Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.

AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO Malignancies of the liver, including hepatocelluar carcinoma and metastatic tumors of different origin, represent a high unmet medical need. Current therapeutic strategies frequently comprise inhibiting more than one molecular pathway contributing to tumor growth and maintenance. We are developing ALN-VSP, an RNAi therapeutic for liver malignancies comprised of lipid particle-formulated small interfering RNAs (siRNAs) targeting VEGF and the mitotic kinesin, KSP (Eg5, Kif11). For each target, potent and specific siRNA duplexes have been identified following extensive screening in cell culture. Single dose screens were conducted initially; then, a subset of siRNAs was evaluated for dose-response and lead siRNAs selected based on potency in vitro . Moreover, silencing of KSP in vitro leads to cell death in multiple tumor cell lines, while silencing of VEGF leads to inhibition of VEGF secretion into cell culture medium. To achieve hepatic delivery, lipid particle formulations have been evaluated and shown to achieve silencing of hepatic gene expression with multiple siRNAs directed against distinct targets, including VEGF and KSP. To evaluate the potential of this approach for treating liver cancer, pre-clinical mouse orthotopic liver tumor model studies have been conducted with ALN-VSP. These tumor models comprised direct injection of human hepatoma cells (Hep3B) into the liver of scid/beige mice, which resulted in tumor growth as well as alpha-fetoprotein secretion. In these studies, ALN-VSP demonstrated dose-dependent mRNA suppression, mRNA cleavage consistent with an RNAi mechanism, monoaster formation consistent with KSP inhibition, inhibition of tumor growth and reduction of circulating AFP, and prolongation of survival. In addition, extension of lifespan with ALN-VSP was superior to the approved multi-kinase inhibitor, Sorafenib, in preliminary studies using this animal model. These studies indicate the potential therapeutic benefit of the RNAi therapeutic ALN-VSP targeting VEGF and KSP for the treatment of liver cancer. An IND application for ALN-VSP was submitted in 2008 and received FDA clearance in January. The Phase I clinical trial of ALN-VSP in patients will be initiated in the first half of 2009. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr DDT01-2.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5'-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia.

L'invention porte sur un acide ribonucleique double brin (ARNdb) pour traiter une infection par le virus du papillome humain (HPV). L'ARNdb comprend un brin antisens ayant une sequence nucleotidique qui a une longueur inferieure a 30 nucleotides, d'une maniere generale une longueur de 19-25 nucleotides, et qui est sensiblement complementaire d'au moins une partie d'un gene cible du HPC selectionne a partir du gene E6AP humain. L'invention porte egalement sur une composition pharmaceutique comprenant l'ARNdb conjointement avec un support pharmaceutiquement acceptable ; sur des procedes de traitement de maladies provoquees par une infection par le HPV et l'expression du gene E6AP a l'aide de la composition pharmaceutique ; et sur des procedes pour inhiber l'expression de genes cibles du HPV dans une cellule.

Ribonucleic acid double stranded (dsRNA) for inhibiting expression of a human PCSK9 gene in a cell, such dsRNA at least two sequences that are complementary to each other and wherein a sense strand comprises a first sequence and an antisense strand comprising comprises a second sequence comprising a region of complementarity which is fully complementary to at least a part of a mRNA encoding PCSK9, and wherein said region of complementarity is less than 30 nucleotides in length and inhibiting said dsRNA, upon contact with a cell expressing said PCSK9 expression of said PCSK9 gene, wherein: (a) said first sequence is the sequence of SEQ ID NO: 1229 and said second sequence is the sequence of SEQ ID NO: 1230; or (b) said first sequence is the sequence of SEQ ID NO: 1227 and said second sequence is the sequence of SEQ ID NO: 1228.

Delivery of small interfering RNAs (siRNAs) in vivo , using clinically relevant modes of administration, is critical for the advancement of RNA interference (RNAi) therapeutics. In this work, we demonstrate systemic delivery of siRNAs and potent in vivo down-modulation of two important disease targets, apolipoprotein B (apoB) and proprotein convertase subtilisin kexin 9 (PCSK9). A single injection of liposomal siRNA resulted in >90% silencing of apoB mRNA expression in the liver 48 h after administration. The effect was demonstrated to occur through cleavage of the apoB mRNA at precisely the site predicted for the RNAi mechanism. Reductions in apoB protein, cholesterol, and low-density lipoprotein (LDLc) levels were observed in 48 hours that lasted for at least 23 days, thus demonstrating an immediate, potent and durable biological effect. In addition to apoB we have also demonstrated the ability to down-modulate other important liver targets such as PCSK9. PCSK9 has been closely implicated in LDLc regulation. We have demonstrated PCSK9 down-modulation in several animal models including, mouse, humanized mouse, rat, and non-human primate. Down-modulation of PCSK9 levels resulted in significant lowering of cholesterol (20 – 60%) in all animal models tested. These findings strongly support the potential of RNAi therapeutics as a new class of drug for metabolic and cardiovascular diseases. Our next steps include selecting the most potent lead molecule and moving it into GLP safety studies.