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

Detecting chromatin interactions along and between sister chromatids with SisterC [preprint]

Accurate chromosome segregation requires chromosome compaction with concordant disentanglement of the two sister chromatids. This process has been studied extensively by microscopy but has remained a challenge for genomic methods, such as Hi-C, because sister chromatids have identical DNA sequences. Here we describe SisterC, a chromosome conformation capture assay that can distinguish interactions between and within sister chromatids. The assay is based on BrdU incorporation during S-phase, which labels the newly replicated strands of the sister chromatids. This is followed by Hi-C, e.g. during different stages of mitosis, and the selective destruction of BrdU containing strands by UV/Hoechst treatment. After PCR amplification and sequencing of the remaining intact strands, this allows for the assignment of Hi-C products as inter- and intra-sister interactions by read orientation. We performed SisterC on mitotically arrested S. cerevisiae cells. As expected, we find prominent interactions and alignment of sister chromatids at their centromeres. Along the arms, sister chromatids are less precisely aligned with inter-sister connections every ~35kb. In many instances, inter-sister interactions do not involve the interaction of two identical loci but occur between cohesin binding sites that can be offset by 5 to 25kb. Along sister chromatids, extruding cohesin forms loops up to 50kb. Combined, SisterC allows the observation of the complex interplay between sister chromatid compaction and sister chromatid segregation as the cell transitions from late S-phase to mitosis. SisterC should be applicable to study mitotic events in a wide range of organisms and cell types

Oligonucleotides targeting TCF4 triplet repeat expansion inhibit RNA foci and mis-splicing in Fuchs\u27 dystrophy

Fuchs\u27 endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has approximately 2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD

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

Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing

RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2\u27-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes

Heavily and Fully Modified RNAs Guide Efficient SpyCas9-Mediated Genome Editing [preprint]

RNA-based drugs depend on chemical modifications to increase potency and nuclease stability, and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. No studies have yet explored chemical modification at all positions of the crRNA guide and tracrRNA cofactor. Here, we have identified several heavily-modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2\u27-OH groups) that are functional in human cells. These designs demonstrate a significant breakthrough for Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides

Universal fractal scaling of self-organized networks

There is an abundance of literature on complex networks describing a variety of relationships among units in social, biological, and technological systems. Such networks, consisting of interconnected nodes, are often self-organized, naturally emerging without any overarching designs on topological structure yet enabling efficient interactions among nodes. Here we show that the number of nodes and the density of connections in such self-organized networks exhibit a power law relationship. We examined the size and connection density of 46 self-organizing networks of various biological, social, and technological origins, and found that the size-density relationship follows a fractal relationship spanning over 6 orders of magnitude. This finding indicates that there is an optimal connection density in self-organized networks following fractal scaling regardless of their sizes

The network topology of a potential energy landscape: A static scale-free network

Here we analyze the topology of the network formed by the minima and transition states on the potential energy landscape of small clusters. We find that this network has both a small-world and scale-free character. In contrast to other scale-free networks, where the topology results from the dynamics of the network growth, the potential energy landscape is a static entity. Therefore, a fundamentally different organizing principle underlies this behaviour: The potential energy landscape is highly heterogeneous with the low-energy minima having large basins of attraction and acting as the highly-connected hubs in the network.Comment: 4 pages, 4 figures, revtex

Effect of aerobic exercise on amyloid accumulation in preclinical Alzheimer’s: A 1-year randomized controlled trial

Background Our goal was to investigate the role of physical exercise to protect brain health as we age, including the potential to mitigate Alzheimer’s-related pathology. We assessed the effect of 52 weeks of a supervised aerobic exercise program on amyloid accumulation, cognitive performance, and brain volume in cognitively normal older adults with elevated and sub-threshold levels of cerebral amyloid as measured by amyloid PET imaging. Methods and findings This 52-week randomized controlled trial compared the effects of 150 minutes per week of aerobic exercise vs. education control intervention. A total of 117 underactive older adults (mean age 72.9 [7.7]) without evidence of cognitive impairment, with elevated (n = 79) or subthreshold (n = 38) levels of cerebral amyloid were randomized, and 110 participants completed the study. Exercise was conducted with supervision and monitoring by trained exercise specialists. We conducted 18F-AV45 PET imaging of cerebral amyloid and anatomical MRI for whole brain and hippocampal volume at baseline and Week 52 follow-up to index brain health. Neuropsychological tests were conducted at baseline, Week 26, and Week 52 to assess executive function, verbal memory, and visuospatial cognitive domains. Cardiorespiratory fitness testing was performed at baseline and Week 52 to assess response to exercise. The aerobic exercise group significantly improved cardiorespiratory fitness (11% vs. 1% in the control group) but there were no differences in change measures of amyloid, brain volume, or cognitive performance compared to control. Conclusions Aerobic exercise was not associated with reduced amyloid accumulation in cognitively normal older adults with cerebral amyloid. In spite of strong systemic cardiorespiratory effects of the intervention, the observed lack of cognitive or brain structure benefits suggests brain benefits of exercise reported in other studies are likely to be related to non-amyloid effects