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.

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.

Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE(-/-) mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR(-/-))-deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver.

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. plasma PCSK9 ͉ tissue LDLR levels P roprotein convertase subtilisin/kexin type 9 (PCSK9) is a member of the mammalian serine proprotein convertase family that typically functions in the proteolytic processing and maturation of secretory proteins (1, 2). PCSK9 was the first family member to be implicated in a dominantly inherited form of hypercholesterolemia (3). Mechanistic studies addressing the function of PCSK9 in mice and humans have demonstrated that overexpression or gain-of-function mutations in PCSK9 reduced low density lipoprotein receptor (LDLR) protein levels in liver, which significantly increased circulating plasma cholesterol both in mice and humans (4). Additional studies showed that the deletion of Pcsk9 in mice resulted in increased LDLR levels, accelerated the clearance of low density lipoprotein cholesterol (LDLc), and reduced circulating cholesterol levels (5). Recently, studies in mice have also shown that lowering PCSK9 transcript levels by antisense oligonucleotides resulted in reduced total cholesterol, LDLc, and HDL cholesterol (HDLc) in blood and increased LDLR levels in liver after 6 weeks of treatment (6). This effect was very similar to that observed in the Pcsk9 Ϫ/Ϫ mice (5). Collectively, these studies have clearly established a role for PCSK9 in cholesterol homeostasis.Validation of PCSK9 as an attractive therapeutic target for the treatment of hypercholesterolemia has come from genetic studies in humans. Cohen et al. (7) first identified loss-of-function mutations in PCSK9...

RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N(12)-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration.

Bu 2 ) 2 C 6 H 3 ) has been prepared by two independent routes that involve deprotonation of Ru-(II) ammine complexes. Complex 2 reacts with phenylacetylene to yield the Ru(II) acetylide complex (PCP)Ru(CO)(CtCPh) (5) and ammonia. In addition, complex 2 rapidly activates dihydrogen at room temperature to yield ammonia and the previously reported hydride complex (PCP)Ru(CO)(H). The ability of the amido complex 2 to cleave the H-H bond is attributed to the combination of a vacant coordination site for binding/activation of dihydrogen and a basic amido ligand. Complex 2 also undergoes an intramolecular C-H activation of a methyl group on the PCP ligand to yield ammonia and a cyclometalated complex. The reaction of (PCP)Ru(CO)(Cl) with MeLi allows the isolation of (PCP)Ru(CO)-(Me) (8), and complex 8 undergoes an intramolecular C-H activation analogous to the amido complex 2 to produce methane and the cyclometalated complex. Determination of activation parameters for the intramolecular C-H activation transformations of 2 and 8 reveal identical ∆H q {18(1) kcal/mol} with ∆S q ) -23(4) eu and -18(4) eu, respectively. Density functional theory has been applied to the study of intermolecular activation of methane and dihydrogen by (PCP′)Ru(CO)(NH 2 ) to yield (PCP′)Ru(CO)(NH 3 )(X) (X ) Me or H; PCP′ ) 2,6-(CH 2 -PH 2 ) 2 C 6 H 3 ). The results indicate that the activation of dihydrogen is both exoergic and exothermic. In contrast, the addition of a C-H bond of methane across the Ru-NH 2 bond has been calculated to be endoergic and endothermic. The surprising endoergic nature of the methane C-H activation has been attributed to a large and unfavorable change in Ru-N bond dissociation energy upon conversion from Ru-amido to Ru-ammine.

Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.

The synthesis and reactivity of late-metal complexes with heteroatomic π-donating ligands have received significant recent attention. [1][2][3][4][5][6] Efforts in this area have been prompted in part due to the expectation that the combination of "soft" late metals with "hard" donor ligands (e.g., oxygen-and nitrogen-based ligands) can result in weak bonds and reactive ligand moieties; however, thermochemical studies of metal-heteroatom bond strengths suggest that homolytic bond strengths between late metals and heteroatom ligands are not inherently weak. 7,8 Additionally, it has been suggested that the scarcity of late-metal systems with π-donating ligands is due to the presence of π-conflict between filled metal dπ orbitals and lone electron pairs residing on the π-donating ligands. 1,9 Recently, application of Drago's E-C bonding theory to understanding the bonding and reactivity of such complexes has been reported and raises questions as to the importance of π-π repulsion in such systems. 3 Bergman et al.'s recent reports of the synthesis and reactivity of the Ru(II) complex trans-(DMPE) 2 Ru(H)-(NH 2 ) (DMPE ) 1,2-bis(dimethylphosphinoethane)) provide a striking example of a highly reactive ruthenium amido moiety. 10,11 The parent amido ligand of this complex exhibits remarkable reactivity, including the ability to deprotonate several C-H bonds. Such extraordinary basicity and reactivity raises several questions, including the following: (1) is the enhanced basicity a general feature of such complexes and (2) what features control the amido reactivity? To begin to answer these questions, complexes with variable ancillary ligands must be accessed. We now report the synthesis and preliminary reactivity of the first example (to our knowledge) of a series of octahedral and d 6 parent amido complexes in which the ancillary ligands are systematically varied. Particularly germane here are CpRu II (Cp ) cyclopentadienyl) phosphine complexes with amido ligands reported by Roundhill et al. 12 {TpRu(L)(L′)} complexes have received significant recent attention. 13 These fragments offer the significant synthetic advantage of being able to systematically control the steric and electronic features of the metal coordination sphere via variation of L and L′. Reflux of the known complex TpRu(PPh 3 ) 2 (Cl) 14 with excess trimethylphosphine or trimethyl phosphite in toluene yields TpRu(PMe 3 ) 2 (Cl) (1) and TpRu{P(OMe) 3 } 2 (Cl) (2) in high yields (95% and 87%, respectively) after workup. Reactions of 1, 2, TpRu(CO)(PPh 3 )(Cl), 15 and TpRu-(PPh 3 ) 2 (Cl) with AgOTf yield the corresponding triflate complexes TpRu(PMe 3 ) 2 (OTf) (3), TpRu{P(OMe) 3 } 2 (OTf) (4), TpRu(CO)(PPh 3 )(OTf) (5), and TpRu(PPh 3 ) 2 (OTf) (6). Complexes 3-5 can be isolated cleanly, while the triphenylphosphine complex 6 has eluded isolation and is generated in situ. Reactions of complexes 3-6 in different solvents at variable temperatures with metal amides (e.g., NaNH 2 or LiNH 2 ) or metal amides in combination with ammonia result in no reaction...