RACE-Seq identifies the Argonaute-2 cleavage products of RNA interference-based oligonucleotides

The recent announcement of the first successful Phase III clinical trial of a RNA interference (RNAi)-based therapeutic is a major achievement in the field. Synthetic RNAi therapeutic oligonucleotides are either first cleaved by Dicer or incorporate directly into the Argonaute-2 RNA-induced silencing complex (AGO2-RISC) and directs the protein complex to homologous RNA. Cleavage of target RNA occurs opposite bases 10-11 when counting from the 5’ end of the hybridized siRNA guide strand. The capture and identification of these cleaved products by 5’ Rapid Amplification of cDNA Ends and Sanger sequencing remains the gold standard for confirming Argonaute-2 mediated RNAi cleavage. Next Generation Sequencing of 5’ RACE has brought new insights into the biological activity of RNAi-based oligonucleotides. This work currently represents the largest undertaking using RACE-Seq to investigate AGO2-RISC-mediated activity. RACESeq reported the expected RISC-cleaved product for each of the oligonucleotides investigated. Additionally, RACE-Seq analysis revealed that some of the oligonucleotides could be processed into multiple active siRNA molecules. Analysis of the activity of a Dicer substrate siRNA targeting transthyretin revealed that this molecule by-passed Dicer processing but still induced RNAi activity. In examining RACE-Seq peak profiles, an on-target mechanism of action (MOA) for up to four active siRNA derived from siRNA19 is proposed. The shRNA19 RACE-Seq assay predicted that this hairpin molecule probably exists as two distinct forms, one with a 7-nucleotide loop and the other with a 5-nucleotide loop. The project also focused on optimising the library preparation, data filtering and data presentation for RACE-Seq. A simplified, low computation data analysis pipeline was designed and used to align the filtered dataset to a reference sequence and to count the 5’ ends. RACE-Seq is presented as a suitable solution for investigating, discriminating and quantifying specific RNA cleavage events and visualizing evidence for an on-target MOA of RNAi based oligonucleotide therapeutics

Clinical potential of oligonucleotide-based therapeutics in the respiratory system

The discovery of an ever-expanding plethora of coding and non-coding RNAs with nodal and causal roles in the regulation of lung physiology and disease is reinvigorating interest in the clinical utility of the oligonucleotide therapeutic class. This is strongly supported through recent advances in nucleic acids chemistry, synthetic oligonucleotide delivery and viral gene therapy that have succeeded in bringing to market at least three nucleic acid-based drugs. As a consequence, multiple new candidates such as RNA interference modulators, antisense, and splice switching compounds are now progressing through clinical evaluation. Here, manipulation of RNA for the treatment of lung disease is explored, with emphasis on robust pharmacological evidence aligned to the five pillars of drug development: exposure to the appropriate tissue, binding to the desired molecular target, evidence of the expected mode of action, activity in the relevant patient population and commercially viable value proposition.</p

Safe and Efficient Silencing with a Pol II, but not a Pol lII, Promoter Expressing an Artificial miRNA Targeting Human Huntingtin

Huntington\u27s disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken β-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CβA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CβA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CβA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CβA promoter can provide an effective and safe dose of a human huntingtin miRNA

miR-196b target screen reveals mechanisms maintaining leukemia stemness with therapeutic potential.

We have shown that antagomiR inhibition of miRNA miR-21 and miR-196b activity is sufficient to ablate MLL-AF9 leukemia stem cells (LSC) in vivo. Here, we used an shRNA screening approach to mimic miRNA activity on experimentally verified miR-196b targets to identify functionally important and therapeutically relevant pathways downstream of oncogenic miRNA in MLL-r AML. We found Cdkn1b (p27Kip1) is a direct miR-196b target whose repression enhanced an embryonic stem cell–like signature associated with decreased leukemia latency and increased numbers of leukemia stem cells in vivo. Conversely, elevation of p27Kip1 significantly reduced MLL-r leukemia self-renewal, promoted monocytic differentiation of leukemic blasts, and induced cell death. Antagonism of miR-196b activity or pharmacologic inhibition of the Cks1-Skp2–containing SCF E3-ubiquitin ligase complex increased p27Kip1 and inhibited human AML growth. This work illustrates that understanding oncogenic miRNA target pathways can identify actionable targets in leukemia

Achilles Is a Circadian Clock-controlled Gene That Regulates the Immune System and Its Rhythmicity in Drosophila

Circadian clock is a transcriptional/translational feedback loop that drives the rhythmic expression of downstream mRNAs. Termed “clock-controlled genes,” these molecular outputs of the circadian clock orchestrate cellular, metabolic, and behavioral rhythms. As part of our on-going work to characterize key upstream regulators of circadian mRNA expression, we have identified a novel clock-controlled gene in Drosophila melanogaster, Achilles (Achl), which is rhythmic at the mRNA level in the brain and represses expression of immune response genes, especially anti-microbial peptides in the immune system. Achl knock-down in the brain dramatically elevates expression of crucial immune response genes, including IM1 (Immune induced molecule 1), Mtk (Metchnikowin), and Drs (Drosomysin). As a result, flies with knocked-down Achl expression are more resistant to bacterial challenges. Meanwhile, no significant change in core clock gene expression and locomotor activity is observed, suggesting that Achl influences rhythmic mRNA outputs rather than directly regulating the core timekeeping mechanism. Additionally, Achl knock-down in the brain disrupts the rhythmicity of the immune system. Flies with knocked-down Achl show altered rhythmicity in both survival towards infection and sensitivity of immune response gene induction upon infection. Using high-throughput RNA-sequencing, we also identified candidate clock controlled genes that are downstream of Achl. Notably, Achl knock-down in the absence of immune challenge significantly diminishes the fly’s overall lifespan and resistance towards starvation, indicating a behavioral or metabolic cost of constitutively activating this pathway. Together, our data demonstrate that (1) Achl is a novel clock-controlled gene that (2) regulates the immune system in a repressive manner. (3) Achl regulates the rhythmicity of the immune system, and (4) Achl participates in signaling from the brain to immunological tissues

HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species

This is the author accepted manuscript. The final version is available from AAAS via the DOI in this recordData and materials availability: The full dataset of the dsRNA screen in Drosophila S2R+cells is available at www.flyrnai.org/screensummary. All other data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.Inactivation of the VHL tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and VHL inactivation in two species (human and Drosophila) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of VHL−/− ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α–dependent VHL−/− ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α–independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors.National Cancer InstituteDana-Farber Cancer InstituteHoward Hughes Medical InstituteNational Institute of General Medical Science

Identification of small interfering RNA targeting Signal Transducer and Activator of Transcription 6: Characterisation and selection of candidates for pre-clinical development

The interleukin (IL)-13 pathway and its associated transcription factor, signal transducer and activator of transcription 6 (STAT6), have been clearly implicated in the pathogenesis of bronchial asthma. We have developed a system to effectively screen the STAT6 gene for targeting with small interfering (si) RNA molecules. By incorporating an in silico and in vitro screening system we were able to identify fourteen siRNA molecules suitable for pre-clinical drug development. Furthermore, we were able to demonstrate that modification of certain siRNAs, designed to improve in vivo longevity, was possible without significant loss of target knockdown efficacy and that the siRNA produced by our selection process did not induce demonstrable interferon responses. These data suggest that several STAT6-targeting siRNA suitable for pre-clinical development are available for potential use in the treatment of asthma

A CNS-Active siRNA Chemical Scaffold for the Treatment of Neurodegenerative Diseases

Small interfering RNAs (siRNAs) are a promising class of drugs for treating genetically-defined diseases. Therapeutic siRNAs enable specific modulation of gene expression, but require chemical architecture that facilitates efficient in vivodelivery. siRNAs are informational drugs, therefore specificity for a target gene is defined by nucleotide sequence. Thus, developing a chemical scaffold that efficiently delivers siRNA to a particular tissue provides an opportunity to target any disease-associated gene in that tissue. The goal of this project was to develop a chemical scaffold that supports efficient siRNA delivery to the brain for the treatment of neurodegenerative diseases, specifically Huntington’s disease (HD). HD is an autosomal dominant neurodegenerative disorder that affects 3 out of every 100,000 people worldwide. This disorder is caused by an expansion of CAG repeats in the huntingtin gene that results in significant atrophy in the striatum and cortex of the brain. Silencing of the huntingtin gene is considered a viable treatment option for HD. This project: 1) identified a hyper-functional sequence for siRNA targeting the huntingtin gene, 2) developed a fully chemically modified architecture for the siRNA sequence, and 3) identified a new structure for siRNA central nervous system (CNS) delivery—Divalent-siRNA (Di-siRNA). Di-siRNAs, which are composed of two fully chemically-stabilized, phosphorothioate-containing siRNAs connected by a linker, support potent and sustained gene modulation in the CNS of mice and non-human primates. In mice, Di-siRNAs induced potent silencing of huntingtin mRNA and protein throughout the brain one month after a single intracerebroventricular injection. Silencing persisted for at least six months, with the degree of gene silencing correlating to guide strand tissue accumulation levels. In Cynomolgus macaques, a bolus injection exhibited significant distribution and robust silencing throughout the brain and spinal cord without detectable toxicity. This new siRNA scaffold opens the CNS for RNAi-based gene modulation, creating a path towards developing treatments for genetically-defined neurological disorders

Locked nucleic acid (LNA) mediated improvements in siRNA stability and functionality

Therapeutic application of the recently discovered small interfering RNA (siRNA) gene silencing phenomenon will be dependent on improvements in molecule bio-stability, specificity and delivery. To address these issues, we have systematically modified siRNA with the synthetic RNA-like high affinity nucleotide analogue, Locked Nucleic Acid (LNA). Here, we show that incorporation of LNA substantially enhances serum half-life of siRNA's, which is a key requirement for therapeutic use. Moreover, we provide evidence that LNA is compatible with the intracellular siRNA machinery and can be used to reduce undesired, sequence-related off-target effects. LNA-modified siRNAs targeting the emerging disease SARS, show improved efficiency over unmodified siRNA on certain RNA motifs. The results from this study emphasize LNA's promise in converting siRNA from a functional genomics technology to a therapeutic platform

A preclinical platform for assessing antitumor effects and systemic toxicities of cancer drug targets

SignificanceMany new cancer drugs fail at the clinical stage owing to poor efficacy and/or excessive toxicity, though whether this reflects shortcomings of the target or the drug is often unclear. To gain earlier insights into factors that can influence the therapeutic index of target inhibition in vivo, we combine inducible RNA interference and somatic engineering technologies to produce a cost-effective platform that enables systemic and inducible suppression of candidate target in normal tissues and tumor cells in the same mouse. By comparing the consequences of genetic and pharmacological CDK9 inhibition, we establish the utility of this platform to predict factors influencing the therapeutic index. Additionally, our studies provide support, and some cautionary notes, for the clinical development of CDK9 inhibitors