Jonathan K. Watts

Chemically modified siRNA: tools and applications.

Chemical modification provides solutions to many of the challenges facing siRNA therapeutics. This review examines the various siRNA modifications available, including every aspect of the RNA structure and siRNA duplex architecture. The applications of chemically modified siRNA are then examined, with a focus on specificity (elimination of immune effects and hybridization-dependent off-target effects) and delivery. We also discuss improvement of nuclease stability and potency.

Silencing disease genes in the laboratory and the clinic

Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past 20 years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology. Copyright

Enhancer RNA Facilitates NELF Release from Immediate Early Genes

Enhancer RNAs (eRNAs) are a class of long noncoding RNAs (lncRNA) expressed from active enhancers, whose function and action mechanism are yet to be firmly established. Here we show that eRNAs facilitate the transition of paused RNA polymerase II (RNAPII) into productive elongation by acting as a decoy for the negative elongation factor (NELF) complex upon induction of immediate early genes (IEGs) in neurons. eRNAs are synthesized prior to the culmination of target gene transcription and interact with the NELF complex. Knockdown of eRNAs expressed at neuronal enhancers impairs transient release of NELF from the specific target promoters during transcriptional activation, coinciding with a decrease in target mRNA induction. The enhancer-promoter interaction was unaffected by eRNA knockdown. Instead, chromatin looping might enable eRNAs to act locally at a specific promoter. Our findings highlight the spatiotemporally regulated action mechanism of eRNAs during early transcriptional elongation.

The chemical evolution of oligonucleotide therapies of clinical utility

After nearly 40 years of development, oligonucleotide therapeutics are nearing meaningful clinical productivity. One of the key advantages of oligonucleotide drugs is that their delivery and potency are derived primarily from the chemical structure of the oligonucleotide whereas their target is defined by the base sequence. Thus, as oligonucleotides with a particular chemical design show appropriate distribution and safety profiles for clinical gene silencing in a particular tissue, this will open the door to the rapid development of additional drugs targeting other disease-associated genes in the same tissue. To achieve clinical productivity, the chemical architecture of the oligonucleotide needs to be optimized with a combination of sugar, backbone, nucleobase, and 3′- and 5′-terminal modifications. A portfolio of chemistries can be used to confer drug-like properties onto the oligonucleotide as a whole, with minor chemical changes often translating into major improvements in clinical efficacy. One outstanding challenge in oligonucleotide chemical development is the optimization of chemical architectures to ensure long-term safety. There are multiple designs that enable effective targeting of the liver, but a second challenge is to develop architectures that enable robust clinical efficacy in additional tissues.

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing

We report that combining a DNA analog (2′F-ANA) with rigid RNA analogs [2′F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1–1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.

2′-Fluoro-4′-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity

The synthesis of oligonucleotides containing 2′-deoxy-2′-fluoro-4′-thioarabinonucleotides is described. 2′-Deoxy-2′-fluoro-5-methyl-4′-thioarabinouridine (4′S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4′S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4′S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4′-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2′-Deoxy-2′-fluoro-4′-thioarabinouridine (4′S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2′F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.

Clinical status of duplex RNA

Double-stranded RNA has become a ubiquitous tool for inhibition of gene expression in the laboratory. If similar success could be achieved in vivo, duplex RNA might provide a new class of therapeutics capable of treating a broad spectrum of disease. Chemists and biologists developing duplex RNA as a drug have made progress but continue to face challenges. This review presents the current status of duplex RNA in the clinic and comments on future prospects for the approach.

Differential stability of 2′F-ANA•RNA and ANA•RNA hybrid duplexes: roles of structure, pseudohydrogen bonding, hydration, ion uptake and flexibility

Hybrids of RNA with arabinonucleic acids 2′F-ANA and ANA have very similar structures but strikingly different thermal stabilities. We now present a thorough study combining NMR and other biophysical methods together with state-of-the-art theoretical calculations on a fully modified 10-mer hybrid duplex. Comparison between the solution structure of 2′F-ANA•RNA and ANA•RNA hybrids indicates that the increased binding affinity of 2′F-ANA is related to several subtle differences, most importantly a favorable pseudohydrogen bond (2′F–purine H8) which contrasts with unfavorable 2′-OH–nucleobase steric interactions in the case of ANA. While both 2′F-ANA and ANA strands maintained conformations in the southern/eastern sugar pucker range, the 2′F-ANA strand’s structure was more compatible with the A-like structure of a hybrid duplex. No dramatic differences are found in terms of relative hydration for the two hybrids, but the ANA•RNA duplex showed lower uptake of counterions than its 2′F-ANA•RNA counterpart. Finally, while the two hybrid duplexes are of similar rigidities, 2′F-ANA single strands may be more suitably preorganized for duplex formation. Thus the dramatically increased stability of 2′F-ANA•RNA and ANA•RNA duplexes is caused by differences in at least four areas, of which structure and pseudohydrogen bonding are the most important.

Effect of chemical modifications on modulation of gene expression by duplex antigene RNAs that are complementary to non-coding transcripts at gene promoters

Antigene RNAs (agRNAs) are small RNA duplexes that target non-coding transcripts rather than mRNA and specifically suppress or activate gene expression in a sequence-dependent manner. For many applications in vivo, it is likely that agRNAs will require chemical modification. We have synthesized agRNAs that contain different classes of chemical modification and have tested their ability to modulate expression of the human progesterone receptor gene. We find that both silencing and activating agRNAs can retain activity after modification. Both guide and passenger strands can be modified and functional agRNAs can contain 2′F-RNA, 2′OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications. The mechanism of agRNA activity appears to be maintained after chemical modification: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded. These data demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications.

Antisense and antigene inhibition of gene expression by cell-permeable oligonucleotide-oligospermine conjugates.

Oligonucleotides and their derivatives are a proven chemical strategy for modulating gene expression. However, their negative charge remains a challenge for delivery and target recognition inside cells. Here we show that oligonucleotide-oligospermine conjugates (Zip nucleic acids or ZNAs) can help overcome these shortcomings by serving as effective antisense and antigene agents. Conjugates containing DNA and locked nucleic acid (LNA) oligonucleotides are active, and oligospermine conjugation facilitates carrier-free cell uptake at nanomolar concentrations. Conjugates targeting the CAG triplet repeat within huntingtin (HTT) mRNA selectively inhibit expression of the mutant huntingtin protein. Conjugates targeting the promoter of the progesterone receptor (PR) function as antigene agents to block PR expression. These observations support further investigation of ZNA conjugates as gene silencing agents.