Stable Nucleic Acid Lipid Particles Research Articles

Self-Assembly of Stable Monomolecular Nucleic Acid Lipid Particles with a Size of 30 nm

The design of efficient nucleic acid complexes is key to progress in genetic research and therapies based on RNA interference. For optimal transport within tissue and across extracellular barriers, nucleic acid carriers need to be small and stable. In this Article, we prepare and characterize mono-nucleic acid lipid particles (mono-NALPs). The particles consist of single short double-stranded oligonucleotides or single siRNA molecules each encapsulated within a closed shell of a cationic-zwitterionic lipid bilayer, furnished with an outer polyethylene glycol (PEG) shield. The particles self-assemble by solvent exchange from a solution containing nucleic acid mixed with the four lipid components DOTAP, DOPE, DOPC, and DSPE-PEG(2000). Using fluorescence correlation spectroscopy, we monitor the formation of mono-NALPs from short double-stranded oligonucleotides or siRNA and lipids into monodisperse particles of approximately 30 nm in diameter. Small angle neutron and X-ray scattering and transmission electron microscopy experiments substantiate a micelle-like core-shell structure of the particles. The PEGylated lipid shell protects the nucleic acid core against degradation by nucleases, sterically stabilizes the mono-NALPs against disassembly in collagen networks, and prevents nonspecific binding to cells. Hence, PEG-lipid shielded mono-NALPs are the smallest stable siRNA lipid system possible and may provide a structural design to be built upon for the development of novel nucleic acid delivery systems with enhanced biodistribution in vivo.

Toward a siRNA-containing nanoparticle targeted to breast cancer cells and the tumor microenvironment

The present work aimed at designing a lipid-based nanocarrier for siRNA delivery toward two cell sub-populations within breast tumors, the cancer and the endothelial cells from angiogenic tumor blood vessels. To achieve such goal, the F3 peptide, which is specifically internalized by nucleolin overexpressed on both those sub-populations, was used as a targeting moiety.The developed F3-targeted stable nucleic acid lipid particles presented adequate features for systemic administration. In addition, the attachment of the F3 peptide onto the liposomal surface enabled an internalization by both cancer and endothelial cells from angiogenic blood vessels that was significantly higher than the one observed with non-cancer cells. Sequence-specific downregulation of enhanced green fluorescent protein (eGFP) in eGFP-overexpressing human cancer cell lines, both at the protein and mRNA levels, was further observed upon delivery of anti-eGFP siRNA by F3-targeted liposomes, in contrast with the non-targeted counterpart. This effect was highly dependent on the content of poly(ethylene glycol) (PEG), as evidenced by the co-localization studies between the siRNA and the lysosomes.Overall, the present work represents an important contribution toward a nanoparticle with multi-targeting capabilities in breast cancer, both at the cellular and molecular level.

Tumor vasculature is a key determinant for the efficiency of nanoparticle-mediated siRNA delivery

Delivering small interfering RNA (siRNA) to tumors using clinically viable formulations remains the primary technical hurdle that prevents the development of siRNA therapy for cancer treatment. Over the past several years, significant effort has been devoted to explore novel delivery strategies, whereas relatively little attention has been paid to understand the impact of physiological constrains such as tumor vasculature on the efficiency of siRNA delivery. Using the previously described positive-readout tumor models where successful siRNA delivery leads to an upregulation of β-galactosidase within tumor sections, we analyzed the spatial distribution of localized target knockdown within tumor sections relative to tumor hypoxia and found that stable nucleic acid lipid particle (SNALP), a lipid nanoparticle-based delivery system, predominantly delivers siRNA to areas adjacent to functional tumor blood vessels. Increasing tumor vascularity by ectopic expression of VEGF resulted in more efficient siRNA delivery to tumors using SNALP. SNALP-mediated delivery of a siRNA-targeting Ran GTPase led to target knockdown and significant antitumor efficacy in the highly vascularized HepG2-derived liver tumors, but not in the poorly vascularized HCT-116-derived liver tumors. These results highlight the significant impact of tumor vasculature on siRNA delivery and call for a more focused effort on addressing tumor penetration after extravasation, an area of only limited attention currently.

Abstract 1644: siRNA targeting of cell cycle kinase Wee1 inhibits hepatocullar carconima growth in vitro and in vivo

Abstract Development of targeted therapy for hepatocellular carcinoma (HCC) remains a major challenge. Previously studies have shown that protein level and kinase activity of Wee1 are significantly elevated in HCC compared with surrounding cirrhotic tissues, although the underlying mechanisms are still unknown. Under normal conditions, Wee1 kinase plays an important role in maintaining G2 arrest through the inhibitory phosphorylation of cdc2 on Tyr-15. In the present study, we explored the possibility of Wee1 being a potential therapeutic target for HCC. To inactivate Wee1, three Wee1-specific small interfering (si) RNAs (Wee1-1, Wee1-2 and Wee1-3) were tested for growth inhibition in HCC cell lines as determined by MTT assay, FACS analysis and microscopy. To obtain insights into molecular changes caused by Wee1 silencing, global changes in gene expression were examined by illumina microarray. For in vivo evaluation of Wee1 as a therapeutic target, we employed orthotopic xenograft model using luciferase-expressing HCC reporter cell lines Huh7- and HepG2-luc+ and stable-nucleic-acid-lipid-particle (SNALP) as an optimal carrier of siRNA into liver. Among the tested siRNA molecules, the Wee1-2siRNA was the most effective in inhibiting Huh7 and HepG2 cell growth (80% and 84%, respectively) which was paralleled by a similar decrease in the levels of target mRNA and protein. Wee1 knockdown by siRNA also caused a block in cell cycle progression and induced apoptosis of HCC cells. The comparison of gene expression profiles in HepG2 cells treated with either control siRNA or Wee1-2siRNA identified 506 differentially expressed genes (P < 0.05 by bootstrap t-test). Genes functionally involved in cell proliferation, such as cdk2, cyclin B1, and Akt1, were down-regulated while cell cycle inhibitor p21 and tumor suppressor TSC2 were up-regulated. Western blotting showed that Wee1 silencing significantly increased the expression of p53 and p21 and decreased cyclin D1 protein levels in Wee1-deficient HepG2 cells, which could contribute to cell cycle arrest and induction of apoptosis. Wee1 5/6, a modified variant of Wee1-2siRNA, was then selected for in vivo application based on the growth inhibitory effect and minimal induction of unwanted immune response. Systemic delivery of Wee1 5/6 variant by SNALP significantly suppressed both Huh7 and HepG2 tumor growth in orthotopic xenograft model. In addition, administration of Wee1 5/6 SNALP increased the survival of mice bearing HepG2-derived tumors bearing mice in a dose-dependent manner. Taken together, these results indicate that Wee1 maybe an attractive molecular target for systemic HCC therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1644. doi:10.1158/1538-7445.AM2011-1644

Definition of Ubiquitination Modulator COP1 as a Novel Therapeutic Target in Human Hepatocellular Carcinoma

The development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. The ubiquitination modulator COP1 regulates p53 activity by ubiquitination and it is frequently overexpressed in human HCC. In this study, we tested the hypothesis that COP1 blockade by short interfering RNA (siRNA)-mediated inhibition could affect the course of HCC progression. The COP1 isoform COP1-1 was selected as the most effective target for siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Growth inhibition occurred in HCC cells that retained wild-type p53 or expressed mutant p53 (Y220C or R249S), whereas p53-null Hep3B cells were resistant. Microarray expression analysis revealed that the antiproliferative effects of COP1 blockade were driven by a common subset of molecular alterations including a p53-associated functional network. In an orthotopic mouse xenograft model of HCC, systemic delivery of a modified COP1 siRNA by stable nucleic acid-lipid particles suppressed neoplastic growth in liver without unwanted immune responses. Our findings offer a first proof of principle that COP1 can be a promising target for systemic therapy of HCC.

Different angiogenesis effect of mini-TyrRS/mini-TrpRS by systemic administration of modified siRNAs in rats with acute myocardial infarction

We aimed to clarify the different angiogenesis effects of mini-tyrosyl-tRNA synthetase (TyrRS)/minitryptophanyl-tRNA synthetase (TrpRS) in rodent primates with acute myocardial infarction, by delivering small interfering RNAs (siRNAs) systemically in a liposomal formulation. Left coronary artery ligation was used to establish the model of acute myocardial infarction in rats; mini-TyrRS/mini-TrpRS-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP), and administered by intravenous injection to rats. Rats were divided into four experiment groups: sham operated group (no left anterior descending artery [LAD] occlusion); negative control group (LAD occlusion + saline injection); mock transfection group (LAD occlusion + mock transfected injection); experiment group (LAD occlusion + mini-TyrRS/mini-TrpRS-specific siRNAs injection). Silencing efficiency was assayed by Western blotting. To determine whether mini-TyrRS/mini-TrpRS affected the angiogenesis activity of rats with myocardial infarction, we measured the myocardial infarction size by TTC staining, and the capillary density using immunohistochemistry staining, to investigate the expression of factor VIII. The myocardial infarction size and the capillary density of mini-TyrRS-siRNA group were respectively 18.89% and 8.64/0.1 mm(2) 1 month after ligation, while in the mini-TrpRS-siRNA group these values were 7.33% and 17.32/0.1 mm(2), significantly different compared with the mock transfection group (14.19%; 13.56/0.1 mm(2)) and negative control group (14.28%; 13.89/0.1 mm(2)), P < 0.05. There were no significant changes between the mock transfection group and the negative control group, P > 0.05. These results indicated that angiogenesis is either stimulated by mini-TyrRS or inhibited by mini-TrpRS in rat models with acute myocardial infarction.

Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study

SummaryBackgroundWe previously showed that small interfering RNAs (siRNAs) targeting the Zaire Ebola virus (ZEBOV) RNA polymerase L protein formulated in stable nucleic acid-lipid particles (SNALPs) completely protected guineapigs when administered shortly after a lethal ZEBOV challenge. Although rodent models of ZEBOV infection are useful for screening prospective countermeasures, they are frequently not useful for prediction of efficacy in the more stringent non-human primate models. We therefore assessed the efficacy of modified non-immunostimulatory siRNAs in a uniformly lethal non-human primate model of ZEBOV haemorrhagic fever.MethodsA combination of modified siRNAs targeting the ZEBOV L polymerase (EK-1 mod), viral protein (VP) 24 (VP24-1160 mod), and VP35 (VP35-855 mod) were formulated in SNALPs. A group of macaques (n=3) was given these pooled anti-ZEBOV siRNAs (2 mg/kg per dose, bolus intravenous infusion) after 30 min, and on days 1, 3, and 5 after challenge with ZEBOV. A second group of macaques (n=4) was given the pooled anti-ZEBOV siRNAs after 30 min, and on days 1, 2, 3, 4, 5, and 6 after challenge with ZEBOV.FindingsTwo (66%) of three rhesus monkeys given four postexposure treatments of the pooled anti-ZEBOV siRNAs were protected from lethal ZEBOV infection, whereas all macaques given seven postexposure treatments were protected. The treatment regimen in the second study was well tolerated with minor changes in liver enzymes that might have been related to viral infection.InterpretationThis complete postexposure protection against ZEBOV in non-human primates provides a model for the treatment of ZEBOV-induced haemorrhagic fever. These data show the potential of RNA interference as an effective postexposure treatment strategy for people infected with Ebola virus, and suggest that this strategy might also be useful for treatment of other emerging viral infections.FundingDefense Threat Reduction Agency.

Are we any closer to combating Ebola infections?

Are we any closer to combating Ebola infections?

Abstract 1576: Molecular targeting of histone deacetylase 2 for treatment of human hepatocellular carcinoma

Abstract Development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. We have recently identified an elevated expression of histone deacetylase 2 (HDAC2) in primary human hepatocellular carcinoma (HCC) and HCC derived cell lines. In this study, we have tested if the downregulation of HDAC2 by siRNA can affect the course of HCC progression in vitro and in vivo. To inactivate HDAC2 gene expression, three HDAC2-specific siRNAs (HDAC2-1, HDAC2-2 and HDAC2-3) were designed and tested for growth inhibition in Huh7 and HepG2 HCC cell lines as determined by MTT assay, FACS analysis and microscopy. To obtain insights into molecular changes caused by HDAC2 silencing, global changes in gene expression were examined by illumina microarray. For in vivo evaluation of HDAC2 as a therapeutic target, we employed orthotopic xenograft model using luciferase-expressing HCC reporter cell line Huh7-luc+ and stable-nucleic-acid-lipid-particle (SNALP) as an optimal carrier of siRNA into liver. The HDAC2-1siRNA was the most effective in inhibiting Huh7 and HepG2 cell growth (68% and 71%, respectively) which was paralleled by a similar decrease in the levels of target mRNA and protein. HDAC2-deficient cells also exhibited a 1.9-fold increase in apoptosis through activation of caspase-3. The comparison of gene expression profiles in HepG2 cells treated with either control siRNA or HDAC2-1siRNA identified 299 differentially expressed genes. Consistent with in vitro observations, genes functionally involved in apoptosis, such as CDKN1A, SOCS2, TP53I3, and BTG2, were up-regulated while genes associated with cellular metabolic process (e.g. HOXD1, PAK2, SMAD9, CDK4, PCK2 and SKP2) were down-regulated. HDAC2 3/7siRNA, a chemically modified variant of HDAC2-1siRNA displaying a minimal induction of IL-6 in murine Flt3L dendricite cultures, was selected for in vivo treatment of Huh7-luc+-derived HCC. Administration of SNALP- HDAC2 3/7siRNA effectively suppressed xenograft growth as compared to the SNALP-control siRNA treatment. Taken together, these results indicate that HDAC2 is an important regulator of HCC cell growth and survival, and may be an attractive target for systemic therapy of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1576.

Rational design of cationic lipids for siRNA delivery

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

Abstract B204: Development of ALN-VSP: An RNAi therapeutic for liver malignancies

Abstract Malignancies of the liver, including primary (hepatocellular carcinoma) and secondary (metastatic) tumors, represent a significant unmet medical need. We are developing a therapeutic for solid tumors involving the liver that is comprised of lipid particle-formulated small interfering RNAs (siRNAs) targeting VEGF and the mitotic kinesin, KSP (Eg5). For each target, potent siRNA duplexes were selected following extensive screening in tissue culture cells. To assess efficacy in vivo, a stable nucleic acid lipid particle (SNALP) formulation was developed based on similar formulations previously shown to silence liver-expressed genes via systemic administration in multiple species. A SNALP-formulated combination of the KSP and VEGF siRNAs (referred to as ALN-VSP) was tested in orthotopic liver tumor models in which human tumor cells are implanted directly into the livers of immunocompromised mice. We show that intravenous administration of ALN-VSP leads to silencing of both KSP and VEGF expression in established liver tumors. This is accompanied by the formation of numerous aberrant mitotic figures (“monoasters”) in tumor cells indicative of the pharmacologic inhibition of KSP. In addition, we demonstrate that ALN-VSP treatment provides a clear survival benefit even when treatment is initiated in animals with a significant tumor burden. A Phase 1 clinical trial of ALN-VSP has recently been initiated. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B204.

Advancement to the clinic of an RNAi therapeutic for solid tumors

3585 Background: Malignancies of the liver, including primary (hepatocellular carcinoma) and secondary (metastatic) tumors, represent a significant unmet medical need. We are developing a therapeutic for solid tumors involving the liver that is comprised of lipid particle-formulated short interfering RNAs (siRNAs) targeting VEGF and the mitotic kinesin, KSP (Eg5). For each target, potent siRNA duplexes were selected following extensive screening in tissue culture cells. Efficacy was demonstrated in a mouse liver tumor model. Methods: To assess efficacy in vivo, a stable nucleic acid lipid particle (SNALP) formulation was developed based on similar formulations previously shown to silence liver-expressed genes via systemic administration in multiple species. A SNALP-formulated combination of the KSP and VEGF siRNAs (referred to as ALN-VSP01) was tested in an orthotopic liver tumor model in which human hepatoma cells (Hep3B) are implanted directly into the livers of immunocompromised mice. Results: Intravenous administration of ALN-VSP01 leads to dose-dependent inhibition of both KSP and VEGF expression in established liver tumors. This was accompanied by the formation of numerous aberrant mitotic figures (“monoasters”) in tumor cells indicative of the pharmacologic inhibition of KSP. In addition, tumor growth was significantly inhibited by a course of ALN-VSP01 treatment, and ALN-VSP01 treatment provided a clear survival benefit even when treatment was initiated in animals with a significant tumor burden. As a control, a SNALP-formulated siRNA targeting Luciferase was administered and shown to have no effect in these studies. Conclusions: Systemic administration of ALN-VSP01 exhibited clear efficacy in a mouse orthotopic liver tumor model. Clinical testing of ALN-VSP01 is expected to initiate in early 2009. [Table: see text]

Stable nucleic acid lipid particles (SNALPs) for small interfering RNA delivery

Stable nucleic acid lipid particles (SNALPs) for small interfering RNA delivery

Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice

siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

Systemic and Specific Delivery of Small Interfering RNAs to the Liver Mediated by Apolipoprotein A-I

Tissue-targeted delivery of small interfering RNA (siRNA) must be achieved before RNA interference (RNAi) technology can be used in practical therapeutic approaches. In this study, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of nucleic acids to the liver is demonstrated using real-time in vivo imaging. As a proof of concept, synthetic siRNAs against hepatitis B virus (HBV) were formulated into complexes of apo A-I and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (DTC-Apo) and injected intravenously (i.v.) into a mouse model carrying replicating HBV. We show that administration of these nanoparticles can significantly reduce viral protein expression by receptor-mediated endocytosis. The advantages of the apo A-I-mediated siRNA delivery method are its liver specificity, its effectiveness at low doses (< or = 2 mg/kg) in only a single treatment, and its persistent antiviral effect up to 8 days. The liver-targeted gene silencing was also shown by in vivo images, in which bioluminescent signals emitted from the liver were efficiently reduced after i.v. administration of luciferase-specific siRNA and DTC-Apo lipoplex. Thus, our unique approach to siRNA delivery creates a foundation for the development of a new class of promising therapeutics against hepatitis viruses or hepatocyte genes related to tumor growth.

Postexposure Protection of Guinea Pigs against a Lethal Ebola Virus Challenge Is Conferred by RNA Interference

BackgroundEbola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs) MethodsFour siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid–lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge ResultsTreatment of guinea pigs with a pool of the L gene–specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge ConclusionsFurther development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats

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.

Fighting viral hepatitis with small RNAs

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Morrissey DV, Lockridge JA, Shaw L, Blanchard K, Jensen K, Breen W, Hartsough K, Machemer L, Radka S, Jadhav V, Vaish N, Zinnen S, Vargeese C, Bowman K, Shaffer CS, Jeffs LB, Judge A, MacLachlan I, Polisky B. The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA–SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3mg/kg/day reduced serum HBV DNA>1.0log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 days after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach. [Abstract reproduced by permission of Nat Biotechnol 2005;23:1002–7]

Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids

An analogous series of cationic lipids (1,2-distearyloxy- N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy- N,N-dimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy- N,N-dimethyl-3-aminopropane (DLinDMA) and 1,2-dilinolenyloxy- N,N-dimethyl-3-aminopropane (DLenDMA)) possessing 0, 1, 2 or 3 double bonds per alkyl chain respectively, was synthesized to determine the correlation between lipid saturation, fusogenicity and efficiency of intracellular nucleic acid delivery. 31P-NMR analysis suggests that as saturation increases, from 2 to 0 double bonds, lamellar (L α) to reversed hexagonal (H II) phase transition temperature increases, indicating decreasing fusogenicity. This trend is largely reflected by the efficiency of gene silencing observed in vitro when the lipids are formulated as Stable Nucleic Acid Lipid Particles (SNALPs) encapsulating small inhibitory RNA (siRNA). Uptake experiments suggest that despite their lower gene silencing efficiency, the less fusogenic particles are more readily internalized by cells. Microscopic visualization of fluorescently labelled siRNA uptake was supported by quantitative data acquired using radiolabelled preparations. Since electrostatic binding is a precursor to uptake, the pKa of each cationic lipid was determined. The results support a transfection model in which endosomal release, mediated by fusion with the endosomal membrane, results in cytoplasmic translocation of the nucleic acid payload.

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs

The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.