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.

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.

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.

Detailed knowledge of the composition and structure of the spliceosome and its assembly intermediates is a prerequisite for understanding the complex process of pre-mRNA splicing. To this end, we have developed a tobramycin affinity-selection method that is generally applicable for the purification of native RNP complexes. By using this method, we have isolated human prespliceosomes that are ideally suited for both biochemical and structural studies. MS identified >70 prespliceosome-associated proteins, including nearly all known U1 and U2 snRNP proteins, and expected non-snRNP splicing factors. In addition, the DEAD-box protein p68, RNA helicase A, and a number of proteins that appear to perform multiple functions in the cell, such as YB-1 and TLS, were detected. Several previously uncharacterized proteins of unknown function were also identified, suggesting that they play a role in splicing and potentially act during prespliceosome assembly. These data provide insight into the complexity of the splicing machinery at an early stage of its assembly. S pliceosomes, the complex enzymes responsible for pre-mRNA splicing, consist of the U1, U2, U5, and U4͞U6 small nuclear ribonucleoproteins (snRNP), plus numerous non-snRNP proteins. During the stepwise assembly of spliceosomes, several distinct complexes, which form in a defined order (i.e., E complex, followed by A, B, and C), can be distinguished biochemically in vitro (reviewed in refs. 1 and 2). Before association of snRNPs and splicing factors, the pre-mRNA is bound by heterogenous nuclear ribonucleoprotein (hnRNP) proteins, forming the H complex. Spliceosome assembly is initiated by the ATP-independent interaction of the U1 snRNP with the 5Ј splice site which, in part, involves base pairing between the U1 snRNA and the pre-mRNA. This initial complex, termed E (early), also contains the U2 snRNP, which at this stage is loosely associated with the pre-mRNA (3). In a subsequent ATP-dependent step, the U2 snRNA base-pairs with the branch site, leading to the stable association of the U2 snRNP and formation of the A complex (also called the prespliceosome). Contacts between snRNA͞proteins and the pre-mRNA that are established during these early stages of spliceosome assembly play crucial roles in the recognition and selection of splice sites. Thus, regulation of alternatively spliced pre-mRNAs is often achieved by modulating the association of spliceosomal components during E and A complex formation. In a subsequent step, the U4͞U6.U5 tri-snRNP binds, leading to the formation of spliceosomal B complex. After a major conformational change and the first transesterification reaction of splicing, complex C is generated. After the second transesterification step, the mRNA and excised intron are released, and the spliceosome dissociates.To date, Ͼ200 spliceosomal proteins have been identified in mammals by MS (4-6). However, in these studies, a mixture of purified splicing complexes was analyzed, preventing the assignment of a particular protein to a specific splicing co...