The Scientist’s Top 10 Innovations in 2017—and Jerry’s Top Pick

  • Multiplexed Assays Are Trending
  • Miniaturization is Big—Pun Intended
  • Jerry’s Top Pick is an Amazing 4 Inch Cube

Taken from the-scientist.com

Welcome to my first blog of the New Year, 2018! My New Year’s resolution is to “double down” by giving my best effort to provide interesting and informative content about what’s trending in nucleic acid research. Having said that, and in keeping with tradition, this first blog of the year pairs my comments on the Top 10 Innovations in 2017, as reported by the The Scientist, with my personal “fav” for the best new product launched last year.

So, with an imaginary loud flourish of trumpets, read on to learn about The Scientist’s 10 winners that I’ve listed from 10th to 1st place—to build your interest—after which I comment on my personal fav.

Top 10 Innovations in 2017 Reported by The Scientist

10. TrueCut Cas9 Protein v2 from Thermo Fisher Scientific is a next-generation CRISPR-Cas9 protein engineered to deliver maximum editing efficiency across a range of genes and standard cell lines, as well as stem cells, T cells, and primary cells.

With regard to this product, I think it’s worth mentioning that TriLink offers mRNA-encoded Cas9, as an alternative to delivering Cas9 protein per se. Also, my several past blogs on CRISPR can be accessed here.

9. TSQ Altis Triple Stage Mass Spectrometer from Thermo Fisher Scientific robustly and reliably quantitates most analyte types, even in complex samples such as plasma and tissue, thus enabling wide applicability, including forensic toxicology and clinical research.

8. Chromium from 10x Genomics for profiling single-cell gene expression, enables deep profiling of complex cell populations, is provided as a complete droplet-based system of reagents, barcodes, hardware and software for sample prep prior to high-throughput sequencing.

7. Edit-R crRNA Library—Human Genome from Dharmacon provides users with an arrayed library of synthetic crRNA guides in a “one-well-per-gene” format, with four distinct guides per gene for redundancy to improve statistical power.

6. HiBiT Protein Tagging System from Promega is a new detection system for quantifying proteins in or on a cell of interest, using a small and easily integrated 11-amino-acid tag (HiBiT) that interacts with a complementary large 156-amino-acid component leading to bioluminescence.

5. SR-X Ultra-Sensitive Biomarker Detection System from Quanterix offers more than 80 different assays to test samples (e.g. blood, serum, cerebral spinal fluid, single-cell lysates) for cytokines or other markers of neurodegeneration or neuroinflammation, and more.

4. Blaze from Intabio is a system for detecting and identifying protein isoforms that aims to save pharma companies time in lab prep work for QC of biologics manufacturing. Launch will be “within the next few months” and “pricing is still yet to be set,” according to The Scientist.

3. QGel Assay Kit for Organoids from QGel provides fully synthetic extracellular matrix to reproducibly grow research organoids, which are miniaturized and simplified version of an organ produced in vitro.

2. i-STAT Alinity from Abbott is a handheld, cartridge-based blood-testing device for user-friendly point-of-care assays on a blood sample of just several drops—including glucose levels and hematocrit—with results directly delivered to a patient’s medical record for clinicians within 2-10minutes. Alinity is available in about 50 countries, but Abbott is waiting for a few more assays to be cleared by the US Food and Drug Administration before selling it stateside.

Regarding this product, I should mention that my several past blogs on point-of-care can be accessed here.

1. IsoCode Chip from IsoCode is new single-cell technology allowing researchers to characterize cells based on the proteins they secrete—as many as 42 different cytokines, chemokines, and other types of molecules. IsoCode chips contain thousands of long microchambers that house only single cells. Within each microchamber, 15 spatially separated slots contain up to three different antibodies targeting specific secreted proteins; upon binding, each antibody fluoresces in a different color to distinguish the proteins. This provides the ability to simultaneously profile thousands of individual T cells or immune cells at one time.

Taken from The Scientist Dec 2017

IsoCode chips come in 10 different panels, ranging from 24 to 42 antibodies per panel, at a cost of $500–$600. The automated IsoLight imaging and workflow platform can be purchased starting at $200,000. But the IsoCode chips can also be paired with other fluorescence microscopy systems.

Jerry’s Fav for the Best Innovation in 2017

My personal pick for this honor goes to the world’s first on-site Legionella DNA test to prevent Legionnaires’ disease, which was released this past November by the Canadian company Spartan Bioscience. According to a press release, it is the first on-site DNA test for Legionella bacteria and it can detect and quantify Legionella in only 45 minutes, compared to 10-14 days for off-site sample analysis using traditional culturing methodology. The system pictured here consists of a coffee-cup-sized portable DNA analyzer called the Spartan Cube, which employs a single-use disposable test cartridge.

Taken from ctvnews.ca

This innovative product stood out for me because it brings together the following topics that I have separately blogged about:

  • Reoccurring outbreaks of potentially fatal Legionnaire’s disease, such as that which recently hit New York City, and also shut down Disneyland.
  • Decentralized analytical testing for more rapid “sample-to-answer” applications on-site, i.e. in the field—wherever that might be—or at point-of-care in hospitals, clinics, and elsewhere.
  • Miniaturization and simplification of qPCR using cleverly engineered devices, such as the Spartan Cube, in conjunction with single-use disposable test cartridges.

Legionella is a common environmental bacterium that can infect the cooling towers of Heating, Ventilation, and Air Conditioning (HVAC) systems of large buildings. Infected cooling towers release aerosolized water droplets contaminated with Legionella into the surrounding air. Globally, there are hundreds of thousands of office-building towers, hospitals, hotels, shopping malls, and other large buildings at risk for infection by Legionella. Weekly testing with the Spartan system can rapidly detect Legionella bacterial growth early, and thus allow cleaning and decontamination of the cooling tower before Legionella reaches dangerous levels to human health.

In addition, and also importantly, traditional culture test methods can underestimate the Legionella concentration on site. The Centers for Disease Control and Prevention (CDC) found that Legionella culture can underestimate actual Legionella levels by a factor of 10 or more. Culture incorrectly reported that water samples were negative for Legionella an average of 11.5 percent of the time when in fact they were positive.

Paul Lem. Provided by Paul Lem

According to Paul Lem, M.D., CEO of Spartan Bioscience, who I contacted about cost to customers, “the price for the Cube and Legionella tests is $5-10K/building/year, depending on the building. It’s a subscription model.”

Regarding my further inquiry about testimonials to date, Dr. Lem provided a copy of a November 26, 2017 article reported in The Globe and Mail which quotes him as saying that “several property managers are testing the device at close to 100 properties, including BGIS and Ottawa’s KRP Properties, owned by tech entrepreneur [Sir] Terry Matthews.”

The article also states that “the market could be worth billions of dollars globally, encompassing office buildings, malls, hospitals, schools, theme parks, spas and so on. ‘Nobody really knows because the market doesn’t really exist yet,’” said Lem.

For the sake of increased public safety toward exposure to Legionella, let’s all hope that this application of the amazing Spartan Cube is indeed very successful. And, moreover, that 2018 is a great year for other nucleic acids-based innovations, many of which I look forward to blogging about here.

As usual, your comments are welcomed.

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Advances in Nucleic Acid-Based Therapeutics Against Alzheimer’s

  • Dementia Develops in Someone in the World Every 3 Seconds
  • Nucleic Acid-Based Approaches for an Alzheimer’s Drug Are Advancing
  • Ionis Pharmaceuticals is First to Begin Clinical Trials with Antisense Oligonucleotide Targeting Tau

The concept of nucleic acid-based therapeutics, as originally conceived by Paul Zamecnik, goes back to his seminal publication in 1978 that I’ve blogged about previously. However, it wasn’t until the advent of automated synthesis of various types of modified oligonucleotide analogs that this “antisense” approach to drug development achieved critical mass of sustained attention. Since then, synthetic oligonucleotides and mechanisms of interference with mRNA expression have greatly expanded to now include a diverse “armory” of alternatives to combat diseases, including:

  • Antisense oligonucleotides (ASOs) that induce cleavage of mRNA by RNase H or other mechanisms
  • Short-interfering RNA (siRNA) that are incorporated into RISC that cleaves mRNA
  • Antagomirs that block microRNA (miR) binding to mRNA
  • Aptamers that bind to target proteins as drugs, or for targeted delivery of other types of drugs
  • Splice-switching oligonucleotides (SSOs) that hybridize with a pre-mRNA and disrupt normal splicing
  • Recently reported RNA-guided RNA-targeted CRISPR-Cas variants that can knockdown RNA systems much more specifically than siRNA

Investigating possible NA-based approaches to currently refractory, or other so-called ‘undruggable’ diseases, has attracted much needed interest from academics and pharma researchers, as well as implementation of GMP procedures. Recognizing this need, and the absence of a conventional drug to treat Alzheimer’s disease (AD)—the leading cause of dementia in adults—kindled my efforts to research the literature and write this blog.

What is AD?

Auguste Deter, who was a patient of German psychiatrist Alois Alzheimer, was the first described case of what became known as Alzheimer’s disease. Taken from Wikipedia.org

According to a U.S. National Institutes of Aging fact sheet, AD is an irreversible, progressive brain disorder that slowly destroys memory and thinking skills, and eventually the ability to carry out the simplest tasks. In most people with AD, symptoms first appear in their mid-60s. Statistics vary, but AD accounts for as much as ~80% of all dementia—an umbrella term for loss of cognitive functioning—which will develop in someone in the world every 3 seconds, and afflict close to 50 million people in 2017. This is based on estimates by Alzheimer’s Disease International.

Brains from bodies of deceased persons who had advanced AD exhibit overall “shrinkage,” macroscopically, compared to persons who did not show indications

Taken from fellowshipoftheminds.com

of AD. Microscopically, AD is characterized by accumulation of toxic “amyloid plaques,” which are sticky buildup that accumulate outside nerve cells, or neurons. Amyloid is a protein that is normally found throughout the body, but in AD the protein is cleaved by beta secretase (BACE1) and gamma secretase to yield amyloid beta (Aβ) peptides of 36–43 amino acids. Aβ molecules can aggregate to form flexible soluble oligomers that may exist in several forms. It is now believed that certain misfolded oligomers can induce other Aβ molecules to also take the misfolded oligomeric form, leading to a chain reaction akin to a prion infection.

Amyloid plaques. Taken from slideshare.net

Since 1993, when a variant of the apolipoprotein E (APOE) gene was found to be strongly associated with increased vascular and plaque Aβ deposits in late-stage AD patients, many researchers have probed APOE connections to Aβ. However, in September 2017 this investigative field was reportedly “stunned” by results published by Shi et al. showing that neurotoxic effects associated with APOE may result from a damaging immune response to a different protein named tau, encoded by the MAPT gene. This late-breaking scientific news is part of an ongoing story of AD molecular pathology that interested readers will want to follow.

“Tangles” of tau protein (green) are visible in a brain cell from someone who had Alzheimer’s disease. Taken from Underwood, Science (2017)

Recent Advances in Nucleic Acid-Based Drugs for AD

My strategy for researching this topic began with querying Google Scholar for “Alzheimer’s” articles since 2013 that are coupled with key words for each of the above nucleic acid-based drug modalities, namely, “antisense,” “siRNA,” etc. This led to literature ranked by Google Scholar according to citation counts. I then perused this information to select items for each of these modalities. I linked to the search results for readers interested in further digging into these topics.

Antisense Oligonucleotides

A report that caught my attention was by Lane et al. from Ionis (formerly Isis) Pharmaceuticals. This company (founded and still led by Stanley Crooke, about whom I’ve blogged) is widely acknowledged to be the leader in clinical development of antisense therapeutics. In a nutshell, Lane et al. hypothesized that, given the critical role for tau (see above) in transducing Aβ-linked neurotoxicity, reducing the synthesis of tau could have a therapeutic effect.

Ionis-MAPTRx, a 2′-O-methoxyethyl chimeric ASO, was found to reduce tau expression in transgenic mice and was tested in FDA/IND-enabling toxicology studies in rodents and non-human primates (NHPs). Intrathecal administration of the highest dose in NHPs resulted in a mean MAPT mRNA reduction of 77% in frontal cortex and 74% in hippocampus without dose-limiting side effects. Following up on this, I found that on October 13, 2017, Ionis announced initiation of a Clinical Study of Ionis-MAPTRx in patients with AD, thus earning a $10 million milestone payment from Biogen.

In another ASO approach, Farr et al. have further investigated their previously reported 20-mer phosphonothioate oligonucleotide (GAO) that had been shown to knockdown levels of glycogen synthase kinase (GSK)-3β, which is a multifunctional protein implicated in the pathological characteristics of AD, including neurofibrillary tangles, Aβ, and neurodegeneration. In the present study, they assessed the impact of peripherally administered GAO on learning and memory—measured by a T-maze (see picture)—in two different mouse models of AD, as well as knockdown of protein expression. GAO-treated mice showed improved acquisition and retention, along with decreased protein levels. They concluded that this study “supports peripherally administered GAO as a viable means to mediate GSK-3β activity within the brain and a possible treatment for AD.”

Taken from ratbehaviour.org

To learn about a T-maze, click here.

siRNA

SLN loaded with siRNA (green). Taken from precisionnanosystems.com

While there are several intriguing studies of ASOs associated with AD therapy, siRNA is much more common in the research and treatment of AD. The report that I found most interesting was published by Rassu et al. in 2017, and is titled Nose-to-brain delivery of BACE1 siRNA loaded in solid lipid nanoparticles for Alzheimer’s therapy. My interest stemmed from the focus on developing delivery technology to achieve “nose-to-brain” as a very convenient, non-invasive route for delivery using solid lipid nanoparticles (SLNs), which are viewed as promising technologies for drug-delivery.

In this report, the siRNA targeted the secretase BACE1, which has been widely investigated because of its involvement in forming neurotoxic Aβ peptides, as mentioned above. To increase the transcellular pathway in neuronal cells, a short cell-penetrating peptide derived from rabies virus glycoprotein known as RVG-9R was used, based on previous studies demonstrating that intravenous treatment with an RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Building on this prior knowledge, Rassu et al. optimized the molar ratio of RVG-9R and BACE1 siRNA, and investigated chitosan-coated and uncoated SLNs as a nasal delivery system capable of exploiting both olfactory and trigeminal nerve pathways.

Taken from researchgate.net

The positive charges from protonated amino (NH2) groups of the coating formulation ensured muco-adhesiveness to the particles, and prolonged residence-time in the nasal cavity. They studied cellular transport of siRNA released from the SLNs using the Caco-2 cells, which is a human epithelial colorectal adenocarcinoma cell line, as a model for epithelial-like phenotypes. It was found that siRNA better permeates the monolayer when released from chitosan-coated SLNs vs. uncoated SLNs or “naked” siRNA.

Chitosan. Taken from Wikipedia.org

Antagomir

MicroRNA-146a (miR-146a) is upregulated in the brains of patients with AD and induces activation of tau (see above). To determine whether reducing miR-146a could ameliorate tau-related AD pathologies, Wang et al. assessed its levels and the use of a miR-146a inhibitor (antagomir) in a validated mouse model of AD. The antagomir and solvent control were delivered into the hippocampus of these mice at three months of age and memory was tested in all mice using several types of mazes (see above) before extracting brain samples for quantitative RT-PCR using measurements of miR-146a and protein targets. In a nutshell, the overall results demonstrated that improvement of memory by intrahippocampal miR-146a antagomir was associated with the predicted alterations in the tau-related neural pathway, confirming that inhibition of miR-146a expression has a therapeutic effect in this mouse model of AD. It was concluded that “this data support (sic) the concept that miR-146a antagomir is a potential efficacious therapeutic target for the tau pathology of AD.”

Aptamers

Aptamer A1 structure reported by Liang et al.

In a study published by Liang et al. in 2015, systematic evolution of ligands by exponential enrichment (SELEX) with random-sequence libraries was used to obtain a DNA aptamer (A1). That aptamer is pictured below, and has been shown to have high-affinity binding to purified human BACE1 extracellular domain. They subsequently confirmed that A1 exhibited a marked inhibitory effect on BACE1 activity in an AD cell model, based on decreased concentrations of Aβ fragments and BASE1 protein. These investigators concluded that “these findings support the preliminary feasibility of an aptamer evolved from a SELEX strategy to function as a potential BACE1 inhibitor. To our knowledge, this is the first study to acquire a DNA aptamer that exhibited binding specificity to BACE1 and inhibited its activity.”

Splice-Switching Oligonucleotides

According to Hinrich et al., apolipoprotein E receptor 2 (ApoER2), which is involved in long‐term potentiation, learning, and memory, has been proposed to be involved in AD, though a role for the receptor in the disease is not clear. ApoER2 signaling requires amino acids encoded by alternatively spliced exon 19. To test the role of deregulated ApoER2 splicing in AD, they designed a splice-switching oligonucleotide (SSO) that increases exon 19 splicing. Treatment of AD mice with a single dose of SSO corrected ApoER2 splicing for up to 6 months and improved synaptic function and learning and memory. They concluded that “these results reveal an association between ApoER2 isoform expression and AD, and provide preclinical evidence for the utility of SSOs as a therapeutic approach to mitigate AD symptoms by improving ApoER2 exon 19 splicing.”

CRISPR

Individuals heterozygous for the Swedish mutation of the amyloid precursor protein (APPswe) display an increased β-secretase cleavage leading to higher Aβ levels—both in brain and peripheral tissues, according to Gyorgy et al. They added that the mutation is a double base change adjacent to each other and has a dominant effect, which led them to hypothesize that the CRISPR system would selectively disrupt the mutated allele without affecting the wild-type (wt) allele.

In a nutshell, human APPswe fibroblasts and non-mutated control fibroblasts from subjects of the same family were grown in vitro, and then transfected with a Cas9 plasmid together with different guide RNAs (gRNAs) designed to bind either the mutated or non-mutated site with the mutation in the gRNA recognition sequence. Sanger sequencing was performed on cells that had been successfully transfected with CRISPR plasmids, and on such cells, both the APPswe mutant and wt alleles could be disrupted with gRNAs designed against the mutated and non-mutated sites, respectively. Moreover, these effects appeared to be highly specific as assayed by deep sequencing as they did not find any random mutations on the wt allele with the gRNA targeting the mutated site or vice versa.

This study reported in 2016 was said to “[provide] the first experimental evidence that the CRISPR/Cas9 method could be used to develop a novel treatment strategy against familial forms of Alzheimer’s disease caused by dominant mutations.”

Closing Comments

From the above sampling of publications reporting promising results for nucleic-acid-based therapeutic approaches to AD, I hope you will agree with me that it seems likely a clinically successful drug will prevail. What and when are uncertain, but I’m betting that something will be Ionis-MAPTRx , which is the most advanced clinical candidate to date.

Addendum

Taken from October 31, 2017 GEN

Alzheimer’s disease may move, cancer-like, from place to place in the body, lodging in the brain after originating in peripheral tissues, according to October 31, 2017 news in GEN. This cancer-like mobility was demonstrated through a technique called parabiosis—the surgical union of two specimens to allow them to share a blood supply, as shown here. This technique was used to keep pairs of mice together for several months, wherein normal mice, which don’t naturally develop AD, were joined to transgenic AD mice, modified to carry a mutant human gene that produces high levels of plaque-forming Aβ. It was reported that human Aβ originating from transgenic AD mice entered the circulation and accumulated in the brains of normal mice, forming cerebral amyloid angiopathy and Aβ plaques after a 12-month period of parabiosis.

Highlights of 2015 Publications Using TriLink BioTechnologies Products

  • Publications Citing TriLink Products Exceed 6,000
  • TriLink Products Showed up at a Rate of One Publication per Work Day
  • Among These Customer Publications, Modified mRNA is Trending
Taken from thetrymovement.com

Taken from thetrymovement.com

From my college classes decades ago, I can still clearly recall—thankfully—many “ah ha” moments. Most importantly is when I crystalized to purity and then confirmed structure by NMR the first compound I synthesized in Organic Chemistry Lab. Another ah ha moment—but on a completely different level—was during a philosophy class when the professor partially paraphrased a quote by Aristotle as “we are what we do.” The full quote given above is even more thought provoking because it ties in the notion of excellence, which I took to heart then, and have attempted to live by ever since.

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We’re Celebrating Click Chemistry In Honor of National DNA Day

  • The Verbification of Click Chemistry 
  • Old Chemistry Morphs into New Applications for DNA and RNA  
  • Amazingly, Phosphorus in DNA and RNA is not Needed for Function 

This post comes only two days after National DNA Day 2015 on April 25th so it’s apropos to feature DNA, but I’d also like to give a nod to the lesser recognized RNA, without which DNA would be akin to music notes in search of a melody.  If you’re a regular reader of this blog, you know my stance on this subject and so I digress…

So-called “Click Chemistry” is trending so “hot” that it has led to a phenomenon known as verbification, which is when a noun becomes a verb by virtue of popularity and linguistic convenience. So, just as Google has become to google for virtually everyone, Click has become to click for synthetic chemists and biotechnologists. Whether or not you’re already familiar with Clicking, I hope to provide herein some interesting snippets about Click, its growing ubiquity, and how it has enabled synthesis of a completely novel, non-phosphorous linkage in DNA that nevertheless functions flawlessly in vivo—a stunning feat never before achieved that has intriguing implications about life. More on that later, but first some snippets about Click.

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DNA’s Forgotten Discoverer: Swiss Scientist Friedrich Miescher 

  • Discovered in 1869 in Pus Cells from Bandages of Crimean War Soldiers
  • Miescher Named this New Matter Nuclein and Intuited that it Played a Fundamental Role in Heredity
  • This put the “N” in DNA—Deoxynucleic Acid
  • Children now Isolate DNA from Fruits & Vegetables in Elementary School 

Truth be told, what led me to writing this post was suddenly realizing one day that, although the vast majority of my professional career involves nucleic acids—and DNA in particular—I did not know anything about the discovery of DNA or its naming. My follow-on thoughts were that this was somewhat embarrassing for a blogger focused on nucleic acids, and should be remedied by some homework! This is also good timing since my mind is currently aflutter with all things DNA in anticipation of National DNA Day coming up on April 25. In the event that you recall my past commentary about the bias toward DNA, yes I am still supporting a National RNA Day to balance the ranks, but I digress…

Friedrich Miescher as young man (taken from cienciaytecnicasuabia.blogspot.com via Bing Images)

Friedrich Miescher as young man (via Bing Images)

In doing my so-called homework, I learned about Swiss scientist Friedrich Miescher’s life story and circumstances surrounding his discovery in the late 1860s of new matter that he named nuclein, which eventually became incorporated into the term nucleic acid. Those circumstances, including Miescher’s unusual source of nuclein, were quite interesting to me so I thought they’d be worth sharing in this post, which draws upon a lengthy article by Ralf Dahm, who has written extensively about Miescher, and has a website worth visiting.

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