. We introduced vectors harboring reporters with miRNA199a binding sites in cells expressing high endogenous levels of miRNA199a and compared the reporter expression in HCC cells with low endogenous miRNA199a. We observed that the expression of reporters with miRNA199a binding sites is significantly inhibited in miRNA199a-positive cells, whereas minimal effect was observed in miRNA199a-negative HCC cells. In addition, we created a post-transcriptionally regulated suicide gene therapeutic system based on cytosine deaminase (CD)/5-fluorocytosine (5-FC) exploiting miRNA199a binding sites and observed significantly lower cell death for miRNA199a-positive cells. Furthermore, we observed a decrease in the levels of miRNA199 in 3D tumorspheres of miRNA199a-positive Hepa1-6 cells and a reduction in the inhibition of reporter expression after transfection in these 3D models when compared with 2D Hepa1-6 cells. In summary, we provide evidence of miRNA199a-based post-transcriptional detargeting with relevance to HCC gene therapy.

miRNA122a regulation of gene therapy vectors targeting hepatocellular cancer stem cells.

In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3'-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs.

Hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation system for cancer gene therapy.

Transcriptional targeting is the best approach for specific gene therapy. Hypoxia is a common feature of the tumour microenvironment. Therefore, targeting gene expression in hypoxic cells by placing transgene under the control of a hypoxia-responsive promoter can be a good strategy for cancer-specific gene therapy. The hypoxia-inducible gene expression system has been investigated more in suicide gene therapy and it can also be of great help in knocking down cancer gene therapy with siRNAs. However, this system needs to be optimised to have maximum efficacy with minimum side effects in normal tissues. The combination of tissue-/tumour-specific promoters with HRE core sequences has been found to enhance the specificity and efficacy of this system. In this review, hypoxia-inducible gene expression system as well as gene therapy strategies targeting tumour hypoxia will be discussed. This review will also focus on hypoxia-inducible tumour-specific promoters as a dual-targeting transcriptional regulation systems developed for cancer-specific gene therapy.

Seek and destroy: targeted adeno-associated viruses for gene delivery to hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer with high incidence globally. Increasing mortality and morbidity rates combined with limited treatment options available for advanced HCC press for novel and effective treatment modalities. Gene therapy represents one of the most promising therapeutic options. With the recent approval of herpes simplex virus for advanced melanoma, the field of gene therapy has received a major boost. Adeno-associated virus (AAV) is among the most widely used and effective viral vectors today with safety and efficacy demonstrated in a number of human clinical trials. This review identifies the obstacles for effective AAV based gene delivery to HCC which primarily include host immune responses and off-target effects. These drawbacks could be more pronounced for HCC because of the underlying liver dysfunction in most of the patients. We discuss approaches that could be adopted to tackle these shortcomings and manufacture HCC-targeted vectors. The combination of transductional targeting by modifying the vector capsid and transcriptional targeting using HCC-specific promoters has the potential to produce vectors which can specifically seek HCC and deliver therapeutic gene without significant side effects. Finally, the identification of novel HCC-specific ligands and promoters should facilitate and expedite this process.

Tissue-Specific Promoters in the CNS.

This chapter outlines some general principles of transcriptional targeting approaches using viral vectors in the central nervous system. Transcriptional targeting is first discussed in the context of vector tropism and appropriate delivery. Then, some of our own attempts to restrict expression of therapeutic factors to distinct brain cell populations are discussed, followed by a detailed description of the setscrews that are available for these experiments. A critical discussion of current stumbling blocks and necessary developments to achieve clinical applicability of advanced targeted vector systems is provided.

Transcriptional targeting of human liver carboxylesterase (hCE1m6) and simultaneous expression of anti-BCRP shRNA enhances sensitivity of breast cancer cells to CPT-11.

BACKGROUND: The major factor limiting the efficacy of breast cancer chemotherapy is multidrug resistance due to overexpression of the breast cancer resistance protein ATP-binding cassette, sub-family G (WHITE), member 2 (ABCG2). We hypothesized that conversion of camptothecin-11 (CPT-11) to its highly cytotoxic metabolite SN-38 by a mutant human carboxyl esterase (hCE1m6) specifically in cancer cells and inhibition of ABCG2 by anti-ABCG2 short hairpin RNA, leads to accumulation of a higher concentration of SN-38, resulting in higher therapeutic efficacy and less toxicity to normal cells. MATERIALS AND METHODS: A mutant human carboxyl esterase hCE1m6 with human telomerase reverse transcriptase promoter was integrated into the VISA (VP16-Gal4-WPRE) amplification system. The plasmid was transfected into MCF-12A, MDA-MB-231, and MCF-7 cells using JetPRIME®. Cancer-specific expression of hCE1m6 in breast cancer cell lines was tested by real-time polymerase chain reaction (real time-PCR) and western blot. In vitro conversion of CPT-11 to SN-38 was evaluated on lysates of transfected cells. Cytotoxicity of CPT-11 against cells transfected with the plasmid was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Real-time PCR and western blot analysis revealed that hCE1m6 was expressed only in breast cancer cells, MCF-7 and MDA-MB-231, but not in the normal MCF-12A breast cell line. From the CPT-11 conversion assay on cell lysates, it was found that expressed hCE1m6 in cancer cells was able to effectively convert CPT-11 to SN-38. CONCLUSION: Breast cancer cell lines transfected with hCE1m6 showed an increased susceptibility to CPT-11 in comparison to MCF-12A cells.