Martin C. Woodle

Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque

Development of therapeutic agents for severe acute respiratory syndrome (SARS) viral infection using short interfering RNA (siRNA) inhibitors exemplifies a powerful new means to combat emerging infectious diseases. Potent siRNA inhibitors of SARS coronavirus (SCV) in vitro were further evaluated for efficacy and safety in a rhesus macaque (Macaca mulatta) SARS model using clinically viable delivery while comparing three dosing regimens. Observations of SARS-like symptoms, measurements of SCV RNA presence and lung histopathology and immunohistochemistry consistently showed siRNA-mediated anti-SARS efficacy by either prophylactic or therapeutic regimens. The siRNAs used provided relief from SCV infection–induced fever, diminished SCV viral levels and reduced acute diffuse alveoli damage. The 10–40 mg/kg accumulated dosages of siRNA did not show any sign of siRNA-induced toxicity. These results suggest that a clinical investigation is warranted and illustrate the prospects for siRNA to enable a massive reduction in development time for new targeted therapeutic agents.

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.

Harnessing in vivo siRNA delivery for drug discovery and therapeutic development

The use of RNA interference (RNAi) is spreading rapidly to nearly every aspect of biomedical research. The gene silencing capability of RNAi is being used to study individual genes biological function and role in biochemical pathways. However, the efficacy of RNAi depends upon efficient delivery of the intermediates of RNAi, short interfering RNA (siRNA) and short hairpin RNA (shRNA) oligonucleotides. The delivery challenge is even greater when the aim is to inhibit the expression of target genes in animal models. Although i n vivo delivery of siRNA is complicated and challenging, recent results are encouraging. In this review, the latest developments of in vivo delivery of siRNA and the crucial issues related to this effort are addressed.

In Vivo Application of RNA Interference: From Functional Genomics to Therapeutics

Abstract RNAi has rapidly become a powerful tool for drug target discovery and validation in cell culture, and now has largely displaced efforts with antisense and ribozymes. Consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Studies on RNAi have resulted in two basic methods for its use for gene selective inhibition: 1) cytoplasmic delivery of short dsRNA oligonucleotides (siRNA), which mimics an active intermediate of an endogenous RNAi mechanism and 2) nuclear delivery of gene expression cassettes that express a short hairpin RNA (shRNA), which mimics the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism. Non‐viral gene delivery systems are a diverse collection of technologies that are applicable to both of these forms of RNAi. Importantly, unlike antisense and ribozyme systems, a remarkable trait of siRNA is a lack of dependence on chemical modifications blocking enzymatic degradation, although chemical protection methods developed for the earlier systems are being incorporated into siRNA and are generally compatible with non‐viral delivery systems. The use of siRNA is emerging more rapidly than for shRNA, in part due to the increased effort required to construct shRNA expression systems before selection of active sequences and verification of biological activity are obtained. In contrast, screens of many siRNA sequences can be accomplished rapidly using synthetic oligos. It is not surprising that the use of siRNA in vivo is also emerging first. Initial in vivo studies have been reported for both viral and non‐viral delivery but viral delivery is limited to shRNA. This review describes the emerging in vivo application of non‐viral delivery systems for RNAi for functional genomics, which will provide a foundation for further development of RNAi therapeutics. Of interest is the rapid adaptation of ligand‐targeted plasmid‐based nanoparticles for RNAi agents. These systems are growing in capabilities and beginning to pose a serious rival to viral vector based gene delivery. The activity of siRNA in the cytoplasm may lower the hurdle and thereby accelerate the successful development of therapeutics based on targeted non‐viral delivery systems.

Long circulating, cationic liposomes containing amino‐PEG‐phosphatidylethanolamine

Ligand attachment to polyethylene glycol (PEG) grafted, long circulating liposomes at the polymer terminus is of interest for targeting but the effect of positively charged groups is unknown. Amino‐polyethylene glycol‐phosphatidylethanolamine (AminoPEG‐PE), prepared in four steps from α‐amino‐ω‐hydroxy‐PEG, was tested for influence on liposome interactions in vivo: blood circulation and biodistribution. Despite surface amines on each liposome conferring cationic behavior, in vivo properties are comparable to those obtained with methoxy‐PEG‐PE. The consequences are profound for targeting and possibly systemic delivery of cationic lipidic‐polynucleotide complexes.

Sterically Stabilized Liposomes: Physical and Biological Properties

Advanced liposomal therapeutics has been attained by liposome surface modification, initially with specific glycolipids and subsequently with surface-grafted PEG, reducing in vivo rapid recognition and uptake, giving prolonged blood circulation, and providing selective localization in tumors and other pathological sites, as described in recent reviews. The result is improved efficacy of encapsulated agents. The surface PEG may produce a steric barrier, as described for colloids. Reduced in vivo uptake may result from inhibition of plasma-protein adsorption, or opsonization, by the steric coating. Several physical studies support this mechanism, including electrophoretic mobility (zeta potential). Our previous results for 2000-dalton PEG indicated a coating thickness about 5 nm, in agreement with independent measurements. We report here results for 750 to 5000-dalton PEGs. The calculated coating thickness increases with molecular weight in a nonlinear fashion. The dependence of blood circulation and tissue distribution on PEG molecular weight correlates with zeta-potential estimates of PEG-coating thickness. Effects on tissue distribution are reported for liver and spleen, the major phagocytic organs. The biological properties of these liposomes depend on the surface polymer rather than the lipid bilayer, yielding important advantages for lipid-mediated control of drug interaction and release without affecting the biodistribution.

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.

Prolonged systemic delivery of peptide drugs by long-circulating liposomes : illustration with vasopressin in the brattleboro rat

The value of novel systemically long-circulating liposomes to prolong the duration of an antidiuretic hormone, arg8-vasopressin (VP), was investigated as a representative of low molecular weight pep-tides with rapid clearance. Cholesterol content was found to have a controlling effect on VP release in serum. Three types of liposomes were selected for urine production measurements in VP deficient Brattleboro rats. One contained phosphatidylserine (PS), which was rapidly cleared from the circulation. In the other two liposomes, the PS component was replaced by either phosphatidylglycerol or a novel phospholipid derivatized with polyethylene glycol (PEG); both showing prolonged circulation. Free VP (up to 8 µg/kg) gave reduced urine production for less than 24 hr. The PG formulation exhibited a dose-dependent prolonged duration of bioactivity of up to 4 days. Substitution of PEG-PE resulted in a 2-day delay followed by a prolonged duration of bioactivity for over 4 days. The duration of the prolonged bioactivity was not dose dependent but the amplitude was. This is attributed to VP release from liposomes which have distributed intact to another compartment without having been taken up by the RES. By balancing liposome circulation time, release rate, and dose, long-circulating liposomes can be applied to prolong the biological activity of a therapeutic peptide.

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.

Modulation of angiogenesis with siRNA inhibitors for novel therapeutics

Cancer and many other serious diseases are characterized by the uncontrolled growth of new blood vessels. Recently, RNA interference (RNAi) has reinvigorated the therapeutic prospects for inhibiting gene expression and promises many advantages over binding inhibitors, including high specificity, which is essential for targeted therapeutics. This article describes the latest developments using small-interfering RNA (siRNA) inhibitors to downregulate various angiogenic and tumor-associated factors, both in cell-culture assays and in animal disease models. The majority of research efforts are currently focused on understanding gene function, as well as proof-of-concept for siRNA-mediated anti-angiogenesis. The prospects for siRNA therapeutics, both advantages and looming hurdles, are evaluated.