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5′-(E)-Vinylphosphonate (VP) is an effective bioisostere of the natural 5′-monophosphate in small interfering RNAs (siRNAs). Solid-phase synthesis of VP-siRNAs requires the use of appropriately protected VP-phosphoramidites in combination with optimal oligonucleotide deprotection conditions. Addition of 3% (v) neat diethylamine to the standard aqueous ammonia deprotection conditions allows clean and rapid one-step deprotection of 5′-[O,O-bis(pivaloyloxymethyl)] (POM)-protected VP oligonucleotides, minimizing side reactions and impurities, which broadly enhances the scope of VP oligonucleotide synthesis.

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.

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.

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 acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology.

The segmentation motor pattern of the intestine, the most dominant pattern in response to eating, designed to optimally absorb nutrients, was described in the late 19th century. How the intestine switches from propulsion to segmentation was revealed recently by us in Nature Communications (in press). Fatty acids induce a low frequency transient rhythmic pacemaker in a network of interstitial cells of Cajal (ICC) associated with the deepmuscular neural plexus (ICC-DMP; 1-4 cycles/min), second to the dominant omnipresent slow wave pacemaker that is generated by ICC associated with the myenteric plexus (ICC-MP; 30-40 cycles/min) in both mouse and rat colon. The introduction of the second pacemaker is associated with slow wave activity changing into a waxing and waning electrical pattern. Not unlike the interaction of waves caused by two stones dropped in water, the pacemaker activities interact with each other, changing a propulsive motor pattern into a non-propagating rhythmic pattern of short-lasting contractions. Our aim of the present study was to understand the mechanism of initiation and control of this trigger for segmentation, and the switch that can change segmentation into propulsion and vice versa. Using intracellular electrical recordings (n=21), we found that the ICC-DMP pacemaker is under tonic inhibitory control by enteric nitrergic nerves and can be evoked by substance P acting on NK1 receptors of ICCDMP at 3.6 ± 0.6 cycles/min. Furthermore, the switch can be activated by medium chain fatty acids through the blood stream at 2.8 ± 0.5 cycles/min, and by short chain fatty acids via the lumen. To further understand the nature of the interaction between the two pacemakers we have developed a model based on the electrophysiological characteristics of the electrical pacemaker activities and based on phase-amplitude coupling of the electrical activities. The motor patterns of the musculature develop because both pacemaker activities propagate into the musculature from opposite sides. The segmentation activity develops when the propagation velocities of the two pacemakers are significantly different from each other. When the propagation velocities are similar, the two pacemakers strengthen each other to create strong low frequency propulsive activity. Hence, the ICC-DMP play a critical role in initiation and the regulation of the nature of the motor pattern of the intestine. It is likely therefore that abnormalities in the ICC-DMP pacemaker activity or its neural or nutrient control may give rise to either the propulsive or segmentation motor pattern becoming too dominant resulting in diarrhea or constipation. The ICC-DMP pacemaker will therefore become a target for rectifying abnormal motor patterns.

Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21(WAF1/ Cip1) (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-of-principle that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease.