Use Of Tissue-specific Promoters Research Articles

5-HT<sub>3</sub> receptors in GtoPdb v.2023.3

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [69]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4 transmembrane (TM) subunits that form an intrinsic cation selective channel [7]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [86, 125], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [127, 66, 161]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [161]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [85]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [9], but this is inconsistent with recent reports which show at least one A-A interface [99, 154]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [35, 44, 59, 88, 143, 132, 82], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([19], but see [44, 33, 38]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [99, 154]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [142, 139, 73]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and HTR3E [67, 21, 127, 126, 123]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [156, 82, 123]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function.

5-HT<sub>3</sub> receptors in GtoPdb v.2021.3

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [69]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4 transmembrane (TM) subunits that form an intrinsic cation selective channel [7]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [86, 125], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [127, 66, 161]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [161]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [85]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [9], but this is inconsistent with recent reports which show at least one A-A interface [99, 154]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [35, 44, 59, 88, 143, 132, 82], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([19], but see [44, 33, 38]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [99, 154]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [142, 139, 73]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and HTR3E [67, 21, 127, 126, 123]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [156, 82, 123]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function.

Transcription initiation mapping in 31 bovine tissues reveals complex promoter activity, pervasive transcription, and tissue-specific promoter usage.

Characterizing transcription start sites is essential for understanding the regulatory mechanisms that control gene expression. Recently, a new bovine genome assembly (ARS-UCD1.2) with high continuity, accuracy, and completeness was released; however, the functional annotation of the bovine genome lacks precise transcription start sites and contains a low number of transcripts in comparison to human and mouse. By using the RAMPAGE approach, this study identified transcription start sites at high resolution in a large collection of bovine tissues. We found several known and novel transcription start sites attributed to promoters of protein-coding and lncRNA genes that were validated through experimental and in silico evidence. With these findings, the annotation of transcription start sites in cattle reached a level comparable to the mouse and human genome annotations. In addition, we identified and characterized transcription start sites for antisense transcripts derived from bidirectional promoters, potential lncRNAs, mRNAs, and pre-miRNAs. We also analyzed the quantitative aspects of RAMPAGE to produce a promoter activity atlas, reaching highly reproducible results comparable to traditional RNA-seq. Coexpression networks revealed considerable use of tissue-specific promoters, especially between brain and testicle, which expressed several genes in common from alternate loci. Furthermore, regions surrounding coexpressed modules were enriched in binding factor motifs representative of each tissue. The comprehensive annotation of promoters in such a large collection of tissues will substantially contribute to our understanding of gene expression in cattle and other mammalian species, shortening the gap between genotypes and phenotypes.

TATA Binding Protein (TBP) Promoter Drives Ubiquitous Expression of Marker Transgene in the Adult Sea Anemone Nematostella vectensis.

Nematostella vectensis has emerged as one as the most established models of the phylum Cnidaria (sea anemones, corals, hydroids and jellyfish) for studying animal evolution. The availability of a reference genome and the relative ease of culturing and genetically manipulating this organism make it an attractive model for addressing questions regarding the evolution of venom, development, regeneration and other interesting understudied questions. We and others have previously reported the use of tissue-specific promoters for investigating the function of a tissue or a cell type of interest in vivo. However, to our knowledge, genetic regulators at the whole organism level have not been reported yet. Here we report the identification and utilization of a ubiquitous promoter to drive a wide and robust expression of the fluorescent protein mCherry. We generated animals containing a TATA binding protein (TBP) promoter upstream of the mCherry gene. Flow cytometry and fluorescent microscopy revealed expression of mCherry in diverse cell types, accounting for more than 90% of adult animal cells. Furthermore, we detected a stable mCherry expression at different life stages and throughout generations. This tool will expand the existing experimental toolbox to facilitate genetic engineering and functional studies at the whole organism level.

5-HT<sub>3</sub> receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The 5-HT3 receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on 5-Hydroxytryptamine (serotonin) receptors [66]) is a ligand-gated ion channel of the Cys-loop family that includes the zinc-activated channels, nicotinic acetylcholine, GABAA and strychnine-sensitive glycine receptors. The receptor exists as a pentamer of 4TM subunits that form an intrinsic cation selective channel [5]. Five human 5-HT3 receptor subunits have been cloned and homo-oligomeric assemblies of 5-HT3A and hetero-oligomeric assemblies of 5-HT3A and 5-HT3B subunits have been characterised in detail. The 5-HT3C (HTR3C, Q8WXA8), 5-HT3D (HTR3D, Q70Z44) and 5-HT3E (HTR3E, A5X5Y0) subunits [83, 122], like the 5-HT3B subunit, do not form functional homomers, but are reported to assemble with the 5-HT3A subunit to influence its functional expression rather than pharmacological profile [124, 63, 157]. 5-HT3A, -C, -D, and -E subunits also interact with the chaperone RIC-3 which predominantly enhances the surface expression of homomeric 5-HT3A receptor [157]. The co-expression of 5-HT3A and 5-HT3C-E subunits has been demonstrated in human colon [82]. A recombinant hetero-oligomeric 5-HT3AB receptor has been reported to contain two copies of the 5-HT3A subunit and three copies of the 5-HT3B subunit in the order B-B-A-B-A [7], but this is inconsistent with recent reports which show at least one A-A interface [96, 150]. The 5-HT3B subunit imparts distinctive biophysical properties upon hetero-oligomeric 5-HT3AB versus homo-oligomeric 5-HT3A recombinant receptors [32, 41, 56, 85, 139, 129, 79], influences the potency of channel blockers, but generally has only a modest effect upon the apparent affinity of agonists, or the affinity of antagonists ([17], but see [41, 30, 35]) which may be explained by the orthosteric binding site residing at an interface formed between 5-HT3A subunits [96, 150]. However, 5-HT3A and 5-HT3AB receptors differ in their allosteric regulation by some general anaesthetic agents, small alcohols and indoles [138, 135, 71]. The potential diversity of 5-HT3 receptors is increased by alternative splicing of the genes HTR3A and E [64, 19, 124, 123, 120]. In addition, the use of tissue-specific promoters driving expression from different transcriptional start sites has been reported for the HTR3A, HTR3B, HTR3D and HTR3E genes, which could result in 5-HT3 subunits harbouring different N-termini [152, 79, 120]. To date, inclusion of the 5-HT3A subunit appears imperative for 5-HT3 receptor function.

Generation and Analysis of Cartilage-Specific CCN2 Overexpression in Transgenic Mice.

Recent progress in gene-editing technology has provided a strong impact for improved our understanding of molecular functions in living organisms. Here we describe our method to generate transgene-overexpressing mouse models, which method involves the use of tissue-specific promoters for analyzing a certain molecule (s) in special tissues. The protocol described in this chapter uses the Col2a1 promoter-enhancer, which is known for driving specific and strong transgene expression in cartilage and is based on several of our studies showing a positive role of the connective tissue growth factor (CCN2) in cartilage-bone development and maintenance of articular cartilage. These mice show strongly accelerated endochondral ossification resulting in enhanced bone elongation, as well as resistance to age-related articular degeneration. This protocol also describes how to analyze the molecular mechanisms of these phenomena by use of chondrocytes isolated from CCN2-overexpressing cartilage.

Phosphorylation Processes Controlling Aromatase Activity in Br east Cancer: An Update.

Local estrogen production from aromatase-mediated conversion of androgens is an important mechanism of autocrine growth stimulation in hormone-dependent breast cancers. The control mechanism of aromatase enzymatic activity in recent years has been demonstrated to be more complex than previously identified. Indeed, it is well known that aromatase expression is regulated at the transcriptional level through the alternative use of tissue-specific promoters, whereas it has become clear that the activity of this enzyme is also controlled by post-transcriptional modifications, such as phosphorylation processes. This paper presents a selective review of the novel findings in this area showing phosphorylation/dephosphorylation of aromatase as a switch to rapidly modulate its enzymatic activity. Particularly, we describe studies conducted in our laboratories, focusing on the role of estrogens in modulating aromatase activity in estrogen-dependent breast cancer cells. Two separate mechanisms are described. First, 17β-estradiol (E2), through c-Src kinase, is able to enhance tyrosine phosphorylation levels of aromatase protein and increases its enzymatic activity and estrogen biosynthesis. Secondly, E2, through the activation of PI3K/Akt pathway, impairs the ability of the tyrosine phosphatase PTP1B to dephosphorylate aromatase, resulting in a consequent enhanced phosphorylation and activity of the aromatase protein itself. These new controls of aromatase function provide insights into the mechanisms through which local estrogen production can be altered in breast cancer tissues. They also offer a vast array of possibilities for identifying different cell signalings that should be targeted in novel therapeutic strategies for breast cancer treatment.

Expression of the β-1,3-glucanase gene bgn13.1 from Trichoderma harzianum in strawberry increases tolerance to crown rot diseases but interferes with plant growth.

The expression of antifungal genes from Trichoderma harzianum, mainly chitinases, has been used to confer plant resistance to fungal diseases. However, the biotechnological potential of glucanase genes from Trichoderma has been scarcely assessed. In this research, transgenic strawberry plants expressing the β-1,3-glucanase gene bgn13.1 from T. harzianum, under the control of the CaMV35S promoter, have been generated. After acclimatization, five out of 12 independent lines analysed showed a stunted phenotype when growing in the greenhouse. Moreover, most of the lines displayed a reduced yield due to both a reduction in the number of fruit per plant and a lower fruit size. Several transgenic lines showing higher glucanase activity in leaves than control plants were selected for pathogenicity tests. When inoculated with Colletotrichum acutatum, one of the most important strawberry pathogens, transgenic lines showed lower anthracnose symptoms in leaf and crown than control. In the three lines selected, the percentage of plants showing anthracnose symptoms in crown decreased from 61 % to a mean value of 16.5 %, in control and transgenic lines, respectively. Some transgenic lines also showed an enhanced resistance to Rosellinia necatrix, a soil-borne pathogen causing root and crown rot in strawberry. These results indicate that bgn13.1 from T. harzianum can be used to increase strawberry tolerance to crown rot diseases, although its constitutive expression affects plant growth and fruit yield. Alternative strategies such as the use of tissue specific promoters might avoid the negative effects of bgn13.1 expression in plant performance.

Endoglin (CD105) Silencing Mediated by shRNA Under the Control of Endothelin-1 Promoter for Targeted Gene Therapy of Melanoma.

Endoglin (CD105), a transforming growth factor (TGF)-β coreceptor, and endothelin-1, a vasoconstrictor peptide, are both overexpressed in tumor endothelial and melanoma cells. Their targeting is therefore a promising therapeutic approach for melanoma tumors. The aim of our study was to construct a eukaryotic expression plasmid encoding the shRNA molecules against CD105 under the control of endothelin-1 promoter and to evaluate its therapeutic potential both in vitro in murine B16F10-luc melanoma and SVEC4-10 endothelial cells and in vivo in mice bearing highly metastatic B16F10-luc tumors. Plasmid encoding shRNA against CD105 under the control of the constitutive U6 promoter was used as a control. We demonstrated the antiproliferative and antiangiogenic effects of both plasmids in SVEC4-10 cells, as well as a moderate antitumor and pronounced antimetastatic effect in B16F10-luc tumors in vivo. Our results provide evidence that targeting melanoma with shRNA molecules against CD105 under the control of endothelin-1 promoter is a feasible and effective treatment, especially for the reduction of metastatic spread.

Cloning and characterization of a multifunctional promoter from maize (Zea mays L.).

The use of tissue-specific promoters to drive the expression of target genes during certain developmental stages or in specific organs can prevent unnecessary gene expression caused by constitutive promoters. Utilizing heterologous promoters to regulate the expression of genes in transgenic receptors can help prevent gene silencing. Here, we engineered heterologous maize promoters that regulate gene-specific expression in rice plant receptors. We performed a histochemical and quantitative β-glucuronidase (GUS) analysis of the Zea mays legumin1 (ZM-LEGF) gene promoter and detailed detection of stably transformed rice expressing the GUS gene under the control of the promoter of ZM-LEGF (pZM-LEGF) and its truncated promoters throughout development. When the promoter sequence was truncated, the location and intensity of GUS expression changed. The results suggest that the sequence from -140 to +41 is a critical region that confers the expression of the entire promoter. Truncation of pZM-LEG (3'-deleted region of pZM-LEGF) markedly increased the GUS activity, with the core cis-elements located in the -273 to -140 regions, namely pZM-LEG6. Detailed analysis of pZM-LEG6::GUS T2 transformant rice seeds and plant tissues at different developmental stages indicated that this promoter is an ideal vegetative tissue-specific promoter that can serve as a valuable tool for transgenic rice breeding and genetic engineering studies.

A Doxycycline-Inducible System for Genetic Correction of iPSC Disease Models.

Patient-derived induced pluripotent stem cells (iPSCs) are valuable tools for the study of developmental biology and disease modeling. In both applications, genetic correction of patient iPSCs is a powerful method to understand the specific contribution of a gene(s) in development or diseased state(s). Here, we describe a protocol for the targeted integration of a doxycycline-inducible transgene expression system in a safe harbor site in iPSCs. Our gene targeting strategy uses zinc finger nucleases (ZFNs) to enhance homologous recombination at the AAVS1 safe harbor locus, thus increasing the efficiency of the site-specific integration of the two targeting vectors that make up the doxycycline-inducible system. Importantly, the use of dual-drug selection in our system increases the efficiency of positive selection for double-targeted clones to >50%, permitting a less laborious screening process. If desired, this protocol can also be adapted to allow the use of tissue-specific promoters to drive gene expression instead of the doxycycline-inducible promoter (TRE). Additionally, this protocol is also compatible with the use of Transcription-Activator-Like Effector Nucleases (TALENs) or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system in place of ZFNs.

Plant tissue-specific promoters can drive gene expression in Escherichia coli

Plant transgenesis often requires the use of tissue-specific promoters to drive the transgene expression exclusively in targeted tissues. Although the eukaryotic promoters are expected to stay silent in Escherichia coli, when the promoter-transgene units within the plant transformation vectors are constructed and propagated, some eukaryotic promoters have been reported to be active in prokaryotes. The potential activity of plant promoter in E. coli cells should be considered in cases of expression of proteins that are toxic for host cells, environmental risk assessment or the stability in E. coli of plant vectors for specific Cre/loxP applications. In this study, DNA fragments harbouring four embryo- and/or pollen-specific Arabidopsis thaliana promoters were investigated for their ability to drive heterologous gene expression in E. coli cells. For this, they were fused to gfp:gus reporter genes in the pCAMBIA1304 vector. Although BPROM, bacterial sigma70 promoter recognition program identified several sequences with characteristics similar to bacterial promoters including -10 and -35 sequences in each of tested fragments, the experimental approach showed that only one promoter fragment was able to drive relatively strong- and one promoter fragment relatively weak-GUS expression in E. coli cells. Remaining two tested promoters did not drive any transgene expression in bacteria. Our results also showed that cloning of a shorter plant promoter sequence into vectors containing lacZ α-complementation system can increase the probability of gene expression driven by upstream located lac promoter. This should be considered when cloning of plant expression units, the expression of which is unwanted in E. coli.

Seminal vesicles and urinary bladder as sites of aromatization of androgens in men, evidenced by a CYP19A1‐driven luciferase reporter mouse and human tissue specimens

The human CYP19A1 gene is expressed in various tissues by the use of tissue-specific promoters, whereas the rodent cyp19a1 gene is expressed mainly in the gonads and brain. We generated a transgenic mouse model containing a >100-kb 5' region of human CYP19A1 gene connected to a luciferase reporter gene. The luciferase activity in mouse tissues mimicked the CYP19A1 gene expression pattern in humans. Interestingly, the reporter gene activity was 16 and 160 times higher in the urinary bladder and seminal vesicles, respectively, as compared with the activity in the testis. Accordingly, CYP19A1 gene and P450arom protein expression was detected in those human tissues. Moreover, the data revealed that the expression of CYP19A1 gene is driven by promoters PII, I.4, and I.3 in the seminal vesicles, and by promoters PII and I.4 in the urinary bladder. Furthermore, the reporter gene expression in the seminal vesicles was androgen dependent: Castration decreased the expression ∼20 times, and testosterone treatment restored it to the level of an intact mouse. This reporter mouse model facilitates studies of tissue-specific regulation of the human CYP19A1 gene, and our data provide evidence for seminal vesicles as important sites for estrogen production in males.

Technical challenges and limitations of current mouse models of ovarian cancer

The development of genetically engineered models (GEM) of epithelial ovarian cancer (EOC) has been very successful, with well validated models representing high grade and low grade serous adenocarcinomas and endometrioid carcinoma (EC). Most of these models were developed using technologies intended to target the ovarian surface epithelium (OSE), the cell type long believed to be the origin of EOC. More recent evidence has highlighted what is likely a more prevalent role of the secretory cell of the fallopian tube in the ontogeny of EOC, however none of the GEM of EOC have demonstrated successful targeting of this important cell type.The precise technologies exploited to develop the existing GEM of EOC are varied and carry with them advantages and disadvantages. The use of tissue specific promoters to model disease has been very successful, but the lack of any truly specific OSE or oviductal secretory cell promoters makes the outcomes of these models quite unpredictable. Effecting genetic change by the administration of adenoviral vectors expressing Cre recombinase may alleviate the perceived need for tissue specific promoters, however the efficiencies of infection of different cell types is subject to numerous biological parameters that may lead to preferential targeting of certain cell populations.One important future avenue of GEM of EOC is the evaluation of the role of genetic modifiers. We have found that genetic background can lead to contrasting phenotypes in one model of ovarian cancer, and data from other laboratories have also hinted that the exact genetic background of the model may influence the resulting phenotype. The different genetic backgrounds may modify the biology of the tumors in a manner that will be relevant to human disease, but they may also be modifying parameters which impact the response of the host to the technologies employed to develop the model.

A molecular approach to combat spatio-temporal variation in insecticidal gene (Cry1Ac) expression in cotton

Spatio-temporal expression of an insecticidal gene (Cry1Ac) in pre existing transgenic lines of transgenic cotton was studied. Seasonal decline in expression of Cry1Ac differed significantly among different cotton lines tested in the field conditions. The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, bolls, anthers and petals. Expression of the gene decreased consistently with the age of plants. Toxin expression in fruiting parts was not enough to confer full resistance against bollworms. The reduction in efficacy of transgenic cotton plants late in the season was attributed to reduction in promoter activity. For this purpose, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) promoter was isolated from Gossypium arboreum that was further cloned upstream of an insecticidal gene (Cry1Ac) in expression vector pCAMBIA 1301. A local cotton cultivar NIAB-846 was transformed with Cry1Ac driven by rbcS promoter. The same cotton cultivar was also transformed with Cry1Ac gene driven by 35SCaMV promoter to compare the expression pattern of insecticidal gene under two different promoters. The results showed that rbcS is an efficient promoter to drive the expression of Cry1Ac gene consistent throughout the life of cotton plant as compared to 35S promoter. The use of tissue specific promoter is also useful for addressing the biosafety issues as the promoter activity is limited to green parts of plants, hence no gene expression in roots, cotton seed and other cotton products and by products.

Immunological perspective of self versus tumor antigens: insights from the RIP‐gp model

Self-reactive T cells in the body are controlled by mechanisms of peripheral tolerance that limit their activation and induction of immune pathology. Our understanding of these mechanisms has been advanced by the use of tissue-specific promoters to express neo-self-antigens. Here, we present findings using the RIP-gp (rat insulin promoter-glycoprotein) transgenic mouse, which expresses the lymphocytic choriomeningitis virus glycoprotein (LCMV-gp) specifically in the pancreatic β islet cells. T cells responsive to this antigen remain ignorant of the LCMV-gp expressed by the islets, and breaking tolerance is dependent upon the maturation status of antigen-presenting cells, the avidity of the T-cell receptor ligation, and the level of major histocompatibility complex expression in the pancreas. Furthermore, decreased activity of Casitas B-lineage lymphoma b, a negative regulator of T-cell receptor signaling, can allow recognition and destruction of the pancreatic islets. This review discusses the roles of these factors in the context of anti-tissue responses, both in the setting of autoimmunity and in anti-tumor immunity.

MicroRNA-regulated transgene expression systems for gene therapy and virotherapy

For safe and effective gene therapy, targeted tissue-restricted transgene expression is desirable. Various methods have been developed to achieve such expression, including the use of tissue-specific promoters. In addition to these approaches, a new system which can regulate transgene expression, including viral gene expression, by exploiting microRNAs (miRNAs) has recently been developed. miRNAs are approximately 22-nucleotide (nt)-long non-coding RNAs that translationally suppress or catalytically degrade target mRNA through binding to imperfectly complementary sequences in the 3'-untranslated region (UTR). In miRNA-regulated transgene expression systems, tandem copies of sequences perfectly complementary to the miRNAs are usually incorporated into the 3'-UTR of the transgene expression cassette, leading to the suppression of transgene expression in cells expressing the corresponding miRNAs. miRNA-mediated regulation of transgene expression was first demonstrated for lentivirus vectors, and subsequently this technology was applied to replication-incompetent adenovirus vectors, tumor-specific oncolytic viruses for cancer therapy, and recombinant live attenuated viruses for vaccine therapy. The aim of this review is to highlight the applications of miRNA-regulated transgene expression systems for gene therapy and virotherapy.

Improving Adenovirus Based Gene Transfer: Strategies to Accomplish Immune Evasion

Adenovirus (Ad) based gene transfer vectors continue to be the platform of choice for an increasing number of clinical trials worldwide. In fact, within the last five years, the number of clinical trials that utilize Ad based vectors has doubled, indicating growing enthusiasm for the numerous positive characteristics of this gene transfer platform. For example, Ad vectors can be easily and relatively inexpensively produced to high titers in a cGMP compliant manner, can be stably stored and transported, and have a broad applicability for a wide range of clinical conditions, including both gene therapy and vaccine applications. Ad vector based gene transfer will become more useful as strategies to counteract innate and/or pre-existing adaptive immune responses to Ads are developed and confirmed to be efficacious. The approaches attempting to overcome these limitations can be divided into two broad categories: pre-emptive immune modulation of the host, and selective modification of the Ad vector itself. The first category of methods includes the use of immunosuppressive drugs or specific compounds to block important immune pathways, which are known to be induced by Ads. The second category comprises several innovative strategies inclusive of: (1) Ad-capsid-display of specific inhibitors or ligands; (2) covalent modifications of the entire Ad vector capsid moiety; (3) the use of tissue specific promoters and local administration routes; (4) the use of genome modified Ads; and (5) the development of chimeric or alternative serotype Ads. This review article will focus on both the promise and the limitations of each of these immune evasion strategies, and in the process delineate future directions in developing safer and more efficacious Ad-based gene transfer strategies.

Low Current-driven Micro-electroporation Allows Efficient In Vivo Delivery of Nonviral DNA into the Adult Mouse Brain

Viral gene transfer or transgenic animals are commonly used technologies to alter gene expression in the adult brain, although these approaches lack spatial specificity and are time consuming. We delivered plasmid DNA locally into the brain of adult C57BL/6 mice in vivo by voltage- and current-controlled electroporation. The low current-controlled delivery of unipolar square wave pulses of 125 µA with microstimulation electrodes at the injection site gave 16 times higher transfection rates than a voltage-controlled electroporation protocol with plate electrodes resulting in currents of about 400 mA. Transfection was restricted to the target region and no damage due to the electric pulses was found. Our current-controlled electroporation protocol indicated that the use of very low currents resulting in applied voltages within the physiological range of the membrane potential, allows efficient transfection of nonviral plasmid DNA. In conclusion, low current-controlled electroporation is an excellent approach for electroporation in the adult brain, i.e., gene function can be influenced locally at a high level with no mortality and minimal tissue damage.

Modification of cardiovascular ion channels by gene therapy

Delivery of genes to the heart and vasculature for therapeutic purposes is an exciting strategy that is approaching clinical reality. Abnormalities of expression or function of ion channels is central to many cardiovascular diseases and gene delivery to modify ion channels is an appealing alternative to traditional therapy with small-molecule drugs. Potential therapeutic targets include hypertrophy and heart failure, atrioventricular node modification in atrial fibrillation, ventricular tachycardia and hypertension. Numerous approaches for gene delivery are under development, including use of tissue-specific promoters in viral vectors. For other applications, such as development of biological pacemakers, cells can be transduced with pacemaker genes in vitro, and then the cells implanted within the heart. There are short-term hurdles to therapeutic gene delivery to modify cardiovascular ion channels, but in the intermediate and longer term, the outlook is promising.