Mutant huntingtin messenger RNA forms neuronal nuclear clusters in rodent and human brains.

Mutant messenger RNA (mRNA) and protein contribute to the clinical manifestation of many repeat-associated neurological disorders, with the presence of nuclear RNA clusters being a common pathological feature. Yet, investigations into Huntington's disease-caused by a CAG repeat expansion in exon 1 of the huntingtin (HTT) gene-have primarily focused on toxic protein gain-of-function as the primary disease-causing feature. To date, mutant HTT mRNA has not been identified as an in vivo hallmark of Huntington's disease. Here, we report that, in two Huntington's disease mouse models (YAC128 and BACHD-97Q-ΔN17), mutant HTT mRNA is retained in the nucleus. Widespread formation of large mRNA clusters (∼0.6-5 µm3) occurred in 50-75% of striatal and cortical neurons. Cluster formation was independent of age and driven by expanded repeats. Clusters associate with chromosomal transcriptional sites and quantitatively co-localize with the aberrantly processed N-terminal exon 1-intron 1 mRNA isoform, HTT1a. HTT1a mRNA clusters are observed in a subset of neurons from human Huntington's disease post-mortem brain and are likely caused by somatic expansion of repeats. In YAC128 mice, clusters, but not individual HTT mRNA, are resistant to antisense oligonucleotide treatment. Our findings identify mutant HTT/HTT1a mRNA clustering as an early, robust molecular signature of Huntington's disease, providing in vivo evidence that Huntington's disease is a repeat expansion disease with mRNA involvement.

PK-modifying anchors significantly alter clearance kinetics, tissue distribution, and efficacy of therapeutics siRNAs.

Effective systemic delivery of small interfering RNAs (siRNAs) to tissues other than liver remains a challenge. siRNAs are small (∼15 kDa) and therefore rapidly cleared by the kidneys, resulting in limited blood residence times and tissue exposure. Current strategies to improve the unfavorable pharmacokinetic (PK) properties of siRNAs rely on enhancing binding to serum proteins through extensive phosphorothioate modifications or by conjugation of targeting ligands. Here, we describe an alternative strategy for enhancing blood and tissue PK based on dynamic modulation of the overall size of the siRNA. We engineered a high-affinity universal oligonucleotide anchor conjugated to a high-molecular-weight moiety, which binds to the 3' end of the guide strand of an asymmetric siRNA. Data showed a strong correlation between the size of the PK-modifying anchor and clearance kinetics. Large 40-kDa PK-modifying anchors reduced renal clearance by ∼23-fold and improved tissue exposure area under the curve (AUC) by ∼26-fold, resulting in increased extrahepatic tissue retention (∼3- to 5-fold). Furthermore, PK-modifying oligonucleotide anchors allowed for straightforward and versatile modulation of blood residence times and biodistribution of a panel of chemically distinct ligands. The effects were more pronounced for conjugates with low lipophilicity (e.g., N-Acetylgalactosamine [GalNAc]), where significant improvement in uptake by hepatocytes and dose-dependent silencing in the liver was observed.

Bis-choline tetrathiomolybdate prevents copper-induced blood-brain barrier damage.

In Wilson disease, excessive copper accumulates in patients' livers and may, upon serum leakage, severely affect the brain according to current viewpoints. Present remedies aim at avoiding copper toxicity by chelation, for example, by D-penicillamine (DPA) or bis-choline tetrathiomolybdate (ALXN1840), the latter with a very high copper affinity. Hence, ALXN1840 may potentially avoid neurological deterioration that frequently occurs upon DPA treatment. As the etiology of such worsening is unclear, we reasoned that copper loosely bound to albumin, that is, mimicking a potential liver copper leakage into blood, may damage cells that constitute the blood-brain barrier, which was found to be the case in an in vitro model using primary porcine brain capillary endothelial cells. Such blood-brain barrier damage was avoided by ALXN1840, plausibly due to firm protein embedding of the chelator bound copper, but not by DPA. Mitochondrial protection was observed, a prerequisite for blood-brain barrier integrity. Thus, high-affinity copper chelators may minimize such deterioration in the treatment of neurologic Wilson disease.

Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep.

siRNAs comprise a class of drugs that can be programmed to silence any target gene. Chemical engineering efforts resulted in development of divalent siRNAs (di-siRNAs), which support robust and long-term efficacy in rodent and nonhuman primate brains upon direct cerebrospinal fluid (CSF) administration. Oligonucleotide distribution in the CNS is nonuniform, limiting clinical applications. The contribution of CSF infusion placement and dosing regimen on relative accumulation, specifically in the context of large animals, is not well characterized. To our knowledge, we report the first systemic, comparative study investigating the effects of 3 routes of administration - intrastriatal (i.s.), i.c.v., and intrathecal catheter to the cisterna magna (ITC) - and 2 dosing regimens - single and repetitive via an implanted reservoir device - on di-siRNA distribution and accumulation in the CNS of Dorset sheep. CSF injections (i.c.v. and ITC) resulted in similar distribution and accumulation across brain regions. Repeated dosing increased homogeneity, with greater relative deep brain accumulation. Conversely, i.s. administration supported region-specific delivery. These results suggest that dosing regimen, not CSF infusion placement, may equalize siRNA accumulation and efficacy throughout the brain. These findings inform the planning and execution of preclinical and clinical studies using siRNA therapeutics in the CNS.

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system.

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

Serum Deprivation of Mesenchymal Stem Cells Improves Exosome Activity and Alters Lipid and Protein Composition.

Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in serum-deprived conditions are enriched in six protein pathways and one lipid class, dilysocardiolipin. Inspired by these findings, we engineer an "artificial exosome," in which the incorporation of one lipid (dilysocardiolipin) and three proteins (Rab7, Desmoplakin, and AHSG) into conventional neutral liposomes produces vesicles that mimic cargo delivering activity of natural exosomes.

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways.

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

RNAi modulation of placental sFLT1 for the treatment of preeclampsia.

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1 mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia.

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin.

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.

Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.

Effective transvascular delivery of therapeutic oligonucleotides to the brain presents a major hurdle to the development of gene silencing technologies for treatment of genetically defined neurological disorders. Distribution to the brain after systemic administrations is hampered by the low permeability of the blood-brain barrier (BBB) and the rapid clearance kinetics of these drugs from the blood. Here we show that transient osmotic disruption of the BBB enables transvascular delivery of hydrophobically modified small interfering RNA (hsiRNA) to the rat brain. Intracarotid administration of 25% mannitol and hsiRNA conjugated to phosphocholine-docosahexanoic acid (PC-DHA) resulted in broad ipsilateral distribution of PC-DHA-hsiRNAs in the brain. PC-DHA conjugation enables hsiRNA retention in the parenchyma proximal to the brain vasculature and enabled active internalization by neurons and astrocytes. Moreover, transvascular delivery of PC-DHA-hsiRNAs effected Htt mRNA silencing in the striatum (55%), hippocampus (51%), somatosensory cortex (52%), motor cortex (37%), and thalamus (33%) 1 week after administration. Aside from mild gliosis induced by osmotic disruption of the BBB, transvascular delivery of PC-DHA-hsiRNAs was not associated with neurotoxicity. Together, these findings provide proof-of-concept that temporary disruption of the BBB is an effective strategy for the delivery of therapeutic oligonucleotides to the brain.

Efficient Gene Silencing in Brain Tumors with Hydrophobically Modified siRNAs.

Glioblastoma (GBM) is the most common and lethal form of primary brain tumor with dismal median and 2-year survivals of 14.5 months and 18%, respectively. The paucity of new therapeutic agents stems from the complex biology of a highly adaptable tumor that uses multiple survival and proliferation mechanisms to circumvent current treatment approaches. Here, we investigated the potency of a new generation of siRNAs to silence gene expression in orthotopic brain tumors generated by transplantation of human glioma stem-like cells in athymic nude mice. We demonstrate that cholesterol-conjugated, nuclease-resistant siRNAs (Chol-hsiRNAs) decrease mRNA and silence luciferase expression by 90% in vitro in GBM neurospheres. Furthermore, Chol-hsiRNAs distribute broadly in brain tumors after a single intratumoral injection, achieving sustained and potent (>45% mRNA and >90% protein) tumor-specific gene silencing. This readily available platform is sequence-independent and can be adapted to target one or more candidate GBM driver genes, providing a straightforward means of modulating GBM biology in vivoMol Cancer Ther; 17(6); 1251-8. ©2018 AACR.

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo.

Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery.

A High-throughput Assay for mRNA Silencing in Primary Cortical Neurons in vitro with Oligonucleotide Therapeutics.

Primary neurons represent an ideal cellular system for the identification of therapeutic oligonucleotides for the treatment of neurodegenerative diseases. However, due to the sensitive nature of primary cells, the transfection of small interfering RNAs (siRNA) using classical methods is laborious and often shows low efficiency. Recent progress in oligonucleotide chemistry has enabled the development of stabilized and hydrophobically modified small interfering RNAs (hsiRNAs). This new class of oligonucleotide therapeutics shows extremely efficient self-delivery properties and supports potent and durable effects in vitro and in vivo. We have developed a high-throughput in vitro assay to identify and test hsiRNAs in primary neuronal cultures. To simply, rapidly, and accurately quantify the mRNA silencing of hundreds of hsiRNAs, we use the QuantiGene 2.0 quantitative gene expression assay. This high-throughput, 96-well plate-based assay can quantify mRNA levels directly from sample lysate. Here, we describe a method to prepare short-term cultures of mouse primary cortical neurons in a 96-well plate format for high-throughput testing of oligonucleotide therapeutics. This method supports the testing of hsiRNA libraries and the identification of potential therapeutics within just two weeks. We detail methodologies of our high throughput assay workflow from primary neuron preparation to data analysis. This method can help identify oligonucleotide therapeutics for treatment of various neurological diseases.

Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

5΄-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.

5΄-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5΄-vinylphosphonate confers novel properties to siRNAs. Specifically, 5΄-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5΄-to-3΄ exonucleases. Delivery of conjugated siRNAs requires extensive chemical modifications to achieve stability in vivo. Because chemically modified siRNAs are poor substrates for phosphorylation by kinases, and 5΄-phosphate is required for loading into RNA-induced silencing complex, the synthetic addition of a 5΄-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5΄-phosphate is removed within 2 hours. The 5΄-phosphate mimic 5΄-(E)-vinylphosphonate stabilizes the 5΄ end of the guide strand by protecting it from phosphatases and 5΄-to-3΄ exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5΄-(E)-vinylphosphonate stabilizes 5΄ phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.

Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain.

Ligand-conjugated siRNAs have the potential to achieve targeted delivery and efficient silencing in neurons following local administration in the central nervous system (CNS). We recently described the activity and safety profile of a docosahexaenoic acid (DHA)-conjugated, hydrophobic siRNA (DHA-hsiRNA) targeting Huntingtin (Htt) mRNA in mouse brain. Here, we report the synthesis of an amide-modified, phosphocholine-containing DHA-hsiRNA conjugate (PC-DHA-hsiRNA), which closely resembles the endogenously esterified biological structure of DHA. We hypothesized that this modification may enhance neuronal delivery in vivo. We demonstrate that PC-DHA-hsiRNA silences Htt in mouse primary cortical neurons and astrocytes. After intrastriatal delivery, Htt-targeting PC-DHA-hsiRNA induces ∼80% mRNA silencing and 71% protein silencing after 1 week. However, PC-DHA-hsiRNA did not substantially outperform DHA-hsiRNA under the conditions tested. Moreover, at the highest locally administered dose (4 nmol, 50 µg), we observe evidence of PC-DHA-hsiRNA-mediated reactive astrogliosis. Lipophilic ligand conjugation enables siRNA delivery to neural tissues, but rational design of functional, nontoxic siRNA conjugates for CNS delivery remains challenging.

Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain.

The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6-60 µg). Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73%) and cortex (up to 51%) after 1 week. Moreover, following a bilateral intrastriatal injection (60 µg), we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders.

Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.

Delivery represents a significant barrier to the clinical advancement of oligonucleotide therapeutics for the treatment of neurological disorders, such as Huntington's disease. Small, endogenous vesicles known as exosomes have the potential to act as oligonucleotide delivery vehicles, but robust and scalable methods for loading RNA therapeutic cargo into exosomes are lacking. Here, we show that hydrophobically modified small interfering RNAs (hsiRNAs) efficiently load into exosomes upon co-incubation, without altering vesicle size distribution or integrity. Exosomes loaded with hsiRNAs targeting Huntingtin mRNA were efficiently internalized by mouse primary cortical neurons and promoted dose-dependent silencing of Huntingtin mRNA and protein. Unilateral infusion of hsiRNA-loaded exosomes, but not hsiRNAs alone, into mouse striatum resulted in bilateral oligonucleotide distribution and statistically significant bilateral silencing of up to 35% of Huntingtin mRNA. The broad distribution and efficacy of hsiRNA-loaded exosomes delivered to brain is expected to advance the development of therapies for the treatment of Huntington's disease and other neurodegenerative disorders.

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing In Vivo.

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene(®) branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo.