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Posts Tagged ‘anticancer drugs’


Rosa’s to like

Curator & Reporter: Larry H. Bernstein, MD, FCAP

 

 

Reality Check: Cancer Experts Discuss Hurdles Facing CAR-T Therapy

http://www.xconomy.com/national/2015/09/18/reality-check-cancer-experts-discuss-hurdles-facing-car-t-therapy/

BEN FIDLER

September 18th, 2015

There’s a lot of excitement these days about a type of cellular immunotherapy known as CAR-T, a method of modifying peoples’ immune cells to fight cancer. But you could also fill a book listing all the problems its makers will have to solve—how to test, manufacture, and even the define the nature of these cancer-killing cells—before the CAR-T story is a successful one.

These hurdles, not the hype, were the subject of a panel of experts from industry, academia, and the FDA at the Inaugural International Cancer Immunotherapy Conference in New York Thursday afternoon. The panelists included University of Pennsylvania professor Carl June, whose work has led to programs now in clinical testing at Novartis; Adaptimmune executive vice president Gwendolyn Binder-Scholl; and GlaxoSmithKline’s head of immuno-oncology Cedrik Britten, among others.

CAR-T stands for chimeric antigen receptor T cell, which describes an engineered version of the immune system’s attack dogs. CAR-T cells are a patient’s own T cells altered outside the body to be cancer killers, then put back in to go after tumor cells.

CAR-T therapies from Novartis, Juno Therapeutics (NASDAQ: JUNO), and Kite Pharma (NASDAQ: KITE) have produced impressive results so far for certain blood cancers, leading to long-lasting remissions in some patients.

But the field is early in its development. Researchers are trying to figure out how to make these therapies useful for more common cancers, such as lung, breast, and ovarian, and how to mitigate the overactive immune responses they can cause. Biotechs and pharma companies developing autologous therapies—which modify the cells of each individual patient—are wrestling with how to manufacture and distribute them at scale.

But a different, larger question looms, and it gets to the heart of why autologous T cell therapy is truly a new medical frontier. The cells that are delivered back into the patient are not what ends up doing the bulk of the therapeutic work.

The panelists Thursday noted how T cell therapies could throw a wrench into a typical, and crucial, clinical strategy. Early in the clinical testing of a drug, companies usually run what are known as dose escalation studies. Different doses of products are tested, low to high, to establish a trend of responses, see what safety issues pop up, and pick the optimal dose to move forward.

But because CAR-T cell populations expand once they’re put back into a patient’s body, doses are harder to define. What’s more, cranking up a dose for such a powerful therapy could be dangerous. “Are classical trial designs applicable, or do they have to be changed?” asked GSK’s Britten. “You can not have a simple dose escalation [study] with a drug that replicates.”

Adaptimmune’s Binder-Scholl called for more guidance from regulators to help figure out a more standardized scheme for dose escalation studies.

“I think the biology is going to make [that type of guidance] awfully challenging,” says Juno’s chief financial officer Steve Harr, who also wasn’t on the panel. “I would like to think over time we get into something a bit more predictable, and maybe we have some type of a standard, but we’re very early in this process.”

 

Medscape Cardiology Black on Cardiology

SPRINT Hypertension Trial: Preliminary Results Discussed

http://www.medscape.com/viewarticle/851134?nlid=88408_2021&src=wnl_edit_medp_card&uac=211176CK&spon=2&impID=829281&faf=1

Henry R. Black, MD; William C. Cushman, MD    Disclosures | Sept 18, 2015

Stopped Early for Benefit

Henry R. Black, MD: Hi. I’m Dr Henry Black. I’m adjunct professor of medicine at the Langone New York University School of Medicine, and I’m here today with my long-term friend and colleague, Dr Bill Cushman. Bill, thank you very much for doing this.

William C. Cushman, MD: Delighted to be here.

Dr Black: What I want to talk about is the SPRINT study,[1]which you’ve been a primary participant in. The top-line resultswere just released. Tell us a little bit about SPRINT: who was in it, what the hypothesis was, and how it compares to the ACCORD study, which you also participated in.

Dr Cushman: Sure. I’m Dr Bill Cushman. I’m from Memphis, Tennessee. And I’m chief of the preventive medicine section at the VA and professor of preventive medicine at the University of Tennessee.

I was a network principal investigator in SPRINT, which meant that I oversaw about a quarter of the sites. SPRINT was a study sponsored by the National Institutes of Health (NIH), primarily the National Heart, Lung, and Blood Institute (NHLBI). But other institutes—the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute on Aging, the National Institute of Neurological Disorders and Stroke—were also involved.

SPRINT was a study of 9361 participants who were randomized to either a lower, more intensive goal of less than 120 mm Hg systolic blood pressure (SBP) compared with a goal of less than 140 mm Hg systolic. That was considered standard when we designed the study, and all guidelines recommended at least getting below 140 mm Hg.

We recruited a participant pool of high-risk hypertensive patients with SBPs of ≥130 mm Hg. They could be on medications (the majority were), but they didn’t have to be. Participants not only had to have elevated blood pressure, but they also had to be above age 50 and they had to have some other indices of risk: known cardiovascular disease, chronic kidney disease, or being above age 75, for example, or having a Framingham risk assessment for cardiovascular disease of ≥ 15% over 10 years.

They were randomly allocated to these two groups, with the intent of being followed for about 5 years. The primary outcome in SPRINT was a combined cardiovascular outcome that included myocardial infarction (MI), acute coronary syndrome other than MI, stroke, heart failure, or cardiovascular death.

Now, there are a lot of other outcomes in SPRINT, including whether this lower blood pressure goal would prevent dementia, changes on MRI, or chronic kidney disease. Those outcomes have not been stopped or announced yet, and we’re still collecting data on that.

The cardiovascular outcomes were viewed as so positive in terms of the benefit that the Data and Safety Monitoring Board recommended to Gary Gibbons, the director of the NHLBI, that the cardiovascular part of the trial—and the intensive intervention in particular—should be stopped and that the investigators and the participants should be unblinded. And that was done.

Dr Black: Were the antihypertensive regimens prescribed, or was it whatever the docs wanted to do?

Dr Cushman: Good point. We actually recommended using the major classes that were proven to be of benefit in cardiovascular outcome trials in hypertension: either thiazide-type diuretics, ACE inhibitors, angiotensin receptor blockers, or calcium blockers. It was primarily those four classes, and they could be combined in whatever way the investigators wanted. We did put a lot of emphasis on using thiazide-type diuretics because of the ALLHAT[2] results.

But the way they could be combined was really up to the investigators. Now, if the participants had known coronary disease or some other indication for a beta-blocker, that could certainly be used. And then other drugs could be added. We had a very large formulary representative of all the major classes of drugs— not only those classes, but also beta-blockers, alpha blockers, aldosterone inhibitors (spironolactone or amiloride, for example).

We had a lot of drugs available. They were predominantly purchased for the study, by NIH. There were only two drugs that were donated by the pharmaceutical companies. The study was entirely funded by NIH.

SPRINT vs ACCORD

Dr Black: How is this different from ACCORD?[3]

Dr Cushman: In ACCORD, we had the same two SBP goals: less than 120 mm Hg compared with a SBP of less than 140 mm Hg. However, SPRINT is twice as large as ACCORD.

As you may remember, we did not show a significant benefit for the lower SBP goal for the overall cardiovascular outcome in the ACCORD trial. We did see a significant reduction in stroke of about 40%, but that was a secondary outcome. The primary outcomes in mortality were not reduced in ACCORD.

However, ACCORD was about half the size of SPRINT. And even though the ACCORD blood pressure study was done in patients with diabetes, on average, they were probably a little lower-risk than our SPRINT participants because of their somewhat younger age (average age, 62 years), the absence of real chronic kidney disease, and several other reasons.

Even though ACCORD didn’t show a statistically significant benefit, it did show a 12% reduction in the cardiovascular outcome with a confidence interval that could have included up to a 27% benefit.

In contrast, SPRINT was twice as large, with a higher-risk population with an older average age. We excluded people with diabetes because that was being looked at in ACCORD. And we excluded people who’d had a prior stroke because that was being looked at in the SPS3[4] post-stroke study in terms of blood pressure goals.

Despite that, we had a very high-risk population. And what we found was about a third of a reduction in the primary cardiovascular events. That was significant.

We also saw, quite importantly, about a 25% reduction in all-cause mortality. That was surprising. The results are quite clear that there’s dramatic benefit in terms of both cardiovascular events and total mortality.

Dr Black: You probably can’t tell us this yet, but what was the blood pressure achieved in the less-than-140 group compared with the less-than-120 group?

We also saw, quite importantly, about a 25% reduction in all-cause mortality. That was surprising. The results are quite clear that there’s dramatic benefit in terms of both cardiovascular events and total mortality.

 

Diabetes Drug Empagliflozin Cuts CV Deaths in Landmark EMPA-REG Trial

http://www.medscape.com/viewarticle/851114?nlid=88408_2021&src=wnl_edit_medp_card&uac=211176CK&spon=2&impID=829281&faf=1

Lisa Nainggolan

STOCKHOLM ( updated with commentary ) — Patients with type 2 diabetes and established cardiovascular disease receiving the glucose-lowering agent empagliflozin (Jardiance, Boehringer Ingelheim/Lilly), a sodium glucose cotransporter-2 (SGLT-2) inhibitor, were less likely to die than those taking placebo in the large, much-anticipated EMPA-REG OUTCOME study, hailed here as a landmark trial.

The benefit on survival was seen regardless of the cause of death — empagliflozin prevented one in three cardiovascular deaths, with a significant 38% relative risk reduction in cardiovascular mortality, as well as a significant 32% relative reduction in all-cause mortality.

CV death was one component of the primary composite outcome, which also included nonfatal myocardial infarction (MI) or nonfatal stroke. It was the CV mortality benefit, however, that primarily drove the reduction in this end point.

“Empagliflozin is reducing death, the ultimate outcome,” senior author of the study, Silvio Inzucchi, MD, of Yale Diabetes Center, New Haven, Connecticut, told Medscape Medical News. “This is a first in my lifetime — a diabetes drug trial that has shown improved outcomes in high-risk cardiovascular patients.”

This is a first in my lifetime — a diabetes drug trial that has shown improved outcomes in high-risk cardiovascular patients.

Dr Inzucchi was given multiple rounds of applause as he presented the findings of EMPA-REG OUTCOME here at the European Association for the Study of Diabetes (EASD) 2015 Meeting, The study was also published simultaneously in the New England Journal of Medicine, by a team led by Bernard Zinman MD, director, Diabetes Centre, Mount Sinai Hospital, Toronto, Ontario.

 

Sept 17, 2015   http://dx.doi.org:/10.1056/NEJMoa1504720

Type 2 diabetes is a major risk factor for cardiovascular disease,1,2 and the presence of both type 2 diabetes and cardiovascular disease increases the risk of death.3 Evidence that glucose lowering reduces the rates of cardiovascular events and death has not been convincingly shown,4-6although a modest cardiovascular benefit may be observed after a prolonged follow-up period.7Furthermore, there is concern that intensive glucose lowering or the use of specific glucose-lowering drugs may be associated with adverse cardiovascular outcomes.8 Therefore, it is necessary to establish the cardiovascular safety benefits of glucose-lowering agents.9

 

Cohen’s Brain Bits: Let the Sunshine in?

http://www.medpagetoday.com/Blogs/CohensBrainBits/53630?xid=nl_mpt_DHE_2015-09-19&eun=g337145d0r    Published: Sep 18, 2015

By Joshua Cohen MD, MPH

Vitamin D is actually not a vitamin at all — it is a group of fat-soluble steroid hormones responsible for a host of important functions in the body. As it is found in low levels in most foods other than fish and dairy, vitamin D is primarily synthesized from cholesterol in the skin upon exposure to UVB radiation.

While the discovery of vitamin D nearly a century ago stemmed from its role in calcium homeostasis and metabolism, an abundance of studies in the past decade have demonstrated the critical role vitamin D plays in neuronal development and protection. Indeed, in the past few years, researchers have uncovered an association between vitamin D deficiency and an array of important neurologic diseases.

study in this week’s JAMA Neurology investigated the relationship between vitamin D levels, as measured in the blood as 25-hydroxyvitamin D, and the rate of cognitive decline in a population of 382 multi-ethnic older adults. Both vitamin D insufficient (12-20 ng/mL) and deficient (<12 ng/mL) participants demonstrated accelerated cognitive decline in multiple functional domains, especially episodic memory and executive function, that are the domains most affected in patients with Alzheimer’s dementia.

Previous studies have emphasized the essential role of vitamin D in the brain and have raised concern about the effect of vitamin D deficiency on the brain. Vitamin D’s neuroprotective roles include stimulation of neurotrophin release, neuroimmunomodulation, and interaction with reactive nitrogen and oxygen species. Vitamin D appears to also play a role in neurodevelopment through its regulation of nerve growth factor synthesis. Imaging studies have found increases in white matter hyperintensities and enlarged ventricles in vitamin D deficient study participants.

 

Channel Molecular Noise to Keep Cells Healthy

http://www.genengnews.com/gen-news-highlights/channel-molecular-noise-to-keep-cells-healthy/81251746/

Molecular fluctuations or noise within and among cells can be manipulated to control the networks that govern the workings of living cells—promoting cellular health and potentially alleviating diseases such as cancer. [Daniel K. Wells]

Complex networks are noisy, whether they constitute food webs, power grids, or cells. And when networks buzz and crackle beyond normal bounds, bad things can happen: ecosystems can collapse, power grids can leave us in the dark, and cells can tumble into cancerous states.

All these networks are amenable to similar mathematical treatments says a scientific team at Northwestern University. The team, led by physicist Adilson E. Motter, Ph.D., substantiated this claim by focusing on a particularly difficult biophysical problem: the rational control of cellular behavior. To date, attempts to exert such control have been frustrated by the high dimensionality and noise that are inherent properties of large intracellular networks.

Dr. Motter and his colleagues noted that the response of biological systems to noise has been studied extensively. Yet they also realized that little had been done to exploit noise, or to at least channel it. They hoped to find a way to do so and thereby demonstrate the possibility of preserving or inducing desirable cell states.

Using a newly developed computational algorithm, Dr. Motter and colleagues showed that molecular-level noise can be manipulated to control the networks that govern the workings of living cells—promoting cellular health and potentially alleviating diseases such as cancer. They presented their results September 16 in the journal Physical Review X, in an article entitled, “Control of Stochastic and Induced Switching in Biophysical Networks.”

“Here we present a scalable, quantitative method based on the Freidlin-Wentzell action to predict and control noise-induced switching between different states in genetic networks that, conveniently, can also control transitions between stable states in the absence of noise,” wrote the authors. “We apply this methodology to models of cell differentiation and show how predicted manipulations of tunable factors can induce lineage changes, and further utilize it to identify new candidate strategies for cancer therapy in a cell death pathway model.”

Essentially, by leveraging noise, the team found that the high-dimensional gene regulatory dynamics could be controlled instead by controlling a much smaller and simpler network, termed a “network of state transitions.” In this network, cells randomly transition from one phenotypic state to another—sometimes from states representing healthy cell phenotypes to unhealthy states where the conditions are potentially cancerous. The transition paths between these states can be predicted, as cells making the same transition will typically travel along similar paths in their gene expression.

The team began by using noise to define the most-likely transition pathway between different system states, and connecting these paths into the network of state transitions. By doing so, the researchers could then focus on just one path between any two states, distilling a multidimensional system to a series of one-dimensional interconnecting paths.

Then, using their computational approach, the team identified optimal modifications of experimentally adjustable parameters, such as protein activation rates, to encourage desired transitions between different states.

 

Mitochondrial Protein Finding May Allow Scientists to Control Apoptosis

http://www.genengnews.com/gen-news-highlights/mitochondrial-protein-finding-may-allow-scientists-to-control-apoptosis/81251742/

http://www.genengnews.com/Media/images/GENHighlight/Sep18_2015_NIH_Mitochondria1481397318.jpg

A protein embedded in the surface of mitochondria opens the door to apoptosis, causing cells to experience severe power failures, according to new work by researchers at Temple University School of Medicine. The study, appearing in Molecular Cell, suggests that blocking the door with a small-molecule inhibitor could be key to the treatment of cardiovascular diseases such as heart attack and stroke, where extensive mitochondrial dysfunction and cell death hinder tissue recovery.

The study (“SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore”), led by Muniswamy Madesh, Ph.D., associate professor in the department of biochemistry, the Cardiovascular Research Center, and the Center for Translational Medicine at Temple University School of Medicine (TUSM), shows that the protein, spastic paraplegia 7 (SPG7), is the central component of the so-called permeability transition pore (PTP), a protein complex in the mitochondrial membrane that mediates necrotic cell death (death caused by cell injury).

The identification of SPG7 marks a major advance in scientists’ understanding of how the PTP affects necrosis. Although first described in 1976, the molecular parts of the pore have eluded discovery. “The only known molecular component of the PTP prior to our discovery of SPG7 was a protein called CypD, which is necessary for pore function,” Dr. Madesh explained.

To identify genes that modulate PTP opening induced by calcium overload or increased levels of reactive oxygen species (ROS), the two primary factors that cause mitochondrial dysfunction and cell death via pore opening, Dr. Madesh’s team devised an RNA interference-based screen in which the activity of each gene under investigation was knocked down, or silenced, to examine its effects on mitochondrial calcium levels.

The researchers began with a panel of 128 different genes but after initial screening narrowed the field to just 14 candidate PTP components. Subsequent experiments showed that the loss of only one of them, SPG7, prevented pore opening.

Much of what is known about the PTP comes from studies of mitochondria in disease. In pathological states, particularly those involving hypoxia, calcium, and ROS accumulate within mitochondria, causing them to swell and prompting the PTP to open. Because pore opening disrupts the flow of electrons and protons across the mitochondrial membranes, which normally sustains energy production, it results in a catastrophic drop in cellular energy levels.

In the absence of disease, precisely how the PTP helps to mediate normal cellular physiology remains unclear. According to Dr. Madesh, “Under physiological conditions, SPG7 may function through transient pore openings to release toxic metabolites that have accumulated in mitochondria.” He plans to explore this possibility with knockout animal models.

 

“See-Through” Brain Developed by Japanese Researchers

http://www.genengnews.com/gen-news-highlights/see-through-brain-developed-by-japanese-researchers/81251727/

Scientists at the RIKEN Brain Science Institute in Japan have developed a new method for creating transparent tissue that can be used to illuminate 3D brain anatomy at high resolutions. Published in Nature Neuroscience, the work showcases the novel technology and its practical importance in clinical science by showing how it has given new insights into Alzheimer’s disease plaques.

“The usefulness of optical clearing techniques can be measured by their ability to gather accurate 3D structural information that cannot be readily achieved through traditional 2D methods,” explains lead scientist Atsushi Miyawaki, M.D., Ph.D. “Here, we achieved this goal using a new procedure, and collected data that may resolve several current issues regarding the pathology of Alzheimer’s disease. While Superman’s x-ray vision is only the stuff of comics, our method, called ScaleS, is a real and practical way to see through brain and body tissue.”

In recent years, generating see-through tissue—a process called optical clearing—has become a goal for many researchers in life sciences because of its potential to reveal complex structural details of our bodies, organs, and cells—both healthy and diseased—when combined with advanced microscopy imaging techniques. Previous methods were limited because the transparency process itself can damage the structures under study.

The original recipe reported by the Miyawaki team in 2011, termed Scale, was an aqueous solution based on urea that suffered from this same problem. The research team spent five years improving the effectiveness of the original recipe to overcome this critical challenge, and the result is ScaleS, a new technique with many practical applications.

“The key ingredient of our new formula is sorbitol, a common sugar alcohol,” notes Dr. Miyawaki. “By combining sorbitol in the right proportion with urea, we could create transparent brains with minimal tissue damage, that can handle both florescent and immunohistochemical labeling techniques, and is even effective in older animals.”

The team has devised several variations of the Scale technique that can be used together. By combining ScaleS with AbScale—a variation for immunolabeling—and ChemScale—a variation for fluorescent chemical compounds—they generated multicolor high-resolution 3D images of amyloid beta plaques in older mice from a genetic mouse model of Alzheimer’s disease developed at the RIKEN BSI by the Takaomi Saido team.

After showing how ScaleS treatment can preserve tissue, the researchers put the technique to practical use by visualizing in 3D the mysterious “diffuse” plaques seen in the postmortem brains of Alzheimer’s disease patients that are typically undetectable using 2D imaging. Contrary to current assumptions, the diffuse plaques proved not to be isolated, but showed extensive association with microglia —mobile cells that surround and protect neurons.

 

GEN Roundup on Cell-Based Assays for Biological Relevancy

Cell-Based Assay Platforms are Evolving to Meet Diverse Challenges

  • Cell-based assay platforms are evolving to meet diverse challenges—mimicking disease states, preserving signaling pathways, modeling drug responses, and recreating environments conducive to tissue development.

GEN recently interviewed a number of experts on cell-based assay technology to get a sense of the state of the art and to find out where this technology might be most valuable to life sciences research.

  • GEN: What are some of the main challenges that are faced when validating cell-based assays?
  • Dr. Kelly: Considerable challenges come from using systems involving a living organism in the validation of cell-based assays. The characteristics of such systems will likely affect the criteria for validation suitability. These criteria might be specific for primary cells, immortalized cell lines, cancerous cell lines, or cells generated de novo from multipotent stem cells.
  • Chemical reagents are generally well characterized by parameters such as molecular weight, solubility, etc., which are unlikely to change between assays.
  • However, characteristics of primary cells or established cell lines, such as viability, growth phase, proliferation rate, level of metabolism, and even cell size are much more vulnerable.
  • Mr. Trinquet: Beyond developing the right cell-based assay, the main challenge remains the relevancy of the cell model for the target being investigated. Generally, a single assay must also be compatible with a broad variety of cell technologies/models, from engineered cells to more complex models, such as 2D, 3D, microtissue, primary culture, and induced pluripotent stem cell models.
  • This certainly adds some difficulty, given that protein expression levels may differ from one model to another. Also, these assays must generally translate well all along the value chain, from high-throughput screening to late stages of lead op, so that end users do not have to switch between too many assay technologies.
  • Dr. Hsu: Cell-based assays provide more biologically relevant information than biochemical assays for high-throughput screening and ADME/Tox. One challenge in developing and validating cell-based assays is to generate cells that reliably express the drug target and give reproducible results with good Z′ over time.
  • We developed and launched the industry’s first cell-based assays and profiling services for G-protein-coupled receptors. The expression of G-protein-coupled receptors has been worked out, but ion channels are challenging. Another challenge is to make sure the assays and readouts are target specific and predictive, with a good dynamic range and signal-to-noise ratio to differentiate compounds with different potencies and efficacies.
  • Dr. Khimani: Cell-based assays provide a complex and physiologically relevant medium to evaluate the effect of novel therapeutic or modulatory candidates. However, unlike traditional assay formats, cell-based assays introduce a number of challenging factors that must be considered—such as cell type, expression level, stability, and passage viability—when optimizing the assay conditions.
  • In addition, with complex cell-based assay systems, data extraction and signal-to-noise optimization can be time-consuming bottlenecks. Other challenges, particularly with high-content screening, include separate investments in instrumentation, training, data analysis, and data management, all leading to a lower throughput.
  • Dr. Fan: Cell-based assays are model systems, and the most critical challenge facing such assays is how well they reflect real biology. Cell-based assays offer great advantages over biochemical assays because they are conducted in cellular contexts. That said, most of the current cell-based assays use a homogeneous population of cells grown from immortalized cell lines, many of which express target proteins or reporters in excessive, nonphysiological amounts via transient transfection or randomly integrated stable clones. These cell models are far from the actual cellular context in normal or diseased tissue such as a tumor.
  • In addition, phenotypical consequences of an analyte of interest to the cell could reflect a combination of effects that a single cell-based assay would not be able to fully address. These factors impact the validation or correlation of the results of a cell-based assay with a phenotypical consequence, an animal model study, or a clinically relevant finding.
  • Dr. Piper: The most formidable challenge in generating and validating cell-based assays is achieving predictability and translatability. Next-generation re-targeting systems (such as the Jump-In™ platform) have made over-expression of genes, even multigene cassettes, fast, reliable, and easy compared to traditional single-cell cloning.
  • While simple overexpression of a target may be sufficient to drive a primary screen and identify hits, it often lacks a sufficiently complex pathophysiological context to robustly convert hits to lead candidates that are meaningful in clinical trials. These systems have value at early stages, but they would benefit from improvements or secondary screens that can better translate to clinical results.
  • Dr. Payne: The choice of a cell system remains a challenge. Cell lines produce reproducible results, but do not accurately model living systems. Although primary cells are more physiologically relevant, they are inherently variable, making it harder to deliver a robust cell-based assay.
  • Choosing appropriate endpoints can be time consuming: measuring one parameter is not enough to accurately determine the functionality of a drug. The ability to analyze several markers in multiplex assays provides greater information on drug efficacy and toxicity, the latter being important for failing flawed drugs earlier. Finally, once validated offline, assays still require revalidation when transferred to automated context.
  • GEN: What is more valuable to researchers with respect to cell-based assays miniaturization or ultra-high throughput?
  • Dr. Kelly: A single cell contains the complete genome of the species and thousands of expressed genes, implying that one cell could provide the same information as millions. High-throughput efforts should be aimed at our ability to multiplex, multivisualize, and microarray the enormous amount of information that one cell can provide.Mr. Trinquet: Miniaturization may be more important because the cells that are used are more complex and costly to produce massively. It comes to be particularly important when several assays need to be run in parallel using the same sample, such as cell lysate after stimulation.

 

Dana-Farber Researchers Use Gene Editing to Short-Circuit Sickle Cell Disease

Sep 16, 2015

a GenomeWeb staff reporter

NEW YORK (GenomeWeb) – Scientists have developed a gene editing strategy that could help treat sickle cell disease by short-circuiting the mutated hemoglobin causing the disease.

“We’ve now targeted the modifier of the modifier of a disease-causing gene,” Stuart Orkin, chairman of pediatric oncology at Dana-Farber Cancer Institute and associate chief of hematology/oncology at Boston Children’s Hospital, said in a statement. “It’s a very different approach to treating disease.”

Using CRISPR/Cas9 gene editing tools to systematically excise stretches of a promoter region of the enhancer gene BCL11A — which selects the type of hemoglobin that blood cells create — the researchers found an edit that inactivated BCL11A in human blood stem cells. The cut leads cells to increase levels of fetal hemoglobin, resulting in a milder form of sickle cell disease.

The scientists, led by Orkin and Daniel Bauer of Dana-Farber and Boston Children’s, and Feng Zhang of the Broad Institute, published their study today in Nature.

The human genome codes for both a fetal version and an adult version of hemoglobin. A mutation in the adult version of the protein causes sickle cell disease. BCL11A became a target of sickle cell disease research after Orkin’s laboratory revealed its direct role in the transition from fetal to adult hemoglobin in a 2009 study published in Nature. In 2013, a study led by Orkin and Bauer found the promoter region which controls expression of BCL11A in red blood cells.

 

Musical Scales

http://www.the-scientist.com//?articles.view/articleNo/43794/title/Musical-Scales/

The quest to document an ancient sea creature reveals a cyclical chorus of fish songs.

By Kerry Grens | September 1, 2015

http://www.the-scientist.com/Sept2015/notebook2.jpg

 

fish songs

fish songs

Several years ago, ichthyologist Eric Parmentier met a French marine biologist and filmmaker, Laurent Ballesta, who was organizing an expedition to South Africa to produce a documentary film on the coelacanth. This ancient fish—one whose fossil record dates back at least 350 million years—has an almost mythical legacy. Although it was widely assumed to have gone extinct 65 million years ago, a live specimen was found in 1938, and scientists have identified two extant species of coelacanth. Both species move in a peculiar way, waggling four lobe-like fins in an alternating pattern, as we do our arms and legs. Their anatomy is also unusual: a tiny brain, a joint at the back of the head that allows the animal to open its jaws widely, and only rudimentary vertebrae. Ballesta’s trip inspired Parmentier, who studies fish acoustics, to collaborate with the team. “I hoped to be the first guy to record [sounds of] the coelacanth.”

In the spring of 2013, divers successfully planted a hydrophone inside the cave and also shot video footage of a coelacanth. (The resulting documentary by Ballesta is available on YouTube. Although it is in French, the footage obviates the need for fluency to enjoy the film.) Day and night, for weeks, the hydrophone dutifully recorded the sounds within the cave. When Parmentier retrieved the files and went to analyze the recordings, there was one big problem: it was filled with dozens of different fish calls. “Maybe the coelacanth is in these sound files, but it’s completely masked by the other sounds,” he says.

Nonetheless, the tape captured ceaseless, never-before-heard chatter among the aquatic organisms within the cave (PNAS, 112:6092-97, 2015). To make some sense of it, Parmentier’s team undertook the laborious task of characterizing the sounds recorded over 19 nonconsecutive days (to make this feasible, the group pared down its analysis to the first nine minutes of every hour). The researchers assigned more than 2,700 sounds to 17 groups, most of which sounded to Parmentier like fish (one group was clearly dolphin, based on its high frequency, he says). These included frog-like croaks, grunts that sounded like a creaking door, a moan, and one that sounded like a whistle blown under water. “It’s fair to say, based on the characteristics of the sounds they were hearing, they are probably fish sounds,” says Erica Staaterman, a postdoc at the Smithsonian who studies fish acoustic communication.

 

NIH Awards Beth Israel Team $3M to Continue Study of Heart Disease Biomarker  Sep 17, 2015

a GenomeWeb staff reporter

NEW YORK (GenomeWeb) – The National Institutes of Health has awarded a Beth Israel Deaconess Medical Center (BIDMC) research team $3 million in funding to support the second phase of an effort to identify microRNAs that can be used to predict clinical outcomes of heart disease patients.

The grant, which was awarded under the NIH’s Extracellular RNA Communication program, follows a $4 million award the group received to kick off the project in 2013.

To date, the team has identified a number of miRNA biomarker candidates including miR-30d, which the researchers reported earlier this year as a predictor of beneficial cardiac remodeling in patients following a heart attack and a key player in preventing cell death.

With the latest grant, the investigators aim to validate miR-30d and other candidate miRNAs in several large patient cohorts.

microRNA-based tests

microRNA-based tests

https://www.youtube.com/watch?v=oMaiIyGfhQw

 

Unraveling determinants of transcription factor binding outside the core binding site

Michal Levo, Einat Zalckvar, Eilon Sharon, Ana Carolina Dantas Machado, Yael Kalma, Maya Lotam-Pompan, Adina Weinberger, Zohar Yakhini, Remo Rohs and Eran Segal. “Unraveling determinants of transcription factor binding outside the core binding site”. Genome Res. July 2015 25: 1018-1029.

http://genome.cshlp.org/content/25/7/1018.abstract

Binding of transcription factors (TFs) to regulatory sequences is a pivotal step in the control of gene expression. Despite many advances in the characterization of sequence motifs recognized by TFs, our ability to quantitatively predict TF binding to different regulatory sequences is still limited. Here, we present a novel experimental assay termed BunDLE-seq that provides quantitative measurements of TF binding to thousands of fully designed sequences of 200 bp in length within a single experiment. Applying this binding assay to two yeast TFs, we demonstrate that sequences outside the core TF binding site profoundly affect TF binding. We show that TF-specific models based on the sequence or DNA shape of the regions flanking the core binding site are highly predictive of the measured differential TF binding. We further characterize the dependence of TF binding, accounting for measurements of single and co-occurring binding events, on the number and location of binding sites and on the TF concentration. Finally, by coupling our in vitro TF binding measurements, and another application of our method probing nucleosome formation, to in vivo expression measurements carried out with the same template sequences serving as promoters, we offer insights into mechanisms that may determine the different expression outcomes observed. Our assay thus paves the way to a more comprehensive understanding of TF binding to regulatory sequences and allows the characterization of TF binding determinants within and outside of core binding sites.

 

Defective Mitochondria Transform Normal Cells into Tumors

http://www.genengnews.com/gen-news-highlights/defective-mitochondria-transform-normal-cells-into-tumors/81251487/

An international research team reports that defects in mitochondria, play a key role in the transition from normal cells to cancerous ones. When the scientists disrupted a key component of mitochondria, otherwise normal cells took on characteristics of cancerous tumor cells.

Their study (“Disruption of cytochrome c oxidase function induces the Warburg effect and metabolic reprogramming”) is published Oncogene and was led by members of the lab of Narayan G. Avadhani, Ph.D., the Harriet Ellison Woodward Professor of Biochemistry in the department of biomedical sciences in the school of veterinary medicine at the University of Pennsylvania. Satish Srinivasan, Ph.D., a research investigator in Dr. Avadhani’s lab, was the lead author.

In 1924, German biologist Otto Heinrich Warburg observed that cancerous cells consumed glucose at a higher rate than normal cells and had defects in their grana, the organelles that are now known as mitochondria. He postulated that the mitochondrial defects led to problems in the process by which the cell produces energy, called oxidative phosphorylation, and that these defects contributed to the cells becoming cancerous.

“The first part of the Warburg hypothesis has held up solidly in that most proliferating tumors show high dependence on glucose as an energy source and they release large amounts of lactic acid,” said Dr. Avadhani. “But the second part, about the defective mitochondrial function causing cells to be tumorigenic, has been highly contentious.”

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RNAi – On Transcription and Metabolic Control

Writer and Curator: Larry H Bernstein, MD, FCAP

 

RNAi

This is the third contribution to a series on transcription and metabolic control. It reveals the enormous complexity in this emerging research.

 

mRNA, small RNAs, long RNAs, RNAi and DicAR

Aberrant mRNA translation in cancer pathogenesis
Pier Paolo Pandolfi
Oncogene (2004) 23, 3134–3137
http://dx.doi.org:/10.1038/sj.onc.1207618

As the molecular processes that control mRNA translation and ribosome biogenesis in the eukaryotic cell are extremely complex and multilayered, their deregulation can in principle occur at multiple levels, leading to both disease and cancer pathogenesis. For a long time, it was speculated that disruption of these processes may participate in tumorigenesis, but this notion was, until recently, solely supported by correlative studies. Strong genetic support is now being accrued, while new molecular links between tumor-suppressive and oncogenic pathways and the control of protein synthetic machinery are being unraveled. The importance of aberrant protein synthesis in tumorigenesis is further underscored by the discovery that compounds such as Rapamycin, known to modulate signaling pathways regulatory of this process, are effective anticancer drugs. A number of fundamental questions remain to be addressed and a number of novel ones emerge as this exciting field evolves.

 

mRNA Translation and Energy Metabolism in Cancer
I. Topisirovic and N. Sonenberg
Cold Spring Harbor Symposia on Quantitative Biology, Volume LXXVI
http://dx.doi.org:/10.1101/sqb.2011.76.010785

A prominent feature of cancer cells is the use of aerobic glycolysis under conditions in which oxygen levels are sufficient to support energy production in the mitochondria (Jones and Thompson 2009; Cairns et al. 2010). This phenomenon, named the “Warburg effect,” after its discoverer Otto Warburg, is thought to fuel the biosynthetic requirements of the neoplastic growth (Warburg 1956; Koppenol et al. 2011) and has recently been acknowledged as one of the hallmarks of cancer (Hanahan and Weinberg 2011). mRNA translation is the most energy-demanding process in the cell (Buttgereit and Brand 1995).In mammalian cells it consumes >20% of cellular ATP, not considering the energy that is required for the biosynthesis of the components of the translational machinery (e.g., ribosome biogenesis; Buttgereit and Brand 1995). Control of mRNA translation plays a pivotal role in the regulation of gene expression (Sonenberg and Hinnebusch 2009). In fact, a recent study demonstrated that mammalian proteome is mostly governed at the mRNA translation level (Schwanhausser et al. 2011). Malfunction of mRNA translation critically contributes to human disease, including diabetes, heart disease, blood disorders, and, most notably, cancer (Fig. 1; Crozier et al. 2006; Narla and Ebert 2010; Silvera et al. 2010; Spriggs et al. 2010). The first account of changes in the translational apparatus in cancer dates back to 1896, showing enlarged and irregularly shaped nucleoli that are the site of ribosome biogenesis (Pianese 1896). Rapidly proliferating cancer cells have more ribosomes than normal cells.

Figure 1. Dysregulated mRNA translation plays a pivotal role in cancer. Malignant cells are characterized by enlarged nucleoli and a larger number of ribosomes than their normal counterparts. Mutations and/or altered expression of ribosomal proteins (e.g., RPS19, RPS 24), rRNA-modifying enzymes (e.g., dyskerin), translation initiation factors (e.g., eIF4E), or the initiator tRNA (tRNAiMet) result in malignant transformation. Signaling pathways whose dysfunction is frequent in cancer (e.g., MAPK, PI3K/AKT) affect mRNA translation. Perturbations in the translatome result in aberrant cellular growth, proliferation, and survival characteristic of tumorigenesis.

 

In stark contrast to normal cells, in cancer cells ribosomal biogenesis is uncoupled from cell proliferation (Stanners et al. 1979). Accordingly, cancer cells exhibit abnormally high rates of protein synthesis (Silvera et al. 2010). That ribosomal dysfunction plays a central role in cancer is further corroborated by the findings that genetic alterations, which encompass the components of the ribosome machinery (i.e., “ribosomopathies”), are characterized by elevated cancer risk (Narla and Ebert 2010).

mRNA translation is the most energy-consuming process in the cell and strongly correlates with cellular metabolic activity. Translation and energy metabolism play important roles in homeostatic cell growth and proliferation, and when dysregulated lead to cancer. eIF4E is a key regulator of translation, which promotes oncogenesis by selectively enhancing translation of a subset of tumor-promoting mRNAs (e.g., cyclins and c-myc). PI3K/AKT and mitogen-activated protein kinase (MAPK) pathways, which are strongly implicated in cancer etiology, exert a number of their biological effects by modulating translation. The PI3K/AKT pathway regulates eIF4E function by inactivating the inhibitory 4E-BPs via mTORC1, whereas MAPKs activate MAP kinase signal-integrating kinases 1 and 2, which phosphorylate eIF4E. In addition, AMP-activated protein kinase, which is a central sensor of the cellular energy balance, impairs translation by inhibiting mTORC1. Thus, eIF4E plays a major role in mediating the effects of PI3K/AKT, MAPK, and cellular energetics on mRNA translation.Figure 2. eIF4E is regulated by multiple mechanisms. The expression of eIF4E is regulated by several transcription factors (e.g., c-myc, hnRNPK, p53) and adenine-uracil-rich element binding proteins (i.e., HuR and AUF1). eIF4E is suppressed by 4E-BPs, which are regulated by mTORC1. MAP kinase signal integrating kinases 1 and 2 (MNKs) phosphorylate eIF4E.

 

Figure 3. Ras/MAPK and PI3K/AKT/mTORC1 regulate the activity of eIF4E. Various stimuli activate phosphoinositide-3-kinase (PI3K) through the receptor tyrosine kinases (RTKs). Upon activation, PI3K converts phosphatidylinositol 4,5-bisphosphate (PIP2) into phosphatidylinositol-3,4,5-triphosphate (PIP3). This reaction is reversed by PTEN. Phosphoinositide-dependent protein kinase 1 (PDK1) and AKT bind to PIP3 via their pleckstrin homology domains, which allows for the phosphorylation and activation of AKT by PDK1. In addition, the mammalian target of rapamycin complex 2 (mTORC2) modulates the activity of AKT by phosphorylating its hydrophobic motif. AKT phosphorylates tuberous sclerosis complex 2 (TSC2) at multiple sites, which results in its inhibition and consequent activation of Ras homolog enriched in brain (Rheb), which is a small GTPase that activates mTORC1. mTORC1 phosphorylates 4E-BPs leading to their dissociation from eIF4E. In addition to the PI3K/AKT pathway, the activity of mTORC1 is regulated by the serine/threonine kinase 11/LKB1/AMP-kinase (LKB1/AMPK) pathway, regulated in development and DNA damage response 1 (REDD1) and Rag GTPases in response to the changes in cellular energy balance, oxygen and amino acid availability, respectively. Ras and the MAPK pathways are activated by various stimuli through receptor tyrosine kinases (RTKs). In addition the MAPK pathway isactivatedthrough theGprotein–coupled receptors(GPCRs) and byproteinkinaseC (PKC;notshown).TheMAPK pathways encompass an initial GTPase-regulated kinase (MAPKKK), which activates an effector kinase (MAPK) via an intermediate kinase (MAPKK). In response to stimuli such as growth factors, hormones, and phorbol-esters, Ras GTPase stimulates Raf kinase (MAPKKK), which activates extracellular signal-regulated kinases 1 and 2 (ERK 1 and 2) via extracellular signal-regulated kinase activator kinases MEK1 and 2 (MAPKK). Cellular stresses, including osmotic shock, inflammatory cytokines, and UV light, activate p38 MAPKs via multiple mechanisms including Rac kinase (MAPKKK) and MKK3 and 6 (MAPKK). p38 MAPK and ERK activate the MAPK signal–integrating kinases 1 and 2 (MNK1/2), which phosphorylate eIF4E. Additional abbreviations are provided in the text.

 

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis
Cancer Cell Nov, 2014; 26: 707–721.
http://dx.doi.org/10.1016/j.ccell.2014.09.005

Breast cancer cells secrete exosomes with specific capacity for cell-independent miRNA biogenesis, while normal cellderivedexosomes lack thisability. Exosomes derivedfrom cancer cellsand serum frompatients withbreast cancer contain the RISC loading complex proteins, Dicer, TRBP, and AGO2, which process pre-miRNAs into mature miRNAs. Cancer exosomes alter the transcriptome of target cells in a Dicer-dependent manner, which stimulate nontumorigenic epithelial cells to form tumors.This study identifies a mechanism whereby cancer cells impart an oncogenic field effect by manipulating the surrounding cells via exosomes. Presence of Dicer in exosomes may serve as biomarker for detection of cancer.


Dicers at RISC. The Mechanism of RNAi

Marcel Tijsterman and Ronald H.A. Plasterk
Cell, Apr 2014; 117:1–4

Figure 1. Model for RNA Silencing in Drosophila In an ordered biochemical pathway, miRNAs (left panel) and siRNAs (right panel) are processed from double-stranded precursor molecules by Dcr-1and Dcr-2, respectively, and stay attached to Dicer-containing complexes, which assemble into RISC. The degree of complementarity between the RNA silencing molecule (in red) and its cognate target determines the fate of the mRNA: blocked translation or immediate destruction.

Argonaute2 Cleaves the Anti-Guide Strand of siRNA during RISC Activation
Cell 2005; 123:621-629
http://www.cell.com/cgi/content/full/123/4/621/DC1/
Dicing and slicing- The core machinery of the RNA interference pathway
Scott C Hammond
FEBS Letters 579 (2005) 5822–5829
http://dx.doi.org:/10.1016/j.febslet.2005.08.079

Fig. 1. Domain organization of RNaseIII gene family. Three classes of RNaseIII genes are shown. The PAZ domain in Dm-Dicer-2 contains mutations in several residues required for RNA binding and may not be functional.

Fig. 2. Model for Dicer catalysis. The PAZ domain binds the 2 nt 30 overhang of a dsRNA terminus. The RNaseIII domains form a pseudo-dimer. Each domain hydrolyzes one strand of the substrate. The binding site of the dsRBD is not defined. The function of the helicase domain is not known.

Fig. 3. Biogenesis pathway of microRNAs. MicroRNA genes are transcribed by RNA polymerase II. The primary transcript is referred to as ‘‘primicroRNA’’. Drosha processing occurs in the nucleus. The resulting precursor, ‘‘pre-microRNA’’, is exported to the cytoplasm for Dicer processing. In a coordinated manner, the mature microRNA is transferred to RISC and unwound by a helicase. mRNA targets that duplex in the Slicer scissile site are cleaved and degraded, if the microRNA is loaded into an Ago2 RISC. Mismatched targets are translationally suppressed. All Ago family members are believed to function in translational suppression.

Fig. 4. Model for Slicer catalysis. The siRNA guide strand is bound at the 50 end by the PIWI domain and at the 30 end by the PAZ domain. The 50 phosphate is coordinated by conserved basic residues. mRNA targets are initially bound by the seed region of the siRNA and pairing is extended to the 30 end. The RNaseH fold hydrolyzes the target in a cation dependent manner. Slicer cleavage is measured from the 50 end of the siRNA. Product is released by an unknown mechanism and the enzyme recycles.

 

 

RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Historically, it was known by other names, including co-suppression, post transcriptional gene silencing (PTGS), and quelling. Only after these apparently unrelated processes were fully understood did it become clear that they all described the RNAi phenomenon. Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in 1998.

 

Two types of small ribonucleic acid (RNA) molecules – microRNA (miRNA) and small interfering RNA (siRNA) – are central to RNA interference. RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from producing a protein. RNA interference has an important role in defending cells against parasitic nucleotide sequences – viruses and transposons. It also influences development.

 

The RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double stranded fragments of ~20 nucleotide siRNAs. Each siRNA is unwound into two single-stranded RNAs (ssRNAs), the passenger strand and the guide strand. The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC). The most well-studied outcome is post-transcriptional gene silencing, which occurs when the guide strand pairs with a complementary sequence in a messenger RNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. In some organisms, this process spreads systemically, despite the initially limited molar concentrations of siRNA.
http://en.wikipedia.org/wiki/RNA_interference

 

http://upload.wikimedia.org/wikipedia/commons/thumb/e/e4/ShRNA_Lentivirus.svg/481px-ShRNA_Lentivirus.svg.png

 

http://www.frontiersin.org/files/Articles/66078/fnmol-06-00040-HTML/image_m/fnmol-06-00040-g001.jpg
http://dx.doi.org:/10.3389/fnmol.2013.00040

The enzyme dicer trims double stranded RNA, to form small interfering RNA or microRNA. These processed RNAs are incorporated into the RNA-induced silencing.
MiRNA biogenesis and function. (A) The canonical miRNA biogenesis pathway is Drosha- and Dicer-dependent. It begins with RNA Pol II-mediated transcription..

 

Dicer Promotes Transcription Termination

Dicer Promotes Transcription Termination

Dicer Promotes Transcription Termination at Sites of Replication Stress to Maintain Genome Stability
Cell Oct 2014; 159(3): 572–583
http://dx.doi.org/10.1016/j.cell.2014.09.031

http://www.cell.com/cms/attachment/2019646604/2039684570/fx1.jpg

 

18-13 miRNA- protein complex ap-chap-18-pp-42-728

18-13 miRNA- protein complex ap-chap-18-pp-42-728

18-13 miRNA- protein complex (a) Primary miRNA transcript Translation blocked Hydrogen bond (b) Generation and function of miRNAs Hairpin miRNA miRNA Dicer …

http://image.slidesharecdn.com/ap-chap-18-pp-1229097198123780-1/95/ap-chap-18-pp-42-728.jpg?cb=1229090143

 

 

Identification and characterization of small RNAs involved in RNA silencing
FEBS Letters 579 (2005) 5830–5840
http://dx.doi.org:/10.1016/j.febslet.2005.08.009

Fig. 1. Small RNA cloning procedure. Outline of the small RNA cloning procedure. RNA is dephosphorylated (step 1) for joining the 30 adapter by T4 RNA ligase 1 in the presence of ATP (step 2). The use of a chemically adenylated adapter and truncated form of T4 RNA ligase 2 (Rnl2) allows eliminating the dephosphorylation step (step 4). If the RNA was dephosphorylated, it is re-phosphorylated (step 3) prior to 50 adapter ligation with T4 RNA ligase 1 and ATP (step 5). After 50 adapter ligation, a standard reverse transcription is performed (step 6). Alternatively, after 30 adapter ligation, the RNA is used directly for reverse transcription simultaneously with 50 adaptor joining (step 7). In this case, the property of reverse transcriptase to add non-templated cytidine residues at the 50 end of synthesized DNA is used to facilitate template switch of the reverse transcriptase to the 30 guanosine residues of the 50 adapter (SMART technology, Invitrogen). Abbreviations: P and OH indicate phosphate and hydroxyl ends of the RNA; App indicates 50 chemically adenylated adapter; L, 30 blocking group; CIP, calf alkaline phosphatase and PNK, polynucleotide kinase.

 

Transcriptional regulatory functions of nuclear long noncoding RNAs
Trends in Genetics, Aug 2014; 30(8):348-356
http://dx.doi.org/10.1016/j.tig.2014.06.001

Cis-acting lncRNAEnhancer-associated lncRNAIntergenic lncRNA

lncRNA

Promoter-associated lncRNA

Proximity transfer

Trans-acting lncRNA

 

Functional interactions among microRNAs and long noncoding RNAs
Sem Cell Dev Biol 2014; 34:9-14
http://dx.doi.org/10.1016/j.semcdb.2014.05.015
Genome-wide application of RNAi to the discovery of potential drug targets
FEBS Letters 579 (2005) 5988–599
http://dx.doi.org://10.1016/j.febslet.2005.08.015

Fig. 1. Schematic representation of gene silencing by an shRNA-expression vector. The shRNA is processed by Dicer. The processed siRNA enters the RNA-induced silencing complex (RISC), where it targets mRNA for degradation.

Fig. 2. Schematic representation of a transcription system for production of siRNA

Fig. 3. (A) Schematic representation of the proposed siRNA-expression system. Three or four C to U or A to G mutations are introduced into the sense strand. (B) Schematic representation of the discovery of a novel gene using an siRNA library.

 

Imperfect centered miRNA binding sites are common and can mediate repression of target mRNAs
Martin et al. Genome Biology 2014, 15:R51 http://genomebiology.com/2014/15/3/R51

 

 

 

 

Table 1 Number of inferred targets for each miRNA tested

miRNA Probes Transcripts Genes
miR-10a 2,206 5,963 1,887
miR-10a-iso 1,648 1,468 4,211
miR-10b 1,588 3,940 1,365
miR-10b-iso 963 2,235 889
miR-17-5p 1,223 2,862 1,137
miR-17-5p-iso 1,656 3,731 1,461
miR-182 2,261 6,423 2,008
miR-182-iso 1,569 4,316 1,444
miR-23b 2,248 5,383 1,990
miR-27a 2,334 5,310 2,069

Probes: number of probes significantly enriched in pull-downs compared to controls (5% FDR). Transcripts: number of transcripts to which those probes map exactly. Genes: number of genes from which those transcripts originate

Figure 2 Biotin pull-downs identify bone fide miRNA targets. (A) Volcano plot showing the significance of the difference in expression between the miR-17-5p pull-down and the mock-transfected control, for all transcripts expressed in HEK293T cells. Both targets predicted by TargetScan or validated previously via luciferase assay were significantly enriched in the pull-down compared to the controls. (B) Results from luciferase assays on previously untested targets predicted using TargetScan and uncovered using the biotin pull-down. The plot indicates mean luciferase activity from either the empty plasmid or from pMIR containing a miRNA binding site in the 3′ UTR, relative to a negative control. Asterisks indicate a significant reduction in luciferase activity (one-sided t-test; P<0.05) and error bars the standard error of the mean over three replicates. (C-E) Targets identified through PAR-CLIP or through miRNA over-expression studies show greater enrichment in the pull-down. Cumulative distribution of log fold-change in the pull-down for transcripts identified as targets by the indicated miRNA over-expression study or not. Red, canonical transcripts found to be miR-17-5p targets in the indicated study (Table S5 in Additional file 1); black, all other canonical transcripts; p, one-sided P-value from Kolmogorov-Smirnov test for a difference in distributions. (F) To confirm that our results were dependent on RISC association, cells were transfected with either single or double-stranded synthetic miRNAs, then subjected to AGO2 immunoprecipitation. The biotin pull-down was performed in the AGO2-enriched and AGO2-depleted fractions. (G-H) Quantitative RT-PCR revealed that, with double-stranded (ds) miRNA (G), four out of five known targets were enriched relative to input mRNA (*P≤0.05, **P<0.01, ***P<0.001) in the AGO2-enriched but not in the AGO2-depleted fractions, but this enrichment was not seen for the cells transfected with a single-stranded (ss) miRNA (H). The numbers on the x-axis correspond to those in Figure 2F. Error bars represent the standard error of mean (sem).

Figure 5 IsomiRs and canonical miRNAs target many of the same transcripts.

Hammerhead ribozymes in therapeutic target discovery and validation
Drug Disc Today 2009; 14(15/16): 776-783
http://dx.doi.org/10.1016/j.drudis.2009.05.003

Figure 1. Features of hammerhead ribozymes. A generic diagram of a hammerhead ribozyme bound to its target substrate: NUH is the cleavage triplet on target sequence, stems I and III are sites of the specific interactions between ribozyme and target, stem II is the structural element connecting separate parts of the catalytic core. Arrows represent the cleavage site, numbering system according to Hertel et al. [60].

hammerhead ribozyme

hammerhead ribozyme

https://www-ssrl.slac.stanford.edu/research/highlights_archive/ribozyme_fig1.jpg

 

Figure 1  Schematic (A) and ribbon (B) diagrams depicting the crystal structure of the full-length hammerhead ribozyme. The sequence and secondary structure

 

TABLE 1 Typical examples of successful applications of hammerhead ribozymes. Most of the data are derived from [10] and [11], the others are expressly specified.

  • Growth factors, receptors, transduction elements
  • Oncogenes, protoncogenes, fusion genes
  • Apoptosis, survival factors, drug resistance
  • Transcription factors
  • Extracellular matrix, matrix modulating factors
  • Circulating factors
  • Viral genome, viral genes

Figure 2.Target–ribozyme interactions. (a) As cheme of ribozyme binding to full substrate. The calculated energy of this binding ensures the formation of a stable complex. At the denaturating temperature, Tm, will allow this complex to survive to biological conditions. Conversely, after cleavage, binding energies calculated on single, (b) and (c), ribozyme arms are very low and no longer stable. These properties will ensure both the efficient release of cleavage fragments and the prevention of binding to unrelated targets. RNAs complementary to one binding arm only will not be bound or cleaved by the hammerhead catalytic sequence.

Figure 3. ‘Chemical omics’ approach. According to this target discovery strategy: (1) a first round of ‘omic’ study (proteomic, genomic, metabolomic, …) will enable the discovery of a set of (2) putative markers. A series of hammerhead ribozymes will then be prepared in order to target each marker. (4) A second ‘omic’ study round will be performed on (3) knocked down samples obtained after ribozymes administration. (5) A new series of markers will then be produced. An expanding analytical process of this type may be further repeated. Finally, a robust bioinformatic algorithm will make it possible to connect the different markers and draw new hypothetical links and pathways.

 

miRNA

ADAR Enzyme and miRNA Story
Sara Tomaselli, Barbara Bonamassa, Anna Alisi, et al.
Int. J. Mol. Sci. 2013, 14, 22796-22816;
http://dx.doi.org:/10.3390/ijms141122796

Adenosine deaminase acting on RNA (ADAR) enzymes convert adenosine (A) to inosine (I) in double-stranded (ds) RNAs. Since Inosine is read as Guanosine, the biological consequence of ADAR enzyme activity is an A/G conversion within RNA molecules. A-to-I editing events can occur on both coding and non-coding RNAs, including microRNAs (miRNAs), which are small regulatory RNAs of ~20–23 nucleotides that regulate several cell processes by annealing to target mRNAs and inhibiting their translation. Both miRNA precursors and mature miRNAs undergo A-to-I RNA editing, affecting the miRNA maturation process and activity. ADARs can also edit 3′ UTR of mRNAs, further increasing the interplay between mRNA targets and miRNAs. In this review, we provide a general overview of the ADAR enzymes and their mechanisms of action as well as miRNA processing and function. We then review the more recent findings about the impact of ADAR-mediated activity on the miRNA pathway in terms of biogenesis, target recognition, and gene expression regulation.

Figure 1. Structure of ADAR family proteins: ADAR1, ADAR2, and ADAR3. The ADAR enzymes contain a C-terminal conserved catalytic deaminase domain (DM), two or three dsRBDs in the N-terminal portion. ADAR1 full-length protein also contains a N-terminal Zα domain with a nuclear export signal (NES) and a Zβ domain, while ADAR3 has a  R-domain. A nuclear localization signal is also indicated.

 

Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites
Doron Betel, Anjali Koppal, Phaedra Agius, Chris Sander, Christina Leslie
Genome Biology 2010, 11:R90 http://genomebiology.com/2010/11/8/R90

microRNAs are a class of small regulatory RNAs that are involved in post-transcriptional gene silencing. These small (approximately 22 nucleotide) single-strand RNAs guide a gene silencing complex to an mRNA by complementary base pairing, mostly at the 3′ untranslated region (3′ UTR). The association of the RNAinduced silencing complex (RISC) to the conjugate mRNA results in silencing the gene either by translational repression or by degradation of the mRNA. Reliable microRNA target prediction is an important and still unsolved computational challenge, hampered both by insufficient knowledge of microRNA biology as well as the limited number of experimentally validated targets.

mirSVR is a new machine learning method for ranking microRNA target sites by a down-regulation score. The algorithm trains a regression model on sequence and contextual features extracted from miRanda-predicted target sites. In a large-scale evaluation, miRanda-mirSVR is competitive with other target prediction methods in identifying target genes and predicting the extent of their downregulation at the mRNA or protein levels. Importantly, the method identifies a significant number of experimentally determined non-canonical and non-conserved sites.
Human RISC – MicroRNA Biogenesis and Posttranscriptional Gene Silencing
Cell 2005; 123:631-640
http://dx.doi.org:/10.1016/j.cell.2005.10.022
Development of microRNA therapeutics
Eva van Rooij & Sakari Kauppinen
EMBO Mol Med (2014) 6: 851–864
http://dx.doi.org:/10.15252/emmm.20110089

MicroRNAs (miRNAs) play key regulatory roles in diverse biological processes and are frequently dysregulated in human diseases. Thus, miRNAs have emerged as a class of promising targets for therapeutic intervention. Here, we describe the current strategies for therapeutic modulation of miRNAs and provide an update on the development of miRNA-based therapeutics for the treatment of cancer, cardiovascular disease and hepatitis C virus (HCV) infection.

Figure 1. miRNA biogenesis and modulation of miRNA activity by miRNA mimics and antimiR oligonucleotides. MiRNA genes are transcribed by RNA polymerase II from intergenic, intronic or polycistronic loci to long primary miRNA transcripts (pri-miRNAs) and processed in the nucleus by the Drosha–DGCR8 complex to approximately 70 nt pre-miRNA hairpin structures. The most common alternative miRNA biogenesis pathway involves short intronic hairpins, termed mirtrons, that are spliced and debranched to form pre-miRNA hairpins. Pre-miRNAs are exported into the cytoplasm and then cleaved by the Dicer–TRBP complex to imperfect miRNA: miRNA* duplexes about 22 nucleotides in length. In the cytoplasm, miRNA duplexes are incorporated into Argonaute-containing miRNA induced silencing complex (miRISC), followed by unwinding of the duplex and retention of the mature miRNA strand in miRISC, while the complementary strand is released and degraded. The mature miRNA functions as a guide molecule for miRISC by directing it to partially complementary sites in the target mRNAs, resulting in translational repression and/or mRNA degradation. Currently, two strategies are employed to modulate miRNA activity: restoring the function of a miRNA using double-stranded miRNA mimics, and inhibition of miRNA function using single-stranded anti-miR oligonucleotides.

Figure 2. Design of chemically modified miRNA modulators. (A) Structures of chemical modifications used in miRNA modulators. A number of different sugar modifications are used to increase the duplex melting temperature (Tm) of anti-miR oligonucleotides. The20-O-methyl(20-O-Me), 20-O-methoxyethyl(20-MOE )and 20-fluoro(20-F) nucleotides are modified at the 20 position of the sugar moiety, whereas locked nucleic acid (LNA) is a bicyclic RNA analogue in which the ribose is locked in a C30-endo conformation by introduction of a 20-O,40-C methylene bridge. To increase nuclease resistance and enhance the pharmacokinetic properties, most anti-miR oligonucleotides harbor phosphorothioate (PS) backbone linkages, in which sulfur replaces one of the non-bridging oxygen atoms in the phosphate group. In morpholino oligomers, a six-membered morpholine ring replaces the sugar moiety. Morpholinos are uncharged and exhibit a slight increase in binding affinity to their cognate miRNAs. PNA oligomers are uncharged oligonucleotide analogues, in which the sugar–phosphate backbone has been replaced by a peptide-like backbone consisting of N-(2-aminoethyl)-glycine units. (B) An example of a synthetic double-stranded miRNA mimic described in this review. One way to therapeutically mimic a miRNA is by using synthetic RNA duplexes that harbor chemical modifications for improved stability and cellular uptake. In such constructs, the antisense (guide) strand is identical to the miRNA of interest, while the sense (passenger) strand is modified and can be linked to a molecule, such as cholesterol, for enhanced cellular uptake. The sense strand contains chemical modifications to prevent mi-RISC loading. Several mismatches can be introduced to prevent this strand from functioning as an anti-miR, while it is further left unmodified to ensure rapid degradation.The20-F modification helps to protect the antisense strand against exonucleases, hence making the guide strand more stable, while it does not interfere with mi-RISC loading. (C) Design of chemically modified anti-miR oligonucleotides described in this review. Antagomirs are30 cholesterol-conjugated,20-O-Me oligonucleotides fully complementary to the mature miRNA sequence with several PS moieties to increase their in vivo stability. The use of unconjugated 20-F/MOE-, 20-MOE- or LNA-modified anti-miR oligonucleotides harboring a complete PS backbone represents another approach for inhibition of miRNA function in vivo. The high duplex melting temperature of LNA-modified oligonucleotides allows efficient miRNA inhibition using truncated, high-affinity 15–16-nucleotide LNA/DNA anti-miR oligonucleotides targeting the 50 region of the mature miRNA. Furthermore, the high binding affinity of fully LNA-modified 8-mer PS oligonucleotides, designated as tiny LNAs, facilitates simultaneous inhibition of entire miRNA seed families by targeting the shared seed sequence.

Human MicroRNA Targets
Bino John, Anton J. Enright, Alexei Aravin, Thomas Tuschl,.., Debora S. Mark
PLoS Biol 2004; 2(11): e363  http://www.plosbiology.org

More than ten years after the discovery of the first miRNA gene, lin-4 (Chalfie et al. 1981; Lee et al. 1993), we know that miRNA genes constitute about 1%–2% of the known genes in eukaryotes. Investigation of miRNA expression combined with genetic and molecular studies in Caenorhabditis elegans, Drosophila melanogaster, and Arabidopsis thaliana have identified the biological functions of several miRNAs (recent review, Bartel 2004). In C. elegans, lin-4 and let-7 were first discovered as key regulators of developmental timing in early larval developmental transitions (Ambros 2000; Abrahante et al. 2003; Lin et al. 2003; Vella et al. 2004). More recently lsy-6 was shown to determine the left–right asymmetry of chemoreceptor expression (Johnston and Hobert 2003). In D. melanogaster, miR-14 has a role in apoptosis and fat metabolism (Xu et al. 2003) and the bantam miRNA targets the gene hid involved in apoptosis and growth control (Brennecke et al. 2003).

MicroRNAs (miRNAs) interact with target mRNAs at specific sites to induce cleavage of the message or inhibit translation. The specific function of most mammalian miRNAs is unknown. We have predicted target sites on the 39 untranslated regions of human gene transcripts for all currently known 218 mammalian miRNAs to facilitate focused experiments. We report about 2,000 human genes with miRNA target sites conserved in mammals and about 250 human genes conserved as targets between mammals and fish. The prediction algorithm optimizes sequence complementarity using position-specific rules and relies on strict requirements of interspecies conservation. Experimental support for the validity of the method comes from known targets and from strong enrichment of predicted targets in mRNAs associated with the fragile X mental retardation protein in mammals. This is consistent with the hypothesis that miRNAs act as sequence-specific adaptors in the interaction of ribonuclear particles with translationally regulated messages. Overrepresented groups of targets include mRNAs coding for transcription factors, components of the miRNA machinery, and other proteins involved in translational regulation, as well as components of the ubiquitin machinery, representing novel feedback loops in gene regulation. Detailed information about target genes, target processes, and open-source software for target prediction (miRanda) is available at http://www.microrna.org. Our analysis suggests that miRNA genes, which are about 1% of all human genes, regulate protein production for 10% or more of all human genes.

Figure 1. Target Prediction Pipeline for miRNA Targets in Vertebrates The mammalian (human, mouse, and rat) and fish (zebra and fugu) 39 UTRs were first scanned for miRNA target sites using position specific rules of sequence complementarity. Next, aligned UTRs of orthologous genes were used to check for conservation of miRNA– target relationships (‘‘target conservation’’) between mammalian genomes and, separately, between fish genomes. The main results (bottom) are the conserved mammalian and conserved fish targets, for each miRNA,as well as a smaller set of super-conserved vertebrate targets.   http://dx.doi.org:/10.1371/journal.pbio.0020363.g00
Figure 2. Distribution of Transcripts with Cooperativity of Target Sites and Estimated Number of False Positives Each bar reflects the number of human transcripts with a given number of target sites on their UTR. Estimated rate of false positives(e.g., 39%for2 targets) is given by the number of target sites predicted using shuffled miRNAs processed in a way identical to real miRNAs, including the use of interspecies conservation filter. http://dx.doi.org:/10.1371/journal.pbio.0020363.g002

Conserved Seed Pairing, Often improved an-Flanked by Adenosines, Indicates Thousands of Human Genes are MicroRNA Targets
Cell, Jan 2005; 120: 15–20
http://dx.doi.org:/10.1016/j.cell.2004.12.035

Integrated analysis of microRNA and mRNA expression. adding biological significance to microRNA target predictions.
Maarten van Iterson, Sander Bervoets, Emile J. de Meijer, et al.
Nucleic Acids Research, 2013; 41(15), e146
http://dx.doi.org:/10.1093/nar/gkt525

Current microRNA target predictions are based on sequence information and empirically derived rules but do not make use of the expression of microRNAs and their targets. This study aimed to improve microRNA target predictions in a given biological context, using in silico predictions, microRNA and mRNA expression. We used target prediction tools to produce lists of predicted targets and used a gene set test designed to detect consistent effects of microRNAs on the joint expression of multiple targets. In a single test, association between microRNA expression and target gene set expression as well as the contribution of the individual target genes on the association are determined. The strongest negatively associated mRNAs as measured by the test were prioritized. We applied our integration method to a well-defined muscle differentiation model. Validation of our predictions in C2C12 cells confirmed predicted targets of known as well as novel muscle-related microRNAs. We further studied associations between microRNA–mRNA pairs in human prostate cancer, finding some pairs that have been recently experimentally validated by others. Using the same study, we showed the advantages of the global test over Pearson correlation and lasso. We conclude that our integrated approach successfully identifies regulated microRNAs and their targets.

Long non-coding RNA and microRNAs might act in regulating the expression of BARD1 mRNAs
Int J Biol & Cell Biol 2014; 54:356-367
http://dx.doi.org/10.1016/j.biocel.2014.06.018

 

Passenger-Strand Cleavage Facilitates Assembly of siRNA into Ago2-Containing RNAi Enzyme Complexes
Cell 2006; 123:607-620
http://dx.doi.org:/10.1016/j.cell.2006.08.044

 

RNAi- RISC Gets Loaded
Cell 2005; 123:543-553
http://dx.doi.org:/10.1016/j.cell.2005.11.006
RNAi- The Nuts and Bolts of the RISC Machine
Cell 2005; 122:17-20
http://dx.doi.org:/10.1016/j.cell.2005.06.023
Structural domains in RNAi
FEBS Letters 579 (2005) 5841–5849
http://dx.doi.org:/10.1016/j.febslet.2005.07.072

Fig. 1. A ‘‘Domain-centric’’ view of RNAi. (A) The conserved pathways of RNA silencing. The domain structure of each protein in (hypothetical) interaction with its RNA is shown. For clarity, the second column lists domains in order N- to C-terminal. Figures are not to scale. In brief, Drosha, an RNase III enzyme, and its obligate binding partner, Pasha recognize pri-mRNA loops, and cut these into 70 nt hairpin pre-miRNAs. Dicer utilizes a PAZ domain to sense the 30 2-nt overhang created, and further processes these, and dsRNAs into miRNAs and siRNAs. Argonaute binds the 50 end of guide RNAs via its PIWI domain, and the 30 end via a PAZ domain, yielding RISCs that effect RNA silencing through several mechanisms. A Viral protein, VP19 can suppress RNA silencing by sequestering siRNAs. (B) A summary of known siRNA structural biology. Listed by domain are solved structures, their protein/organism of origin, and ligands, where applicable. Also shown are PDB codes.

Fig. 2. Novel modes of RNA recognition. (A) A typical dsRBD: Xenopus binding protein A (1DI2). A RNA helix is modeled pink, and the protein is rendered in transparent electrostatic contours (blue is basic, red acidic). Note the interaction of helices along the major groove, and the position of helix 1. A second dsRBD protein is visible, in the lower right. (B) A dsRBD, Saccharomyces Rnt1P (1T4L), recognizes hairpin loops. A novel third helix (top) pushes helix one into the loop of a hairpin RNA. (C) 30-OH recognition by PAZ. Human Eif2c1 (1SI3) bound to RNA (pink) is shown. PAZ is green, with transparent electrostatic surface plot. The OB-fold (nucleotide binding fold) and the insertion domain are labeled. Note the glove-and-thumb like cleft they form, that the 30-OH is inserted into. A basic groove (blue) the RNA binds along outside the cleft is visible. (D) A close-up view of PAZ, as in C (surface not-transparent, slightly rotated). See white arrows for orientation, and location of 30-OH binding site. RNA is shown red in sticks. The terminal –OH is barely visible, buried in a cleft. It and the carbon it bonds have been colored yellow for clarity. (E) The PIWI domain (2BGG). Note the insertion of the 50P red (labeled) into the binding site. Its complimentary strand (pink) is not annealed to it, and the 30 overhang and first complimentary bases sit on the protein surface. (F) An enlarged view of (E), with protein in slate and RNA modeled as red sticks. The coordinated magnesium is a grey sphere, which is coordinated by the terminal carboxylate of the protein, protein side chains, and RNA phosphate oxygens. The 50 base stacks against a conserved Tyr. Several other sidechain contacts are shown.

Fig. 3. Argonaute/RISC. (A) P. furiosus Argonaute (PDB 1Z26). A color-guided key to the domains is presented. PAZ sits over the PIWI/N/MID bowl and active site. The liganding atoms for the catalytic metal are depicted as yellow balls for clarity. The tungstate binding site (50P surrogate) is shown as tan spheres. (B) A guide strand channel. Looking down from the PAZ domain towards the active site, Z-sections are clipped off. Colors of domains are as in the key in (A). Wrapping down along a basic cleft from the PAZ 30OH binding site (approximate position labeled), a RNA binding groove passes the active site (yellow), and runs down to the 50P binding site (tan balls). A second cleft running perpendicular to this one at its entry may accommodate target strand RNA. For more detail, and models of siRNA placed into the grooves, see [27,29].

Fig. 4. VP19 sequestration of siRNA. (A) CIRV VP19 (1RPU, RNA removed). Two monomers (blue and cyan) form an 8 strand, concave b-sheet with bracketing helices at the ends. (B) Tombus viral VP19 bound to siRNA (1 monomer shown). RNA strands are modeled as sticks, with one strand pink and one red. The bracketing helix places two tryptophans in position to stack over the terminal RNA bases. On the b-sheet surface, and Arg and a Lys interact with the phosphate backbone, and at the center of the RNA binding surface, a number of Ser and Thr mediate an extensive hydrogen bond network. Both the Trp brackets and RNA binding by an extended b-sheet are unique.

 

Small RNA asymmetry in RNAi- Function in RISC assembly and gene regulation
FEBS Letters 579 (2005) 5850–5857
http://dx.doi.org:/10.1016/j.febslet.2005.08.071

 

The role of the oncofetal IGF2 mRNA-binding protein 3 (IGF2BP3) in cancer
Seminars in Cancer Biol 2014; 29:3-12
http://dx.doi.org/10.1016/j.semcancer.2014.07.006

Table 1 – Target mRNAs of IGF2BP3.

Target cis-Element Regulation
CD44 3’ -utr Control of mRNA stability
IGF2 5’ -utr Translational control
H19 ncRNA Unknown
ACTB 3’ -utr Unknown
MYC CRD Unknown
CD164 Unknown Control of mRNA stability
MMP9 Unknown Control of mRNA stability
ABCG2 Unknown Unknown
PDPN 3’ -utr Control of mRNA stability
HMGA2 3’ -utr Protection from miR directed degradation
CCND1 3’ -utr translational control
CCND3 3’ -utr translational control
CCNG1 3’ -utr translationalcontrol

 

Targeting glucose uptake with siRNA-based nanomedicine for cancer therapy
Biomaterials 2015; 51:1-11
http://dx.doi.org/10.1016/j.biomaterials.2015.01.068
The therapeutic potential of RNA interference
FEBS Letters 579 (2005) 5996–6007
http://dx.doi.og:/10.1016/j.febslet.2005.08.004

Table 1 Companies developing RNAi therapeutics that includes cancer

Company name Primary areas of interest
Atugen AG Metabolic disease; cancer ocular disease; skin disease
Benitec Australia Limited Hepatitis C virus; HIV/AIDS; cancer; diabetes/obesity
Calando Pharmaceuticals Nanoparticle technology
Genta Incorporated Cancer
Intradigm Corporation Cancer; SARS; arthritis
Sirna Therapeutics, Inc. AMD; Hepatitis C virus; asthma; diabetes; cancer; Huntington s disease; hearing loss

 

The Noncoding RNA Revolution—Trashing Old Rules to Forge New Ones
Cell 2014; 157:77-94
http://dx.doi.org/10.1016/j.cell.2014.03.008

Figure 1. Noncoding RNAs Function in Diverse Contexts Noncoding RNAs function in all domains of life, regulating gene expression from transcription to splicing to translation and contributing to genome organization and stability. Self-splicing RNAs, ribosomes, and riboswitches function in both eukaryotes and bacteria. Archaea (not shown) also utilize ncRNA systems including ribosomes, riboswitches, snoRNPs, and CRISPR. Orange strands, ncRNA performing the action indicated; red strands, the RNA acted upon by the ncRNA. Blue strands, DNA. Triangle, small-molecule metabolite bound by a riboswitch. Ovals indicate protein components of an RNP, such as the spliceosome (white oval), ribosome (two purple subunits), or other RNPs (yellow ovals). Because of the importance of RNA structure in these ncRNAs, some structures are shown but they are not meant to be realistic.

 

miRNAs and cancer targeting

Table 1 of targets

miRNA Cancer type reference
NA GI cancer Current status of miRNA-targeting therapeutics and preclinical studies against gastroenterological carcinoma
NA Renal cell Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis
NA urothelial
cancer
A microRNA expression ratio defining the invasive phenotype in bladder tumors
miR-31 breast A Pleiotropically Acting MicroRNA, miR-31, inhibits breast cancer growth
miR-512-3p NSCLC Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer
miR-495 gastric Methylation-associated silencing of miR-495 inhibit the migration and invasion of human gastric cancer cells
microRNA-218 prostate microRNA-218 inhibits prostate cancer cell growth and promotes apoptosis by repressing TPD52 expression
MicroRNA-373 cervical cancer MicroRNA-373 functions as an oncogene and targets YOD1 gene in cervical cancer
miR-25 NSCLC miR-25 modulates NSCLC cell radio-sensitivity – inhibiting BTG2 expression
miR-92a cervical cancer miR-92a. upregulated in cervical cancer & promotes cell proliferation and invasion by targeting FBXW7
MiR-153 NSCLC MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19
miR-203 melanoma miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1
miR-204-5p Papillary thyroid miR-204-5p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma
miR-342-3p Hepato-cellular miR-342-3p affects hepatocellular carcinoma cell proliferation via regulating NF-κB pathway
miR-1271 NSCLC miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5
miR-203 pancreas Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203
miR-203 metastatic SCC Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human SCC Metastasis
miR-204 RCC TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma
NA urologic MicroRNAs and cancer. Current and future perspectives in urologic oncology
NA RCC MicroRNAs and their target gene networks in renal cell carcinoma
NA osteoSA MicroRNAs in osteosarcoma
NA urologic MicroRNA in Prostate, Bladder, and Kidney Cancer
NA urologic Micro-RNA profiling in kidney and bladder cancers

 

Current status of miRNA-targeting therapeutics and preclinical studies against gastroenterological carcinoma
Shibata et al. Molecular and Cellular Therapies 2013, 1:5 http://www.molcelltherapies.com/content/1/1/5

Differential expression profiling of microRNAs and their potential involvement in renal cell carcinoma pathogenesis
Clinical Biochemistry 43 (2010) 150–158
http://dx.doi.org:/10.1016/j.clinbiochem.2009.07.020

A microRNA expression ratio defining the invasive phenotype in bladder tumors
Urologic Oncology: Seminars and Original Investigations 28 (2010) 39–48
http://dx.doi.org:/10.1016/j.urolonc.2008.06.006

A Pleiotropically Acting MicroRNA, miR-31, inhibits breast cancer growth
Cell 137, 1032–1046, June 12, 2009
http://dx.doi.org:/10.1016/j.cell.2009.03.047

Inhibition of RAC1-GEF DOCK3 by miR-512-3p contributes to suppression of metastasis in non-small cell lung cancer
Intl JBiochem & Cell Biol 2015; 61:103-114
http://dx.doi.org/10.1016/j.biocel.2015.02.005

Methylation-associated silencing of miR-495 inhibit the migration and invasion of human gastric cancer cells by directly targeting PRL-3
Biochem Biochem Res Commun 2014; 456:344-350
http://dx.doi.org/10.1016/j.bbrc.2014.11.083

microRNA-218 inhibits prostate cancer cell growth and promotes apoptosis by repressing TPD52 expression
Biochem Biophys Res Commun 2015; 456:804-809
http://dx.doi.org/10.1016/j.bbrc.2014.12.026

MicroRNA-373 functions as an oncogene and targets YOD1 gene in cervical cancer
BBRC 2015; xx:1-6
http://dx.doi.org/10.1016/j.bbrc.2015.02.138

miR-25 modulates NSCLC cell radio-sensitivity – inhibiting BTG2 expression
BBRC 2015; 457:235-241
http://dx.doi.org/10.1016/j.bbrc.2014.12.094

miR-92a. upregulated in cervical cancer & promotes cell proliferation and invasion by targeting FBXW7
BBRC 2015; 458:63-69
http://dx.doi.org/10.1016/j.bbrc.2015.01.066

MiR-153 inhibits migration and invasion of human non-small-cell lung cancer by targeting ADAM19
BBRC 2015; 456:381-385
http://dx.doi.org/10.1016/j.bbrc.2014.11.093

miR-203 inhibits melanoma invasive and proliferative abilities by targeting the polycomb group gene BMI1
BBMC 2015; 456: 361-366
http://dx.doi.org/10.1016/j.bbrc.2014.11.087

miR-204-5p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma
BBRC 2015; 457:621-627
http://dx.doi.org/10.1016/j.bbrc.2015.01.037

miR-342-3p affects hepatocellular carcinoma cell proliferation via regulating NF-κB pathway
BBRC 2015; 457:370-377
http://dx.doi.org/10.1016/j.bbrc.2014.12.119

miR-1271 promotes non-small-cell lung cancer cell proliferation and invasion via targeting HOXA5
BBRC 2015; 458:714-719
http://dx.doi.org/10.1016/j.bbrc.2015.02.033

Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203
Cell Immunol 2014; 292:65-69
http://dx.doi.org/10.1016/j.cellimm.2014.09.004

Rewiring of an Epithelial Differentiation Factor, miR-203, to Inhibit Human Squamous Cell Carcinoma Metastasis
Cell Reports 2014; 9:104-117
http://dx.doi.org/10.1016/j.celrep.2014.08.062

TRPM3 and miR-204 Establish a Regulatory Circuit that Controls Oncogenic Autophagy in Clear Cell Renal Cell Carcinoma
Cancer Cell Nov 10, 2014; 26: 738–753
http://dx.doi.org/10.1016/j.ccell.2014.09.015

MicroRNA in Prostate, Bladder, and Kidney Cancer
Eur Urol 2011; 59:671-681
http://dx.doi.org/10.1016/j.eururo.2011.01.044

Micro-RNA profiling in kidney and bladder cancers
Urologic Oncology: Seminars and Original Investigations 2007; 25:387–392
http://dx.doi.org:/10.1016/j.urolonc.2007.01.019

MicroRNAs and cancer. Current and future perspectives in urologic oncology
Urologic Oncology: Seminars and Original Investigations 2010; 28:4–13
http://dx.doi.org:/10.1016/j.urolonc.2008.10.021

MicroRNAs and their target gene networks in renal cell carcinoma
BBRC 2011; 405:153-156
http://dx.doi.org/10.1016/j.bbrc.2011.01.019

MicroRNAs in osteosarcoma
Clin Chim Acta 2015; 444:9-17
http://dx.doi.org/10.1016/j.cca.2015.01.025

 

Table 2. miRNA cancer therapeutics

 

 

  • miRNA and mRNA cancer signatures determined by analysis of expression levels in large cohorts of patients
    | PNAS | Nov 19, 2013; 110(47): 19160–19165
    http://www.pnas.org/cgi/doi/10.1073/pnas.1316991110The study of mRNA and microRNA (miRNA) expression profiles of cells and tissue has become a major tool for therapeutic development. The results of such experiments are expected to change the methods used in the diagnosis and prognosis of disease. We introduce surprisal analysis, an information-theoretic approach grounded in thermodynamics, to compactly transform the information acquired from microarray studies into applicable knowledge about the cancer phenotypic state. The analysis of mRNA and miRNA expression data from ovarian serous carcinoma, prostate adenocarcinoma, breast invasive carcinoma, and lung adenocarcinoma cancer patients and organ specific control patients identifies cancer-specific signatures. We experimentally examine these signatures and their respective networks as possible therapeutic targets for cancer in single cell experiments.

 

 

RNA editing is vital to provide the RNA and protein complexity to regulate the gene expression. Correct RNA editing maintains the cell function and organism development. Imbalance of the RNA editing machinery may lead to diseases and cancers. Recently,RNA editing has been recognized as a target for drug discovery although few studies targeting RNA editing for disease and cancer therapy were reported in the field of natural products. Therefore, RNA  editing may be a potential target for therapeutic natural products

 

Aberrant microRNA (miRNA) expression is implicated in tumorigenesis. The underlying mechanisms are unclear because the regulations of each miRNA on potentially hundreds of mRNAs are sample specific.

 

We describe a novel approach to infer Probabilistic Mi RNA–mRNA  Interaction Signature (‘ProMISe’) from a single pair of miRNA–mRNA expression profile. Our model considers mRNA and miRNA competition as a probabilistic function of the expressed seeds (matches). To demonstrate ProMISe, we extensively exploited The Cancer Genome Atlas data. As a target predictor, ProMISe identifies more confidence/validated targets than other methods. Importantly, ProMISe confers higher cancer diagnostic power than using expression profiles alone.

Gene set enrichment analysis on averaged ProMISe uniquely revealed respective target enrichments of oncomirs miR-21 and 145 in glioblastoma and ovarian cancers. Moreover, comparing matched breast (BRCA) and thyroid (THCA) tumor/normal samples uncovered thousands of tumor-related interactions. For example, ProMISe– BRCA network involves miR-155/183/21, which exhibits higher ProMISe coupled with coherently higher miRNA expression and lower target expression; oncomirs miR-221/222 in the ProMISe–THCA network engage with many downregulated target genes. Together, our probabilistic approach of integrating expression and sequence scores establishes a functional link between the aberrant miRNA and mRNA expression, which was previously under-appreciated due to the methodological differences.

 

 

 

 

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Larry H. Bernstein, MD, FCAP, Reviewer and Curator

http://pharmaceuticalinnovation.com/2012-12-09/larryhbern/Silencing Cancers with Synthetic siRNAs

The challenge of cancer drug development has been marker by less than a century of development of major insights into the know of biochemical pathways and the changes in those pathways in a dramatic shift in enrgy utilization and organ development, and the changes in those pathways with the development of malignant neoplasia.  The first notable change is the Warburg Effect (attributed to the 1860 obsevation by Pasteur that yeast cells use glycolysis under anaerobic conditions).  Warburg also referred to earlier work by Meyerhoff, in a ratio of CO2 release to O2 consumption, a Meyerhoff ratio.  Much more was elucidated after the discovery of the pyridine nucleotides, which gave understanding of glycolysis and lactate production with a key two enzyme separation at the forward LDH reaction and the back reentry to the TCA cycle.  But the TCA cycle could be used for oxidative energy utilization in the mitochondria by oxidative phosphorylation elucidated by Peter Mitchell, or it can alternatively be used for syntheses, like proteins and lipid membrane structures.

A brilliant student in Leloir’s laboratory in Brazil undertook a study of isoenzyme structure in 1971, at a time that I was working under Nathan O. Kaplan on the mechanism of inhibition of mitochondrial malate dehydrogenase. In his descripton, taking into account the effect of substrates upon protein stability (FEBS) could be, in a prebiotic system, the form required in order to select protein and RNA in parallel or in tandem in a way that generates the genetic code (3 bases for one amino acid). Later, other proteins like reverse transcriptase, could transcribe it into the more stable DNA. Leloir had just finished ( a few years before 1971 but, not published by these days yet) a somehow similar reasoning about metabolic regions rich in A or in C or .. G or T.  He later spent time in London to study the early events in the transition of growing cells linked to ion fluxes, which he was attracted to by the idea that life is so strongly associated with the K (potassium) and Na (sodium) asymmetry.   Moreover, he notes that while DNA is the same no matter the cell is dead or alive,  and therefore,  it is a huge mistake to call DNA the molecule of life. In all life forms, you will find K reach inside and Na rich outside its membrane. On his return to Brazil, he accepted a request to collaborate with the Surgery department in energetic metabolism of tissues submitted to ischemia and reperfusion. This led me back to Pasteur and Warburg effects and like in Leloir´s time, he worked with a dimorphic yeast/mold that was considered a morphogenetic presentation of the Pasteur Effect.  His findings were as follows. In absence of glucose, a condition that prevents the yeast like cell morphology, which led to the study of an enzyme “half reaction”. The reaction that on the half, “seen in our experimental conditions did not followed classical thermodynamics” (According to Collowick & Kaplan (of your personal knowledge) vol. I See Utter and Kurahashi in it). This somehow contributed to a way of seeing biochemistry with modesty. The second and more strongly related to the Pasteur Effect was the use an entirely designed and produced in our Medical School Coulometer spirometer that measures oxygen consumption in a condition of constant oxygen supply. At variance with Warburg apparatus and Clark´s electrode, this oxymeters uses decrease in partial oxygen pressure and decrease electrical signal of oxygen polarography to measure it (Leite, J.V.P. Research in Physiol. Kao, Koissumi, Vassali eds Aulo Gaggi Bologna, 673-80-1971). “With this, I was able to measure the same mycelium in low and high “cell density” inside the same culture media. The result shows, high density one stops mitochondrial function while low density continues to consume oxygen (the internal increase or decrease in glycogen levels shows which one does or does not do it). Translation for today: The same genome in the same chemical environment behave differently mostly likely by its interaction differences. This previous experience fits well with what  I have to read by that time of my work with surgeons.  Submitted to total ischemia tissues mitochondrial function is stopped when they already have enough oxyhemoglobin (1) Epstein, Balaban and Ross Am J Physiol.243, F356-63 (1982) 2) Bashford , C. L, Biological membranes a practical approach Oxford Was. P 219-239 (1987).”

Of course, the world of medical and pharmaceutical engagement with this problem, though changed in focus, has benefitted hugely from “The Human Genome Project”, and the events since the millenium, because of technology advances in instrumental analysis, and in bioinformatics and computational biology.  This has lead to recent advances in regenerative biology with stem cell “models”, to advances in resorbable matrices, and so on.  We proceed to an interesting work that applies synthetic work with nucleic acid signaling to pharmacotherapy of cancer.

Synthetic RNAs Designed to Fight Cancer

Fri, 12/06/2013 Biosci Technology
Xiaowei Wang and his colleagues have designed synthetic molecules that combine the advantages of two experimental RNA therapies against cancer. (Source: WUSTL/Robert J. Boston)In search of better cancer treatments, researchers at Washington University School of Medicine in St. Louis have designed synthetic molecules that combine the advantages of two experimental RNA therapies.  The study appears in the December issue of the journal RNA.
 RNAs play an important role in how genes are turned on and off in the body. Both siRNAs and microRNAs are snippets of RNA known to modulate a gene’s signal or shut it down entirely. Separately, siRNA and microRNA treatment strategies are in early clinical trials against cancer, but few groups have attempted to marry the two.   “These are preliminary findings, but we have shown that the concept is worth pursuing,” said Xiaowei Wang, assistant professor of radiation oncology at the School of Medicine and a member of the Siteman Cancer Center. “We are trying to merge two largely separate fields of RNA research and harness the advantages of both.”
 “We designed an artificial RNA that is a combination of siRNA and microRNA, The showed that the artificial RNA combines the functions of the two separate molecules, simultaneously inhibiting both cell migration and proliferation. They designed and assembled small interfering” RNAs, or siRNAs,  made to shut down– or interfere with– a single specific gene that drives cancer.  The siRNA molecules work extremely well at silencing a gene target because the siRNA sequence is made to perfectly complement the target sequence, thereby
  • silencing a gene’s expression.
Though siRNAs are great at turning off the gene target, they also have potentially dangerous side effects:
  • siRNAs inadvertently can shut down other genes that need to be expressed to carry out tasks that keep the body healthy.
 According to Wang and his colleagues, siRNAs interfere with off-target genes that closely complement their “seed region,” a short but important
  • section of the siRNA sequence that governs binding to a gene target.
 “We can never predict all of the toxic side effects that we might see with a particular siRNA,” said Wang. “In the past, we tried to block the seed region in an attempt to reduce the side effects. Until now,
  • we never tried to replace the seed region completely.”
 Wang and his colleagues asked whether
  • they could replace the siRNA’s seed region with the seed region from microRNA.
Unlike siRNA, microRNA is a natural part of the body’s gene expression. And it can also shut down genes. As such, the microRNA seed region (with its natural targets) might reduce
  • the toxic side effects caused by the artificial siRNA seed region. Plus,
  • the microRNA seed region would add a new tool to shut down other genes that also may be driving cancer.
 Wang’s group started with a bioinformatics approach, using a computer algorithm to design
  • siRNA sequences against a common driver of cancer,
  • a gene called AKT1 that encourages uncontrolled cell division.
They used the program to select siRNAs against AKT1 that also had a seed region highly similar to the seed region of a microRNA known to inhibit a cell’s ability to move, thus
  • potentially reducing the cancer’s ability to spread.
In theory, replacing the siRNA seed region with the microRNA seed region also would combine their functions
  • reducing cell division and
  • movement with a single RNA molecule.
 Of more than 1,000 siRNAs that can target AKT1,
  • they found only three that each had a seed region remarkably similar to the seed region of the microRNA that reduces cell movement.
 They then took the microRNA seed region and
  • used it to replace the seed region in the three siRNAs that target AKT1.
The close similarity between the two seed regions is required because
  • changing the original siRNA sequence too much would make it less effective at shutting down AKT1.
 They dubbed the resulting combination RNA molecule “artificial interfering” RNA, or aiRNA. Once they arrived at these three sequences using computer models,
  1. they assembled the aiRNAs and
  2. tested them in cancer cells.
 One of the three artificial RNAs that they built in the lab
  • combined the advantages of the original siRNA and the microRNA seed region that was transplanted into it.
This aiRNA greatly reduced both
  1. cell division (like the siRNA) and
  2. movement (like the microRNA).
And to further show proof-of-concept, they also did the reverse, designing an aiRNA that
  1. both resists chemotherapy and
  2. promotes movement of the cancer cells.
 “Obviously, we would not increase cell survival and movement for cancer therapy, but we wanted to show how flexible this technology can be, potentially expanding it to treat diseases other than cancer,” Wang said.
Source: WUSTL

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Reported by: Dr. V.S. Karra, Ph.D

Transcription is a cellular process by which genetic information from DNA is copied to messenger RNA for protein production. But anticancer drugs and environmental chemicals can sometimes interrupt this flow of genetic information by causing modifications in DNA.

Chemists at the University of California, Riverside have now developed a test in the lab to examine how such DNA modifications lead to aberrant transcription and ultimately a disruption in protein synthesis.

The chemists report that the method, called “competitive transcription and adduct bypass” or CTAB, can help explain how DNA damage arising from anticancer drugs and environmental chemicals leads to cancer development.

“Aberrant transcription induced by DNA modifications has been proposed as one of the principal inducers of cancer and many other human diseases,” said Yinsheng Wang, a professor of chemistry, whose lab led the research. “CTAB can help us quantitatively determine how a DNA modification diminishes the rate and fidelity of transcription in cells. These are useful to know because they affect how accurately protein is synthesized. In other words, CTAB allows us to assess how DNA damage ultimately impedes protein synthesis, how it induces mutant proteins.”

Study results appeared online in Nature Chemical Biology on Aug. 19.

Wang explained that the CTAB method can be used also to examine various proteins involved in the repair of DNA. One of his research group’s goals is to understand how DNA damage is repaired—knowledge that could result in the development of new and more effective drugs for cancer treatment.

“This, however, will take more years of research,” Wang cautioned.

His lab has a long-standing interest in understanding the biological and human health consequences of DNA damage. The current research was supported by the National Cancer Institute, the National Institute of Environmental Health Sciences and the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health.

Wang was joined in the research by UC Riverside’s Changjun You (a postdoctoral scholar and the research paper’s first author), Xiaoxia Dai, Bifeng Yuan, Jin Wang and Jianshuang Wang; Philip J. Brooks of the National Institute on Alcohol Abuse and Alcoholism, Md.; and Laura J. Niedernhofer of the University of Pittsburgh School of Medicine, Penn.

Next, the researchers plan to use CTAB to investigate how other types of DNA modifications compromise transcription and how they are repaired in human cells.

A quantitative assay for assessing the effects of DNA lesions on transcription

Source:

http://www.rdmag.com

University of California, Riverside

 

 

 

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