Puthupparampil V. Scaria

Inhibition of Ocular Angiogenesis by siRNA Targeting Vascular Endothelial Growth Factor Pathway Genes : Therapeutic Strategy for Herpetic Stromal Keratitis

Ocular neovascularization often results in vision impairment. Frequently vascular endothelial cell growth factors (VEGFs) are mainly responsible for the pathological neovascularization as in the case in neovascularization induced by CpG oligodeoxynucleotides and herpes simplex virus infection in this report. siRNAs targeting either VEGFA, VEGFR1, VEGFR2, or a mix of the three were shown to significantly inhibit neovascularization induced by CpG when given locally or systemically. The efficacy of systemic administration was facilitated by the use of a polymer delivery vehicle. Additional experiments showed a significant inhibitory effect of the siRNAs mix when given either locally or systemically in vehicle against herpes simplex virus-induced angiogenesis as well as against lesions of stromal keratitis. These results indicate that the use of VEGF pathway-specific siRNAs represents a useful therapy against neovascularization-related eye diseases.

Pharmaceutical prospects for RNA interference

RNA interference has proven to be a powerful tool in gene function validation. Recently, the first studies were published reporting a disease-modulating activity of the technique, suggesting a promise for RNA interference as a novel therapeutic strategy. This review discusses the recent advancements in realizing the clinical utility of RNA-interference.

Sterically stabilized polyplex: ligand-mediated activity

Synthetic vectors have been considered as a safer and more versatile alternative to viral-based gene delivery systems. A variety of very simple synthetic vector systems, e.g., cationic lipid- and polymer-complexed plasmid DNA have activity in vivo but it appears to be mediated by non-specific electrostatic interactions limiting targeting. In order to avoid these problems, we designed a sterically stabilized layered colloidal system. The steric polymer coating reduces non-specific interactions. We have synthesized a PEG conjugate of PEI that complexes DNA to form small, stable colloids with a steric polymer coat on their surface. The polymer enhances colloidal stability and reduces non-specific binding and toxicity. It also renders the complex inactive presumably due to reduced binding. Ligands are then appended to the distal end of the steric polymer to restore cell binding and expression at target cells. We prepared conjugates with RGD peptide ligands appended to the distal end of the steric polymer. The resulting conjugates also form complexes but with ligands exposed on their surface restoring binding and activity. Labeled oligonucleotides and DNA were used to measure intracellular distribution. Oligonucleotides are found localized in the nucleus, whereas the labeled plasmid DNA remained in the cytoplasm. Import of plasmid DNA into the nucleus appears to be very inefficient yet sufficient for expression.

Cationic liposomes and nucleic acids

Abstract During the past decade, cationic lipids have emerged as the primary choice for gene delivery in vitro, i.e. transfection of cultured cells. A number of lipids with cationic head groups have been synthesized and evaluated. However, their success in vivo for gene therapy has been limited. To date, simple electrostatic complexes of cationic lipid mixtures with DNA have been hampered in numerous aspects: lack of colloidal stability, relatively low efficiency observed as expression levels or % of transfected cells, short duration of expression, and most importantly, non-specific interactions with many cells and tissues. Appreciation of the complexity of in vivo requirements, and especially opposing requirements for extra- and intracellular trafficking, is leading to engineered designs of gene delivery vectors containing cationic lipids. These designs attempt to assemble layered colloidal systems that accommodate the multiple functions required to traverse the various extra- and intracellular barriers. Successful development of such systems will depend on the ability to characterize and optimize each step rather than rely only on reporter gene expression, in addition to the obvious need to characterize the layered nature of the complexes. Importantly, many pharmacological aspects must be considered, especially control of the biodistribution and toxicity. Initial reports on such systems appear to provide at least a proof of the concept.

Polyethyleneimine (PEI) Mediated siRNA Gene Silencing in the Schistosoma mansoni Snail Host, Biomphalaria glabrata

An in vivo, non-invasive technique for gene silencing by RNA interference (RNAi) in the snail, Biomphalaria glabrata, has been developed using cationic polymer polyethyleneimine (PEI) mediated delivery of long double-stranded (ds) and small interfering (si) RNA. Cellular delivery was evaluated and optimized by using a ‘mock’ fluorescent siRNA. Subsequently, we used the method to suppress expression of Cathepsin B (CathB) with either the corresponding siRNA or dsRNA of this transcript. In addition, the knockdown of peroxiredoxin (Prx) at both RNA and protein levels was achieved with the PEI-mediated soaking method. B. glabrata is an important snail host for the transmission of the parasitic digenean platyhelminth, Schistosoma mansoni that causes schistosomiasis in the neotropics. Progress is being made to realize the genome sequence of the snail and to uncover gene expression profiles and cellular pathways that enable the snail to either prevent or sustain an infection. Using PEI complexes, a convenient soaking method has been developed, enabling functional gene knockdown studies with either dsRNA or siRNA. The protocol developed offers a first whole organism method for host-parasite gene function studies needed to identify key mechanisms required for parasite development in the snail host, which ultimately are needed as points for disrupting this parasite mediated disease.

Surface-modified HK:siRNA nanoplexes with enhanced pharmacokinetics and tumor growth inhibition.

We characterized in this study the pharmacokinetics and antitumor efficacy of histidine-lysine (HK):siRNA nanoplexes modified with PEG and a cyclic RGD (cRGD) ligand targeting αvβ3 and αvβ5 integrins. With noninvasive imaging, systemically administered surface-modified HK:siRNA nanoplexes showed nearly 4-fold greater blood levels, 40% higher accumulation in tumor tissue, and 60% lower luciferase activity than unmodified HK:siRNA nanoplexes. We then determined whether the surface-modified HK:siRNA nanoplex carrier was more effective in reducing MDA-MB-435 tumor growth with an siRNA targeting Raf-1. Repeated systemic administration of the selected surface modified HK:siRNA nanoplexes targeting Raf-1 showed 35% greater inhibition of tumor growth than unmodified HK:siRNA nanoplexes and 60% greater inhibition of tumor growth than untreated mice. The improved blood pharmacokinetic results and tumor localization observed with the integrin-targeting surface modification of HK:siRNA nanoplexes correlated with greater tumor growth inhibition. This investigation reveals that through control of targeting ligand surface display in association with a steric PEG layer, modified HK: siRNA nanoplexes show promise to advance RNAi therapeutics in oncology and potentially other critical diseases.

A branched histidine/lysine peptide, H2K4b, in complex with plasmids encoding antitumor proteins inhibits tumor xenografts

In this study we investigated whether a particular branched HK polymer, H2K4b, was an effective in vivo carrier of plasmids expressing the antiangiogenic kringle 1–5 or the tumor suppressor p53.

New cationic lipids form channel-like pores in phospholipid bilayers.

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.

Increased tumor distribution and expression of histidine-rich plasmid polyplexes

Selecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays.

Enhancement of antifungal activity by integrin-targeting of branched histidine rich peptides

Abstract The treatment of invasive candidiasis associated with growing numbers of immunocompromised patients remains a major challenge complicated by increasing drug resistance. A novel class of branched histidine-lysine (bHK) peptides has promising antifungal activity, and exhibits a mechanism similar to natural histatins, and thus may avoid drug resistance. The present studies evaluate ligand targeting of bHK peptides to fungal surface integrins by determining whether a cyclic RGD (cRGD) peptide with a large PEG linker could enhance bHK peptide antifungal activity. Whereas conjugates containing only the PEG linker reduced bHK peptide activity, conjugates with the cRGD-PEG ligand resulted in marked enhancement of activity against Candida albicans. This study provides the first demonstration of benefit from ligand targeting of antifungal agents to fungal surface receptors.