Circadian-Coupled Genes Expression and Regulation in HIV-Associated Chronic Obstructive Pulmonary Disease (COPD) and Lung Comorbidities.

People living with HIV (PLWH) have an elevated risk of chronic obstructive pulmonary disease (COPD) and are at a higher risk of asthma and worse outcomes. Even though the combination of antiretroviral therapy (cART) has significantly improved the life expectancy of HIV-infected patients, it still shows a higher incidence of COPD in patients as young as 40 years old. Circadian rhythms are endogenous 24 h oscillations that regulate physiological processes, including immune responses. Additionally, they play a significant role in health and diseases by regulating viral replication and its corresponding immune responses. Circadian genes play an essential role in lung pathology, especially in PLWH. The dysregulation of core clock and clock output genes plays an important role in chronic inflammation and aberrant peripheral circadian rhythmicity, particularly in PLWH. In this review, we explained the mechanism underlying circadian clock dysregulation in HIV and its effects on the development and progression of COPD. Furthermore, we discussed potential therapeutic approaches to reset the peripheral molecular clocks and mitigate airway inflammation.

Sphingosine-1-Phosphate Receptor 3 Induces Endothelial Barrier Loss via ADAM10-Mediated Vascular Endothelial-Cadherin Cleavage.

Mechanical ventilation (MV) is a life-supporting strategy employed in the Intensive Care Unit (ICU). However, MV-associated mechanical stress exacerbates existing lung inflammation in ICU patients, resulting in limited improvement in mortality and a condition known as Ventilator-Induced Lung Injury (VILI). Sphingosine-1-phosphate (S1P) is a circulating bioactive lipid that maintains endothelial integrity primarily through S1P receptor 1 (S1PR1). During VILI, mechanical stress upregulates endothelial S1PR3 levels. Unlike S1PR1, S1PR3 mediates endothelial barrier disruption through Rho-dependent pathways. However, the specific impact of elevated S1PR3 on lung endothelial function, apart from Rho activation, remains poorly understood. In this study, we investigated the effects of S1PR3 in endothelial pathobiology during VILI using an S1PR3 overexpression adenovirus. S1PR3 overexpression caused cytoskeleton rearrangement, formation of paracellular gaps, and a modified endothelial response towards S1P. It resulted in a shift from S1PR1-dependent barrier enhancement to S1PR3-dependent barrier disruption. Moreover, S1PR3 overexpression induced an ADAM10-dependent cleavage of Vascular Endothelial (VE)-cadherin, which hindered endothelial barrier recovery. S1PR3-induced cleavage of VE-cadherin was at least partially regulated by S1PR3-mediated NFκB activation. Additionally, we employed an S1PR3 inhibitor TY-52156 in a murine model of VILI. TY-52156 effectively attenuated VILI-induced increases in bronchoalveolar lavage cell counts and protein concentration, suppressed the release of pro-inflammatory cytokines, and inhibited lung inflammation as assessed via a histological evaluation. These findings confirm that mechanical stress associated with VILI increases S1PR3 levels, thereby altering the pulmonary endothelial response towards S1P and impairing barrier recovery. Inhibiting S1PR3 is validated as an effective therapeutic strategy for VILI.

A Neutralizing Aptamer to TGFBR2 and miR-145 Antagonism Rescue Cigarette Smoke- and TGF-ß-Mediated CFTR Expression.

Transforming growth factor ß (TGF-ß), signaling induced by cigarette smoke (CS), plays an important role in the progression of airway diseases, like chronic bronchitis associated with chronic obstructive pulmonary disease (COPD), and in smokers. Chronic bronchitis is characterized by reduced mucociliary clearance (MCC). Cystic fibrosis transmembrane conductance regulator (CFTR) plays an important role in normal MCC. TGF-ß and CS (via TGF-ß) promote acquired CFTR dysfunction by suppressing CFTR biogenesis and function. Understanding the mechanism by which CS promotes CFTR dysfunction can identify therapeutic leads to reverse CFTR suppression and rescue MCC. TGF-ß alters the microRNAome of primary human bronchial epithelium. TGF-ß and CS upregulate miR-145-5p expression to suppress CFTR and the CFTR modifier, SLC26A9. miR-145-5p upregulation with a concomitant CFTR and SLC26A9 suppression was validated in CS-exposed mouse models. While miR-145-5p antagonism rescued the effects of TGF-ß in bronchial epithelial cells following transfection, an aptamer to block TGF-ß signaling rescues CS- and TGF-ß-mediated suppression of CFTR biogenesis and function in the absence of any transfection reagent. These results demonstrate that miR-145-5p plays a significant role in acquired CFTR dysfunction by CS, and they validate a clinically feasible strategy for delivery by inhalation to locally modulate TGF-ß signaling in the airway and rescue CFTR biogenesis and function.

Role of Non-Coding RNAs in Lung Circadian Clock Related Diseases.

Circadian oscillations are regulated at both central and peripheral levels to maintain physiological homeostasis. The central circadian clock consists of a central pacemaker in the suprachiasmatic nucleus that is entrained by light dark cycles and this, in turn, synchronizes the peripheral clock inherent in other organs. Circadian dysregulation has been attributed to dysregulation of peripheral clock and also associated with several diseases. Components of the molecular clock are disrupted in lung diseases like chronic obstructive pulmonary disease (COPD), asthma and IPF. Airway epithelial cells play an important role in temporally organizing magnitude of immune response, DNA damage response and acute airway inflammation. Non-coding RNAs play an important role in regulation of molecular clock and in turn are also regulated by clock components. Dysregulation of these non-coding RNAs have been shown to impact the expression of core clock genes as well as clock output genes in many organs. However, no studies have currently looked at the potential impact of these non-coding RNAs on lung molecular clock. This review focuses on the ways how these non-coding RNAs regulate and in turn are regulated by the lung molecular clock and its potential impact on lung diseases.

Aptamers in HIV research diagnosis and therapy.

Aptamers are high affinity single-stranded nucleic acid or protein ligands which exhibit specificity and avidity comparable to, or exceeding that of antibodies and can be generated against most targets. The functionality of aptamers is based on their unique tertiary structure, complexity and their ability to attain unique binding pockets by folding. Aptamers are selected in vitro by a process called Systematic Evolution of Ligands by Exponential enrichment (SELEX). The Kd values for the selected aptamer are often in the picomolar to low nanomolar range. Stable and nontoxic aptamers could be selected for a wide range of ligands including small molecules to large proteins. Aptamers have shown tremendous potential and have found multipurpose application in the field of therapeutic, diagnostic, biosensor and bio-imaging. While their mechanism of action can be similar to that of monoclonal antibodies, aptamers provide additional advantages in terms of production cost, simpler regulatory approval and lower immunogenicity as they are synthesized chemically. Human immunodeficiency virus (HIV) is the primary cause of acquired immune deficiency syndrome (AIDS), which causes significant morbidity and mortality with a significant consequent decrease in the quality of patient's lives. While cART has led to good viral control, people living with HIV now suffer from non-HIV comorbidities due to viral protein expression that cannot be controlled by cART. Hence pathophysiological mechanisms that govern these comorbidities with a focus on therapies that neutralize these HIV effects gained increased attention. Recent advances in HIV/AIDS research have identified several molecular targets and for the development of therapeutic and diagnostic using aptamers against HIV/AIDS. This review presents recent advances in aptamers technology for potential application in HIV diagnostics and therapeutics towards improving the quality of life of people living with HIV.

Soluble adenylyl cyclase mediates hydrogen peroxide-induced changes in epithelial barrier function.

BACKGROUND: Elevated H2O2 levels are associated with inflammatory diseases and H2O2 exposure is known to disrupt epithelial barrier function, leading to increased permeability and decreased electrical resistance. In normal human bronchial epithelial (NHBE) cells, fully differentiated at the air liquid interface (ALI), H2O2 activates an autocrine prostaglandin pathway that stimulates transmembrane adenylyl cyclase (tmAC) as well as soluble adenylyl cyclase (sAC), but the role of this autocrine pathway in H2O2-mediated barrier disruption is not entirely clear. METHODS: To further characterize the mechanism of H2O2-induced barrier disruption, NHBE cultures were treated with H2O2 and evaluated for changes in transepithelial resistance and mannitol permeability using agonist and inhibitors to dissect the pathway. RESULTS: A short (<10 min) H2O2 treatment was sufficient to induce resistance and permeability changes that occurred 40 min to 1 h later and the changes were partially sensitive to EP1 but not EP4 receptor antagonists. EP1 receptors were localized to the apical compartment of NHBE. Resistance and permeability changes were sensitive to inhibition of sAC but not tmAC and were partially blocked by PKA inhibition. Pretreatment with a PLC inhibitor or an IP3 receptor antagonist reduced changes in resistance and permeability suggesting activation of sAC occurred through increased intracellular calcium. CONCLUSION: The data support an important role for prostaglandin activation of sAC and PKA in H2O2-induced barrier disruption.

Transforming growth factor-ß1 and cigarette smoke inhibit the ability of ß2-agonists to enhance epithelial permeability.

Chronic bronchitis, caused by cigarette smoke exposure, is characterized by mucus hypersecretion and reduced mucociliary clearance (MCC). Effective MCC depends, in part, on adequate airway surface liquid. Cystic fibrosis transmembrane conductance regulator (CFTR) provides the necessary osmotic gradient for serosal to mucosal fluid transport through its ability to both secrete Cl(-) and regulate paracellular permeability, but CFTR activity is attenuated in chronic bronchitis and in smokers. ß2-adrenergic receptor (ß2-AR) agonists are widely used for managing chronic obstructive pulmonary disease, and can activate CFTR, stimulate ciliary beat frequency, and increase epithelial permeability, thereby stimulating MCC. Patients with chronic airway diseases and cigarette smokers demonstrate increased transforming growth factor (TGF)-ß1 signaling, which suppresses ß2-agonist-mediated CFTR activation and epithelial permeability increases. Restoring CFTR function in these diseases can restore the ability of ß2-agonists to enhance epithelial permeability. Human bronchial epithelial cells, fully redifferentiated at the air-liquid interface, were used for (14)C mannitol flux measurements, Ussing chamber experiments, and quantitative RT-PCR. ß2-agonists enhance epithelial permeability by activating CFTR via the ß2-AR/adenylyl cyclase/cAMP/protein kinase A pathway. TGF-ß1 inhibits ß2-agonist-mediated CFTR activation and epithelial permeability enhancement. Although TGF-ß1 down-regulates both ß2-AR and CFTR mRNA, functionally it only decreases CFTR activity. Cigarette smoke exposure inhibits ß2-agonist-mediated epithelial permeability increases, an effect reversed by blocking TGF-ß signaling. ß2-agonists enhance epithelial permeability via CFTR activation. TGF-ß1 signaling inhibits ß2-agonist-mediated CFTR activation and subsequent increased epithelial permeability, potentially limiting the ability of ß2-agonists to facilitate paracellular transport in disease states unless TGF-ß1 signaling is inhibited.

Nanomedicine for the Treatment of Viral Diseases: Smaller Solution to Bigger Problems.

The continuous evolution of new viruses poses a danger to world health. Rampant outbreaks may advance to pandemic level, often straining financial and medical resources to breaking point. While vaccination remains the gold standard to prevent viral illnesses, these are mostly prophylactic and offer minimal assistance to those who have already developed viral illnesses. Moreover, the timeline to vaccine development and testing can be extensive, leading to a lapse in controlling the spread of viral infection during pandemics. Antiviral therapeutics can provide a temporary fix to tide over the time lag when vaccines are not available during the commencement of a disease outburst. At times, these medications can have negative side effects that outweigh the benefits, and they are not always effective against newly emerging virus strains. Several limitations with conventional antiviral therapies may be addressed by nanotechnology. By using nano delivery vehicles, for instance, the pharmacokinetic profile of antiviral medications can be significantly improved while decreasing systemic toxicity. The virucidal or virus-neutralizing qualities of other special nanomaterials can be exploited. This review focuses on the recent advancements in nanomedicine against RNA viruses, including nano-vaccines and nano-herbal therapeutics.

Emerging roles of senolytics/senomorphics in HIV-related co-morbidities.

Human immunodeficiency virus (HIV) is known to cause cellular senescence and inflammation among infected individuals. While the traditional antiretroviral therapies (ART) have allowed the once fatal infection to be managed effectively, the quality of life of HIV patients on prolonged ART use is still inferior. Most of these individuals suffer from life-threatening comorbidities like chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), and diabetes, to name a few. Interestingly, cellular senescence is known to play a critical role in the pathophysiology of these comorbidities as well. It is therefore important to understand the role of cellular senescence in the disease progression and co-morbidity development in HIV-infected individuals. In this respect, use of senolytic/senomorphic drugs as combination therapy with ART would be beneficial for HIV patients. This review provides a critical analysis of the current literature to determine the potential and efficacy of using senolytics/senotherapeutics in managing HIV infection, latency, and associated co-morbidities in humans. The various classes of senolytics have been studied in detail to focus on their potential to combat against HIV infections and associated pathologies with advancing age.

Inflammatory lung injury is associated with endothelial cell mitochondrial fission and requires the nitration of RhoA and cytoskeletal remodeling.

Higher levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a TLR4 agonist, are associated with poor clinical outcomes in sepsis-induced acute lung injury (ALI). Little is known regarding the mechanisms by which eNAMPT is involved in ALI. Our recent work has identified a crucial role for mitochondrial dysfunction in ALI. Thus, this study aimed to determine if eNAMPT-mediated inflammatory injury is associated with the loss of mitochondrial function. Our data show that eNAMPT disrupted mitochondrial bioenergetics. This was associated with cytoskeleton remodeling and the loss of endothelial barrier integrity. These changes were associated with enhanced mitochondrial fission and blocked when Rho-kinase (ROCK) was inhibited. The increases in mitochondrial fission were also associated with the nitration-mediated activation of the small GTPase activator of ROCK, RhoA. Blocking RhoA nitration decreased eNAMPT-mediated mitochondrial fission and endothelial barrier dysfunction. The increase in fission was linked to a RhoA-ROCK mediated increase in Drp1 (dynamin-related protein 1) at serine(S)616. Another TLR4 agonist, lipopolysaccharide (LPS), also increased mitochondrial fission in a Drp1 and RhoA-ROCK-dependent manner. To validate our findings in vivo, we challenged C57BL/6 mice with eNAMPT in the presence and absence of the Drp1 inhibitor, Mdivi-1. Mdivi-1 treatment protected against eNAMPT-induced lung inflammation, edema, and lung injury. These studies demonstrate that mitochondrial fission-dependent disruption of mitochondrial function is essential in TLR4-mediated inflammatory lung injury and identify a key role for RhoA-ROCK signaling. Reducing mitochondrial fission could be a potential therapeutic strategy to improve ARDS outcomes.

Metabolic reprogramming, oxidative stress, and pulmonary hypertension.

Mitochondria are highly dynamic organelles essential for cell metabolism, growth, and function. It is becoming increasingly clear that endothelial cell dysfunction significantly contributes to the pathogenesis and vascular remodeling of various lung diseases, including pulmonary arterial hypertension (PAH), and that mitochondria are at the center of this dysfunction. The more we uncover the role mitochondria play in pulmonary vascular disease, the more apparent it becomes that multiple pathways are involved. To achieve effective treatments, we must understand how these pathways are dysregulated to be able to intervene therapeutically. We know that nitric oxide signaling, glucose metabolism, fatty acid oxidation, and the TCA cycle are abnormal in PAH, along with alterations in the mitochondrial membrane potential, proliferation, and apoptosis. However, these pathways are incompletely characterized in PAH, especially in endothelial cells, highlighting the urgent need for further research. This review summarizes what is currently known about how mitochondrial metabolism facilitates a metabolic shift in endothelial cells that induces vascular remodeling during PAH.

A conditional RNA Pol II mono-promoter drives HIV-inducible, CRISPR-mediated cyclin T1 suppression and HIV inhibition.

Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) targeted to HIV proviral DNA has shown excision of HIV from infected cells. However, CRISPR-based HIV excision is vulnerable to viral escape. Targeting cellular co-factors provides an attractive yet risky alternative to render viral escape irrelevant. Cyclin T1 is a critical modulator of HIV transcription and mediates recruitment of positive transcription elongation factor-b (P-TEFb) kinase for transcriptional elongation. Hence, a CRISPR-mediated cyclin T1 inactivation will silence HIV transcription, locking it in an inactive form in the cell and thereby serving as an effective antiviral and possibly effecting a functional cure. However, cellular genes play important roles, and their uncontrolled inhibition can promote undesirable effects. Here, we demonstrate a conditional inducible RNA polymerase II (RNA Pol II) mono-promoter-based co-expression of a CRISPR system targeting cyclin T1 from a single transcription unit. Co-expression of guide RNA (gRNA) and CRISPR-associated protein (Cas9) is observed only in HIV-infected cells and leads to sustained HIV suppression in stringent chronically infected cell lines as well as in T cell lines. We further show that incorporation of cis-acting ribozymes immediately upstream of the gRNA further enhances HIV silencing.

Albuterol modulates its own transepithelial flux via changes in paracellular permeability.

Although inhaled bronchodilators are commonly used in the treatment of airway disease to dilate airway smooth muscle, little is known regarding the mechanisms that regulate albuterol movement across the epithelium to reach its target, the airway smooth muscle. Because the rate of onset depends on the transepithelial transport of albuterol, to determine the mechanisms that regulate the transepithelial movement of albuterol is essential. Human bronchial epithelial cells, fully redifferentiated in culture at the air-liquid interface, were used to study the cellular uptake and total transepithelial flux of (3)H-albuterol from the apical to the basolateral surfaces. (3)H-mannitol and transepithelial electrical resistance were used to quantify changes in paracellular permeability. The majority of albuterol flux across the epithelium occurred via the paracellular route. The cellular uptake of albuterol was found to be saturable, whereas transepithelial flux was not. Cellular uptake could be inhibited by the amino acids lysine and histidine, with no effect on net transepithelial flux. Transepithelial flux was altered by maneuvers that collapsed or disrupted intercellular junctions. Acidification, usually seen in exacerbations of airway disease, decreased albuterol flux. In addition, albuterol increased its own paracellular permeability. The ability of albuterol to modulate paracellular permeability was blocked by the ß(2)-adrenergic receptor-selective antagonist ICI 118551. Albuterol mainly crosses the epithelium via the paracellular pathway, but has the ability to modulate its own permeability through changes in the leakiness of tight junctions, which is modulated through the signaling of the ß(2)-adrenergic receptor.

Dysregulation of mitochondrial complexes and dynamics by chronic cigarette smoke exposure Utilizing MitoQC reporter mice.

Cigarette smoke (CS) is known to cause impaired mitophagy and mitochondrial dysregulation in the pathogenesis of chronic obstructive pulmonary disease (COPD)/emphysema. Mitochondrial complexes and dynamics are affected by acute CS exposure in lung epithelium and mouse lung. We hypothesize that chronic CS exposure (4 months) will induce lung mitochondrial dysregulation and abnormal mitophagy. In this study, we employed the mitoQC reporter mice, a mitochondrial reporter strain, which can reflect the mitophagy based on the fluorescence-tagged mitochondria. Chronic CS exposure induced lung inflammatory cell infiltration, airspace enlargement, and lung cellular senescence. We showed the higher occurrence of mitophagy (GFP/mCherry) in the lung cells by CS exposure, associated with more mitochondrial fluorescence signals (GFP+/mCherry+). After chronic CS exposure, the mitochondrial complexes and function related genes were inhibited, while protein levels of complexes I and III slightly changed. Additionally, chronic CS exposure down-regulated most of the mitochondrial dynamic markers at gene expression level, included mitochondrial fusion/fission and mitochondrial translocate/transfer markers. For the markers related to mitophagy, Pink1 and Parkin, decreased gene and protein levels of Parkin, and decreased gene expression of Pink1, were identified in the CS exposure group. Hence, CS-induced mitophagy is mediated by Pink1-Parkin independent mechanism. Thus, we have shown that the chronic CS epxosure dysregulated mitochondrial complexes and dynamics and induced mitophagy, using the state-of-the art mitoQC reporter mouse model. Our results suggested that dysregulated mitochondrial function and dynamics are associated with CS-induced lung injury and phenotypic development of chronic lung diseases, such as COPD/ emphysema.

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.

Cellular stress responses and dysfunctional Mitochondrial-cellular senescence, and therapeutics in chronic respiratory diseases.

The abnormal inflammatory responses due to the lung tissue damage and ineffective repair/resolution in response to the inhaled toxicants result in the pathological changes associated with chronic respiratory diseases. Investigation of such pathophysiological mechanisms provides the opportunity to develop the molecular phenotype-specific diagnostic assays and could help in designing the personalized medicine-based therapeutic approaches against these prevalent diseases. As the central hubs of cell metabolism and energetics, mitochondria integrate cellular responses and interorganellar signaling pathways to maintain cellular and extracellular redox status and the cellular senescence that dictate the lung tissue responses. Specifically, as observed in chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis, the mitochondria-endoplasmic reticulum (ER) crosstalk is disrupted by the inhaled toxicants such as the combustible and emerging electronic nicotine-delivery system (ENDS) tobacco products. Thus, the recent research efforts have focused on understanding how the mitochondria-ER dysfunctions and oxidative stress responses can be targeted to improve inflammatory and cellular dysfunctions associated with these pathologic illnesses that are exacerbated by viral infections. The present review assesses the importance of these redox signaling and cellular senescence pathways that describe the role of mitochondria and ER on the development and function of lung epithelial responses, highlighting the cause and effect associations that reflect the disease pathogenesis and possible intervention strategies.

Use of a U16 snoRNA-containing ribozyme library to identify ribozyme targets in HIV-1.

Hammerhead ribozymes have been shown to silence human immunodeficiency virus-1 (HIV-1) gene expression by site-specific cleavage of viral mRNA. The two major factors that determine whether ribozymes will be effective for post-transcriptional gene silencing are colocalization of the ribozyme and the target RNAs, and the choice of an appropriate target site on the mRNA. An effective screening strategy for potential targets on the viral genome is the use of ribozyme libraries in cell culture. Capitalizing on previous findings that HIV-1 and ribozymes can be colocalized in the nucleolus, we created a novel hammerhead ribozyme library by inserting hammerhead ribozymes with fully randomized stems 1 and 2 into the body of the U16 small nucleolar RNA (snoRNA). Following three rounds of cotransfection with an HIV-1 proviral DNA harboring the herpes simplex virus thymidine kinase (HSV-TK) gene, we selected for gancyclovir-resistant cells and identified a ribozyme sequence that could potentially target both the U5 and gag genes of HIV-1 regions on the HIV-1 genome through partial homologies with these targets. When the ribozymes were converted to full complementarity with the targets, they provided potent inhibition of HIV-1 replication in cell culture. These results provide a novel approach for identifying ribozyme targets in HIV-1.

Aberrant MicroRNAomics in Pulmonary Complications: Implications in Lung Health and Diseases.

Over the last few decades, evolutionarily conserved molecular networks have emerged as important regulators in the expression and function of eukaryotic genomes. Recently, miRNAs (miRNAs), a large family of small, non-coding regulatory RNAs were identified in these networks as regulators of endogenous genes by exerting post-transcriptional gene regulation activity in a broad range of eukaryotic species. Dysregulation of miRNA expression correlates with aberrant gene expression and can play an essential role in human health and disease. In the context of the lung, miRNAs have been implicated in organogenesis programming, such as proliferation, differentiation, and morphogenesis. Gain- or loss-of-function studies revealed their pivotal roles as regulators of disease development, potential therapeutic candidates/targets, and clinical biomarkers. An altered microRNAome has been attributed to several pulmonary diseases, such as asthma, chronic pulmonary obstructive disease, cystic fibrosis, lung cancer, and idiopathic pulmonary fibrosis. Considering the relevant roles and functions of miRNAs under physiological and pathological conditions, they may lead to the invention of new diagnostic and therapeutic tools. This review will focus on recent advances in understanding the role of miRNAs in lung development, lung health, and diseases, while also exploring the progress and prospects of their application as therapeutic leads or as biomarkers.

A guanylurea-functionalized conjugated polymer enables RNA interference in ex vivo human airway epithelium.

We demonstrate a successful target gene knockdown in ex vivo normal human bronchial epithelium (NHBE) cells covered with mucus layers using the guanylurea functionalization technique modulating the chemical environment at the positive charge of a gene carrier.

Potent knock down of HIV-1 replication by targeting HIV-1 Tat/Rev RNA sequences synergistically with catalytic RNA and DNA.

OBJECTIVE: Ribozymes (Rzs) and DNA-enzymes (Dzs) possess the ability to prevent gene expression by cleaving target RNA in a catalytic and sequence-specific manner. Although Rzs or Dzs have been used earlier for HIV-1 gene suppression, the present study explored the possibility of using catalytic RNA and DNA simultaneously in a synergistic manner with the hope that this novel approach will allow more potent inhibition for a longer duration. METHODS: In order to achieve long-term inhibition of HIV-1 replication, a novel non-GUX hammerhead Rz was designed by standard recombinant DNA technology and cloned it under the powerful CMV promoter containing expression vector. A 10-23 catalytic motif containing Dz that was targeted against the conserved second exon of HIV-1 Tat/Rev region was also assembled. RESULTS: Both Rz and Dz possessed sequence-specific cleavage activities individually and simultaneously cleaved target RNA in a synergistic manner under the same in vitro cleavage conditions. These catalytic molecules inhibited HIV-1 replication in macrophages individually and exhibited potent inhibitory effects when used in combination. CONCLUSIONS: The combination strategy described here can be widely used against any target RNA to achieve more effective gene inhibition that exploits the simultaneous sequence-specific cleavage potentials of catalytic RNA and DNA.