Hans-Peter Vornlocher

Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs

RNA interference (RNAi) holds considerable promise as a therapeutic approach to silence disease-causing genes, particularly those that encode so-called ‘non-druggable’ targets that are not amenable to conventional therapeutics such as small molecules, proteins, or monoclonal antibodies. The main obstacle to achieving in vivo gene silencing by RNAi technologies is delivery. Here we show that chemically modified short interfering RNAs (siRNAs) can silence an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chemically modified siRNAs resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. We also show that these siRNAs can silence human apoB in a transgenic mouse model. In our in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation, and we determined that cleavage of the apoB mRNA occurred specifically at the predicted site. These findings demonstrate the therapeutic potential of siRNAs for the treatment of disease.

RNAi-mediated gene silencing in non-human primates.

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg-1. A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.

Interfering with disease: a progress report on siRNA-based therapeutics

RNA interference (RNAi) quietly crept into biological research in the 1990s when unexpected gene-silencing phenomena in plants and flatworms first perplexed scientists. Following the demonstration of RNAi in mammalian cells in 2001, it was quickly realized that this highly specific mechanism of sequence-specific gene silencing might be harnessed to develop a new class of drugs that interfere with disease-causing or disease-promoting genes. Here we discuss the considerations that go into developing RNAi-based therapeutics starting from in vitro lead design and identification, to in vivo pre-clinical drug delivery and testing. We conclude by reviewing the latest clinical experience with RNAi therapeutics.

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2′-O-methyl (2′-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

Host Scavenger Receptor SR-BI Plays a Dual Role in the Establishment of Malaria Parasite Liver Infection

An obligatory step of malaria parasite infection is Plasmodium sporozoite invasion of host hepatocytes, and host lipoprotein clearance pathways have been linked to Plasmodium liver infection. By using RNA interference to screen lipoprotein-related host factors, we show here that the class B, type I scavenger receptor (SR-BI) is the strongest regulator of Plasmodium infection among these factors. Inhibition of SR-BI function reduced P. berghei infection in Huh7 cells, and overexpression of SR-BI led to increased infection. In vivo silencing of liver SR-BI expression in mice and inhibition of SR-BI activity in human primary hepatocytes reduced infection by P. berghei and by P. falciparum, respectively. Heterozygous SR-BI(+/-) mice displayed reduced P. berghei infection rates correlating with liver SR-BI expression levels. Additional analyses revealed that SR-BI plays a dual role in Plasmodium infection, affecting both sporozoite invasion and intracellular parasite development, and may therefore constitute a good target for malaria prophylaxis.

Heme Oxygenase-1 Is an Anti-Inflammatory Host Factor that Promotes Murine Plasmodium Liver Infection

The clinically silent Plasmodium liver stage is an obligatory step in the establishment of malaria infection and disease. We report here that expression of heme oxygenase-1 (HO-1, encoded by Hmox1) is upregulated in the liver following infection by Plasmodium berghei and Plasmodium yoelii sporozoites. HO-1 overexpression in the liver leads to a proportional increase in parasite liver load, and treatment of mice with carbon monoxide and with biliverdin, each an enzymatic product of HO-1, also increases parasite liver load. Conversely, mice lacking Hmox1 completely resolve the infection. In the absence of HO-1, the levels of inflammatory cytokines involved in the control of liver infection are increased. These findings suggest that, while stimulating inflammation, the liver stage of Plasmodium also induces HO-1 expression, which modulates the host inflammatory response, protecting the infected hepatocytes and promoting the liver stage of infection.

Nanosized Multifunctional Polyplexes for Receptor-Mediated SiRNA Delivery

Although our understanding of RNAi and our knowledge on designing and synthesizing active and safe siRNAs significantly increased during the past decade, targeted delivery remains the major limitation in the development of siRNA therapeutics. On one hand, practical considerations dictate robust chemistry reproducibly providing precise carrier molecules. On the other hand, the multistep delivery process requires dynamic multifunctional carriers of substantial complexity. We present a monodisperse and multifunctional carrier system, synthesized by solid phase supported chemistry, for siRNA delivery in vitro and in vivo. The sequence-defined assembly includes a precise cationic (oligoethanamino)amide core, terminated at the ends by two cysteines for bioreversible polyplex stabilization, at a defined central position attached to a monodisperse polyethylene glycol chain coupled to a terminal folic acid as cell targeting ligand. Complexation with an endosomolytic influenza peptide-siRNA conjugate results in nanosized functional polyplexes of 6 nm hydrodynamic diameter. The necessity of each functional substructure of the carrier system for a specific and efficient gene silencing was confirmed. The nanosized polyplexes showed stability in vivo, receptor-specific cell targeting, and silencing of the EG5 gene in receptor-positive tumors. The nanosized appearance of these particles can be precisely controlled by the oligomer design (from 5.8 to 8.8 nm diameter). A complete surface charge shielding together with the high stability result in good tolerability in vivo and the absence of accumulation in nontargeted tissues such as liver, lung, or spleen. Due to their small size, siRNA polyplexes are efficiently cleared by the kidney.

Polyethylenimine/small interfering RNA-mediated knockdown of vascular endothelial growth factor in vivo exerts anti-tumor effects synergistically with Bevacizumab

RNA interference is a powerful method for the knockdown of pathologically relevant genes. The in vivo delivery of siRNAs, preferably through systemic, nonviral administration, poses the major challenge in the therapeutic application of RNAi. Small interfering RNA (siRNA) complexation with polyethylenimines (PEI) may represent a promising strategy for siRNA‐based therapies and, recently, the novel branched PEI F25‐LMW has been introduced in vitro. Vascular endothelial growth factor (VEGF) is frequently overexpressed in tumors and promotes tumor growth, angiogenesis and metastasis and thus represents an attractive target gene in tumor therapy.

Defined Folate-PEG-siRNA Conjugates for Receptor-specific Gene Silencing

Gene silencing mediated by small interfering RNA (siRNA) is a novel approach in the development of new cancer therapeutics. Polycations used for nucleic acid delivery still remain heterogeneous compounds, despite continuous progress in polymer synthetic technologies. Here we report the development of a structural defined folic acid polyethylene glycol (PEG) siRNA conjugate accessible via click chemistry yielding a monodisperse ligand-PEG-siRNA conjugate. The folic acid targeting ligand was synthesized by solid phase supported peptide chemistry. The conjugate was shown to be specifically internalized into folic acid receptor expressing cells. When combined with a structurally defined polycation, again synthesized with the precision of solid phase chemistry, efficient receptor specific gene silencing is achieved.

Small interfering RNA targeting bcl-2 sensitizes malignant melanoma.

Malignant melanoma is a prime example of a treatment-resistant tumor with poor prognosis. Even with innovative treatment regimens, response rates remain low, and the duration of responses is short. More than 90% of all melanomas express the antiapoptotic protein Bcl-2, shown to contribute to a chemoresistant phenotype in melanoma. We previously demonstrated that antisense-mediated inhibition of Bcl-2 sensitizes malignant melanoma to apoptosis-inducing treatment modalities. In the present study, we evaluated synthetic small interfering RNA (siRNA) compounds targeting Bcl-2 as a novel approach to downregulate Bcl-2 expression in melanoma cells. siRNA treatment led up to a 19-fold reduction of bcl-2 mRNA levels and only barely detectable Bcl-2 protein expression at low nanomolar concentrations. Silencing of Bcl-2 in melanoma cells by specific siRNA led to a moderate increase in apoptotic cell death and inhibition of cell growth. However, if siRNA compounds targeting Bcl-2 were combined with the apoptosis-inducing chemotherapeutic agent cisplatin, a massive increase in apoptotic cell death compared with controls was observed. Notably, the combination of Bcl2 siRNA and low-dose cisplatin resulted in a supra-additive effect, with nearly complete suppression of cell growth, whereas cell growth in cisplatin-only-treated cells was only moderately affected (96% vs. 25%, p < 0.001). These findings underline a key role for Bcl-2 in conferring chemoresistance to melanoma and highlight Bcl-2 siRNA strategies as novel and highly effective tools, with the potential for future targeted therapy of malignant melanoma.