CasX Enzymes: A New Family of RNA-Guided Genome Editors

  • Search of Unusual Microbes Yields New CRISPR-Cas Systems
  • Tiny Life Forms Have Smallest Working CRISPR-Cas Systems
  • Novel CasX Structure and Mechanism Characterized by Cryo-Electron Microscopy

In 2012, a Science magazine publication by Doudna, Charpentier, and coworkers describedCas9, the CRISPR-associated (Cas) protein, as a programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. This work has already been cited ~6,500 times, alongside two other Cas9 studies listed in PubMed that year. They have been followed by a steadily increasing number of annual Cas9-related publications, as show in the chart below. A large part of this growing interest is due to the proven utility of CRISPR-Cas9, and variants thereof, for gene editing, which I have previously blogged about.

Given the broad scientific, clinical, and commercial utility of CRISPR-Cas systems, it is not surprising that there has been considerable effort directed toward either engineering analogs of known Cas enzymes, or discovering new homologs in unexplored organisms. With regards to the latter approach, Doudna, Banfield and coworkers noted in 2017 that the then available CRISPR-Cas technologies were based solely on systems from isolated, cultured bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped.

They added that metagenomics—sequencing DNA extracted directly from natural microbial communities—provides access to the genetic material of a huge array of uncultured organisms. For this reason, through use of metagenomics, the researchers were able to discover two previously unknown CRISPR-Cas systems. These new Cas proteins, named CasX and CasY to designate as yet unknown specifics, are said to be among the most compact systems yet discovered. In February 2019, as a follow-up to these discoveries, Doudna and collaborators published the mechanistic details for CRISPR-CasX in Nature magazine. This will be the focus of this blog, but before that story, here are some introductory comments about metagenomics, a transformative technology in its own right.

Putting Together All of the Pieces

In a review by Chen and Pachter, metagenomics is described as “the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species.” They add that “metagenomics has revolutionized microbiology by shifting focus away from clonal isolates towards the estimated 99% of microbial species that cannot currently be cultivated.”

A typical metagenomics project begins with the construction of a DNA library derived from a minimally processed environmental sample that is usually comprised of multiple different genomes with different copy numbers. The increasing capacity of factory-like sequencing centers has facilitated whole-genome shotgun sequencing and genome assembly of these complex mixtures. At the risk of oversimplification, this to me is conceptually akin to simultaneously putting together correctly all of the pieces of multiple different jigsaw puzzles.

There are many technical variations for these sequencing and bioinformatic procedures, but at a high-level, these can be categorized as either using extracted DNA per se (metagenomics) or cDNA derived from reverse transcription of extracted RNA (metatranscriptomics). Both of these approaches were used in the aforementioned discovery of CasX and CasY, starting with quite unusual sample sources: (1) acid-mine drainage samples (from the Richmond Mine at Iron Mountain in California); (2) river water and sediment samples (from a site along the Colorado River in Colorado); and (3) cold, CO2-driven geyser water (from Crystal Geyser on the Colorado Plateau in Utah pictured here). Presumably, these relatively unusual sample sources increased the discovery-probability, as scientists were able to examine the previously unknown organisms present in each sample.

Discovery of CasX and CasY

Using metagenomics, Doudna, Banfield and coworkers found a number of CRISPR-Cas systems, including what they believed to be the first Cas9 in the in the archaeal domain of life. Archaea constitute a domain of single-celled microorganisms. These microbes are prokaryotes, meaning they have no cell nucleus. Archaeal cells have unique properties that separate them from the other two domains of life, bacteria and eukarya, as depicted here. Archaea are further divided into multiple recognized phyla, but classification is difficult, as most have not been isolated in the laboratory.

This divergent Cas9 protein was found in little studied nanoarchaea, as part of an active CRISPR-Cas system. Incidentally, nanoarchaea are “nano” indeed, only ~400 nm in diameter—about 5% of the volume of your archetypical 1 μm3prokaryote, according to one estimate—andNanoarchaeum equitansharbors a genome that is only 480 kb. Also discovered were two previously unknown Cas proteins unlike all the previous Cas proteins. These were named CasX and CasY, since it was not clear what they actually did. CasX and CasY are among the most compact systems yet discovered, according to these researchers, who concluded that “interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.”

Cryo-Electron Microscopy (cryo-EM) Characterization of CasX

In February 2019, a follow-up report in Natureby Doudna and collaborators focused on the mechanistic details for CRISPR-CasX. Although RNA-guided DNA binding and cutting proteins have proven to be transformative tools for genome editing across a wide range of cell types and organisms, only two kinds of CRISPR-Cas nucleases—Cas9 (depicted here) and Cas12a (aka Cpf1)—provide the foundation for this revolutionary technology.

The only conserved part of CasX, the RuvC domain, shares less than 16% identity with RuvC domains in either Cas9 or Cas12a. This evolutionary ambiguity in CasX hinted that this enzyme may have a structure and molecular mechanism distinct from that of other CRISPR-Cas enzymes. These structural and mechanistic questions were investigated by use of cryo-EM, a specialized method recently catapulted into widespread view by the co-awarding of the 2017 Nobel Prize in Chemistry to its three pioneers.

As discussed in an introductory YouTube video on cryo-EM, scientists traditionally used X-ray crystallography to obtain biomolecular structures, which requires growing suitable crystals that are oftentimes extremely difficult or not possible to obtain. However, as seen here, freezing a thin layer of a solution of the sample for cryo-EM enables the technique to handle structures for which crystallography is not a viable option. In addition, cryo-EM can visualize much larger structures than crystallography can—100-fold larger according to one cryo-EM expert. By way of example, a 1.8-Å-resolution structure of 334-kDa glutamate dehydrogenase, and 3.6-Å-resolution structure for 11,200-kDa Dengue virus have been reported.

Scientist preparing samples for cryo-EM under liquid nitrogen temperature

Doudna and collaborators took advantage of cryo-EM to obtain eight molecular structures of CasX in different states, which interested readers can view by consulting the 2019 Nature publication (unfortunately, copyright restrictions prevent reproduction here). The researchers’ verbal description of what was found highlights the following structural elements:

“An unanticipated quaternary structure in which the RNA scaffold dominates the architecture and organization of the enzyme. Phylogenetic, biochemical and structural data show that CasX contains domains distinct from—but analogous to—those found in Cas9 and Cas12a, as well as novel RNA and protein folds; thus establishing the CasX enzyme family as the third CRISPR-Cas platform that is effective for genetic manipulation. Finally, distinct conformational states observed for CasX suggest an ordered non-target- and target-strand cleavage mechanism that may explain how CRISPR–Cas enzymes with a single active site, such as Cas12a, achieve double-stranded DNA (dsDNA) cleavage. The small size of CasX (<1,000 amino acids), its DNA cleavage characteristics, and its derivation from non-pathogenic microorganisms offer important advantages over other CRISPR–Cas genome-editing enzymes.”


On the basis of their functional and structural data, Doudna and collaborators propose a model of CasX activation and DNA cleavage that includes the following steps: (1) guide RNA binding-induced CasX structural stabilization and DNA search; (2) non-target-strand binding-assisted DNA unwinding, R-loop formation and nontarget-strand loading into the RuvC active site; (3) RNA-DNA hybrid duplex bending with the aid of the proposed target-strand loading (TSL) domain to position the target DNA strand for cleavage; and (4) product release after the cleavage of both DNA strands.

They added that two distinct target DNA-bound states indicate that CasX coordinates sequential dsDNA cleavage by its single RuvC nuclease, using the zinc-finger-containing TSL domain. Also, the TSL domain appears to confer a convergent mechanism of acute target-strand DNA bending that is central to all type V single-nuclease CRISPR-Cas enzymes.

Looking forward, they speculated that “[t]he compact size, dominant RNA content and minimal trans-cleavage activity of CasX differentiate this enzyme family from Cas9 and Cas12a, and provide opportunities for therapeutic delivery and safety that may offer important advantages relative to existing genome-editing technologies.”

In my opinion, it will likely take some time and considerable experimentation by the scientific community to assess whether any of these potential advantages offered by CasX will actually pan out and lead to widespread adoption. In the meantime, mRNA-encoding Cas9 has firmly established its utility and enjoys extensive adoption, as exemplified by many diverse applications that I found among the search results for “TriLink AND Cas9” in Google Scholar.

As usual, your comments are welcomed.

You and Your Microbiome—Part 4

  • Your Microbiome Is Modulated by Your Diet
  • Probiotic Dietary Supplements Are Big Business but Lack Scientific Rigor
  • ‘Personalized Nutrition’ for Controlling Glucose Levels Is Supported by a New Study

My 2013 blog, Meet Your Microbiome: The Other Part of You, constituted part 1 of this series. The intent was to underscore scientific recognition that trillions of microbes reside in and on each of us, and influence our health. Moreover, the compositions of these microbiomes change with our diet, what we drink or breath, and who we come into contact with, whether that be family members, pets, or close friends.

Part 2 of this series was my 2014 blog, which focused on links between the global obesity epidemic and the gut microbiome, and cautioned microbiome-based therapies. In part 3, which followed in 2017, I commented on the ‘Top Ten Cited’ microbiome publications in Google Scholar. As shown by the chart below, there is an exponential increase in the annual number of publications in PubMed with terms ‘microbiome(s)’ in all fields. The ~8,900 papers that appeared in 2017 give an average of ~24 papers per day, non-stop 7 days per week, which is truly an impressive torrent of new information.

Credit: Jerry Zon

Cindy D. Davis, PhD

Part 4 of my series on You and Your Microbiome is largely a synopsis of Diet, Microbiome and Health: Past, Present & Future, a recent seminar by Cindy D. Davis, PhD, a Director in the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH). The mission of ODS includes strengthening the common understanding of dietary supplements by evaluating scientific information, supporting research, and educating the public in an effort to foster an enhanced quality of life and health for the U.S. population.

The importance of the ODS mission, and the NIH’s highly regarded reputation as a source of reliable health-related information, led me to select this seminar by Dr. Davis.


Dr. Davis presented her seminar as part of the 2018 Dietary Supplement Research Practicum sponsored by the NIH ODS. This 2.5-day annual event for faculty, students, and health practitioners provides a thorough overview of the issues, concepts, unknowns, and controversies surrounding dietary supplements. This seminar, followed by a Q&A session, is available as a video on YouTube.

In her seminar, Dr. Davis introduces the human microbiome and describes evidence on how diet and dietary supplements can modulate the gastrointestinal (GI) microbial community structure. She also describes evidence on how the GI microbiome can influence the response to dietary components, and the relationship between dietary components, the microbiome, and chronic diseases such as obesity, cardiovascular disease, and cancer.

The Human Microbiome

Perhaps the biggest surprise of the Human Genome Project (HGP) was the discovery that the human genome contains only 20,000 – 25,000 protein-coding genes, about a fifth of the number researchers had expected to find. To search for the missing genes that could account for this discrepancy, researchers started looking toward other sources of genetic material that contribute to human function. One of these sources was the human microbiome.

The human microbiome is defined as the collective genomes of the microbes (composed of bacteria, bacteriophage, fungi, protozoa, and viruses) that live inside and on the human body. There are about 10 times as many microbial cells as human cells. So, to study the human as a “supraorganism,” composed of both non-human and human cells, the NIH launched the Human Microbiome Project (HMP) in 2007, as a conceptual extension of the Human Genome Project.

It is now known that, when compared to the total number of human genes, the genetic contribution of the microbiome to the human supraorganism may be many hundreds of times greater than the genetic contribution of the human genome.

There are estimated to be ~100 trillion (!) gut microbiota, defined as the microbes in our GI tract. Most of the microbes in the microbiome do not cause disease. In fact, humans rely on microbes to perform many important functions that we cannot perform ourselves. Microbes digest food to generate nutrients for host cells, synthesize vitamins, metabolize drugs, detoxify carcinogens, stimulate renewal of cells in the gut lining, and activate and support the immune system.

Establishing what constitutes a healthy microbiome is important because high or low microbial diversity can have different implications for health or disease, depending on the body site. For example, it has been shown that low microbial diversity in the gut is associated with obesity, inflammatory bowel disease, and Crohn’s disease; whereas high microbial diversity in the vagina is often associated with bacterial vaginosis, the most common type of vaginal infection.

Dietary Modulation of Gut Microbiota

There is growing concern that recent lifestyle trends, most notably the high-fat/high-sugar “Western” diet, have altered the genetic composition and metabolic activity of our human gut microbiomes. Such diet-induced changes to gut-associated microbial communities are suspected of contributing to growing epidemics of chronic illnesses in the developed world, including obesity and inflammatory bowel disease. Yet, it remains unclear how quickly and reproducibly gut bacteria respond to dietary change.

David et al. have addressed this question in a study of human volunteers, and found that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression.

Microbial activity mirrored the differences between carnivorous and herbivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi, and even viruses. David et al. concluded that, in concert, these results demonstrate that the gut microbiome can rapidly respond to an altered diet, potentially facilitating the diversity of human dietary lifestyles.

Similar findings have been reported by De Filippo et al., who evaluated the impact of diet in shaping gut microbiota in a comparative study in children from Europe and rural Africa. More recently, there was widespread media coverage on a publication in the highly respected Cell journal by Vangay et al. They used metagenomic DNA sequencing and found that migration from a non-Western country to the U.S. is associated with immediate loss of gut microbiome diversity and function, in which U.S.-associated strains and functions displace native strains and functions. These effects increase with duration of U.S. residence and are associated with increased obesity.

Probiotic Dietary Supplements

In her seminar, Dr. Davis emphasized that usage of probiotics, which by definition contain live bacteria, is by far the fastest growing (~17% annually) dietary supplement. She added that U.S. sales of probiotics in 2016 was $1.8 billion, which ranked third among sales of other dietary supplements, following B vitamins ($2.1 billion) and multivitamins ($5.9 billion). I later found that the global probiotic market is estimated to be $64 billion by 2023.

Dr. Davis emphasized the need for caution in accepting reports purporting linkage between probiotics and parameters for obesity such as BMI, shown here in easy-to-use graphical form. She said this caution is based on the results of a systematic review and meta‐analysis of randomized controlled trials, published by Borgeraas et al. in 2017. In a nutshell, these investigators searched for placebo-controlled trials published between 1946 and September 2016. A meta-analysis was performed to calculate the weighted mean difference between the intervention and control groups. Of 800 studies identified through the literature search, only 15 met standards for inclusion in the final analysis.

The meta‐analysis showed that short‐term (≤12 weeks) probiotic supplementation reduced body weight, BMI and fat percentage, but the effect sizes were small. Overall, the risk of bias within included studies was low. Dr. Davis stated that the “findings [of the 15 trials] were not clinically significant,” and agreed with the conclusion of Borgeraas et al. that “further long‐term studies are required to examine the effects of probiotic supplementation on various measures of body weight.”

‘Probiotics—Where is the Science?’

This section’s title is the rhetorical question posed by Dr. Davis in her concluding opinions, which were grouped into “what we know” followed by “what we don’t know.”

What we know:

  • There’s preliminary evidence that some probiotics are helpful in preventing diarrhea caused by infections and antibiotics, and improving symptoms of irritable bowel syndrome.
  • The U.S. FDA has not approved any probiotic for preventing or treating any health problem.
  • Probiotic supplements are probably better if they have multiple strains of bacteria.
  • Probiotic supplements should provide at least 1 billion live cells per gram.
  • If people are generally healthy, probiotics have a good safety record; if not, a doctor’s advice should be obtained.

What we don’t know:

  • Which probiotics are helpful and which are not; not all probiotics have the same effect, and effects are likely strain-specific.
  • The amount of a probiotic a person should use.
  • Who would most likely benefit from taking probiotics.

Progress in Identifying Helpful Probiotics

Credit: By Sebastian Kaulitzk

In response to Dr. Davis’s opinion that we don’t yet know “which probiotics are helpful and which are not,” I found that progress is being made toward obtaining such information. For example, Piewngam et al. recently reported in the highly regarded journal Nature that probiotic Bacillus bacteria, commonly ingested with vegetables, protect against the pathogen Staphylococcus aureus, which is known to mutate into antibiotic-resistant variants. Feeding mice B. subtilis spores, shown here, completely abrogated colonization of all tested S. aureus strains in the feces and intestines in experimental set-ups with or without antibiotic pretreatment to eliminate the pre-existing microbiota. Interested readers can consult Piewngam et al. for details on the molecular mechanism that underlies this protective effect.

Another example is the recent report by Bodogai et al., published in the equally prestigious Science Translational Medicine. They identified the health-promoting activity of a bacterium called Akkermansia muciniphila. In brief, it was found that in mice and monkeys whose metabolisms had grown dysfunctional with age, taking steps to boost A. muciniphila in the gut reduced the animals’ insulin resistance. Insulin resistance is the gradual impairment of the body’s ability to efficiently use food for fuel. It is best known as a way station on a patient’s path to developing type 2 diabetes.

Bodogai et al. noted that insulin resistance is also linked to a variety of ills, from obesity and inflammation to the sagging immunity and frailty that comes with advancing age. If A. muciniphila can be experimentally established as a probiotic supplement to slow or reverse insulin resistance, it might have broad and powerful anti-aging effects, in addition to its ability to protect obese adults from developing type 2 diabetes.

It will of course take many years of carefully conducted research on large study populations to determine additional answers to the questions still surrounding probiotics. Moreover, these studies will need to consider genetic differences among ethnic groups, as well as the role of ‘personalized nutrition,’ which I comment on in the next section.

‘A Good Diet for You May Be Bad for Me’

This section’s catchy title is how a news story referred to a publication by Zeevi et al. on the need for ‘personalized nutrition’ when monitoring glycemic responses. Zeevi et al., who were cited by Dr. Davis, state that elevated postprandial blood glucose levels constitute a global epidemic and a major risk factor for prediabetes and type II diabetes, but existing dietary methods for controlling them have limited efficacy. To address this problem, Zeevi et al. continuously monitored week-long glucose levels in an 800-person cohort, measured responses to 46,898 meals, and found high variability in the response to identical meals, suggesting that universal dietary recommendations may have limited utility.

Zeevi et al. devised a machine-learning algorithm that integrated blood parameters, dietary habits, anthropometrics, physical activity, and gut microbiota in this cohort. The algorithm showed accurate predictions to personalized postprandial glycemic response to real-life meals. The researchers validated these predictions in an independent 100-person cohort.

Finally, a blinded randomized controlled dietary intervention based on the algorithm resulted in significantly lower postprandial responses and consistent alterations to gut microbiota configuration. It was concluded that, “together, these results suggest that personalized diets may successfully modify elevated postprandial blood glucose and its metabolic consequences.”

Concluding Remarks

I encourage interested readers to peruse the NIH ODS website tab for Health Information, which is loaded with helpful links, such as the extensive alphabetized list of Dietary Supplement Fact Sheets. There is also a tab For Researchers that includes numerous funding opportunities.

On a different note, while writing this blog I became intrigued on discovering how TriLink products may have been used in microbiome-related research. I searched Google Scholar for “microbiome” and “TriLink” in the same article and found 39 results. Perusing these led me to select the following three exemplary snippets, each of which is linked to the original article that can be consulted by interested readers for further details:

(1.) Selective microbial genomic DNA isolation using restriction endonucleases. HE Barnes, G Liu, CQ Weston, P King, LK Pham… – PloS one, 2014 – … The technique can enable the targeted enrichment of genomes from various microbiomes or the specific identification of pathogens from … oligonucleotide containing one G m6 ATC site with the top strand sequence FAM-GCAGG m6 ATCAACAGTCACACT (TriLink, San Diego, CA …

(2.) Peripherally-induced regulatory T cells contribute to the control of autoimmune diabetes. C Schuster, F Zhao, S Kissler – bioRxiv, 2017 – … The effects of the microbiome on T1D may derive from the capacity of different microbial communities to promote the generation of pTregs relevant to pancreatic autoimmunity. In light of our finding … Cas9 mRNA was purchased from TriLink Technologies …

(3.) Fecal Microbiota Transplantation Is Associated With Reduced Morbidity and Mortality in Porcine Circovirus Associated Disease. MC Niederwerder, LA Constance… – Frontiers in …, 2018 – … The LLMDA was used to analyze microbiome composition and diversity of the transplant material and fecal samples … Briefly, the samples were labeled using nick translation with Cy3-labeled random nonamer primers (TriLink Biotechnologies, San Diego, CA, United States) and …

From these three examples, it is evident to me that the TriLink product-reach indeed extends into various interesting and important aspects of microbiome research.

As usual, your comments are welcomed.

An Antisense Oligonucleotide Is the First Drug to Demonstrate Reduction of Mutant Huntingtin Protein in Humans

  • Mutant Huntingtin Protein Causes Huntington Disease (HD), Which Afflicts 30,000 People in the U.S.
  • More Than 200,000 People at Risk of Inheriting HD in the U.S.
  • Developed by Ionis Pharmaceuticals, This Antisense Drug Will Undergo Pivotal Clinical Trials Conducted by Roche

Credit: A Luna Blue

My blog from August 7th, 2018, heralded the clinical efficacy of two oligonucleotide drugs for transthyretin-related amyloidosis. One is an antisense oligonucleotide (ASO) drug, and the other is a short-interfering RNA drug. Five days before this notable achievement, Ionis Pharmaceuticals announced equally important news, stating that the European Medicines Agency granted accelerated review timelines for an ASO (IONIS-HTTRx) for the treatment of people with Huntington’s disease (HD), a neurodegenerative illness.

IONIS-HTTRx is the first drug to demonstrate reduction of mutant huntingtin protein, the underlying cause of HD, which is the focus of the present blog. It should be noted that Ionis and Roche have a long-standing alliance when it comes to HD, under which IONIS-HTTRx (designated RG6042 by Roche) will be evaluated in a pivotal study of a larger patient population to further characterize its safety and efficacy profile in adults with HD.

Basic Facts About HD

Description: According to the NIH, HD (aka Huntington’s disease or Huntington’s chorea) is a progressive brain disorder that causes uncontrolled movements, emotional problems, and loss of thinking ability (cognition). Adult-onset HD, the most common form of this disorder, usually appears in a person’s 30s or 40s. Early signs and symptoms can include irritability, depression, small involuntary movements, poor coordination, and trouble learning new information or making decisions. Affected individuals may have trouble walking, speaking, and swallowing. People with HD also experience changes in personality and a decline in thinking and reasoning abilities. Individuals with the adult-onset form of HD usually live about 15 to 20 years after signs and symptoms begin.

A less common form of HD known as the juvenile form begins in childhood or adolescence. It also involves movement problems and mental and emotional changes. Additional signs of the juvenile form include slow movements, clumsiness, frequent falling, rigidity, slurred speech, and drooling. Juvenile HD tends to progress more quickly than the adult-onset form; affected individuals usually live 10 to 15 years after signs and symptoms appear.

Frequency: HD affects an estimated 3 to 7 per 100,000 people of European ancestry. The disorder appears to be less common in some other populations, including people of Japanese, Chinese, and African descent.

Causes: As depicted here, mutations in the HD gene/HTT gene on chromosome 4 cause Huntington disease. The HTT gene provides instructions for making a protein called huntingtin. Although the function of this protein is unknown, it appears to play an important role in nerve cells (neurons) in the brain, primarily a group of nerve cells at the base of the brain known collectively as the basal ganglia.

Credit: Meletios Verras

The HTT mutation that causes HD involves a DNA segment known as a CAG trinucleotide repeat. Normally, the CAG segment is repeated 10 to 35 times within the gene. In people with HD, the CAG segment is repeated 36 to more than 120 times. People with 36 to 39 CAG repeats may or may not develop the signs and symptoms of Huntington disease, while people with 40 or more repeats almost always develop the disorder.

Neuron structure

CAG codes for glutamine, therefore an increase in the size of the CAG segment leads to the production of an abnormally long version of the huntingtin protein. The elongated protein is cut into smaller, toxic fragments that bind together and accumulate in neurons, disrupting the normal functions of these cells. The dysfunction and eventual death of neurons in certain areas of the brain underlie the signs and symptoms of HD.

Inheritance: As depicted here, HD is inherited in an autosomal dominant pattern wherein one copy of the altered gene in each cell is sufficient to cause the disorder. An affected person usually inherits the altered gene from one affected parent. In rare cases, an individual with HD does not have a parent with the disorder.

As the altered HTT gene is passed from one generation to the next, the CAG trinucleotide repeat often increases in size. A larger number of repeats is usually associated with an earlier onset of signs and symptoms. People with the adult-onset form of HD typically have 40 to 50 CAG repeats in the HTT gene, while people with the juvenile form of the disorder tend to have more than 60 CAG repeats.

Individuals who have 27 to 35 CAG repeats in the HTT gene do not develop Huntington disease, but they are at risk of having children who will develop the disorder. As the gene is passed from parent to child, the size of the CAG trinucleotide repeat may lengthen into the range associated with HD (36 repeats or more).

Genetic Testing: There are 64 available genetic tests for HD listed at a NIH website that you can access here. Among these 64, I am most interested in the 7 tests described as “sequence analysis of the entire coding region,” which to me seem to be the most definitive approach. Of these 7 tests, I clicked on this link for the test named “HTT,” which was the only one of these tests offered by a U.S.-based company, namely, Fulgent Genetics near Los Angeles, CA. While this HTT test is described as using “massively parallel sequencing,” which Fulgent specifies as an Illumina® test, this test’s clinical validity and clinical utility are described as “not provided.” I assume this means that this HTT test can be ordered by a doctor for informational purposes. In any case, there is a tab given for “How To Order” that interested readers can consult.

PCR aficionados will recognize HHT’s CAG repeats to be GC-rich, making them difficult to faithfully amplify on Illumina platforms or through other types of ensemble sequencing methods. However, this can be mitigated by using TriLink CleanAmp® 7-deaza-dGTP. Amplification-free methodology is also available. For example, a poster abstract by Pacific Biosystems (PacBio) describes how a novel approach using CRISPR/Cas9 for specific targeting of individual human genes, followed by PacBio’s single-molecule long-read sequencing methods, enables sequencing of complex genomic regions that cannot be investigated with other technologies. HTT CAG repeat-regions were successfully sequencing in this manner.

ASO Studies Targeting Huntington

Use of an ASO to interfere with the expression of mutant HTT can be traced back to 20 years ago, through the following series of publications by various research groups:

Of these, interested readers can consult the last item by Kordasiewicz et al. (2012); a lengthy, detailed report by collaborators at four sites, including Ionis (then named Isis Pharmaceuticals). In brief, this study demonstrated that transient infusion of ASOs into the cerebral spinal fluid of symptomatic HD mouse models not only delays disease progression, but mediates a sustained reversal of disease phenotype that persists longer than the huntingtin knockdown. Reduction of wild type huntingtin, along with mutant huntingtin, produces the same sustained disease reversal.

Rhesus monkey eating

Similar ASO infusion into non-human primates (Rhesus monkeys) was shown to effectively lower huntingtin in many brain regions targeted by HD pathology. Rather than requiring continuous treatment, these findings established a therapeutic strategy for sustained HD disease reversal produced by transient ASO-mediated diminution of huntingtin synthesis.

Finally, Kordasiewicz et al. note that huntingtin is reportedly essential for one or more early developmental steps. However, no evidence to date has demonstrated toxicity following suppression of huntingtin in the adult brain. In fact, their ASO-mediated simultaneous suppression of mutant and normal huntingtin by 60% in the adult rodent striatum, and suppression of normal huntingtin by 45% in the non-human primate striatum, were both well tolerated.

Readers who are practiced in synthetic or medicinal chemistry are likely interested in the structure of IONIS-HTTRx. After considerable research, I found a SlideShare on LinkedIn, shown below. The SlideShare shows a 5-10-5 20-base gapmer comprised of ‘Generation 2+’ ASO with all-phosphorothioate- and 2’ MOE-modifications. It should be noted, however, that this is only an exemplary generic structure, as the actual sequence is proprietary, according to Anne Smith, the Director of Clinical Development at Ionis, who I contacted for permission to show this image.

Exemplary generic structure of IONIS-HTTRx. With permission from Anne Smith, Ionis

Before outlining future clinical studies of this ASO in the next section, I’ll conclude this section by providing a link to more than 100 items listed in Google Scholar for “Huntington and TriLink,” which can be perused later, as there are many interesting finds. One exemplary item that caught my attention was a patent for single-domain antibodies, which can be used in therapeutic methods to inhibit huntingtin protein aggregation.

Roche’s Clinical Trials of IONIS-HTTRx Renamed as RG6042

In December 2017, Roche acquired development and marketing rights to RG6042 from Ionis. According to an article from September 24, 2018 in Huntington’s Disease News, two new clinical studies by Roche of IONIS-HTTRx—now RG6042—for HD are planned to start by the end of 2018, and will begin enrolling participants by early 2019. These studies will help researchers understand progression of HD and the therapeutic effectiveness of RG6042, which may ‘potentially be the biggest breakthrough in neurodegenerative disease in the past 25 years,’ according to an interview with C. Frank Bennett, PhD, Ionis’ Senior Vice President of Research and franchise leader for the neurological programs.

The upcoming HD Natural History study and the Phase 3 GENERATION HD1 trial were announced at the recent 2018 European Huntington’s Disease Network plenary meeting in Vienna. The 15-month HD Natural History study will assess the correlation between mutant huntingtin protein in cerebral spinal fluid and in other clinical measures of HD, and will also evaluate wearable devices to measure disease burden. There is no therapeutic treatment in this study, as the goal is to understand the natural progression of the disease. The study will include up to 100 early-stage symptomatic patients at sites in the U.S., U.K., Canada, and Germany, and its results are expected to provide valuable information for the Phase 3 GENERATION HD1 study.

The two-year, global GENERATION HD1 trial will evaluate the long-term safety and effectiveness of RG6042 in up to 660 patients with symptoms of HD. It will be ‘the world’s first Phase 3 study to measure the effect’ of a therapy that lowers the amount of mutant huntingtin protein, according to Bennett. The trial will be conducted at 80 to 90 sites in 15 countries around the world.

2019 Breakthrough Prize for Bennett

On October 17, 2018, The Breakthrough Prize Foundation and its well-known sponsors—Sergey Brin, Priscilla Chan and Mark Zuckerberg, Ma Huateng, Yuri and Julia Milner, and Anne Wojcicki, announced the recipients of the 2019 Breakthrough Prize, awarding a collective total of $22 million to nine researchers for important achievements in the Life Sciences, and in Fundamental Physics and Mathematics. Considered the world’s most generous science prize, each Breakthrough Prize is for $3 million.

C. Frank Bennett. With his permission

By remarkable coincidence, The Breakthrough Prize in Life Sciences this year was jointly awarded to C. Frank Bennett at Ionis, and Adrian R. Krainer at Cold Spring Harbor Laboratory. The citation reads “for the development of an effective antisense oligonucleotide therapy for children with the neurodegenerative disease spinal muscular atrophy.”

Spinal muscular atrophy (SMA) is a rare but devastating disease, and the leading genetic cause of infant death. Many children with SMA die before their second birthday. Now, it is no longer a death sentence. Frank Bennett, a pharmacologist, and Adrian Krainer, a biochemist, built upon their discoveries on antisense technology and the natural process of RNA splicing to produce the first drug to treat SMA—Nusinersen (marketed by Biogen as Spinraza). It was approved by the FDA in 2016.

Those who are interested can learn more about SMA and Nusinersen by reading my October 2016 blog, which discusses this exciting breakthrough.

The work by Bennett on an ASO for treatment of SMA, and now his principal involvement in the development of IONIS-HTTRx as a promising drug for HD, are extraordinary contributions to science and society.

I’m more than pleased to have had the opportunity to collaborate with Frank in the early days of Isis Pharmaceuticals.

As usual, your comments are welcomed.


After writing this blog, I came across some important news regarding the identification of sensitive indicators of HD progression and outcome of therapeutic intervention. Byrne et al. assessed mutant huntingtin (mHTT) and neurofilament light (NfL) protein concentrations in cerebrospinal fluid (CSF), as well as blood in parallel with clinical evaluation and magnetic resonance imaging in premanifest and manifest HD mutation carriers. The concentration of CSF mHTT accurately distinguished between controls and HD mutation carriers, whereas NfL concentration, in both CSF and plasma, was able to segregate premanifest from manifest HD. These findings were said to provide evidence that biofluid concentrations of mHTT and NfL have potential for early and sensitive detection of alterations in HD, and could be integrated into both clinical trials and the clinic itself.

Highlights from the 2018 International Roundtable (IRT) on Nucleosides, Nucleotides, and Nucleic Acids

  • Over 400 Attendees from Around the World Congregate at UCSD
  • Four Days Full of Topics Spanning Basic Chemistry Through Therapeutics
  • Jerry Comments on His Five Favorites

According to Dr. Yogesh Sanghvi, this IRT 2018 logo was created by Prof. Yitzhak Tor, who artistically used the elements of XXIII to symbolize sunshine and waves typical to the venue of La Jolla, California.

The 23rd (XXIII) International Roundtable (IRT) on Nucleosides, Nucleotides, and Nucleic Acids was held at the University of California, San Diego (UCSD) in La Jolla, California on August 26 – 30, 2018. This 23rd biannual event, which was sponsored by the International Society of Nucleosides, Nucleotides, and Nucleic Acids (IS3NA) was attended by over 400 researchers in all levels of academia and industry from around the world. Award lectures (2), invited lectures (17), oral presentations (26), and posters (198) spanned a plethora of cutting edge scientific topics, ranging from the origins of life to the development of novel therapeutics. Local organizers were Prof. Yitzhak Tor, Chair (UCSD), Dr. Yogesh Sanghvi (Rasayan, Inc.) and Dr. Rick Hogrefe (TriLink BioTechnologies).

I thoroughly enjoyed attending this 2018 IRT, where I had the opportunity to completely immerse myself in diverse aspects of chemistry, biochemistry, molecular biology, medicinal chemistry, and drug development—all related to nucleosides, nucleotides, and nucleic acids. The meeting gave me the opportunity—and challenge—of selecting five noteworthy presentations, shared here in random rather than rank order. There are several other presentors with fantastic work that I apologize for not being able to discuss at this time. For example, Dr. Alexandre Lebedev at TriLink Biotechnologies, gave an excellent oral presentation titled: Efficient initiation of in vitro mRNA transcription with Cap 0, Cap 1 and Cap 2 oligonucleotide primers (CleanCap®). The presentation highlighted TriLink groundbreaking CleanCap Technology, a chemical solution that provides high mRNA capping efficiency, and avoids self/non-self intracellular responses. If you would like a copy of this presentation, please contact TriLink here.

CRISPR Cloaking

An ongoing “hot topic” is sequence-specific cutting of DNA with CRISPR-Cas9 for gene editing as a research tool or therapeutic modality. I have previously blogged about this here. Given the widespread interest on the subject, it’s apropos to start with a poster titled Reversible RNA acylation for CRISPR-Cas9 gene editing control in cells presented by Maryam Habibian, a postdoc in Eric Kool’s group at Stanford University.

As shown here, Kool’s lab has recently published on the reaction of RNA in aqueous buffer with an azide-substituted acylating agent, which yields several 2′-OH acylations per RNA strand in as little as 10 minutes. This poly-acylated (“cloaked”) RNA is strongly blocked from hybridization with complementary nucleic acids, from cleavage by RNA-processing enzymes, and from folding into active aptamer structures. Importantly, treatment with a water-soluble phosphine results in spontaneous loss of acyl groups (“uncloaking”) that fully restores RNA folding and biochemical activity.

Taken from Kadina et al. Angew Chem Int Ed Engl. (2018)

Data in this poster showed that an azide-substituted reagent efficiently acylates CRISPR single guide RNAs (sgRNAs) in 20 minutes in buffer. These cloaked sgRNAs completely inhibit the endonuclease activity of Cas9 in vitro and in living HeLa cells. However, the sgRNA activity is efficiently recovered both in vitro and in cells by treatment with water-soluble phosphines. This study highlights the utility of reversible RNA acylation as a novel method for temporal control of genome-editing function.

Chemically Modified DNAzymes

Prof. David Perrin at the University of British Columbia in Canada gave an oral presentation titled Chemically modified DNAzymes as sequence-specific ribonuclease-A mimics—from potential therapeutics to the origin of life. He noted that the use of a synthetic RNAzyme or DNAzyme to cut a particular sequence target mRNA for use as a possible therapeutic agent has been a concept for ~40 years, but has not yet come to realization. The principal challenge, he added, is to find a suitably structured nucleic acid that catalyzes efficient phosphodiester bond cleavage in RNA in the absence of Mg+2 or at the relatively low Mg+2 concentrations in cells.

As shown here, this presentation described the in vitro selection of novel RNA cleaving DNAzymes that are selected using 8-histaminyl-deoxyadenosine (imidazole-A), 5-guanidinoallyl-deoxyuridine (guanodino-U), and 5-aminoallyl-deoxycytidine (amino-C), along with dGTP. These modified dNTPs provide key functionalities reminiscent of the active sites of ribonucleases, notably RNase A.

Taken from Perrin and coworkers OP9 Abstract IRT 2018

Remarkably, these exceptional catalysts display classic enzymatic properties of Michaelis-Menten kinetics in the absence of Mg+2. Interested readers can access complete details on this exciting work here. Perrin added that, in honor of the late Stanley Miller (UCSD), whose pioneering work on the origin of life included the possibility of a highly-decorated RNA world, this work represents a chemist’s approach to biomimicry for testing hypotheses of the origin of life in an RNA-world that must of co-opted synthetic modifications, and underscores the use of modified dNTPs for the selection of modified aptamers. You can read more about aptamers in several of my previous blogs.

Direct Sequencing of N6-Methyladenosine in RNA

N6-Methyladenosine. Taken from

Enzyme-mediated post-transcriptional RNA modifications are dynamic, and may have functions beyond fine-tuning the structure and function of RNA. Understanding these epitranscriptomic RNA modification pathways and their functions may allow researchers to identify new layers of gene regulation at the RNA level, according to a “grand challenge” discussed in a previous blog. N6-Methyladenosine (m6A), shown here, is the most abundant modification in eukaryotic mRNA and long noncoding RNA (lncRNA). It is found at 3-5 sites on average in mammalian mRNA, and up to 15 sites in some viral RNA.

In addition to this relatively low density, specific loci in a given mRNA are a mixture of unmodified- and methylated-A residues, thus making it very difficult to detect, locate, and quantify m6A patterns. Importantly, there is now an elegant solution to this problem. In an invited lecture by Prof. Andreas Marx at the University of Konstanz in Germany titled Elucidating the information layer beyond the genome sequence, an engineered polymerase was said to differentiate between unmodified- and methylated-A residues.

Taken from Marx and coworkers Angew Chem Int Ed Engl. (2018)

This novel method, which was recently published, involves in vitro evolution and screening to evolve a reverse-transcription (RT)-active KlenTaq DNA polymerase mutant (RT‐KTQ G668Y Y671A) that delivers prominent RT signatures at m6A sites in different sequence contexts. As shown here, this novel polymerase exhibits increased misincorporation opposite m6A compared to unmodified A. Application of this DNA polymerase in next-generation sequencing allowed for identification of m6 A sites directly from the sequencing data of untreated RNA samples.

Phosphorothioate-Modified Oligo Therapeutics

Pioneering investigations of phosphorthioate (PS)-modified nucleic acids by Prof. Fritz Eckstein, followed by fully automated synthesis of PS-modified oligodeoxynucleotides by Prof. Wojciech Stec and yours truly, enabled many other researchers to develop PS-ODNs as therapeutic agents. Although I have previously blogged about this topic, the utility and prevalence of PS-modifications in ODN-based therapeutics was a common theme throughout many presentations at IRT 2018.

Most prominently, in my opinion, Dr. Punit Seth at Ionis Pharmaceuticals in Carlsbad, California gave an invited lecture titled Engineering selectivity into therapeutic oligonucleotides through chemical design. The talk largely dealt with PS-ODNs and included a slide with the following summary:

  • PS-ODNs interact with several plasma proteins with a range of binding affinities
    • PS content and single-stranded nature are important for binding
    • Binding can be rationalized by an avidity model wherein each PS contributes a fraction to overall binding
  • Interaction with plasma proteins can have functional consequences
    • Binding to α-2-macroglobulin can reduce uptake pathways
    • Strong binding to HRG can reduce activity
    • Lipid conjugation enhances potency in muscle through interactions with albumin and lipoproteins
  • PS-ODNs interact with cell-surface proteins such as Stabilin scavenger receptors
    • Stabilins clear anionic polymers of the extra-cellular matrix suggesting a common pharmacophore with anionic PS-ODNs

The influence of antisense PS-ODN Sp and Rp stereochemistry on such pharmacological factors, including RNase-H activity, has been reported by Wave Life Sciences using stereoselective synthesis methodology introduced by Wada (see also Baran). Extending this approach, Troels Koch at the Roche Innovation Center in Copenhagen gave an invited lecture titled Stereodefined LNA Phosphorothioates: Design, synthesis and properties. In particular, he described investigations of 2′-O, 4′-C methylene bridged moieties commonly referred to as “locked nucleic acids” (LNAs), shown here:

Taken from  //  2′-O, 4′-C methylene LNA. Taken from wikipedia

Koch stated that LNAs have, over the last 15 years, been intensively used in RNA therapeutics because LNAs offer high affinity that translates into higher potency for RNA targeting. He added that nearly all of these LNA oligonucleotides have PS linkages. His presentation illustrated the diversity of measurable properties of stereodefined PS-LNAs. Importantly, it was shown that identifying the best diastereomers from a large random mixture is not trivial. Several identification tactics were described, including the use of quantum mechanical modelling as a guide towards finding the best use of stereodefined LNA.

In striking contrast to the aforementioned focus on inclusion and improvement of PS linkages in therapeutic oligonucleotides, Prof. Jesper Wengel at the University of Southern Denmark in Odense gave an oral presentation titled Novel DNA-mimicking monomers for gapmer antisense oligonucleotides, wherein the objective is to increase gene knock-down specificity by complete removal or substantial reduction of PS linkages and other strategies. His current design is to use phosphodiester (PO)-linked “3-10-3” LNA-DNA-LNA gapmers with palmitic acid-derived moieties attached, as shown below, and bridging N or O in the LNA residues.

Taken from Wengel et al. OP24 at IRT 2018 (Photo by Jerry Zon)

Discovery of a Nucleotide Analog Drug for Ebola Virus

In my blog on the 2014 outbreak of the deadly Ebola virus, I indicated the need for more resources allocated towards the development of a prophylactic vaccine. While such work continues, I was very pleased to learn of promising results obtained for a new drug against Ebola, which would provide treatment for individuals already infected with the virus.

Dr. William Lee at Gilead Sciences, Inc. in Foster City, California, reported in an invited lecture titled Remdesivir (GS-5734): An Antiviral Nucleotide Analog for the Treatment of Ebola Virus that this nucleotide prodrug (shown here) of a novel nucleoside analog has shown broad spectrum in vitro activity against filoviruses, corona viruses, paramyxoviruses, and flaviviruses. Importantly, Remdesivir has demonstrated potent in vivo efficacy against multiple strains of the Ebola virus in the rhesus monkey infection model. His talk reviewed the data in rhesus, the manufacturing challenges, and the limited exposure in patients exposed to the Ebola viruses.

Taken from Lee IL17 Abstract IRT 2018

Concluding Comments

In my opinion, IRT 2018 was indeed jam-packed with innovative and interesting presentations by a diverse array of researchers from around the world, all united by the common thread of nucleosides, nucleotides, and nucleic acids. In addition to the science, there was ample opportunity to renew friendships and, more importantly, network and exchange contact information with new people for potential collaborations on mutually interesting projects.

Presented by Prof. Roger Stromberg, Karolinska Institutet, Sweden and Secretary of IS3NA (Photo by Jerry Zon)

Every IRT meeting includes an announcement (shown here) of the next venue, which for the 24th (XXIV) IRT in 2020 will be Stockholm, Sweden, locally organized by Prof. Roger Stromberg and held at the Karolinska Institutet.

I hope to see you there!

As usual, your comments are welcomed.


While walking through the UCSD campus to the “kick off” Keynote Lecture by Prof. Gerald Joyce (Salk Institute) titled RNA-Targeted drug discovery, to be followed by an all-attendees reception sponsored by TriLink, I came across and photographed the large, brightly colored statue shown here.

UCSD Sun God (Photo by Jerry Zon)

My curiosity about this eye-catching, fanciful figure led me to learn that it is called Sun God, and is an art object created by Niki de Saint Phalle (1930-2002), who is best known for her oversized figures that embrace contradictory qualities such as good and evil. She lived in New York in the 1960s when she was prominent in the development of “happenings” and other artistic efforts involving the integration of art and life. She lived and worked in La Jolla from 1992 until her death in 2002.

De Saint Phalle’s Sun God was the first work commissioned by the Stuart Collection of UCSD and was her first outdoor commission in America. The exuberantly colored, fourteen-foot bird is placed atop a fifteen-foot concrete arch and sits on a grassy area between near the Faculty Club. The students started the Sun God Festival in 1984. It has become one of the largest annual campus events.

The Sun God has become a landmark on the UCSD campus. Students have embellished the statue at various times with giant sunglasses, a cap and gown, a UCSD ID card, and a nest of hay with eggs. Sun God has also been adorned with earphones and a radio/tapeplayer, turning the statue into a “Sony Walkbird,” and has sported a machete and headband for its disguise as “Rambird.” It appears on T-shirts and mugs. The grassy area beneath it is a popular site for rendezvous and celebrations.


Deluge of mRNA Delivery Publications

  • Strong Interest in mRNA Therapeutics Drives Increased Numbers of Delivery Publications
  • Novel Charge-Altering Releasable Transporters (CARTs) Undergo “Self-Immolation”
  • CARTs Outperform Widely Used Lipofectamine In Vitro and Enable In Vivo Delivery

Devotees of this blog may recall my past post in 2013 titled Modified mRNA Mania, which intentionally used the word “mania” to provoke reading about the trending topic on base-modified mRNA as therapeutic agents. My metrics for this mania were a flurry of scientific publications, patent applications staking out intellectual property, and massive investments by venture capitalists and established pharma companies in mRNA therapeutics startups.

As with antisense, siRNA, and antagomir RNA drugs, efficient delivery is widely recognized as a critical technical challenge to overcome. And, not surprisingly, past lipid-based approaches of various sorts are being reinvestigated for repurposing for mRNA delivery.

The focus of the present blog is a new strategy for mRNA delivery developed by a team of collaborators at Stanford University. Although I’ve chosen to highlight this report by McKinlay et al. in prestigious Proc. Natl. Acad. Sci., a search of PubMed for publications indexed to “mRNA delivery” in the title and/or abstract for the period 2005 to 2017 gave articles that can be perused at this link. The graph shown below supports my characterization of this level of activity as “deluge”-like in that there are more than 100 publications, mostly in the last few years, with 40 to 50 more during 2018, by my estimate.

Challenges for mRNA Delivery

Simply stated, the key challenge associated with the use of therapeutic mRNA is an inability to efficiently deliver functionally intact mRNA into cells. Like all nucleic acid-based drugs, mRNA is a macromolecular polyanion and thus it does not readily cross nonpolar cellular and tissue barriers. Moreover, it is also susceptible to rapid degradation by nucleases and ideally it should be protected during the delivery process, even though some success has been reported using intradermal injection of “naked” unmodified mRNA. Finally, after cell entry, rapid release of mRNA in the cytosol and appropriate association with the protein synthesis apparatus is required for translation.

Each of these is a potential point of failure for functional mRNA delivery. In addition to the challenges associated with complexation, protection, delivery, and release, an ideal delivery system would also need to be synthetically accessible, readily tuned for optimal efficacy, and safe.

Charge-Altering Releasable Transporters (CARTs)

McKinlay et al. have successfully addressed each of the challenges mentioned above by developing a highly effective mRNA delivery system comprising charge-altering releasable transporters (CARTs). Since a picture is worth a thousand words, I’ve reproduced here the diagram used by McKinlay et al. to describe their multistep approach with CARTs, namely complexation (1), intracellular delivery (2), and cytosolic release (3) of mRNA transcripts, resulting in induction of protein expression (4).

Taken from McKinlay et al. Proc. Natl. Acad. Sci (2017)

Readers interested in the clever chemistry that underlies CARTs should consult the publication by McKinlay et al. for details. In brief, these dynamic materials, specifically oligo(carbonate-b-α-amino ester)s (1) shown below function initially as polycations that noncovalently complex, protect, and deliver polyanionic mRNA and then subsequently lose their cationic charge through a controlled degradation to a neutral small molecule (2). The proposed mechanism for this degradation mechanism, which McKinlay et al. refer to as “self-immolative,” is pH-dependent.

Proposed rearrangement mechanism for n-mer oligo(α-amino ester)s 1 through tandem five-membered (5) then six-membered (6) transition states to afford an n-2-mer and diketopiperazine 2. Taken from McKinlay et al. Proc. Natl. Acad. Sci (2017)

As exemplified below, CARTs for cellular uptake were synthesized with hydrophobic blocks (n = 15) and cationic blocks (n = 12) such that 11b in physiological phosphate buffered saline (PBS) at pH 7.4 undergoes degradation to form 11c and small molecule 2.

Taken from McKinlay et al. Proc. Natl. Acad. Sci (2017)

These researchers hypothesize that this charge alteration reduces or eliminates the electrostatic anion-binding ability of the originally cationic material, thereby facilitating endosomal escape and enabling free mRNA release into the cytosol for translation. Readers interested in learning more about the complexities of endosomal escape can consult a (free, via Google) book chapter by Uyechi-O’Brien and Szoka titled Mechanisms for Cationic Lipids published in 2003, and a 2012 review by Nguyen and Szoka rhetorically titled Nucleic Acid Delivery: The Missing Pieces of the Puzzle?

Regardless of the actual mechanistic details for CARTs, McKinlay et al. demonstrate the efficacy of these materials to complex, deliver, and release mRNA in various lines of cultured cells including primary mesenchymal stem cells and in animal models, via both intramuscular (i.m.) injection and intravenous (i.v.) administration, resulting in robust gene expression. I’ll briefly outline these findings in what follows; however, the full paper and its supplemental material should be consulted for details.

Incidentally, I’m pleased to add that these CARTs were used to deliver the following base-modified [5-methylcytidine (5meC ) and pseudouridine (Ψ)] reporter mRNAs and dye-labeled mRNA obtained from TriLink BioTechnologies: Enhanced Green Fluorescent Protein (EGFP) mRNA, Firefly Luciferase (Fluc) mRNA, and Cyanine 5 (Cy5)-labeled EGFP mRNA.

Mechanism of Uptake and Release

Using a Cy5-labeled EGFP mRNA it was determined that the mechanism of cell entry for CART mRNA polyplexes is predominantly endocytic by comparing cellular uptake at 4 °C, a condition known to inhibit endocytotic processes, to normal uptake at 37 °C. Consistent with the expected endocytotic mechanism for ∼250-nm particles, HeLa cells displayed a significant (85%) reduction in Cy5 fluorescence at 4 °C.

Cellular uptake and mRNA translation following treatment with CART/mRNA polyplexes were then directly compared with polyplexes formed with non-immolative oligomers. By delivering a mixture of EGFP mRNA and Cy5-labeled EGFP mRNA, analysis of mRNA internalization and expression can be decoupled and simultaneously quantified: Cy5 fluorescence indicates internalized mRNA, irrespective of localization, and EGFP fluorescence denotes cytosolic release and subsequent expression of mRNA.

TriLink Cy5-labeled EGFP mRNA is transcribed with Cy5-UTP and an analog of UTP at a ratio which results in mRNA that is easily visualized and can still be translated in cell culture. Translation efficiency correlates inversely with Cyanine 5-UTP substitution.

This method was used in conjunction with confocal microscopy to compare cellular uptake and mRNA expression of two oligomers, namely, CART D13:A11 (7) and non-immolative, guanidinium-containing D13:G12 (13). Detection included dansylated transporter, Cy5-mRNA, and tetramethylrhodamine (TRITC)-Dextran4400, a stain for endosomal compartments. When cells were imaged 4 h after treatment with CART 7/Cy5-mRNA complexes diffuse fluorescence was observed for both the Cy5 and dansyl fluorophores, indicating that those materials successfully escaped the endosome and dissociated from the polyplexes (i).

Confocal microscopy of HeLa cells treated with Cy5-mRNA complexes using CART 7 or non-immolative oligomer 13 after 4 h. Cells were cotreated TRITC-Dextran4400. Scale bar, 10 μm. Taken from McKinlay et al. Proc. Natl. Acad. Sci (2017)

The two observed puncta in the dansyl signal (ii) was attributed to some intracellular aggregation of the dansyl-labeled lipidated oligocarbonate blocks, resulting from self-immolative degradation of the cationic segments of CART 7. Diffuse fluorescence from (TRITC)-Dextran4400 was also observed and attributed to endosomal rupture and release of the entrapped dextran.

However, when cells are treated with non-immolative 13/Cy5-mRNA complexes, both the Cy5 and dansyl fluorescence remain punctate and colocalized (iii). These signals also strongly overlap with punctate TRITC-Dextran4400, indicative of endosomal entrapment.

Taken together, according to McKinlay et al., these data strongly suggest that the charge altering behavior of CART 7 enables endosomal rupture and mRNA release, contributing to the high performance of these materials for mRNA delivery.

Applications and Animal Experiments

Oligo(carbonate-b-α-amino ester) D13:A11 7 was evaluated in applications to explore the versatility of CART-mediated mRNA delivery. EGFP mRNA expression following delivery by CART 7 was assayed in a panel of cell lines and compared to widely used Lipofectamine 2000 (Lipo). HeLa cells, murine macrophage (J774), human embryonic kidney (HEK-293), CHO, and human hepatocellular carcinoma (HepG2) cells all showed that the percentage of cells expressing EGFP using the CART 7 was >90%, whereas treatment with Lipo induced expression in only 22–55% of the cells. Importantly, in addition to these various immortalized cell lines, mRNA expression was also observed in primary CD1 mouse-derived mesenchymal stem cells (MSCs) with high transfection efficiency.

In vivo bioluminescence imaging (BLI) enables localization and quantification of expression following mRNA delivery in living animals. To assess the efficacy of CART/mRNA complexes following local (i.m.) or systemic (i.v.) routes of administration, CART 7-complexed Fluc mRNA (7.5 μg ) in PBS (75 μL) was given to anesthetized BALB/c mice in the right thigh muscle. As a direct control, naked mRNA was similarly injected in the opposite flank. D-luciferin was systemically administered i.p. at 15 min before imaging for each time point, and luciferase expression was evaluated over 48 h, starting at 1 h after the administration of mRNA complexes.

As shown here, when Fluc mRNA was delivered with polyplexes derived from 7 into the muscle, high levels of luciferase activity were observed at the site of injection. This expression peaked at 4 h and was still observable after 24 h but barely so after 48 h (see publication for percentages). In contrast, i.m. injection of naked mRNA afforded only low levels of luciferase expression, as measured by photon flux, in all five mice (see publication for percentages).

Representative BLI images following i.m. injection of naked mRNA (left flank) or CART/mRNA complexes (right flank).Taken from McKinlay et al. Proc. Natl. Acad. Sci (2017)

Following i.v. injections, the localization of mRNA polyplexes in tissues along the reticuloendothelial system pictured here provides many opportunities in inducing immunotherapeutic responses. According to McKinlay et al., spleen localization is “particularly exciting for future studies involving mRNA-based immunotherapy due to large numbers of dendritic and immune cells in that tissue.” Liver localization was also apparent in these animals, and expression in this tissue “may have applicability for treatment of hereditary monogenic hepatic diseases requiring protein augmentation or replacement such as hereditary tyrosinemia type I, Crigler–Najjar syndrome type 1, alpha-1-antityrpsin deficiency, Wilson disease, and hemophilia A and B, or acquired liver diseases such as viral hepatitis A–E and hepatocellular carcinoma.”

Overview of the reticuloendothelial system. ©Frazier et al. (1996)

Future Perspectives

Rather than paraphrase the future perspectives envisaged by McKinlay et al., here are those views, which to me seem warranted by the promising results summarized above:

“The effectiveness of mRNA delivery using these CARTs represents a strategy for mRNA delivery that results in functional protein expression in both cells and animals. The success of these materials will enable widespread exploration into their utilization for vaccination, protein replacement therapy, and genome editing, while augmenting our mechanistic understanding of the molecular requirements for mRNA delivery.”

As usual, your comments are welcomed.



CRISPR in the Clinic…Coming Soon

  • Trio of CRISPR Discoverers Awarded a $1 Million Kavli Prize
  • CRISPR Therapeutics, a Startup Company, Will Soon Start Clinical Trials
  • New Issue: Concerns for Cancer

Over the past few years, I have periodically blogged about CRISPR-based gene editing, which has been arguably the hottest trending topic in nucleic acid-targeted therapy for about the past five years or so. The catalyst for this burst of publications was a 2012 report in Science on a study led by Doudna and Charpentier (see below). The study focused on the potential utility of CRISPR-Cas9 for genome editing, and it currently has over 5000 citations in Google Scholar. There are ~7400 articles in PubMed indexed to CRISPR, and it is evident from my chart shown here that there is strong growth in the annual number of CRISPR publications in the PubMed database.

Number of CRISPR publications in PubMed

In May 2018, three pioneers in CRISPR technology—Emmanuelle Charpentier of the Max Planck Institute for Infection Biology in Berlin, Virginijus Šikšnys (see Footnote) of Vilnius University in Lithuania, and Jennifer Doudna of the University of California, Berkeley—were awarded the $1 million Kavli Prize in Nanomedicine. This highly prestigious prize from The Norwegian Academy of Science and Letters was awarded “for the invention of CRISPR-Cas9, a precise nanotool for editing DNA, causing a revolution in biology, agriculture, and medicine.”

Emmanuelle Charpentier, Virginijus Šikšnys and Jennifer Doudna (left to right). Taken from

As far as invention goes, there has been continued litigation, recently summarized in a GEN interview with law professor Jacob Sherkow titled CRISPR in the Courthouse. The University of California, Berkeley (UC) and the Broad Institute of MIT and Harvard are at odds over foundational patents covering CRISPR-Cas9. Interested readers should consult “late breaking news,” which covers the recent issuance of a US patent to UC and its partners.

Notwithstanding unresolved intellectual property matters, so-called “surrogate companies” for the holders of these key patents include Editas Medicine (MIT/Harvard), Caribou Biosciences/Intellia Therapeutics (UC and University of Vienna), and CRISPR Therapeutics (Emmanuelle Charpentier), the latter of which is the focus of the present blog. As you’ll read below, CRISPR Therapeutics is developing a “CTX001” approach for the treatment of Sickle cell disease and β-thalassemia for clinical trials this year, which is a highly anticipated milestone—both scientifically and commercially.

CRISPR Therapeutics CTX001

Sickle cell disease and β-thalassemia are caused by genetic mutations in the β-globin gene, which codes for the β subunit of hemoglobin that, as depicted below, is the oxygen carrying component of red blood cells. In these diseases, hemoglobin is missing or defective, which results in devastating medical problems. The approach developed by CRISPR Therapeutics is designed to mimic the presence of fetal hemoglobin (HbF; aka γ-globin) that is present in newborn babies. HbF is a form of hemoglobin that is quickly replaced by adult hemoglobin. However, in rare cases where HbF persists in adults, it provides a protective effect for those who have Sickle cell disease and β-thalassemia.

Taken from

CTX001 is an ex vivo therapy in which autologous (i.e. self-donated) cells are harvested directly from the patient. CRISPR Therapeutics then applies its gene-editing technology to the cells outside of the body, making a single genetic change designed to increase HbF levels in a patient’s own blood cells. The edited cells are then reinfused and are expected to produce red blood cells that contain HbF in the patient’s body, thus overcoming the hemoglobin deficiencies caused by these diseases.

The gene-editing mechanism for CTX001 presented by CRISPR Therapeutics at the American Society of Hematology (ASH) in December 2017 is depicted below. In researching this CRISPR-based mechanism, I found a publication by Bjurström et al. that helps to better understand this depiction. In brief, the zinc-finger transcriptional factor BCL11A has been shown to silence HbF genes in human cells during development, and thus directly regulates HbF switching.

Taken from CRISPR Therapeutics

BCL11A silences HbF by associating with other known γ-globin transcriptional repressors. The gene binds to the locus control region as well as other intergenic sites, which prevents the interaction between the locus control region and the HbF globin gene required for fetal globin expression. Using a guide RNA and Cas9 to enable permanent site-specific genome engineering through a DNA repair pathway, knockdown of the BCL11A gene can be an effective strategy for reactivating HbF and restoring functional erythrocytes.

The aforementioned ASH presentation by CRISPR Therapeutics also includes an overview of Sickle cell disease and β-thalassemia, as shown here. According to an informative historical article that I found, Sickle cell disease and β-thalassemia are related genetic disorders that can cause fatigue, jaundice, and episodes of pain ranging from mild to very severe. They are inherited, and usually both parents must pass on an abnormal gene in order for a child to have the disease. Much more genetic information on these two disorders is available on the NIH Genetics Home Reference.

Taken from CRISPR Therapeutics

CRISPR Therapeutics Clinical Studies Status

The December 2017 ASH presentation by CRISPR Therapeutics received widespread media coverage that heralded the highly anticipated “bench-to-bedside” transition for CRISPR technology. CTX001 was able to efficiently edit the target gene in more than 90 percent of hematopoietic stem cells to achieve about 40 percent of HbF production, which investigators believe is sufficient to improve a patient’s symptoms. Study results also showed that CTX001 affects only cells at the target site and that it has no off-target effects on hematopoietic stem cells, thereby appearing to be a safe potential treatment.

These positive results prompted CRISPR Therapeutics to start a collaboration with Vertex Pharmaceuticals to develop and commercialize CTX001 treatment of Sickle cell disease and β-thalassemia. It was also announced that CRISPR Therapeutics and Vertex are planning to submit an investigational new drug (IND) application to the Food and Drug Administration (FDA) to start a Phase 1/2 clinical trial in Sickle cell disease in the United States in 2018. In addition, CRISPR Therapeutics also submitted a clinical trial application (CTA) for CTX001 to advance into a Phase 1/2 clinical trial in patients with β-thalassemia in Europe in 2018. This trial will evaluate the safety and effectiveness of CTX001 in adult patients with transfusion-dependent β-thalassemia.

After the above announcement, News Atlas reported that the FDA placed a clinical hold on this Phase1/2 trial of CTX001 pending, according to CRISPR Therapeutics, ‘the resolution of certain questions that will be provided by the FDA as part of its review of the IND.’

Concerns for Cancer

In studies published in June 2018 in venerable Nature Medicine, researchers from Sweden’s Karolinska Institute and, separately, Novartis, found that cells whose genomes are successfully edited by CRISPR-Cas9 have the potential to seed tumors inside a patient. CRISPR-Cas9 works by cutting both strands of the DNA double helix. That “injury” causes a cell to activate a gene called p53, which has been called the “Guardian Angel of the Genome” and is the most studied of all human genes, which you can read about in one of my previous blogs.

Whichever action p53 takes, the consequence is the same: CRISPR doesn’t work as intended because the genome edit is mended, or the cell dies. The flip-side of p53 repairing CRISPR edits, or killing cells that accept the edits, is that cells that survive with the edits do so because they have a dysfunctional p53. The reason why that could be a problem is that p53 dysfunction can cause cancer. The p53 gene is reported to be the most frequently mutated gene in human cancer: about 50% of all human cancers have lost p53 or express an inactive, mutant p53.

As a result, the Novartis paper concludes that “it will be critical to ensure that [genome-edited cells] have a functional p53 before and after [genome] engineering.” The Karolinska team warns that p53 and related genes “should be monitored when developing cell-based therapies utilizing CRISPR-Cas9.”

An article in quotes the CEO of CRISPR Therapeutics, Sam Kulkarni, as saying that these p53 findings are “something we need to pay attention to, especially as CRISPR expands to more diseases. We need to do the work and make sure edited cells returned to patients don’t become cancerous.”

Closing Comments

Taken from

Many years ago, I was among the early investigators of antisense therapeutics, which at the time was viewed as a new paradigm that would enable faster bench-to bedside, compared to traditional small molecule drug development. In reality, the antisense approach encountered unforeseen complications and required ~30 years of development to reach demonstrable clinical utility, which I previously wrote about in another blog. Short-interfering RNA (siRNA)-based therapeutics also encountered similar struggles.

While past history is not a predictor of the future, in my humble opinion, CRISPR-based clinical strategies will continue to have to deal with unexpected issues, such as the above p53 situation. While I remain hopefully optimistic about future clinical successes for CRISPR, I won’t be surprised if some of these achievements come slower than currently anticipated.

As usual, your comments are welcomed.


According to June 8, 2018 Science News at a Glance, Virginijus Šikšnys, whose role in the invention of the revolutionary genome editor CRISPR has often been overlooked, received some vindication when he was named a co-winner of the prestigious Kavli Prize in Nanoscience. Šikšnys will share the $1 million award with Doudna and Charpentier, who have received far more attention. Šikšnys first showed that the CRISPR system could be transferred from one bacterium to another. And like Doudna and Charpentier, he independently designed a way to steer the CRISPR complex to specific targets on a genome, which he called “directed DNA surgery.”




Nature’s Number One

  • David Liu is Nature Magazine’s Number One Person Who Mattered in 2017
  • Liu’s Team Has Modified CRISPR to Achieve Single-Base Editing of the Human Genome
  • This Opens the Door for Developing Therapies for Tens of Thousands of Human Genetic Diseases

Nature magazine is considered by many—including yours truly—to be among the best sources of scientifically related news, publications, and editorials. Consequently, when I read the cover of a recent issue of Nature featuring its picks for the “ten people who mattered” in 2017, I was immediately intrigued, and felt compelled to read about who and why these persons were selected—especially for the uber-prestigious number one pick. You can read about all these folks later by clicking here, but for now I’ll focus only on David Liu, who was chosen by Nature as the number one person who mattered in 2017, which is a very special accolade.

The “Gene Corrector”

Referring to Liu as the “Gene Corrector” was Nature magazine’s way of concisely encapsulating the fact that Liu’s laboratory was able to take the already well-known CRISPR system, which I’ve blogged about extensively, to its highest possible pinnacle of performance. Namely, editing only a single nucleotide in the entire human genome comprised of six billion nucleotides, which is the ultimate in specificity for human genome editing. How this was achieved is briefly outlined in the next section, but before getting to that I think it’s nice to put a face with a name, and also include key contributors to this remarkable feat.

According to news from the Broad Institute, which along with Harvard and Howard Hughes Medical Institute are Liu’s multi-academic affiliations, this recent landmark achievement involved contributions by Nicole Gaudelli, currently a postdoctoral fellow in Liu’s lab; Alexis Komor, a former postdoctoral fellow in Liu’s lab who is now an assistant professor at the University of California San Diego; current graduate student Holly Rees; former graduate students Michael Packer and Ahmed Badran, and former postdoctoral fellow David Bryson.

Holly Rees, David Liu, and Nicole Gaudelli (Credit: Casey Atkins). Taken from

Evolution of Base Editing

Evolution of a “base editor” by David Liu has involved a progression of investigations that began in 2013 when Liu joined a host of other luminaries in founding a company now called Editas Medicine to develop treatments based on CRISPR technology. It became evident that despite the great potential for gene editing using CRISPR technology, clinical applications could be limited by the unpredictability of CRISPR/Cas9. Although the Cas9 enzyme cuts DNA where directed by guide RNA, researchers must rely on the cells’ own DNA-repair systems to fix the break, which can create a variety of different edits to the genome.

Liu’s lab looked for ways to improve on that. In 2016, then postdoc Alexis Komor and others on Liu’s team reported its first base editor. They used engineered fusions of CRISPR/dCas9 (a catalytically “dead” Cas9 mutant) and a naturally occurring cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA. These fusion constructs do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, which is copied by polymerases into DNA as T, thereby thus converting a C•G base pair into a T•A within a window of approximately five nucleotides.

Taken from Komor et al. Nature 2016.

The approach has since been deployed in a range of organisms, from wheat to zebrafish and mice. And in September 2017, researchers in China reported that they had used Liu’s base editor to correct a single-letter mutation, or point mutation, for a blood disorder (β-thalassemia) in human embryos, although the edited embryos were not allowed to develop further.

According to the Nature article, Liu postdoc Nicole Gaudelli was eager to build on that work and create an analogous system that could instead deaminate adenine to produce inosine (I). Within the constraints of a polymerase active site, inosine pairs with C and therefore is read or replicated as G. The original A•T base pair is thus replaced with a G•C base pair at the target site. However, no naturally occurring enzymes are known to deaminate adenine in DNA. Nature adds that Gaudelli was therefore proposing to “break a cardinal rule in the Liu lab: no one takes on a project if the first step is to create a new enzyme. The risk of lost time and failure is too high.” Fortunately, Liu nevertheless encouraged her to pursue this risky challenge.

Taken from Gaudelli et al. Nature 2017.

The Broad news article quotes Gaudelli as saying the main challenge for her while developing an adenine base editor (ABE) was “overcoming the psychological hurdle of whether or not ABE could go from concept to reality, since the key component of the editor did not exist naturally and had to be evolved in our lab. It was important to keep the faith that we could not only dream of such a molecular machine, but also build it.”

Building the required hypothetical deoxyadenosine deaminase turned out to be a difficult task that necessitated considerable, sophisticated molecular biological engineering that is more than I can succinctly summarize here. Interested readers will have to consult details in the publication by Guadelli et al. that describes the strategy and execution of numerous rounds of this enzyme evolution and engineering.

During this process they characterized the most promising ABEs from later rounds in depth by choosing a set of 17 human genomic targets that place a target A at position 5 or 7 of the protospacer (see above scheme) and collectively included all possible NAN sequence contexts, wherein N = A, G, C or T. The base editing efficiency of the most active editor (ABE7.10) overall averaged 53 ± 4% at the 17 sites tested, exceeded 50% at 11 of these sites, and ranged from 34–68%. These results were said to compare favorably to the typical C•G to T•A editing efficiency reported by Komor et al.

Guadelli et al. determined that the activity windows of late-stage variants are approximately 4–6 nucleotides wide, from approximately protospacer positions 4 to 7 for ABE7.10, and positions 4 to 9 for several other ABEs. Importantly, they concluded that “the precise editing window boundaries can vary in a target-dependent manner,” which to me implies that application of this methodology will require case-by-case genetic disease-specific optimization with potentially varying degrees of success.

Guadelli et al. did not detect any apparent ABE-induced A•T to G•C DNA editing outside on-target or off-target protospacers following ABE treatment. Although additional studies were said to be needed to examine possible untoward RNA editing by ABEs, they observed no elevated adenine mutation rate among four abundant mRNAs in ABE7.10-treated HEK293T cells compared to untreated cells, nor any apparent ABE toxicity in bacterial or human cells under the conditions investigated.

Editing Disease-Relevant Mutations

Taken from

Finally, Guadelli et al. tested the potential of ABEs to introduce disease-suppressing mutations to correct pathogenic mutations in human cells. In one example, the genetic iron storage disorder hereditary hemochromatosis (HHC) was tested. HHC is commonly caused by a G to A mutation at nucleotide 845 in the human HFE gene, resulting in a C282Y substitution in the HFE protein that leads to excessive iron absorption and potentially life-threatening elevation of serum ferritin.

They transfected DNA encoding ABE7.10 and a guide RNA that places the target adenine at protospacer position 5 into an immortalized lymphoblastoid cell line harboring the HFE C282Y genomic mutation. Editing efficiency was measured by high-throughput DNA sequencing of genomic DNA, which showed clean conversion of the Tyr282 codon to Cys282 in 28% of sequencing reads from transfected cells, with no evidence of undesired editing or indels at the on-target locus.

Concluding Remarks

While the development of ABE is an exciting step forward in base editing, more work remains before base editing can be used to treat patients with genetic diseases, including tests of safety, efficacy, and side effects. “Creating a machine that makes the genetic change you need to treat a disease is an important step forward, but it’s only one part of what’s needed to treat a patient,” said Liu in the Broad news item. “We still have to deliver that machine, we have to test its safety, we have to assess its beneficial effects in animals and patients and weigh them against any side effects—we need to do many more things.”

“But having the machine is a good start.”

I fully agree, and hope that you will too.

As usual, your comments are welcomed.


After writing this blog, it was announced that there is now a specialty journal dedicated to CRISPR-related investigations. The February 2018 inaugural issue of this new journal—aptly named The CRISPR Journal—is shown below.


Aptamers and Clinical Applications

  • Gov Lists 28 Clinical Studies, Mostly Ocular, for Aptamers
  • Only 3 On This List Are Currently Active Studies
  • NOXXON Pharma’s Mirror-Image L-RNA Aptamer is in the Clinic for Cancers

Devoted readers of Zone In With Zon who have photographic memories—or anyone who simply uses this blog’s search engine—will know that my first blog on aptamers touted these nucleic acids as being better than antibodies in many applications. That deliberately provocative proclamation was followed by a second blog on aptamers featuring top-cited publications, and also included aptamer studies which cited TriLink products in the methods section. The present, third blog on aptamers focuses entirely on clinical applications.

In the interest of full disclosure, picking this blog’s titled topic was catalyzed, so to speak, by my being invited to contribute a chapter devoted to clinical aspects of aptamers for a book on nucleic acids therapeutics to be published in 2019. After this book becomes available, I’ll be able to comment on its well-known co-editors, many contributors, and comprehensive contents. However, for now, here are “sneak previews” of some items that will be covered in my chapter.

Overview of Aptamer Functional Versatility

Taken from

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. Clinical applications of aptamers, with or without chemical modifications, are all predicated on target-specific binding.

However, as depicted below for diverse examples, aptamers can function as therapeutic agents per se, or as targeting agents. Direct agency of an aptamer drug can include binding to either extracellular protein, cell-surface protein, or viral surface protein, wherein each target is associated with a specific disease or clinical indication of interest for achieving therapeutic intervention. These three targets are also addressable by conventional small molecules or antibodies; however, it was recognized early on that aptamers might provide advantages in specificity or cost, respectively.

Cartoon depicting various modes for RNA aptamers (shown as stem-loop structures) functioning as therapeutic agents (left panel) or as cell-targeting agents (right panel). Figure from Poolsup & Kim with permission.

Use of aptamers as targeting agents is depicted as conjugates for delivery of either a nanoparticle loaded with drug, therapeutic oligonucleotide, such as short-interfering RNA (siRNA), or small molecule drug, such as a cytotoxic agent. Although the present blog is devoted to the first modality, namely, aptamers as therapeutic (i.e. clinical) agents, a review by Poolsup & Kim can be consulted for information on the second mode, namely, aptamers as targeting agents.

Aptamer Drugs Listed in ClinicalTrials.Gov is my “go to” website for obtaining reliable information on clinical investigations in general. This freely accessible website is a comprehensive registry and results database of publicly and privately supported clinical studies of human participants conducted around the world. This trove of information, which is provided as a service of the U.S. National Institutes of Health, is currently comprised of 261,814 clinical research studies in all 50 states and in 201 countries.

Basic information for each study listed in includes the sponsor, participating investigators and hospitals, aptamer drug, targeted disease(s), and type of study (aka Phase), i.e. safety (Phase 1), efficacy (Phase 2), and better than standard of care (Phase 3). Geographical mapping on global, country, or state/regional bases for participating hospitals is also provided.

Taken from

The website is relatively easy to navigate, and has built-in help “buttons” as well as dropdown menus as aids to find and filter the extensive amount of available information. My search results from several months ago using the term “aptamer” provided information for 32 different clinical studies, which when sorted by medical conditions gave 87 items covering a wide range of diseases, with ocular being the most prevalent general category. However, my reading of procedural details for all 32 studies revealed that, in 4 studies, samples from subjects treated with non-aptamer drugs were to be used for in vitro investigations such as screening for aptamer biomarkers. The remaining 28 clinical studies involve aptamer drugs per se, about which I’ll comment below.

Status of Aptamer Studies in ClinicalTrials.Gov

Among the various ways the search results for 28 aptamer drug studies in can be sorted for perusal and commentary, I chose to use study “status,” which defines as follows:

  • Completed—clinical study has ended normally, and participants are no longer being examined or treated (that is, the “last subject, last visit” has occurred).
  • Terminated—clinical study has stopped recruiting or enrolling participants early and will not start again and participants are no longer being examined or treated.
  • Withdrawn—clinical study stopped before enrolling its first participant.
  • Active—clinical study is ongoing (that is, participants are receiving an intervention or being examined), but potential participants may not be being currently recruited.

Taken from

These categories can be used as filters to easily group studies for perusal, which when I did so, led to several general observations. The overwhelming majority of the 17 Completed clinical studies involve ocular diseases, of which age-related macular degeneration (ARMD) was a frequently studied indication. ARMD is the leading cause of blindness for people over the age of 55 years, in the U.S. and Europe, and occurs as either “dry” or “wet” ARMD, as described elsewhere.

While ARMD is a significant medical problem, my guess as to why it was chosen for initial clinical studies with aptamers is that clinical development had fewer practical challenges. Ocular administration required much smaller amounts of drug, which made manufacturing scale-up less problematic, and there were likely less toxicity issues to worry about. In any case, the sponsors included Eyetech Pharma, Ophthotech Corp, Achemix and NOXXON.

Terminated or Withdrawn studies, which numbered 8, were also mostly ARMD-related but also included aptamer for hemophilia and acute myeloid leukemia sponsored by Baxalta and Antisoma Research. Perhaps most important are the Active studies, which were only 3 in number, and I’ve put into tabular form here with some basic information including the National Clinical Trials (NCT) identification number that is searchable in any search engine. You can click on each NCT number to access detailed information provided in; however, following are some snippets for each of these Active Studies.

Table of Active Studies Listed in ClinicalTrials.Gov in 2017

Sponsor (Study ID) Aptamer Drug Disease(s) Type of Study
Eyetech Pharma (NCT01487044) pegaptanib sodium (Macugen) Diabetic Macular Edema (DME) Phase 1
Ophthotech Corp (NCT02686658) ARC1905 (Anti-C5 Aptamer), Zimura® Dry ARMD Phase 2


Ophthotech Corp (NCT02591914) E10030 (Anti-PDGF Pegylated Aptamer, Fovista®) ARMD Phase 1

Pegaptanib structure taken from DrugBank. Light blue nucleotides are 2’-hydroxyl, dark blue nucleotides are 2’-fluoro, and red nucleotides are 2′-O-methyl.

Pegaptanib sodium (aka Macugen), which originated at NeXstar Pharma and was later taken on by Ophthotech via Eyetech, is a 5’-PEG (40kDa)/3’-inverted dT blocked 2′-fluoro/2′-O-methyl modified 27-mer RNA aptamer targeted against vascular endothelial growth factor (VEGF), and is the only FDA-approved aptamer drug. This Active Study is a single-center trial at the Retina Institute of Hawaii, and involves loading of intravitreal pegaptanib bi-weekly during the initial treatment period for diabetic macular edema (DME) when levels of VEGF, its protein target, are the greatest. This is followed by gradually extending the administration frequency to monthly as homeostasis ensues for the treatment of DME. This study was registered in 2011 and is currently recruiting patients.

ARC1905 (aka Anti-C5 Aptamer and Zimura) is reported to be a 38-mer aptamer sequence that was originally identified from a 2′-fluoro-pyrimidine-modified library, and all but three purines could be replaced by 2′-O-methyl purine nucleotides. For in vivo applications, an inverted dT and a 40 kDa PEG moiety were added to the 3′ and 5′ end, respectively, according to a review. Opthotech’s study of ARC1905 assesses the safety and efficacy of intravitreous administration in subjects with geographic atrophy secondary to dry ARMD. This study was registered in 2016 and is currently recruiting participants in numerous locations in the U. S. as well as some sites in Hungary.

Ophthotech’s E10030 (aka ARC127, Fovista), which is an anti-PDGF-BB aptamer, is reported to be a 36-mer published in 1996 that was derived from a DNA library, and was only modified with a 3′-inverted dT moiety to inhibit exonuclease degradation. Further shortening and post-SELEX modifications (2′fluoro pyrimidines, 2′-O-methyl purines, internal hexyl-linkers, all wherever possible) is reported to improve nuclease resistance and resulted in a 32-mer that was later named ARC126, and then ARC127 when pegylated at the 5′ terminus. Ophthotech’s Active Study of E10030 is an open-label trial located at Retinal Consultants of Arizona for safety, tolerability and development of subfoveal fibrosis by intravitreal administration of altering regimens of this aptamer drug and anti-VEGF therapy in subjects with neovascular ARMD. This study is ongoing but not recruiting patients.

Conclusions and Prospects

Oligonucleotides aptamers as possible therapeutics were scientifically “birthed,” so to speak, nearly 30 years ago, shortly after the “birth” of antisense oligonucleotides for therapeutics. Consequently, on a relative basis, I think it’s telling that my search of lists only 28 studies of aptamer drugs compared to 152 studies found for “antisense.” In addition to this difference in total number of studies for these two classes of oligonucleotide drugs, filtering the data by Study Phase gave 7 aptamer studies as Phase 3 (all ocular diseases) compared to 14 antisense studies as Phase 3 (various diseases). The number of FDA-approved or soon-to-be-approved drugs mirrors the same inequality: only one (Macugen) for aptamers and a handful for antisense (see Ionis Pharma, Alnylam Pharma, and Sarepta Therapeutics).

Having said this, I hasten to add that I’m not “anti-aptamers” and instead “pro-antisense.” In fact, I’m “bullish” on both strategies for drug development, and expect more clinical progress with aptamers as new chemistries are pursued. Past focus on 2′-fluoro/2′-O-methyl modified oligonucleotide as aptamers represents, in effect, exploration of very limited structural diversity, which can now be expanded to hydrophobic SOMAmers and thioaptamers, or C5-modified dU X-Aptamers.

Finally, let’s consider the graph shown here, which I constructed using PubMed search results for the number of annual publications indexed to “clinical development of aptamers” as a search phrase. To me, there is clearly a trend toward increased numbers of such publications with time. Factors could arise which negatively impact this trend going forward, but absent that eventuality the future looks promising.

As usual, your comments are welcomed.


There are additional Active Studies not registered in, such as those with NOXXON’s “Spiegelmers,” which are mirror-image L-nucleic acids stable toward nuclease degradation in vivo. NOXXON states that its lead candidate, NOX-A12, which is an L-RNA aptamer, “is under development as a combination therapy for multiple cancer indications

Taken from

where its impact on the tumor microenvironment is intended to significantly enhance the effectiveness of anti-cancer treatments without adding significant side effects for patients. NOX-A12 is currently in a Phase 1/2 trial in metastatic pancreatic and colorectal cancer patients who are not expected to respond to checkpoint inhibition alone. NOX-A12 has also completed two Phase 2a trials, one in chronic lymphocytic leukemia and the other in in multiple myeloma.”






Jerry’s Favs from the Recent 7th Cambridge Symposium

  • DNA Can Function as an Enzyme
  • RNA Polymerase Activity Without Proteins
  • Systemic Brain Delivery of Therapeutic Oligos

The 7th Cambridge Symposium on Nucleic Acids Chemistry and Biology took place September 3-6, 2017 at historic Queens’ College, Cambridge, which was founded in 1448 by Margaret of Anjou (who was then Queen of England by marriage to King Henry VI). Yours truly had the honor of participating in this event and presenting one of TriLink’s posters on the company’s new types of chemically modified mRNA for mRNA therapeutics. As done for other conferences I’ve attended on behalf of TriLink, I wish to share here my personal favorites among the many lectures, which are still fresh in my mind. However, I hasten to emphasize that, while choosing these “favs” is biased a bit by my scientific interests, all the lectures topics are worth looking at later by perusal of the symposium program of nearly forty presentations.

Taken from

By the way, the symposium’s logo artistically depicts a DNA helix under a wooden bridge, better seen in the accompanying picture of the actual bridge over the River Cam at Queens’ College. Built in 1749, it has become known as the Mathematical Bridge for reasons you can read later, and appears to be an arch but is composed entirely of straight timbers. The historical connection between the DNA double helix and Cambridge is that in 1953 Watson & Crick proposed this now famous deoxynucleic acid structure as the molecular basis for genetics, which I’ll comment on again at the end of this post.

Taken from

Overview of the Symposium

Mike Gait. Taken from

This symposium is the 7th in a popular conference series going way back to 1981 that brings together nucleic acids scientists across a broad area but with emphasis on chemistry, biochemistry and structure. Michael (Mike) Gait, who is at the Medical Research Council Laboratory of Molecular Biology in Cambridge, originated this series and has been a key organizer for all seven conferences. Participants come from all over the world and include professors, students, and companies—as well as Nobel Laureates (this year Jack Szostak of telomeres fame).

In addition to Mike, the organizing committee included Sir Shankar Balasubramanian, who was recently knighted for his contributions to next-generation sequencing and research on G-quadruplexes, the latter of which I featured here a few years ago. Other committee members were Rick Cosstick, Phil Holliger, and Chris Lowe.

Subject areas this year included:

  • Nucleic acids as therapeutics (including antisense, RNAi, aptamers, immune recognition, cell delivery)
  • RNA and DNA structures and their protein complexes (duplexes, quadruplexes, RNA and DNA enzymes, riboswitches, protein complexes + assemblies)
  • Nucleic acids chemistry applied to cells and cell mechanisms (genomes, evolution, repair, cell manipulation)
  • Nucleic acids as tools, structural assemblies and devices (nanostructures, cages, arrays, supra-molecular chemistry)

Marv Caruthers. Taken from

The showcased and highly prestigious Nucleic Acids Award was presented to Marvin (“Marv”) Caruthers in recognition of his seminal contributions to the synthesis of oligodeoxynucleotides (aka “oligos”) based on the use of phosphoramidite chemistry. This mechanistically elegant chemistry enabled much faster and more efficient coupling for automated synthesis of oligos, which fundamentally transformed all manner of basic and applied research with DNA. His award lecture was titled Synthesis, Biochemistry, and Biology of New DNA Analogues, some of which has been recently published. My previous several posts commenting on Marv, who has been a professor at the University of Colorado in Boulder since 1973, can be read later here.

Jerry’s Favs from the Symposium

To encourage inclusion of unpublished results and other types of “late breaking news” from the lab, the organizers forbade use of Twitter or other real-time social media, blogging, or taking pictures of slides being shown. Consequently, what I can say here is restricted to published papers related to my favs. Keeping this limitation in mind, here are my personal top-three talks that I consider to be tied (i.e., have equivalent scientific importance).

DNA Can Function as an Enzyme!

This lecture by Scott Silverman at the University of Illinois, Urbana-Champaign, dealt with DNA enzymes (aka deoxyribozymes), which were first reported in 1996 by Carmi et al., and are of interest because they expand enzymatic functionality from naturally occurring proteins to synthetic nucleic acids. DNA enzymes can be evolved in vitro starting with random sequences of DNA and applying suitable selection.

In a published account titled Pursuing DNA Catalysts for Protein Modification, Silverman has provided a lengthy and chemically detailed description of his use of in vitro selection to develop DNA catalysts for many different covalent modification reactions of peptide and protein substrates. While interested readers can consult Silverman’s account for various examples, it’s illustrative to consider the molecular design strategy depicted below that was used to evolve a synthetic DNA functional-equivalent of naturally occurring protein kinases that, by definition, carry out protein phosphorylation.

Taken from Silverman Acc Chem Res (2015)

In this case, modular deoxyribozyme design involved a stretch of 40 randomized bases (N = A/G/C/T) having a hairpin loop conjugated to a tyrosine (Tyr)-containing peptide on one end, and an ATP-binding aptamer on the other end. This was intended to experimentally assess whether it would be helpful to provide a predetermined small-molecule binding site in the form of an aptamer, which would cooperate functionally with an initially random catalytic region (N40) from the onset of selection. The selection outcome established that while modular deoxyribozymes that utilize a distinct predefined aptamer domain can indeed be identified, such DNA catalysts do not have any functional advantage relative to nonmodular analogues selected simultaneously for binding and catalysis, at least for this test case of tyrosine kinase activity using an ATP phosphoryl donor.

RNA Polymerase Activity Without Proteins!

By analogy to use of in vitro selection to evolve DNA enzymes from complex pools of random sequences of DNA, complex mixtures of unrelated RNA sequences can also be subjected to in vitro selection to evolve RNA enzymes (aka ribozymes). Indeed, as noted and cited in a talk by co-organizer Philipp Holliger, the emergence of an RNA catalyst capable of self-replication is considered a key transition in the origin of life in the prebiotic “RNA World” first hypothesized by Walter Gilbert in the 1980s. How such self-replicating (replicase) ribozymes emerged from the pools of short RNA oligomers arising from prebiotic chemistry and non-enzymatic replication, however, is unclear.

In a published version of Holliger’s talk addressing this important open question, his laboratory carried out an elegant series of experiments demonstrating that RNA polymerase ribozymes can assemble from catalytic networks of RNA oligomers that are each no longer than 30 nucleotides. Additionally, they found that entropically disfavored assembly reactions are driven by iterative freeze-thaw cycles. Such cooling (to freeze)-warming (to melt) cycles for aqueous solutions of RNA oligo reactants are notionally opposite to heating (to dissociate)-cooling (to hybridize) cycles used for amplification by PCR.

Interested readers can peruse Holliger’s publication for details about these novel findings, but for the purposes of this blog the schematic shown below depicts and describes the mechanism for assembly wherein relatively short RNA oligomers undergo serial ligations and “grow” into a self-replicating RNA polymerase. To me, these results provide an amazing glimpse backward in time to how the RNA World may have evolved!

Assembly of a RNA polymerase ribozyme (RPR 1234) from oligonucleotides devoid of pre-activation. (a) Schematic representation of the assembly trajectory involving (anti-clockwise from top left), ribozyme (blue) cleavage of a short 3′ tail (red) generating a 2′, 3′ cyclic phosphate (>p) (red dot), dissociation of the cleaved tail and strand exchange to cognate substrate (orange) followed by ligation of substrate 5′ OH with >p. (b) Network diagram of RPR 1234 assembly from 4 tailed fragments 1, 2, 3 and 4. Tailed input fragments can ligate to their cognate 5′fragments but must be cleaved (red lines) before ligation to 3′fragments. Taken from Holliger Nat Chem (2015).

Systemic Brain Delivery of Therapeutic Oligos!

Taken from

A talk by Fazel Shabanpoor titled Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy described work that he had just published with a group of collaborators that included symposium co-organizer Mike Gait, whose lab interests have recently focused on cell-penetrating peptides. This was a fav for me because it had multiple interesting elements: (1.) systemic brain delivery, which is a widely recognized challenge; (2.) “weirdly” structured morpholino oligos, which have backbone structures quite unlike DNA that I’ve commented on here previously; and (3.) splice-switching antisense oligos (SSOs). The latter class of molecules (SSOs) base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Readers interested in SSOs—currently a “hot topic”—can consult a recent comprehensive review, while SSOs for spinal muscular atrophy (SMA) has been featured in previous blog here.

Shabanpoor’s lecture highlighted the fact that development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, Shabanpoor et al. investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching 20-mer phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. They identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript.

Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. It was concluded that this work provides proof of principle for the ability to select new “peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform” for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases.

Parting Thoughts and The Eagle

I hope that you can now appreciate why these three lectures were my favs from the 7th Cambridge Symposium. Silverman’s conversion of DNA into an enzyme that can phosphorylate a protein is an exciting demonstration of the power of bio-organic chemistry to manipulate DNA to do things it can’t do naturally. Holliger’s demonstration of how RNA polymerase ribozymes may have evolved gives credence to the RNA World hypothesis, and indicates that this supposed prebiotic environment may have provided critical freezing and thawing cycles over many millennia of molecular evolution. In the present biotic world, humans afflicted with neuromuscular and neurodegenerative diseases may benefit from Shabanpoor & Gaits’ new peptide paradigms to enhance CNS delivery and activity of therapeutic oligos.

In conclusion, I should mention that researchers who work with DNA will invariably visit The Eagle when in Cambridge, which is a pub only a short walk from Queens’ College. This pub, which dates back to 1667, is quite famous because it is known with certainty to be the place where Francis Crick interrupted patrons’ lunchtime on February 28, 1953 to announce that he and James Watson had ‘discovered the secret of life’ after they had come up with their proposal for the structure of DNA. Today the pub serves a special ale dubbed “Eagle’s DNA” to commemorate the discovery. Trust me when I say that this ale is mighty tasty, because I enjoyed a pint of it, and while standing in the que for that brew, was inspired to capture this image to share here.

As usual, your comments are welcomed.

Personal photo using a Samsung Galaxy S8




Advances in Aptamer Applications – Part 2

  • Top Cited Aptamer Publications Over the Past Three Years
  • Jerry’s Picks for Top 3 Aptamer Publications So Far This Year
  • TriLink Products Cited in Numerous Aptamer Publications

Aptamers are highly structured nucleic acids that bind to a specific target molecule. RNA or DNA aptamers are usually selected from a very large pool (aka library) of random sequences, and can be comprised of either natural and/or chemically modified nucleotides. My first blog on aptamers was titled Aptamers: Chemistry Bests Mother Nature’s Antibodies. This purposefully provocative claim was intended to emphasize the growing body of evidence that collectively indicates aptamers can perform better than antibodies in many applications.

NMR-derived structures of aptamers binding to either a large protein or small molecule. Taken from genelink .com

Because it has been nearly four years since that boastful blog in 2013, I thought it was time to survey aptamer applications published since then to comment on what has been trending or is otherwise notable. I found more than 1,500 articles in PubMed for 2014 through 2017 (estimate) that have the search term “aptamer” in the title or abstract. Given this huge number of publications, I used Google Scholar citation frequency as a numerical indicator of interest, importance and/or impact for these publications in each year. I also decided to focus on original publications that, by definition, excludes review articles. 

Top 3 Cited Publications in 2014

  1. Activatable fluorescence/MRI bimodal platform for tumor cell imaging via MnO2 nanosheet–aptamer nanoprobe (109 citations)

This Chinese team of researchers led by uber-prolific Weihong Tan, about whom I’ve previously blogged, designed a novel methodology for imaging tumor cells using quenched-fluorescent aptamers. In the presence of target cells, the binding of these “dark” aptamers to cell surface markers weakens the adsorption of aptamers on MnO2 nanosheets causing partial fluorescence recovery (i.e., unquenching), thus illuminating the target cells, as well as facilitating endocytosis into target cells. After endocytosis, reduction of MnO2 nanosheets by glutathione further activates the fluorescence signals and generates large amounts of Mn2+ ions as a contrast agent for magnetic resonance imaging (MRI).

Taken from

  1. A phase II trial of the nucleolin-targeted DNA aptamer AS1411 in metastatic refractory renal cell carcinoma (88 citations)

Taken from

The anticancer mechanism of action for DNA aptamer AS1411, which has multiple G-quadruplex moieties that disrupt cancer cell replication following nucleolin-mediated uptake, is depicted below and detailed elsewhere. In this clinical study, it was found that AS1411 appears to have limited activity in patients with metastatic renal cell carcinoma. However, rare, dramatic and durable responses can be observed and toxicity is low. Further studies with AS1411 and other nucleolin-targeted compounds may benefit from efforts to discover predictive biomarkers for response.

  1. An aptamer-based dipstick assay for the rapid and simple detection of aflatoxin B1 (61 citations)

Aflatoxin B₁ structure. Taken from

Aflatoxin B₁ (AFB1) produced by Aspergillus flavus and A. parasiticus is considered the most toxic aflatoxin and it is highly implicated in hepatocellular carcinoma in humans. In this work by Korean researchers, a rapid and simple dipstick assay based on an aptamer has been developed for determination of AFB1 contamination in food. The dipstick assay format was based on a competitive reaction of a biotin-modified aptamer specific to AFB1 between target and Cy5-modified DNA probes. Streptavidin and anti-Cy5 antibody as capture reagents were immobilized at test and control lines on a membrane of the dipstick assay. The method was confirmed to be specific to AFB1, and the entire process of the assay can be completed within 30 min.

Top 3 Cited Publications in 2015

  1. Aptamer-conjugated silver nanoparticles for electrochemical dual-aptamer-based sandwich detection of staphylococcus aureus (63 citations)

Taken from sciencedirect .com

Staphylococcus aureus (S. aureus) is one of the most important human pathogens and causes numerous illnesses. This report by Iranian researchers describes a sensitive and highly selective dual-aptamer-based sandwich immunosensor for the detection of S. aureus. As depicted below, a biotinylated primary anti-S.aureus aptamer was immobilized on streptavidin coated magnetic beads (MB), which serves as a capture probe. A secondary anti-S.aureus aptamer was conjugated to silver (Ag) nanoparticles such that, in the presence of target bacterium, a sandwich complex is formed on the MB surface and the electrochemical signal of Ag is measured by anodic stripping voltammetry.

  1. Aptamer-based fluorescence biosensor for chloramphenicol determination using upconversion nanoparticles (59 citations)

Chloramphenicol. Taken from Wikipedia .com

Chloramphenicol (CAP) shown below is a naturally occurring antibiotic that is artificially manufactured for use in veterinary and human medicine. Due to its adverse effects in humans, use of the antibiotic is restricted and, in Europe, ‘zero tolerance’ for CAP in food products has been legislated. In this report by Chinese researchers, detection of CAP uses aptamer-conjugated magnetic nanoparticles for both recognition and concentration, together with upconversion nanoparticles for detection. The method was validated for measurement of CAP in milk vs. a commercially available enzyme-linked immunosorbent assay (ELISA) method.

  1. A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus (48 citations)

Taken from mdpi .com

This work by Chinese investigators describes a novel aptamer/graphene interdigitated gold electrode piezoelectric sensor for detecting S. aureus by binding to the aptamer, which is immobilized on the graphene via the π–π stacking of DNA bases, as depicted below. When S. aureus is present, aptamer dissociates from the graphene and thus leads to change of oscillator frequency of the piezoelectric sensor.

Top 3 Cited Publications in 2016

  1. Aptamer–MIP hybrid receptor for highly sensitive electrochemical detection of prostate specific antigen (38 citations)

This study in the UK uses a thiolated DNA aptamer for prostate specific antigen (PSA) immobilized on the surface of a gold electrode. Controlled electropolymerization of dopamine around the complex served to create an imprint of the complex following removal of PSA. This molecularly imprinted polymer (MIP) cavity was found to act synergistically with the embedded aptamer to provide recognition properties superior to that of aptamer alone. A generalized depiction for producing a MIP is shown below.

Taken from sigmaaldrich .com

  1. Aptamer-functionalized nanoparticles for surface immobilization-free electrochemical detection of cortisol in a microfluidic device (34 citations)

Taken from

Monitoring the periodic diurnal variations in cortisol (aka hydrocortisone, show below) from small volume samples of serum or saliva is of great interest, due to the regulatory role of cortisol within various physiological functions and stress symptoms. This publication from China reports use of aptamer-functionalized gold nanoparticles pre-bound with electro-active triamcinolone for detection of cortisol based on its competitive binding to the aptamer by monitoring a signal from the displaced triamcinolone using square wave voltammetry at graphene-modified electrodes. The assay was benchmarked vs. ELISA and radioimmunoassays.

  1. Multifunctional aptamer-based nanoparticles for targeted drug delivery to circumvent cancer resistance (32 citations)

Taken from Liu et al. Biomaterials (2016)

In yet another publication from China, Liu et al. report use of a G-quadruplex nanostructure to target cancer cells by binding with nucleolin, in a manner analogous to that mentioned above. A second component is double-stranded DNA (dsDNA), which is rich in GC base pairs that can be applied for self-assembly with doxorubicin (Dox) for delivery to resistant cancer cells. These nanoparticles were found to effectively inhibit tumor growth with less cardiotoxicity.

Jerry’s Top 3 Publication Picks for 2017-to-Date

Here are my Top 3 “fav” aptamer articles published during the first half of 2017, and my reasons for these aptamer selections—pun intended. Interested readers can consult the original publication for technical details.

  1. Targeted delivery of CRISPR/Cas9 to prostate cancer by modified gRNA using a flexible aptamer-cationic liposome

CRISPR/Cas9 is unquestionably—in my opinion—the hottest topic in nucleic acid-based R&D these days, as I have previously blogged about. Off-target effects of CRISPR/Cas9 can be problematic, so using targeted delivery to cells of interest is an important approach for mitigating this problem. In this study, an aptamer-liposome-CRISPR/Cas9 chimera was designed to combine efficient delivery with adaptability to other situations. The chimera incorporated an RNA aptamer that specifically binds prostate cancer cells expressing the prostate-specific membrane antigen as a ligand, and the approach “provides a universal means of cell type-specific CRISPR/Cas9 delivery, which is a critical goal for the widespread therapeutic applicability of CRISPR/Cas9 or other nucleic acid drugs.”

  1. A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva

This report claims the first ever development of a split aptamer that achieves enhanced target-binding affinity through cooperative binding. In this instance, a split cocaine-binding aptamer incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. This system afforded specific, ultra-sensitive, one-step fluorescence detection of cocaine in saliva without signal amplification. This limit of detection meets the standards recommended by the European Union’s Driving under the Influence of Drugs, Alcohol and Medicines program.

  1. Detection of organophosphorus pesticide–Malathion in environmental samples using peptide and aptamer based nanoprobes

Environmental contamination with pesticide residues has necessitated the development of rapid, easy and highly sensitive approaches for the detection of pesticides such as malathion, a toxic organophosphorus pesticide, widely used in agricultural fields. These Indian investigators employed an aptamer, cationic peptide and unmodified gold nanoparticles. The peptide, when linked to the aptamer renders the gold nanoparticles free and therefore, red in color. When the aptamer is associated with malathion, however, the peptide remains available to cause the aggregation of the nanoparticles and turn the suspension blue. The sensitivity was tested in real samples and the results implied the high practicability of the method.

Aptamer Publications in 2014-Present Citing TriLink Products

I was pleasantly surprised to find more than 250 publications on aptamers in Google Scholar citing the use of TriLink products since 2014. This volume of literature is way too large to summarize succinctly, so I decided to do a quick scan to select the following items that provide an indication of the broad diversity of applications partially enabled by TriLink products:

2’-F-UTP. Taken from trilinkbiotech .com

In closing, I should first mention that, while scanning the aptamer/TriLink publications mentioned above, it was evident that the most frequently cited TriLink products were 2’-F-CTP and 2’-F-UTP, which are incorporated into aptamers to impart nuclease resistance, as discussed on a TriLink webpage.

My second and last comment is that, as you may have noticed, there seems to be a high proportion of aptamer publications coming out of China and/or coauthored by Chinese investigators collaborating with researchers in other countries. This despite the fact that Chinese publications in Life Sciences are ~6-times fewer that those from the US, according to reliable statistics. I have no idea why this is so, but thought it’s an intriguing factoid.

As usual, your comments are welcomed.