Lipid nanoparticle-mediated siRNA delivery for safe targeting of human CML in vivo.

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.

Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection.

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway.

Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipoprotein B and factor VII) that achieved efficient (approximately 80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA.

Delivery of siRNA to the mouse lung via a functionalized lipopolyamine.

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.

Lipid nanoparticle-encapsulated, chemically modified anti-adenoviral siRNAs inhibit hepatic adenovirus infection in immunosuppressed Syrian hamsters.

RNA interference has demonstrated its potential as an antiviral therapy for treatment of human adenovirus (hAd) infections. The only existing viral vector-based system for delivery of anti-adenoviral artificial microRNAs available for in vivo use, however, has proven to be inefficient in therapeutic applications. In this study, we investigated the potential of stabilized small interfering RNA (siRNA) encapsulated in lipid nanoparticles (LNPs) for treatment of hepatic hAd serotype 5 (hAd5) infection in an hAd infection model using immunosuppressed Syrian hamsters. The siRNA sipTPmod directed against the adenoviral pre-terminal protein (pTP) and containing 2'-O-methyl modifications as well as phosphorothioate linkages effectively inhibited hAd5 infection in vitro. In light of this success, sipTPmod was encapsulated in LNPs containing the cationic lipid XL-10, which enables hepatocyte-specific siRNA transfer, and injected intravenously into hAd5-infected immunosuppressed Syrian hamsters. This resulted in a significant reduction of liver hAd5 titers, a trend toward reduced liver injury and inflammation, and reduction of viral titers in the blood and spleen compared with hAd5-infected animals that received a non-silencing siRNA. These effects were demonstrated in animals infected with low and moderate doses of hAd5. These data demonstrate that hepatic hAd5 infection can be successfully treated with anti-adenoviral sipTPmod encapsulated in LNPs.

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.

Characterization of side reactions during the annealing of small interfering RNAs.

Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions-such as 2',3'-isomerization, strand scission, and HF elimination-depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.

Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates.

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.

c-FLIP mediates resistance of Hodgkin/Reed-Sternberg cells to death receptor-induced apoptosis.

Resistance to death receptor-mediated apoptosis is supposed to be important for the deregulated growth of B cell lymphoma. Hodgkin/Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin's lymphoma (cHL), resist CD95-induced apoptosis. Therefore, we analyzed death receptor signaling, in particular the CD95 pathway, in these cells. High level CD95 expression allowed a rapid formation of the death-inducing signaling complex (DISC) containing Fas-associated death domain-containing protein (FADD), caspase-8, caspase-10, and most importantly, cellular FADD-like interleukin 1beta-converting enzyme-inhibitory protein (c-FLIP). The immunohistochemical analysis of the DISC members revealed a strong expression of CD95 and c-FLIP overexpression in 55 out of 59 cases of cHL. FADD overexpression was detectable in several cases. Triggering of the CD95 pathway in HRS cells is indicated by the presence of CD95L in cells surrounding them as well as confocal microscopy showing c-FLIP predominantly localized at the cell membrane. Elevated c-FLIP expression in HRS cells depends on nuclear factor (NF)-kappaB. Despite expression of other NF-kappaB-dependent antiapoptotic proteins, the selective down-regulation of c-FLIP by small interfering RNA oligoribonucleotides was sufficient to sensitize HRS cells to CD95 and tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Therefore, c-FLIP is a key regulator of death receptor resistance in HRS cells.

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

BACKGROUND: 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. METHODS: In subcutaneous tumor xenograft mouse models, we established the therapeutic efficacy and safety of PEI F25-LMW/siRNA-mediated knockdown of VEGF. In biodistribution and siRNA quantification studies, we optimized administration strategies and, employing chemically modified siRNAs, compared the anti-tumorigenic efficacies of: (i) PEI/siRNA-mediated VEGF targeting; (ii) treatment with the monoclonal anti-VEGF antibody Bevacizumab (Avastin); and (iii) a combination of both. RESULTS: Efficient siRNA delivery is observed upon systemic administration, with the biodistribution being dependent on the mode of injection. Toxicity studies reveal no hepatotoxicity, proinflammatory cytokine induction or other side-effects of PEI F25-LMW/siRNA complexes or polyethylenimine, and tumor analyses show efficient VEGF knockdown upon siRNA delivery, leading to reduced tumor cell proliferation and angiogenesis. The determination of anti-tumor effects reveals that, in pancreas carcinoma xenografts, single treatment with PEI/siRNA complexes or Bevacizumab is already highly efficacious, whereas, in prostate carcinoma, synergistic effects of both treatments are observed. CONCLUSIONS: PEI F25-LMW/siRNA complexes, which can be stored frozen as opposed to many other carriers, represent an efficient, safe and promising avenue in anti-tumor therapy, and PEI/siRNA-mediated, therapeutic VEGF knockdown exerts anti-tumor effects.

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.

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.

Targeted Inhibition of the NUP98-NSD1 Fusion Oncogene in Acute Myeloid Leukemia.

NUP98-NSD1-positive acute myeloid leukemia (AML) is a poor prognostic subgroup that is frequently diagnosed in pediatric cytogenetically normal AML. NUP98-NSD1-positive AML often carries additional mutations in genes including FLT3, NRAS, WT1, and MYC. The purpose of our study was to characterize the cooperative potential of the fusion and its associated Neuroblastoma rat sarcoma (NRAS) mutation. By constitutively expressing NUP98-NSD1 and NRASG12D in a syngeneic mouse model and using a patient-derived xenograft (PDX) model from a NUP98-NSD1-positive AML patient, we evaluated the functional role of these genes and tested a novel siRNA formulation that inhibits the oncogenic driver NUP98-NSD1. NUP98-NSD1 transformed murine bone marrow (BM) cells in vitro and induced AML in vivo. While NRASG12D expression was insufficient to transform cells alone, co-expression of NUP98-NSD1 and NRASG12D enhanced the leukemogenicity of NUP98-NSD1. We developed a NUP98-NSD1-targeting siRNA/lipid nanoparticle formulation that significantly prolonged the survival of the PDX mice. Our study demonstrates that mutated NRAS cooperates with NUP98-NSD1 and shows that direct targeting of the fusion can be exploited as a novel treatment strategy in NUP98-NSD1-positive AML patients.

Ligand Conjugated Multimeric siRNAs Enable Enhanced Uptake and Multiplexed Gene Silencing.

Small interfering RNAs (siRNAs) conjugated to N-acetylgalactosamine (GalNAc) ligands have been used to treat disease in patients. However, conjugates with other ligands deliver siRNA less efficiently, limiting the development of new targeted therapies. Most approaches to enhancing the potency of such conjugates have concentrated on increasing ligand effectiveness and/or the chemical stability of the siRNA drug. One complementary and unexplored alternative is to increase the number of siRNAs delivered per ligand. An ideal system would be a single chemical entity capable of delivering multiple copies of an oligonucleotide drug and/or several such drugs simultaneously. Here we report that siRNAs can be stably linked together under neutral aqueous conditions to form chemically defined siRNA "multimers," and that these multimers can be delivered in vivo by a GalNAc ligand. Conjugates containing multiple copies of the same siRNA showed enhanced activity per unit of ligand, whereas siRNAs targeting different genes linked to a single ligand facilitated multigene silencing in vivo; this is the first demonstration of silencing several genes simultaneously in vivo using ligand-directed multimeric siRNA. Multimeric oligonucleotides represent a powerful and practical new approach to improve intracellular conjugate delivery.

Silencing the CSF-1 Axis Using Nanoparticle Encapsulated siRNA Mitigates Viral and Autoimmune Myocarditis.

Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms.

Antibody-directed cell-type-specific delivery of siRNA.

Over the past four years, chemically synthesized short interfering RNA (siRNA) has become the standard tool for specific silencing of gene expression in vitro. The most difficult task in transferring this technology to an in vivo setting is to develop appropriate delivery strategies. With this aim, Song et al. recently reported the development of antibody-protamine fusion proteins as vehicles for receptor-directed delivery of siRNA. When a mixture of siRNA targeting tumor-related genes was administered in this way, tumor growth was inhibited in an engineered melanoma model, demonstrating the therapeutic potential of this technology. However, several challenges remain to be overcome before targeted gene silencing can become a reality for patients.

Effective silencing of EGFR with RNAi demonstrates non-EGFR dependent proliferation of glioma cells.

The epidermal growth factor receptor (EGFR, ErbB1) is frequently dysregulated in a variety of solid human tumors, including malignant glioma. EGFR expression has been associated with disease progression, resistance to standard therapies and poor survival. The application of small interfering RNAs (siRNAs) has become an effective and highly specific tool to modulate gene expression, and a wide range of oncogenes have been silenced successfully. Here we show the siRNA-mediated down-regulation of EGFR in two established glioma cell lines with different EGFR expression levels (U373 MG, LN18). The expression of EGFR mRNA and protein was down-regulated by 70-90%. However, siRNA treatment had no inhibitory effect on cell proliferation, migration and activation status of EGFR-coupled signaling cascades. In accordance with these results, gene expression analysis with microarrays revealed only small, albeit specific changes in expression patterns. In conclusion, these data indicate that the specific down-regulation of EGFR might not be sufficient for a single agent therapeutic approach in malignant glioma.

Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen.

The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.

RNA interference in vivo: toward synthetic small inhibitory RNA-based therapeutics.

Small interfering RNA (siRNA) mediated inhibition of gene expression has rapidly become a major tool for in vitro analysis of protein function. In vivo gene silencing by siRNAs will play an important role for target validation and is the first step towards the development of siRNA-based therapeutics. This chapter reviews the early and intriguing successes in using siRNAs for in vivo gene silencing. The impact of chemical modification on siRNA efficacy in vitro and the potential for employing such modifications to alter the pharmacokinetic properties of siRNAs is also summarized. A protocol describing siRNA-based gene silencing in tumor models can serve as guide for the design of individual in vivo RNA interference experiments.

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.