Inhibition Of Reporter Gene Expression Research Articles

Characterization of RNA G-quadruplexes in porcine epidemic diarrhea virus genome and the antiviral activity of G-quadruplex ligands

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there are still no anti-PEDV drugs with accurate targets. G-quadruplexes (G4s) are non-canonical secondary structures formed within guanine-rich regions of DNA or RNA, and have attracted great attention as potential targets for antiviral strategy. In this study, we reported two putative G4-forming sequences (PQS) in S and Nsp5 genes of PEDV genome based on bioinformatic analysis, and identified that S-PQS and Nsp5-PQS were enabled to fold into G4 structure by using circular dichroism spectroscopy and fluorescence turn-on assay. Furthermore, we verified that both S-PQS and Nsp5-PQS PQS could form G4 structure in live cells by immunofluorescence microscopy. In addition, G4-specific compounds, such as TMPyP4 and PDS, could significantly inhibit transcription, translation and proliferation of PEDV in vitro. Importantly, these compounds exert antiviral activity at the post-entry step of PEDV infection cycle, by inhibiting viral genome replication and protein expression. Lastly, we demonstrated that TMPyP4 can inhibit reporter gene expression by targeting G4 structure in Nsp5. Taken together, these findings not only reinforce the presence of viral G-quadruplex sequences in PEDV genome but also provide new insights into developing novel antiviral drugs targeting PEDV RNA G-quadruplexes.

Mapping Regulatory Elements within 5' and 3' UTRs of SIGLEC15 with a Use of Reporter System

Siglec-15 is an immune suppressor with broad upregulation on various cancer types and has emerged as a potential target for cancer immunotherapy. However, it remains unclear how SIGLEC15 expression is controlled in normal or cancer cells. In this work, we utilized reporter assays to evaluate the impact of the 5' UTR and the 3' UTR of SIGLEC15 mRNA on gene expression. We found that the 3' UTR dramatically reduced reporter protein production, whereas the 5' UTR showed modest inhibitory effect. Quantification of steady-state mRNA revealed the good coupling of protein amount and mRNA abundance that was associated with the 3' UTR. In contrast, the 5' UTR had little effect on mRNA abundance compared with the empty control. By measuring mRNA half-life, we showed that the 3' UTR markedly promoted mRNA degradation. Testing shortened 3' UTR fragments demonstrated five out of the six having notable inhibitory effect, with the one spanning 993-1317 had the most robust activity. More interestingly, the 993-1317 region contains a predicted 43-nt stem-loop structure that showed apparent inhibitory activity in four cell lines tested. These results suggested that the 3' UTR inhibited reporter gene expression by accelerating mRNA decay possibly via multiple cis-regulatory elements, but the 5' UTR repressed gene expression by inhibiting translation. Thus, our findings provided a clue to the molecular mechanism underlying the regulation of SIGLEC15 expression.

The VSV matrix protein inhibits NF-κB and the interferon response independently in mouse L929 cells

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in immune evasion. While VSV has been thought to suppress the interferon (IFN) response primarily by inhibiting host cell transcription and translation, our recent findings indicate that the M protein also targets NF-κB activation. Therefore, the M protein may utilize two distinct mechanisms to limit expression of antiviral genes, inhibiting both host gene expression and NF-κB activation. Here we characterize a recently reported mutation in the M protein [M(D52G)] of VSV isolate 22–20, which suppressed IFN mRNA and protein production despite activating NF-κB. 22–20 inhibited reporter gene expression from multiple promoters, suggesting that 22-20 suppressed the IFN response via M-mediated inhibition of host cell transcription. We propose that suppression of the IFN response and regulation of NF-κB are independent, genetically separable functions of the VSV M protein.

Epigenetic repression of androgen receptor transcription in mutation-negative androgen insensitivity syndrome (AIS type II)

Context: Inactivating mutations within the AR gene are present in only similar to 40% of individuals with clinically and hormonally diagnosed androgen insensitivity syndrome (AIS). Previous studies revealed the existence of an AR gene mutation-negative group of patients with AIS who have compromised androgen receptor (AR) function (AIS type II). Objective: To investigate whether AIS type II can be due to epigenetic repression of AR transcription. Design: Quantification of AR mRNA and AR proximal promoter CpG methylation levels in genital skin-derived fibroblasts (GFs) derived from patients with AIS type II and control individuals. Setting: University hospital endocrine research laboratory. Patients: GFs from control individuals (n = 11) and patients with AIS type II (n = 14). Main Outcome Measure(s): Measurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro. Results: Fifty-seven percent of individuals with AIS type II (n = 8) showed a reduced AR mRNA expression in their GFs. A significant inverse correlation was shown between AR mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158-bp-long region containing these CpGs was sufficient to severely reduce reporter gene expression. This region was bound by the runt related transcription factor 1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells was able to inhibit reporter gene expression through this region. Conclusions: Aberrant CpGs methylation within the proximal AR promoter plays an important role in the control of AR gene expression and may result in AIS type II. We suggest that transcriptional modifiers, such as RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases.

A secondary structure within a human piRNA modulates its functionality

The piwi-interacting RNAs (piRNAs) are small non-coding RNAs, mostly 24–32 nucleotides in length. The piRNAs are not known to have any conserved secondary structure or sequence motifs. Using bioinformatics analysis, we discovered the presence of putative G-quadruplex (GQ) forming sequences in human piRNAs. We studied human piR-48164/piR-GQ containing a potential GQ forming sequence and using biochemical and biophysical techniques confirmed its ability to form a GQ. Using EMSA, we discovered that the formation of GQ structure led to inhibition of the piRNA binding to the HIWI-PAZ domain as well as the complementary base pairing to a target RNA. The inability of the piR-GQ to interact with the PIWI protein might be detrimental to the function of the piRNA. To investigate if the formation of a GQ structure in piRNA prevents its target gene silencing in vivo, we used a reporter assay. The piR-GQ failed to inhibit the reporter gene expression while a mutated version that lacked the ability to form GQ inhibited reporter gene expression indicating that the presence of GQ in piRNA is detrimental to its function. These studies unraveled the dependence of a piRNA's functionality on an RNA secondary structure and added a new layer of regulation to their function.

Epigenetic Repression of Androgen Receptor Transcription in Mutation-Negative Androgen Insensitivity Syndrome (AIS Type II).

Inactivating mutations within the AR gene are present in only ~40% of individuals with clinically and hormonally diagnosed androgen insensitivity syndrome (AIS). Previous studies revealed the existence of an AR gene mutation-negative group of patients with AIS who have compromised androgen receptor (AR) function (AIS type II). To investigate whether AIS type II can be due to epigenetic repression of AR transcription. Quantification of AR mRNA and AR proximal promoter CpG methylation levels in genital skin-derived fibroblasts (GFs) derived from patients with AIS type II and control individuals. University hospital endocrine research laboratory. GFs from control individuals (n = 11) and patients with AIS type II (n = 14). Measurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro. Fifty-seven percent of individuals with AIS type II (n = 8) showed a reduced AR mRNA expression in their GFs. A significant inverse correlation was shown between AR mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158-bp-long region containing these CpGs was sufficient to severely reduce reporter gene expression. This region was bound by the runt related transcription factor 1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells was able to inhibit reporter gene expression through this region. Aberrant CpGs methylation within the proximal AR promoter plays an important role in the control of AR gene expression and may result in AIS type II. We suggest that transcriptional modifiers, such as RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases.

A novel family of transcription factors conserved in angiosperms is required for ABA signalling.

The plant hormone abscisic acid (ABA) plays a crucial role in regulating plant responses to environmental stresses. Interplay of several different proteins including the PYR/PYL/RCAR receptors, A-group PP2C protein phosphatases, SnRK2 protein kinases, and downstream transcription factors regulates ABA signalling. We report here the identification of a family of ABA-induced transcription repressors (AITRs) that act as feedback regulators in ABA signalling. We found that the expression of all the 6 Arabidopsis AITR genes was induced by exogenously ABA, and their expression levels were decreased in ABA biosynthesis mutant aba1-5. BLAST searches showed that AITRs are exclusively present in angiosperms. When recruited to the promoter region of a reporter gene by a fused DNA binding domain, all AITRs inhibited reporter gene expression in transfected protoplasts. In Arabidopsis, aitr mutants showed reduced sensitivity to ABA and to stresses such as salt and drought. Quantitative RT-PCR analysis demonstrated that the ABA-induced response of PP2C and some PYR/PYL/RCAR genes was reduced in AITR5 transgenic plants but increased in an aitr2 aitr5 aitr6 triple mutant. These results provide important new insights into the regulation of ABA signalling in plants, and such information may lead to the production of plants with enhanced resistance to environmental stresses.

An ABA down-regulated bHLH transcription repressor gene, bHLH129 regulates root elongation and ABA response when overexpressed in Arabidopsis.

Plant hormone abscisic acid (ABA) plays a crucial role in modulating plant responses to environmental stresses. Basic helix-loop-helix (bHLH) transcription factors are one of the largest transcription factor families that regulate multiple aspects of plant growth and development, as well as of plant metabolism in Arabidopsis. Several bHLH transcription factors have been shown to be involved in the regulation of ABA signaling. We report here the characterization of bHLH129, a bHLH transcription factor in Arabidopsis. We found that the expression level of bHLH129 was reduced in response to exogenously applied ABA, and elevated in the ABA biosynthesis mutant aba1-5. Florescence observation of transgenic plants expressing bHLH129-GFP showed that bHLH129 was localized in the nucleus, and transient expression of bHLH129 in protoplasts inhibited reporter gene expression. When expressed in Arabidopsis under the control of the 35S promoter, bHLH129 promoted root elongation, and the transgenic plants were less sensitivity to ABA in root elongation assays. Quantitative RT-PCR results showed that ABA response of several genes involved in ABA signaling, including ABI1, SnRK2.2, SnRK2.3 and SnRK2.6 were altered in the transgenic plants overexpressing bHLH129. Taken together, our study suggests that bHLH129 is a transcription repressor that negatively regulates ABA response in Arabidopsis.

Sa2059 Afferent Activation in the Proximal Colon by Enteroendocrine Mediators and Nutrients

Background: Despite the importance of gastric inhibitory polypeptide (GIP) as a physiological incretin, postprandial circulating glucose levels in GIP receptor null (GIPR-/-) and wild-type (wt) mice are similar. However, unlike wt mice, GIPR-/mice do not develop diet-induced obesity, suggesting a potential role for GIPR antagonists in the treatment of obesity. While specific GIPR antagonists, such as N-terminal truncated peptides and peptides with a Glu to Pro substitution at position 3 (Pro3-GIP), have been identified, their short circulating T1/2 has hampered their development. We hypothesize that the T1/2 of these antagonists can be increased significantly by fusing them to the Fc-fragment of IgG, a strategy that has been employed previously for this purpose. Aim: To develop long-acting antagonists to GIPR by fusion to the IgG Fc-fragment. Methods: PCR and molecular cloning were used to produce chimeric genes encoding GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG. After expression in HEK 293 cells, protein A agarose chromatography was used to purify protein from conditioned media. Fc-fusions were identified by Western analysis using a specific GIP antibody, and biological activity was assessed in the presence and absence of 1 nM synthetic GIP(1-42) using a GIP-specific reporter cell line. Results: Transgenic HEK 293 cells produced protein with the predicted molecular size for the Fc fusion and that possessed GIP-specific immunoreactivity. Purified Pro3-GIP-Fc-IgG induced reporter expression with an EC50 of 0.5 μM, whereas GIP(6-42)-Fc-IgG did not. Both GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG antagonized report gene expression induced by 1 nM GIP, with IC50 values of 4 μM and 20 μM, respectively. Summary and Conclusion: Pro3-GIP-Fc-IgG, but not GIP(6-42)-FcIgG, induced reporter gene expression, while both fusion products inhibited reporter gene expression induced by 1 nM GIP, indicating that the former is a partial agonist, while only the latter functions as an antagonist. The IC50 values for GIP(6-42)-Fc-IgG and Pro3-GIPFc-IgG were higher than the corresponding values reported for synthetic GIP(6-42) and Pro3-(GIP), indicating the possibility of partial interference of ligand binding by the Fc fragment. Nevertheless, the increase in T1/2 imparted by the fusion of N-terminal truncated GIP peptides the Fc fragment may thereby provide viable candidates for treating obesity and related disorders.

The 14q32 MicroRNA-487b Targets the Antiapoptotic Insulin Receptor Substrate 1 in Hypertension-Induced Remodeling of the Aorta

To study the role of microRNAs in hypertension-induced vascular pathology before the onset of symptoms of severe cardiovascular disease. MicroRNAs play a crucial role in cardiovascular disease. However, microRNAs are often studied in full-blown cardiovascular disease models, not during development of cardiovascular pathology. Angiotensin II was infused into healthy adult rats, inducing chronic hypertension, and microRNA expression profiles were obtained. The most prominently regulated microRNA, miR-487b, was further investigated, using primary cultures of rat aortic and human umbilical cord arterial cells. MiR-487b is predicted to target insulin receptor substrate 1 (IRS1). IRS1 plays an important role in both insulin signaling and cell proliferation and survival. IRS1 mRNA and protein levels were downregulated in aortae of hypertensive rats. MiR-487b binds directly to both rat and human IRS1 3'UTR and inhibits reporter gene expression in vitro. In primary rat and human arterial adventitial fibroblasts, inhibition of miR-487b leads to upregulation of IRS1 expression. Upregulation of miR-487b had the opposite effect, confirming direct targeting of IRS1 by miR-487b.Immunohistochemistry of aortic cross sections and rt/qPCR analyses of the separate aortic wall layers showed that both IRS1 and miR-487b were present mainly in the adventitia and less or not at all in the intima and tunica media. IRS1 expression in adventitial fibroblasts was predominantly nuclear and nuclear IRS1 is known to have antiapoptotic effects. Indeed, inhibition of miR-487b protected adventitial fibroblasts, and also medial smooth muscle cells, against serum starvation-induced apoptosis and increased cell survival. Angiotensin II-induced hypertension leads to upregulation of miR-487b, which targets IRS1. Via downregulation of IRS1, miR-487b can contribute to cell death and loss of adventitial and medial integrity during hypertension-induced vascular pathology.

Attenuation of Mouse Hepatitis Virus by Deletion of the LLRKxGxKG Region of Nsp1

Coronaviruses are a family of large positive-sense RNA viruses that are responsible for a wide range of important veterinary and human diseases. Nsp1 has been shown to have an important role in the pathogenetic mechanisms of coronaviruses in vivo. To assess the function of a relatively conserved domain (LLRKxGxKG) of MHV nsp1, a mutant virus, MHV-nsp1-27D, with a 27 nts (LLRKxGxKG) deletion in nsp1, was constructed using a reverse genetic system with a vaccinia virus vector. The mutant virus had similar growth kinetics to MHV-A59 wild-type virus in 17CI-1 cells, but was highly attenuated in vivo. Moreover, the mutant virus completely protected C57BL/6 mice from a lethal MHV-A59 challenge. To further analyze the mechanism of the attenuation of the mutant virus, changes in reporter gene expression were measured in nsp1- or nsp1-27D-expressing cells; the results showed that nsp1 inhibited reporter gene expression controlled by different promoters, but that this inhibition was reduced for nsp1-27D. The research in vivo and in vitro suggests that the LLRKxGxKG region of nsp1 may play an important role in this process.

Regulation of androgen receptor expression by Z‐isochaihulactone mediated by the JNK signaling pathway and might be related to cytotoxicity in prostate cancer

The androgen receptor (AR) is a main therapeutic target for treatment of prostate cancer (PCa). The natural compound isochaihulactone (K8), which has a chiral center ring and two racemic forms (E-K8 and Z-K8), has anti-tumor effects on multiple cancer types both in vitro and in vivo. Here, we determined which form of K8 contains significant tumor cytotoxicity and examined how this form regulates AR expression in PCa cells and xenografts. We chose the androgen-dependent human PCa cell line LNCaP and the androgen-independent cell lines DU145 and PC-3 to study the anti-tumor potency and AR regulation mediated by Z-K8. We measured cell viability and used flow cytometry, RT-PCR, and Western blotting. Growth inhibition in vivo was evaluated with an LNCaP xenograft animal model. In LNCaP cells, Z-K8 significantly repressed cell proliferation, induced apoptosis, repressed AR mRNA and protein expression in a time-dependent manner, and induced JNK phosphorylation. Furthermore, treatment with a JNK inhibitor significantly abolished Z-K8-induced AR downregulation. Z-K8 did not significantly inhibit reporter gene expression of constructs containing the AR promoter when it contained a mutated Sp1 binding site. Z-K8 also showed anti-tumor effects in the xenograft animal model. Z-K8 not only induced LNCaP apoptosis but also reduced AR expression. These results indicate that Z-K8 may be a potential anti-tumor drug for PCa therapy.

Inhibition of CD44 Gene Expression in Human Skin Models, Using Self-Delivery Short Interfering RNA Administered by Dissolvable Microneedle Arrays

Treatment of skin disorders with short interfering RNA (siRNA)-based therapeutics requires the development of effective delivery methodologies that reach target cells in affected tissues. Successful delivery of functional siRNA to the epidermis requires (1) crossing the stratum corneum, (2) transfer across the keratinocyte membrane, followed by (3) incorporation into the RNA-induced silencing complex. We have previously demonstrated that treatment with microneedle arrays loaded with self-delivery siRNA (sd-siRNA) can achieve inhibition of reporter gene expression in a transgenic mouse model. Furthermore, treatment of human cultured epidermal equivalents with sd-siRNA resulted in inhibition of target gene expression. Here, we demonstrate inhibition of CD44, a gene that is uniformly expressed throughout the epidermis, by sd-siRNA both in vitro (cultured human epidermal skin equivalents) and in vivo (full-thickness human skin equivalents xenografted on immunocompromised mice). Treatment of human skin equivalents with CD44 sd-siRNA markedly decreased CD44 mRNA levels, which led to a reduction of the target protein as confirmed by immunodetection in epidermal equivalent sections with a CD44-specific antibody. Taken together, these results demonstrate that sd-siRNA, delivered by microneedle arrays, can reduce expression of a targeted endogenous gene in a human skin xenograft model.

Rational design of a small molecule-responsive intramer controlling transgene expression in mammalian cells

Aptamers binding proteins or small molecules have been shown to be versatile and powerful building blocks for the construction of artificial genetic switches. In this study, we present a novel aptamer-based construct regulating the Tet Off system in a tetracycline-independent manner thus achieving control of transgene expression. For this purpose, a TetR protein-inhibiting aptamer was engineered for use in mammalian cells, enabling the RNA-responsive control of the tetracycline-dependent transactivator (tTA). By rationally attaching the theophylline aptamer as a sensor, the inhibitory TetR aptamer and thus tTA activity became dependent on the ligand of the sensor aptamer. Addition of the small molecule theophylline resulted in enhanced binding to the corresponding protein in vitro and in inhibition of reporter gene expression in mammalian cell lines. By using aptamers as adaptors in order to control protein activity by a predetermined small molecule, we present a simple and straightforward approach for future applications in the field of Chemical Biology. Moreover, aptamer-based control of the widely used Tet system introduces a new layer of regulation thereby facilitating the construction of more complex gene networks.

Expression of Multiple Artificial MicroRNAs from a Chicken miRNA126-Based Lentiviral Vector

BackgroundThe use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector.Methods/Principal FindingsWe have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector.Conclusions/SignificanceThis novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.

FMR1 CGG repeat lengths mediate different regulation of reporter gene expression in comparative transient and locus specific integration assays

The Fragile X mental retardation (FMR1) gene contains a polymorphic CGG trinucleotide repeat in the 5′-untranslated region. The repeat length in the normal population is between 5 and 54 repeats. A repeat length between 55 and 200 is defined as the pre-mutation repeat size. Elderly carriers of the pre-mutation can develop the progressive neurodegenerative disease Fragile X-associated tremor/ataxia syndrome (FXTAS). In FXTAS the FMR1 mRNA levels are increased and it is hypothesized that FXTAS is caused by a RNA gain of function mechanism. Repeat lengths beyond 200 CGGs are defined as the full-mutation and causes Fragile X-syndrome which is the most common inherited form of mental retardation. The full-mutation results in the absence of the FMR1 mRNA and protein, FMRP, through abnormal CpG methylation and FMR1 gene silencing. In this report we have used the Flp-In T-REx system to generate locus directed stable cell lines harboring the FMR1 5′-UTR with varying CGG repeat lengths in front of a reporter gene. By this system the influence of various CGG repeat lengths for reporter gene expression can be comparatively examined in cell lines where the only genetic difference is CGG repeat lengths. In such cell lines we find that a full-mutation CGG repeat confers inhibition of reporter gene expression, whereas a pre-mutation CGG repeat did not increase reporter gene expression. In transient transfection assays using the same expression vectors the pre-mutation and full-mutation CGG repeats increased reporter gene expression. This study shows that locus directed integration of model FMR1 CGG transgenes could be a new basic tool to further elucidating the basic molecular mechanisms behind transcriptional deregulation of the FMR1 gene in fragile X-syndrome and FXTAS.

Hepatitis C virus NS2 protein triggers endoplasmic reticulum stress and suppresses its own viral replication

We previously reported that the NS2 protein of hepatitis C virus (HCV) inhibits the expression of reporter genes driven by a variety of cellular and viral promoters. The aim of the study was to determine whether the broad transcriptional repression is caused by endoplasmic reticulum (ER) stress. Phosphorylation of the translation initiation factor eIF2α and HCV replication was detected by Western and Northern blot, respectively. De novo protein synthesis was measured by metabolic labeling. Activation of ER stress responsive genes was determined by promoter reporter assay, as well as mRNA and protein measurement by real time PCR and Western blot. Transient or inducible NS2 protein expression increased eIF2α phosphorylation and reduced de novo protein synthesis. It up-regulated promoter activities and transcript levels of ER stress inducible genes including GRP78, ATF6, and GADD153, as well as GRP78 protein level. The same effect was observed when NS2 was synthesized as part of the core-E1-E2-p7-NS2 polypeptide. NS2 protein also inhibited reporter gene expression from the HCV internal ribosome entry site and consequently reduced HCV replication. The full-length HCV replicon activated GRP78, ATF6, and GADD153 promoters more efficiently than the subgenomic replicon lacking the coding sequence for both the structural proteins and NS2. Abrogation of HCV infection/replication, by an inhibitor of the NS3 protease, relieved ER stress. HCV infection can induce ER stress, with NS2 protein being a major mediator. The stress can be relieved by a feedback mechanism.

A negative element in the downstream region of the Rice tungro bacilliform virus promoter is orientation- and position-independent and is active with heterologous promoters

The promoter of an Indian isolate of the pararetrovirus Rice tungro bacilliform virus (RTBV-WB) contains a negative element downstream of the transcription start site (TSS), between nucleotide residues +58 and +195 ( Mathur and Dasgupta, 2007). To further characterize the element, we show, by using transient gus reporter gene assays in the cells of onion peel, rice calli and Arabidopsis leaves, that it down-regulates heterologous promoters CaMV35S and Maize ubiquitin. Quantitative measurements of transient GUS activity indicated more than 90% inhibition of reporter gene expression by the negative element. We also show, by reversing the orientation of the element downstream and by placing it in a position upstream to a constitutively expressing RTBV promoter, that the negative element is orientation- and position-independent, pointing towards its activity at the transcriptional and not post-transcriptional level.

A dual function TAR Decoy serves as an anti-HIV siRNA delivery vehicle

The TAR RNA of HIV was engineered as an siRNA delivery vehicle to develop a combinatorial therapeutic approach. The TAR backbone was found to be a versatile backbone for expressing siRNAs. Upon expression in human cells, pronounced and specific inhibition of reporter gene expression was observed with TARmiR. The resulting TARmiR construct retained its ability to bind Tat and mediate RNAi. TARmiR was able to inhibit HIV gene expression as a TAR decoy and by RNA interference when challenged with infectious proviral DNA. The implications of this dual function therapeutic would be discussed.

Neuronal gene delivery by negatively charged pullulan–spermine/DNA anioplexes

Nonviral gene transfer to neurons remains unreliable due to a lack of effective and nontoxic vectors. Here, we achieved effective neuronal gene delivery through salt-free complexation of plasmid DNA and pullulan–spermine, a conjugate prepared from a naturally derived polysaccharide and polyamine. Specifically, at low spermine nitrogen:DNA phosphate (N:P) ratios, complexes formed with ζ-potential and diameter of approximately−40 mV and 350 nm, respectively. Higher N:P ratios increased the ζ-potential to approximately +10 mV. All complexes were stable for at least 1 week and protected DNA from degradation. In vitro transfection of rat sensory neurons occurred at all N:P ratios, but uniquely, efficiency was highest for anionic complexes (anioplexes). Subsequent analyses revealed the inhibition of reporter gene expression by asialofetuin (1 mg/ml) and methyl-beta-cyclodextrin (5 m m), indicating utilization of glycoprotein-specific interactions and lipid rafts for uptake and intracellular trafficking. In marked contrast to a commercial cationic lipid reagent, anioplexes did not exhibit measurable cytotoxicity at up to 20 μg/ml DNA. Additionally, transfection efficiency was maintained in the presence of serum and antibiotics. Based on these favorable properties, we successfully established two transfection methods for cultured adult sensory neurons and tissue explants. Collectively, these data suggest that negatively charged pullulan–spermine/DNA anioplexes could represent an effective gene delivery technology, particularly for neurons.