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Although 2'-deoxy-2'-α-F-2'-β

A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5′ end and Sp diastereomers at the 3′ end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5′ end and Sp isomer at the 3′ end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2'-O-hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer's disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing.

We recently reported that RNAi-mediated off-target effects are important drivers of the hepatotoxicity observed for a subset of GalNAc–siRNA conjugates in rodents, and that these findings could be mitigated by seed-pairing destabilization using a single GNA nucleotide placed within the seed region of the guide strand. Here, we report further investigation of the unique and poorly understood GNA/RNA cross-pairing behavior to better inform GNA-containing siRNA design. A reexamination of published GNA homoduplex crystal structures, along with a novel structure containing a single (S)-GNA-A residue in duplex RNA, indicated that GNA nucleotides universally adopt a rotated nucleobase orientation within all duplex contexts. Such an orientation strongly affects GNA-C and GNA-G but not GNA-A or GNA-T pairing in GNA/RNA heteroduplexes. Transposition of the hydrogen-bond donor/acceptor pairs using the novel (S)-GNA-isocytidine and -isoguanosine nucleotides could rescue productive base-pairing with the complementary G or C ribonucleotides, respectively. GalNAc-siRNAs containing these GNA isonucleotides showed an improved in vitro activity, a similar improvement in off-target profile, and maintained in vivo activity and guide strand liver levels more consistent with the parent siRNAs than those modified with isomeric GNA-C or -G, thereby expanding our toolbox for the design of siRNAs with minimized off-target activity.

Aim: The electrophoretic mobility shift assay (EMSA) was evaluated as an alternative to ultrafiltration (UF) to assess plasma protein binding (PPB) of small interfering RNAs (siRNA) and antisense oligonucleotides (ASO). Results & methodology: EMSA analysis showed that PPB depended on siRNA and plasma concentration. Conversely, when analyzed by ultrafiltration, siRNA bound the filtration device nonspecifically and PPB remained >98% across physiologically relevant siRNA concentrations. Using EMSA, siRNA exhibited charge-based interactions with plasma proteins, while ASO remained highly bound to plasma proteins or albumin in the presence of 500 mM salt. Conclusion: PPB characteristics of siRNA and ASO can be distinguished using EMSA. Characterization of siRNA PPB by EMSA enhances our knowledge of siRNA absorption, distribution, metabolism and excretion and advanced development of RNA interference therapeutics.

For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2′-deoxy-2′-fluoro (2′-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2′-F-monomer metabolites was low and transient in rats and humans. In vitro, 2′-F-nucleoside 5′-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2′-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2′-F-monomers were observed after weekly administration of two GalNAc–siRNA conjugates in rats for ∼2 years. Taken together, the results support the conclusion that 2′-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc–siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.

Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-Cα- and 4'-Cβ-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and β-epimers of 4'-C-OMe-uridine and the α-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'β-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F,4'-Cα-OMe-U, 2'-F,4'-Cβ-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cβ-OMe-U or 2'-F,4'-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'β-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-Cα-OMe-U or 2'-F,4'-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.

(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5'-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5'-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5'-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2'-ribosugar substitutions such as 2'-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5'-(E)-VP.

The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced., Willoughby and colleagues show that GalNAc-siRNA conjugate uptake is specifically mediated through the hepatic expressed asialoglycoprotein receptor and that potent conjugates are capable of robust gene silencing in reduced receptor settings. These data combined support the therapeutic potential in disease(s) where receptor levels may be reduced due to liver injury.

We designed novel 4′-modified 2′-deoxy-2′-fluorouridine (2′-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4′-C-methoxy (4′-OMe) as the alpha (C4′α) or beta (C4′β) epimers. The C4′α epimer was synthesized by a stereoselective route in six steps; however, both α and β epimers could be obtained by a nonstereoselective approach starting from 2′-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4′-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the β-epimer led to significant destabilization. Both 4′-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4′-OMe substituent and the vicinal 5′- and 3′-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs con...

Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5′-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5′-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc–siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.

The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.

5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.

A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ~5–15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required

We recently demonstrated that siRNAs conjugated to triantennary N-acetylgalactosamine (GalNAc) induce robust RNAi-mediated gene silencing in the liver, owing to uptake mediated by the asialoglycoprotein receptor (ASGPR). Novel monovalent GalNAc units, based on a non-nucleosidic linker, were developed to yield simplified trivalent GalNAc-conjugated oligonucleotides under solid-phase synthesis conditions. Synthesis of oligonucleotide conjugates using monovalent GalNAc building blocks required fewer synthetic steps compared to the previously optimized triantennary GalNAc construct. The redesigned trivalent GalNAc ligand maintained optimal valency, spatial orientation, and distance between the sugar moieties for proper recognition by ASGPR. siRNA conjugates were synthesized by sequential covalent attachment of the trivalent GalNAc to the 3'-end of the sense strand and resulted in a conjugate with in vitro and in vivo potency similar to that of the parent trivalent GalNAc conjugate design.

Asialoglycoprotein receptor (ASGPR) mediated delivery of triantennary N-acetylgalactosamine (GalNAc) conjugated short interfering RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics. Robust and durable gene silencing upon subcutaneous administration at therapeutically acceptable dose levels resulted in the advancement of GalNAc-conjugated oligonucleotide-based drugs into preclinical and clinical developments. To systematically evaluate the effect of display and positioning of the GalNAc moiety within the siRNA duplex on ASGPR binding and RNAi activity, nucleotides carrying monovalent GalNAc were designed. Evaluation of clustered and dispersed incorporation of GalNAc units to the sense (S) strand indicated that sugar proximity is critical for ASGPR recognition, and location of the clustered ligand impacts the intrinsic potency of the siRNA. An array of nucleosidic GalNAc monomers resembling a trivalent ligand at or near the 3' end of the S strand retained in vitro and in vivo siRNA activity, similar to the parent conjugate design. This work demonstrates the utility of simple, nucleotide-based, cost-effective siRNA-GalNAc conjugation strategies.

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis., American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship, National Science Foundation (U.S.), Massachusetts Institute of Technology. Presidential Fellowship

Traditional metabolic engineering approaches, including homologous recombination, zinc-finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody-Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 415–424, 2013

Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed “Dicer-substrate siRNA” (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2′-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.

Huntington's disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntington's disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntington's disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntington's disease.

During RNA-induced silencing complex (RISC) assembly the guide (or antisense) strand has to separate from its complementary passenger (or sense) strand to generate the active RISC complex. Although this process was found to be facilitated through sense strand cleavage, there is evidence for an alternate mechanism, in which the strands are dissociated without prior cleavage. Here we show that the potency of siRNA can be improved by modulating the internal thermodynamic stability profile with chemical modifications. Using a model siRNA targeting the firefly luciferase gene with subnanomolar IC50, we found that placement of thermally destabilizing modifications, such as non-canonical bases like 2,4-difluorotoluene or single base pair mismatches in the central region of the sense strand (9–12 nt), significantly improve the potency. For this particular siRNA, the strongest correlation between the decrease in thermal stability and the increase in potency was found at position 10. Controls with stabilized sugar-phosphate backbone indicate that enzymatic cleavage of the sense strand prior to strand dissociation is not required for silencing activity. Similar potency-enhancing effects were observed as this approach was applied to other functional siRNAs targeting a different site on the firefly luciferase transcript or endogenously expressed PTEN.

Gene silencing induced by short interfering RNA (siRNA) has proven to be useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug product from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be determined accurately. We have developed a stability-indicating, ion-pair, reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385mM hexafluoro-2-propanol, 14.5mM triethylamine, and 5% methanol (mobile phase A) and 385mM hexafluoro-2-propanol, 14.5mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50x2.1mm i.d., 2.5microm particle size), and separation was performed at 60 degrees C. Quantitation was achieved with ultraviolet (UV) detection at 260nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110microg/ml. Accuracy of the method was determined by replicate analysis (n=5) at four concentrations (R(2)>0.996 and relative standard deviations [RSDs] of 1-4%). The use of an ion-pairing reagent that is compatible with mass spectrometry detection makes this method amenable to liquid chromatography-mass spectrometry (LC-MS) impurity profiling.

The most significant challenge remaining in the development of small interfering RNAs (siRNAs) as a new class of therapeutic drugs is successful delivery in vivo. The majority of reported studies describing delivery of siRNA or short hairpin RNA (shRNA) to the central nervous system (CNS) have focused on RNA interference (RNAi) in neurons. Here we show direct CNS delivery of siRNA to a different cell type-oligodendrocytes-using convection-enhanced delivery, and demonstrate robust silencing of an endogenous oligodendrocyte-specific gene, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) with siRNA formulated in saline. The silencing is not sequence-dependent as several different siRNAs are effective in inhibiting target gene expression. Furthermore, we show that CNPase mRNA reduction is dose-dependent, durable for up to 1 week, and mediated by an RNAi mechanism. Increasing the flow rate of siRNA infusion increased the distribution of mRNA suppression to encompass white matter regions distant from the infusion site. Finally, we demonstrate suppression of CNPase mRNA in the nonhuman primate CNS. Taken together, these results show for the first time robust RNAi within oligodendrocytes in vivo and demonstrate the important potential of siRNAs in the treatment of CNS disorders involving oligodendrocyte pathology.

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.

© 2014 American Chemical Society. Conjugation of small interfering RNA (siRNA) to an asialoglycoprotein receptor ligand derived from N-acetylgalactosamine (GalNAc) facilitates targeted delivery of the siRNA to hepatocytes in vitro and in vivo. The ligands derived from GalNAc are compatible with solid-phase oligonucleotide synthesis and deprotection conditions, with synthesis yields comparable to those of standard oligonucleotides. Subcutaneous (SC) administration of siRNA-GalNAc conjugates resulted in robust RNAi-mediated gene silencing in liver. Refinement of the siRNA chemistry achieved a 5-fold improvement in efficacy over the parent design in vivo with a median effective dose (ED50) of 1 mg/kg following a single dose. This enabled the SC administration of siRNA-GalNAc conjugates at therapeutically relevant doses and, importantly, at dose volumes of ≤1 mL. Chronic weekly dosing resulted in sustained dose-dependent gene silencing for over 9 months with no adverse effects in rodents. The optimally chemically modified siRNA-GalNAc conjugates are hepatotropic and long-acting and have the potential to treat a wide range of diseases involving liver-expressed genes.

Nanoparticles are employed for delivering therapeutics into cells1,2. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell specific internalization, excretion, toxicity, and efficacy3-7. A variety of materials have been explored for delivering small interfering RNAs (siRNAs) - a therapeutic agent that suppresses the expression of targeted genes8,9. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, lack of tissue specificity and potential toxicity10-12. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer targeting ligands (such as peptides and folate) on the nanoparticle surface can be precisely controlled. We show that at least three folate molecules per nanoparticle is required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ∼ 24.2 min) than the parent siRNA (t1/2 ∼ 6 min).

With little or no negative impact on the activity of small interfering RNAs (siRNAs), regardless of the number of modifications or the positions within the strand, the 2'-deoxy-2'-fluoro (2'-F) modification is unique. Furthermore, the 2'-F-modified siRNA (see crystal structure) was thermodynamically more stable and more nuclease-resistant than the parent siRNA, and produced no immunostimulatory response.