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

Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters

Synthetic small duplex RNAs that are fully complementary to gene promoters can silence transcription in mammalian cells. microRNAs (miRNAs) are endogenous small regulatory RNAs that sequence specifically regulate gene expression. We have developed a computational method to identify potential miRNA target sites within gene promoters. Ten candidate miRNAs predicted to target the human progesterone receptor (PR) gene promoter were tested for their ability to modulate gene expression. Several miRNA mimics inhibited PR gene expression and miR-423-5p, which targets a highly conserved region of the PR promoter, was chosen for detailed analysis. Chromatin immunoprecipitation revealed that the miR-423-5p mimic decreased RNA polymerase II occupancy and increased histone H3 lysine 9 dimethylation (H3K9me2) at the PR promoter, indicative of chromatin-level silencing. Transcriptional silencing was transient, independent of DNA methylation, and associated with recruitment of Argonaute 2 (AGO2) to a non-coding RNA (ncRNA) transcript that overlaps the PR gene promoter. The miR-423-5p mimic also silenced expression of immunoglobulin superfamily member 1 (IGSF1), an additional gene with a predicted target site within its promoter. While additional investigations of endogenous miRNA function will be necessary, these observations suggest that recognition of gene promoters by miRNAs may be a natural and general mechanism for regulating gene transcription

Cellular Delivery of Locked Nucleic Acids (LNAs)

Locked nucleic acids (LNAs) are RNA derivatives that have an O‐methylene linkage between the 2 and 4 positions of the ribose. This leads to exceptionally high‐affinity binding to complementary sequences. They are synthesized using standard DNA/RNA synthesis methods, and have a negatively charged backbone that confers good solubility. This unit describes a method for the introduction of LNA oligomers into cells. A support protocol also describes the determination of melting temperatures for LNA oligomers.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143781/1/cpnc0413.pd

\u27The World is Too Messy\u27: The Challenge of Historical Literacy in a General-Education Course

The problem with implementing writing-intensive (or writing-emphasis) liberal arts general education (GE) courses is as evident as it is underacknowledged; that is, this seemingly well-bounded, modest reform involves significant change-on multiple levels and for multiple players in complex institutional and disciplinary activity systems. We know a little about such issues: Writing-across-the-curriculum (WAC) programs are often said to present new challenges for teachers, such as a felt tension between the demands of teaching writing and covering content, and the need for substantial background in writing pedagogy (Russell, Writing in the Academic Disciplines). Although such tensions are both urgent and real, they do not reflect the depth and range of changes a movement to discipline-based writing can entail-in terms of institutional traditions, departmental modes of operation, classroom practice, and students\u27 understandings. And it is crucial to remember that liberal arts courses are almost always in some discipline. Michael Fullan\u27s work on educational change suggests that although these processes may appear technically simple, they are in fact socially complex (The New Meaning 5), invoking individual and collective feelings of loss, anxiety, and struggle (The New Meaning 31; Fullan Change Forces). When attempting change, cautions Fullan, a good rule of thumb is to assume that changing the culture of institutions is the real agenda, not implementing single innovations (The New Meaning 107)

Shrinkage Estimation of Expression Fold Change As an Alternative to Testing Hypotheses of Equivalent Expression

Research on analyzing microarray data has focused on the problem of identifying differentially expressed genes to the neglect of the problem of how to integrate evidence that a gene is differentially expressed with information on the extent of its differential expression. Consequently, researchers currently prioritize genes for further study either on the basis of volcano plots or, more commonly, according to simple estimates of the fold change after filtering the genes with an arbitrary statistical significance threshold. While the subjective and informal nature of the former practice precludes quantification of its reliability, the latter practice is equivalent to using a hard-threshold estimator of the expression ratio that is not known to perform well in terms of mean-squared error, the sum of estimator variance and squared estimator bias. On the basis of two distinct simulation studies and data from different microarray studies, we systematically compared the performance of several estimators representing both current practice and shrinkage. We find that the threshold-based estimators usually perform worse than the maximum-likelihood estimator (MLE) and they often perform far worse as quantified by estimated mean-squared risk. By contrast, the shrinkage estimators tend to perform as well as or better than the MLE and never much worse than the MLE, as expected from what is known about shrinkage. However, a Bayesian measure of performance based on the prior information that few genes are differentially expressed indicates that hard-threshold estimators perform about as well as the local false discovery rate (FDR), the best of the shrinkage estimators studied. Based on the ability of the latter to leverage information across genes, we conclude that the use of the local-FDR estimator of the fold change instead of informal or threshold-based combinations of statistical tests and non-shrinkage estimators can be expected to substantially improve the reliability of gene prioritization at very little risk of doing so less reliably

Synthesis and Purification of Peptide Nucleic Acids

Peptide nucleic acids (PNAs) are DNA analogs in which the normal phosphodiester backbone is replaced by 2‐aminoethyl glycine linkages. Hybridization of PNAs with RNA or DNA follows normal rules for Watson‐Crick base pairing and occurs with high affinity. Thus, PNAs are a promising choice for applications that benefit from high‐affinity hybridization. They are assembled using techniques adapted from peptide chemistry. Protocols are given for both automated and manual synthesis of PNAs as well as their purification. The advantages of each method are discussed, as are the different monomers and reagents that are required. Additionally, protocols are given for adding peptides to PNAs (which can enhance hybridization or cell uptake of the PNA) and for adding a biotin label.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143745/1/cpnc0411.pd

Levo-Tetrahydropalmatine Attenuates Cocaine Self-Administration under a Progressive-Ratio Schedule and Cocaine Discrimination in Rats

Levo-tetrahydropalmatine (l-THP) is an alkaloid found in many traditional Chinese herbal preparations and has a unique pharmacological profile that includes dopamine receptor antagonism. Previously we demonstrated that l-THP attenuates fixed-ratio (FR) cocaine self-administration (SA) and cocaine-induced reinstatement in rats at doses that do not alter food-reinforced responding. This study examined the effects of l-THP on cocaine and food SA under progressive-ratio (PR) schedules of reinforcement and the discriminative stimulus effects of cocaine. In adult male Sprague–Dawley rats self-administering cocaine (0.5 or 1.0 mg/kg/inf), l-THP significantly reduced breaking points at the 1.875, 3.75 and 7.5 mg/kg doses. l-THP also reduced the breaking point and response rate for PR SA of sucrose-sweetened food pellets, although the decrease was significant only at the 7.5 mg/kg l-THP dose. In rats trained to discriminate cocaine (10 mg/kg, ip) from saline, l-THP (1.875, 3.75 and 7.5 mg/kg) produced a rightward shift in the dose–response curve for cocaine generalization. During generalization testing, l-THP reduced response rate, but only at the 7.5 mg/kg dose. l-THP also prevented substitution of the dopamine D2/D3 receptor agonist, (±) 7-OH-DPAT, for cocaine suggesting a potential role for antagonism of D2 and/or D3 receptors in the effects of l-THP. These data further demonstrate that l-THP attenuates the reinforcing and subjective effects of cocaine at doses that do not produce marked motor effects and provide additional evidence that l-THP may have utility for the management of cocaine addiction

Five Facets of Mindfulness and Psychological Health: Evaluating a Psychological Model of the Mechanisms of Mindfulness

There has been an increasing focus on determining the psychological mechanisms underlying the broad effects of mindfulness on psychological health. Mindfulness has been posited to be related to the construct of reperceiving or decentering, defined as a shift in perspective associated with decreased attachment to one\u27s thoughts and emotions. Decentering is proposed to be a meta-mechanism that mobilizes four psychological mechanisms (cognitive flexibility, values clarification, self-regulation, and exposure), which in turn are associated with positive health outcomes. Despite preliminary support for this model, extant studies testing this model have not examined distinct facets of mindfulness. The present study used a multidimensional measure of mindfulness to examine whether this model could account for the associations between five facets of mindfulness and psychological symptoms (depressive symptoms, stress, anxiety symptoms, alcohol-related problems) in a sample of college students (N = 944). Our findings partially support this model. We found significant double-mediated associations in the expected directions for all outcomes (stress, anxiety symptoms, and depressive symptoms) except alcohol-related problems, and for each of the facets of mindfulness except observing. However, decentering and the specific mechanisms did not fully mediate the associations among mindfulness facets and psychological health outcomes. Experimental and ecological momentary assessment designs are needed to understand the psychological processes that account for the beneficial effects of mindfulness