Impact of guanidine-containing backbone linkages on stereopure antisense oligonucleotides in the CNS.

Attaining sufficient tissue exposure at the site of action to achieve the desired pharmacodynamic effect on a target is an important determinant for any drug discovery program, and this can be particularly challenging for oligonucleotides in deep tissues of the CNS. Herein, we report the synthesis and impact of stereopure phosphoryl guanidine-containing backbone linkages (PN linkages) to oligonucleotides acting through an RNase H-mediated mechanism, using Malat1 and C9orf72 as benchmarks. We found that the incorporation of various types of PN linkages to a stereopure oligonucleotide backbone can increase potency of silencing in cultured neurons under free-uptake conditions 10-fold compared with similarly modified stereopure phosphorothioate (PS) and phosphodiester (PO)-based molecules. One of these backbone types, called PN-1, also yielded profound silencing benefits throughout the mouse brain and spinal cord at low doses, improving both the potency and durability of response, especially in difficult to reach brain tissues. Given these benefits in preclinical models, the incorporation of PN linkages into stereopure oligonucleotides with chimeric backbone modifications has the potential to render regions of the brain beyond the spinal cord more accessible to oligonucleotides and, consequently, may also expand the scope of neurological indications amenable to oligonucleotide therapeutics.

In this study, the authors explore the impact of nitrogen-containing (PN) backbones on oligonucleotides that promote RNase H-mediated degradation of a transcript in the central nervous system (CNS). Using Malat1, a ubiquitously expressed non-coding RNA that is predominately localized in the nucleus, and C9orf72, a challenging RNA target requiring a more nuanced targeting strategy, as benchmarks, they show that chimeric oligonucleotides containing stereopure PS and one of the more promising PN backbones (PN-1) have more potent and durable activity throughout the CNS compared with more traditional PS-modified molecules in mouse models. They demonstrate that potency and durability benefits in vivo derive at least in part from increased tissue exposure, especially in more difficult to reach regions of the brain. Ultimately, these benefits enabled the authors to demonstrate pharmacodynamic effects on Malat1 and C9orf72 RNAs in multiple brain regions with relatively low doses.

Stereochemistry Enhances Potency, Efficacy, and Durability of Malat1 Antisense Oligonucleotides In Vitro and In Vivo in Multiple Species.

Purpose: Antisense oligonucleotides have been under investigation as potential therapeutics for many diseases, including inherited retinal diseases. Chemical modifications, such as chiral phosphorothioate (PS) backbone modification, are often used to improve stability and pharmacokinetic properties of these molecules. We aimed to generate a stereopure MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) antisense oligonucleotide as a tool to assess the impact stereochemistry has on potency, efficacy, and durability of oligonucleotide activity when delivered by intravitreal injection to eye. Methods: We generated a stereopure oligonucleotide (MALAT1-200) and assessed the potency, efficacy, and durability of its MALAT1 RNA-depleting activity compared with a stereorandom mixture, MALAT1-181, and other controls in in vitro assays, in vivo mouse and nonhuman primate (NHP) eyes, and ex vivo human retina cultures. Results: The activity of the stereopure oligonucleotide is superior to its stereorandom mixture counterpart with the same sequence and chemical modification pattern in in vitro assays, in vivo mouse and NHP eyes, and ex vivo human retina cultures. Findings in NHPs showed durable activity of the stereopure oligonucleotide in the retina, with nearly 95% reduction of MALAT1 RNA maintained for 4 months postinjection. Conclusions: An optimized, stereopure antisense oligonucleotide shows enhanced potency, efficacy, and durability of MALAT1 RNA depletion in the eye compared with its stereorandom counterpart in multiple preclinical models. Translational Relevance: As novel therapeutics, stereopure oligonucleotides have the potential to enable infrequent administration and low-dose regimens for patients with genetic diseases of the eye.

Effect of Porosity on Strength Distribution of Microcrystalline Cellulose.

Fracture strength of pharmaceutical compacts varies even for nominally identical samples, which directly affects compaction, comminution, and tablet dosage forms. However, the relationships between porosity and mechanical behavior of compacts are not clear. Here, the effects of porosity on fracture strength and fracture statistics of microcrystalline cellulose compacts were investigated through diametral compression tests. Weibull modulus, a key parameter in Weibull statistics, was observed to decrease with increasing porosity from 17 to 56 vol.%, based on eight sets of compacts at different porosity levels, each set containing ∼ 50 samples, a total of 407 tests. Normal distribution fits better to fracture data for porosity less than 20 vol.%, whereas Weibull distribution is a better fit in the limit of highest porosity. Weibull moduli from 840 unique finite element simulations of isotropic porous materials were compared to experimental Weibull moduli from this research and results on various pharmaceutical materials. Deviations from Weibull statistics are observed. The effect of porosity on fracture strength can be described by a recently proposed micromechanics-based formula.

Real time measurement of PEG shedding from lipid nanoparticles in serum via NMR spectroscopy.

Small interfering RNA (siRNA) is a novel therapeutic modality that benefits from nanoparticle mediated delivery. The most clinically advanced siRNA-containing nanoparticles are polymer-coated supramolecular assemblies of siRNA and lipids (lipid nanoparticles or LNPs), which protect the siRNA from nucleases, modulate pharmacokinetics of the siRNA, and enable selective delivery of siRNA to target cells. Understanding the mechanisms of assembly and delivery of such systems is complicated by the complexity of the dynamic supramolecular assembly as well as by its subsequent interactions with the biological milieu. We have developed an ex vivo method that provides insight into how LNPs behave when contacted with biological fluids. Pulsed gradient spin echo (PGSE) NMR was used to directly measure the kinetics of poly(ethylene) glycol (PEG) shedding from siRNA encapsulated LNPs in rat serum. The method represents a molecularly specific, real-time, quantitative, and label-free way to monitor the behavior of a nanoparticle surface coating. We believe that this method has broad implications in gaining mechanistic insights into how nanoparticle-based drug delivery vehicles behave in biofluids and is versatile enough to be applied to a diversity of systems.

Biodistribution and metabolism studies of lipid nanoparticle-formulated internally [3H]-labeled siRNA in mice.

Absorption, distribution, metabolism, and excretion properties of a small interfering RNA (siRNA) formulated in a lipid nanoparticle (LNP) vehicle were determined in male CD-1 mice following a single intravenous administration of LNP-formulated [(3)H]-SSB siRNA, at a target dose of 2.5 mg/kg. Tissue distribution of the [(3)H]-SSB siRNA was determined using quantitative whole-body autoradiography, and the biostability was determined by both liquid chromatography mass spectrometry (LC-MS) with radiodetection and reverse-transcriptase polymerase chain reaction techniques. Furthermore, the pharmacokinetics and distribution of the cationic lipid (one of the main excipients of the LNP vehicle) were investigated by LC-MS and matrix-assisted laser desorption ionization mass spectrometry imaging techniques, respectively. Following i.v. administration of [(3)H]-SSB siRNA in the LNP vehicle, the concentration of parent guide strand could be determined up to 168 hours p.d. (post dose), which was ascribed to the use of the vehicle. This was significantly longer than what was observed after i.v. administration of the unformulated [(3)H]-SSB siRNA, where no intact parent guide strand could be observed 5 minutes post dosing. The disposition of the siRNA was determined by the pharmacokinetics of the formulated LNP vehicle itself. In this study, the radioactivity was widely distributed throughout the body, and the total radioactivity concentration was determined in selected tissues. The highest concentrations of radioactivity were found in the spleen, liver, esophagus, stomach, adrenal, and seminal vesicle wall. In conclusion, the LNP vehicle was found to drive the kinetics and biodistribution of the SSB siRNA. The renal clearance was significantly reduced and its exposure in plasma significantly increased compared with the unformulated [(3)H]-SSB siRNA.

Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes.

The transcription factor TORC2 [transducer of regulated cAMP-responsive element-binding protein (CREB) activity 2] is a major regulator of hepatic gluconeogenesis and is increased in hyperglycemic rodent models. Because chronic hyperglycemia and increased hepatic glucose production, via increased gluconeogenesis, is a key feature of type 2 diabetes, an effective in vivo method to efficiently knock down TORC2 could provide a potential therapy for treating hyperglycemia and type 2 diabetes. To assess this, primary mouse hepatocytes, high-fat diet (HFD)-fed mice, and Zucker diabetic fatty (ZDF) rats were treated with a siRNA against TORC2 (siTORC2), which was delivered via a novel lipid nanoparticle system, or control siRNA (siCON). Compared with siCON, administration of siTORC2 resulted in highly efficient, sustained (1-3 wk) knockdown of TORC2 and its gluconeogenic target genes phosphoenolpyruvate carboxykinase and glucose-6-phophatase in primary mouse hepatocytes and in the livers of HFD-fed mice. In mice, this knockdown was specific to the liver and did not occur in kidney, skeletal muscle, or adipose tissue. In HFD-fed mice, siTORC2 reduced in vivo gluconeogenic capacity, fasting hepatic glucose production, and hyperglycemia, and led to improved hepatic and skeletal muscle insulin sensitivity. siTORC2 treatment also improved systemic hyperglycemia in ZDF rats. In conclusion, these results demonstrate the importance of TORC2 in modulating HGP in vivo and highlight a novel, liver-specific siRNA approach for the potential treatment of hyperglycemia and type 2 diabetes.

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.

Activity of stabilized short interfering RNA in a mouse model of hepatitis B virus replication.

To develop synthetic short interfering RNA (siRNA) molecules as therapeutic agents for systemic administration in vivo, chemical modifications were introduced into siRNAs targeted to conserved sites in hepatitis B virus (HBV) RNA. These modifications conferred significantly prolonged stability in human serum compared with unmodified siRNAs. Cell culture studies revealed a high degree of gene silencing after treatment with the chemically modified siRNAs. To assess activity of the stabilized siRNAs in vivo initially, an HBV vector-based model was used in which the siRNA and the HBV vector were codelivered via high-volume tail vein injection. More than a 3 log10 decrease in levels of serum HBV DNA and hepatitis B surface antigen, as well as liver HBV RNA, were observed in the siRNA-treated groups compared with the control siRNA-treated and saline groups. Furthermore, the observed decrease in serum HBV DNA was 1.5 log10 more with stabilized siRNA compared with unmodified siRNA, indicating the value of chemical modification in therapeutic applications of siRNA. In subsequent experiments, standard systemic intravenous dosing of stabilized siRNA 72 hours after injection of the HBV vector resulted a 0.9 log10 reduction of serum HBV DNA levels after 2 days of dosing. In conclusion, these experiments establish the strong impact that siRNAs can have on the extent of HBV infection and underscore the importance of stabilization of siRNA against nuclease degradation.