Ibrahim F. Benter

Nonviral delivery of synthetic siRNAs in vivo

Sequence-specific gene silencing using small interfering RNA (siRNA) is a Nobel prize-winning technology that is now being evaluated in clinical trials as a potentially novel therapeutic strategy. This article provides an overview of the major pharmaceutical challenges facing siRNA therapeutics, focusing on the delivery strategies for synthetic siRNA duplexes in vivo, as this remains one of the most important issues to be resolved. This article also highlights the importance of understanding the genocompatibility/toxicogenomics of siRNA delivery reagents in terms of their impact on gene-silencing activity and specificity. Collectively, this information is essential for the selection of optimally acting siRNA delivery system combinations for the many proposed applications of RNA interference.

Angiotensin-(1-7) prevents activation of NADPH oxidase and renal vascular dysfunction in diabetic hypertensive rats.

Background/Aim: We examined the influence of chronic treatment with angiotensin-(1–7) [Ang-(1–7)] on renox (renal NADPH oxidase, NOX-4) and the development of renal dysfunction in streptozotocin-treated spontaneously hypertensive rats (diabetic SHR). Methods:Mean arterial pressure, urinary protein and vascular responsiveness of the isolated renal artery to vasoactive agonists were studied in vehicle- or Ang-(1–7)-treated SHR and diabetic SHR. Results: Ang-(1–7) decreased the elevated levels of renal NADPH oxidase (NOX) activity and attenuated the activation of NOX-4 gene expression in the diabetic SHR kidney. Ang-(1–7) treatment increased sodium excretion but did not affect mean arterial pressure in diabetic SHR. There was a significant increase in urinary protein (266 ± 22 mg/24 h) in the diabetic compared to control SHR (112 ± 13 mg/24 h) and treatment of diabetic SHR with Ang-(1–7) reduced the degree of proteinuria (185 ± 23 mg/24 h, p < 0.05). Ang-(1–7) treatment also attenuated the diabetes-induced increase in renal vascular responsiveness to endothelin-1, norepinephrine, and angiotensin II in SHR, but significantly increased the vasodilation of the renal artery of SHR and diabetic SHR to the vasodilator agonists. Conclusion: These results suggest that treatment with Ang-(1–7) constitutes a potential therapeutic strategy to alleviate NOX-mediated oxidative stress and to reduce renal dysfunction in diabetic hypertensive rats.

Sustained Polymeric Delivery of Gene Silencing Antisense ODNs, siRNA, DNAzymes and Ribozymes: In Vitro and In Vivo Studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (d,l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

Toxicogenomics of non-viral vectors for gene therapy: A microarray study of lipofectin- and oligofectamine-induced gene expression changes in human epithelial cells

Of the non-viral vectors, cationic lipid (CL) formulations are the most widely studied for the delivery of genes, antisense oligonucleotides and gene silencing nucleic acids such as small interfering RNAs. However, little is known about the impact of these delivery systems on global gene expression in target cells. In an attempt to study the geno-compatibility of CL formulations in target cells, we have used microarrays to examine the effect of Lipofectin and Oligofectamine on the gene expression profiles of human A431 epithelial cells. Using the manufacturers recommended CL concentrations routinely used for gene delivery, cDNA microarray expression profiling revealed marked changes in the expression of several genes for both Lipofectin- and Oligofectamine-treated cells. Data from the 200 spot arrays housing 160 different genes indicated that Lipofectin or Oligofectamine treatment of A431 cells resulted in more than 2-fold altered expression of 10 and 27 genes, respectively. The downstream functional consequences of CL-induced gene expression alterations led to an increased tendency of cells to enter early apoptosis as assessed by annexin V-FITC flow cytometry analyses. This effect was greater for Oligofectamine than Lipofectin. Observed gene expression changes were not sufficient to induce any significant DNA damage as assessed by single cell gel electrophoresis (COMET) assay. These data highlight the fact that inadvertent gene expression changes can be induced by the delivery formulation alone and that these may, ultimately, have important safety implications for the use of these non-viral vectors in gene-based therapies. Also, the induced non-target gene changes should be taken into consideration in gene therapy or gene silencing experiments using CL formulations where they may potentially mask or interfere with the desired genotype and/or phenotype end-points.

Toxicogenomics of drug delivery systems: Exploiting delivery system-induced changes in target gene expression to enhance siRNA activity

Synthetic siRNAs are typically formulated with drug delivery systems (DDS) that improve cellular uptake for optimal gene silencing activity. Here, we show that two PAMAM dendrimer DDS, differing only in their structural architecture, elicit many different gene expression changes in human cells including opposing effects on the expression of epidermal growth factor receptor (EGFR), a gene targeted for silencing by siRNA. Despite providing similar improvements in siRNA uptake, these two formulations led to a ∼10-fold variation in anti-EGFR siRNA activity. These data show that gene expression changes induced by DDS, separate from their ability to enhance cell uptake, determine ‘apparent’ siRNA potency and thus offer the possibility of tailoring delivery system-siRNA combinations for additive or synergistic effects on gene silencing.

Gene Silencing Nucleic Acids Designed by Scanning Arrays: Anti-EGFR Activity of siRNA, Ribozyme and DNA Enzymes Targeting a Single Hybridization-accessible Region using the Same Delivery System

Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.

Angiotensin‐(1–7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2‐ and NF‐κB‐dependent pathways

BACKGROUND AND PURPOSE Angiotensin‐(1–7) [Ang‐(1–7)] has anti‐inflammatory effects in models of cardiovascular disease and arthritis, but its effects in asthma are unknown. We investigated whether Ang‐(1–7) has anti‐inflammatory actions in a murine model of asthma.

Endogenous angiotensin-(1-7) reduces cardiac ischemia-induced dysfunction in diabetic hypertensive rats

Angiotensin-(1-7) [Ang-(1-7)] is a vasodilator peptide with cardiac and vascular protective properties. We examined the influence of Ang-(1-7), both endogenous and after chronic treatment with the peptide (576microg/(kgday)), on ischemia/reperfusion (I/R)-induced cardiac dysfunction in streptozotocin-treated spontaneously hypertensive rats (diabetic SHR). In isolated perfused hearts, recovery of left ventricular function from 40min of global ischemia was improved significantly in Ang-(1-7)- or captopril-treated diabetic SHR and worsened in animals treated with A779, an Ang-(1-7) receptor (AT((1-7))) antagonist. The beneficial effect of captopril on cardiac recovery was reduced when co-administered with A779. Cardiac NF-kappaB activity appears to be higher in diabetic SHR and treatment with Ang-(1-7) or captopril decreased NF-kappaB activity in diabetic SHR, an effect partially reversed by co-administration of A779. Real-time PCR-based gene array analysis of cardiac tissue revealed that Ang-(1-7) or captopril treatment may reduce expression of several genes of inflammation involved in the NF-kappaB signalling pathway. The data provide for the first time a role for endogenous Ang-(1-7) as well as confirmation that exogenous treatment with the peptide produces cardioprotection. Whether potential anti-inflammatory and transcriptional factor changes are directly linked to the cardioprotection produced by Ang-(1-7) in diabetic SHR remains to be determined.

Angiotensin-(1–7) prevents diabetes-induced attenuation in PPAR-γ and catalase activities

The mechanisms by which angiotensin-(1-7) [Ang-(1-7)] exerts its beneficial effects on end-organ damage associated with diabetes and hypertension are not well understood. The purpose of this study was A) to compare the effects of apocynin with Ang-(1-7) on renal vascular dysfunction and NADPH oxidase activity in a combined model of diabetes and hypertension and B) to further determine whether chronic treatment with Ang-(1-7) can modulate renal catalase, and peroxisome proliferator activated receptor- gamma (PPAR-gamma) levels in streptozotocin-induced diabetes in both normotensive Wistar Kyoto rats (WKY) and in spontaneously hypertensive rats (SHR). Apocynin or Ang-(1-7) treatment for one month starting at the onset of diabetes similarly attenuated elevation of renal NADPH oxidase activity in the diabetic SHR kidney and reduced the degree of proteinuria and hyperglycemia, but had little or modest effect on reducing mean arterial pressure. Both drugs also attenuated the diabetes-induced increase in renal vascular responsiveness to endothelin-1. Induction of diabetes in WKY and SHR animals resulted in significantly reduced renal catalase activity and in PPAR-gamma mRNA and protein levels. Treatment with Ang-(1-7) significantly prevented diabetes-induced reduction in catalase activity and the reduction in PPAR-gamma mRNA and protein levels in both animal models. Taken together, these data suggest that activation of Ang-(1-7)-mediated signaling could be an effective way to prevent the elevation of NADPH oxidase activity and inhibition of PPAR-gamma and catalase activities in diabetes and/or hypertension.

Activation of EGFR/ERBB2 via pathways involving ERK1/2, P38 MAPK, AKT and FOXO enhances recovery of diabetic hearts from ischemia-reperfusion injury.

This study characterized the effects of diabetes and/or ischemia on epidermal growth factor receptor, EGFR, and/or erbB2 signaling pathways on cardiac function. Isolated heart perfusion model of global ischemia was used to study the effect of chronic inhibition or acute activation of EGFR/erbB2 signaling on cardiac function in a rat model of type-1 diabetes. Induction of diabetes with streptozotocin impaired recovery of cardiac function (cardiac contractility and hemodynamics) following 40 minutes of global ischemia in isolated hearts. Chronic treatment with AG825 or AG1478, selective inhibitors of erbB2 and EGFR respectively, did not affect hyperglycemia but led to an exacerbation whereas acute administration of the EGFR ligand, epidermal growth factor (EGF), led to an improvement in cardiac recovery in diabetic hearts. Diabetes led to attenuated dimerization and phosphorylation of cardiac erbB2 and EGFR receptors that was associated with reduced signaling via extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 mitogen activated protein (MAP) kinase and AKT (protein kinase B). Ischemia was also associated with reduced cardiac signaling via these molecules whereas EGF-treatment opposed diabetes and/or ischemia induced changes in ERK1/2, p38 MAP kinase, and AKT-FOXO signaling. Losartan treatment improved cardiac function in diabetes but also impaired EGFR phosphorylation in diabetic heart. Co-administration of EGF rescued Losartan-mediated reduction in EGFR phosphorylation and significantly improved cardiac recovery more than with either agent alone. EGFR/erbB2 signaling is an important cardiac survival pathway whose activation, particularly in diabetes, ischemia or following treatment with drugs that inhibit this cascade, significantly improves cardiac function. These findings may have clinical relevance particularly in the treatment of diabetes-induced cardiac dysfunction.