Highlights from the 14th Annual Meeting of the Oligonucleotide Therapeutics Society (OTS 2018)

  • Nearly 600 Attendees from Around the Globe Converge in Seattle
  • Four Full Days of Nucleic Acid-Based Therapeutics Talks and Posters by Academics and Companies
  • Jerry Comments on His OTS 2018 Favorites

The idea for the OTS was conceived in 2001 by an international group of renowned oligonucleotide scientists, who hoped to bring together expertise from different angles of oligonucleotide research and create synergies to help the field accomplish its full therapeutic potential. The vision included a new era of oligonucleotide drugs that would change the landscape of therapeutic modalities.

This prescient view of a “new era of oligonucleotide drugs” has indeed become reality, and has expanded to include much longer polynucleotides, namely mRNA. The OTS 2018 meeting held on September 30 – October 3 in Seattle, WA, attracted nearly 600 participants from around the world, pictured in this group photo. There were many excellent talks and posters, and before commenting on my favorites among them, I have attempted to distill into a few words my overall impressions about what was “hot,” “new”, and “different” at OTS 2018 compared to past meetings.

  • Credit: Prof. Steven Dowdy, Event Chair

    CRISPR is definitely “hot,” based on the large number of CRISPR talks and posters, which complemented CRISPR “rock star” Dr. Feng Zhang’s exciting Keynote Presentation titled Genome Editing Technologies and Beyond. Here are links to his 83 CRISPR-related publications, and my previous blogs related to CRISPR.

  • What struck me as “new” are the advances in discovery and development of innovative compositions for improved and/or more specific delivery of oligonucleotide and siRNA therapeutics, as well as longer mRNA as drugs. In the next section, I’ll provide an example of research that deals with both of these “new” and “hot” topics.
  • As for what’s “different,” I fully agree with session chairperson Laura Sepp-Lorenzino (Vertex Pharmaceuticals, Inc.), who openly stated that previous meetings, especially early ones, were peppered with “challenges and issues.” Now, there is a preponderance of positive preclinical results and—most importantly—FDA approval of oligonucleotide drugs, along with a promising pipeline.

OTS Young Investigator Award

Prof. Yizhou Dong. With his permission.

This year’s Young Investigator Award was presented to Prof. Yizhou Dong, an Associate Professor at the Ohio State University College of Pharmacy. His presentation titled Development of Nanomaterials for mRNA Therapeutics and Genome Editing described the development of novel nanoparticles for delivery of Cas9 mRNA for improved CRISPR editing of genomic DNA. This exciting talk encompassed both the “new” and “hot” topics at OTS 2018 that I mentioned above.

Prof. Dong designed N1, N3, N5-tris(2-aminoethyl)benzene-1,3,5-tricarboxamide (TT) and applied an orthogonal experimental design to investigate the impacts of formulation components on delivery efficiency. Lead materials comprised of TT, lipid/lipid-like nanoparticles (LLNs), and mRNA encoding human Factor IX (hFIX), fully recovered the level of human Factor IX to normal physiological values in FIX-knockout mice. In addition, he presented results demonstrating that these TT-LLNs were capable of effectively delivering Cas9 mRNA and guide RNA to the mouse liver for genome editing.

Jessica Madigan. Photo by Jerry Zon.

I’m pleased to say that a portion of Prof. Dong’s work was enabled by mRNA gifted by TriLink, and I am doubly pleased that TriLink presented two posters on its mRNA technology and products. One TriLink poster titled Key Critical Quality Attributes for the cGMP Production of Therapeutic Messenger RNA was presented by Jessica Madigan, pictured here, and Craig Dobbs presented a second TriLink poster titled Considerations for the Design and cGMP Manufacturing of mRNA, which featured a novel method for improved synthesis of 5’-cap analogs of mRNA that is referred to as CleanCap®.

Enhanced Delivery of mRNA-Protein Complexes

In the multi-talk Session on mRNA and CRISPR, Prof. Paula Hammond, Head of the Department of Chemical Engineering at Massachusetts Institute of Technology, gave a presentation titled Designer Polypeptides and Electrostatic Assembly for RNA-Protein Enhanced Delivery. The title alone piqued my interest because, in general, macromolecular self-assembly to form functional complexes in a predetermined manner is a trending topic in nucleic acid chemistry.

Prof. Hammond started off by saying that the traditional approach to mRNA delivery is the encapsulation of mRNA within a polycation. This approach requires mRNA to escape the polycation following endosomal uptake of the cargo, and to ultimately access the cytosol, where it can be translated. The translation step requires binding of the mRNA capping protein, EIF4E, Eukaryotic translation initiation factor 4E, and without this binding step, the mRNA is marked for degradation within the cell.

Photo by Jerry Zon

By contrast, Prof. Hammons has demonstrated that the pre-encapsulation of the EIF4E cap pre-complexed with based-modified (5’-methylcytidine and pseudouridine) mRNA—obtained from TriLink—can enhance mRNA translation within cells, multifold in comparison to release of mRNA alone. Furthermore, it was found that a unique series of polypeptides with variable charged side chain structures can enhance encapsulation of mRNA with EIF4E, with optimal systems yielding 70 to 80 times that of the mRNA alone. A key to these polymers is their ability to cooperatively bind RNA to the associated protein machinery needed for translation, in order to enhance translation.

More recently, Prof. Hammond has found similar kinds of enhancements for the delivery of siRNA via co-complexation with the Ago-2 protein to create a pre-assembled version of RISC complex. Finally, the layer-by-layer approach can be used to generate finely tuned release surfaces that can release small molecule, proteins, nucleic acids and other biologic drugs over sustained time periods, and with significant control of release characteristics. According to Prof. Hammond, this approach is particularly attractive for the delivery of proteins and nucleic acids such as siRNA.

Stereopure Phosphorothioate (PS)-Modified Antisense Oligonucleotides

In my recent blog I wrote about the highlights of the 23rd International Roundtable on Nucleosides, Nucleotides, and Nucleic Acids, and I commented on the virtually ubiquitous use of PS-modified linkages in all types of strategies for oligonucleotide-based therapeutics. I added that attention was now being given to stereoselective synthesis as a means of evaluating the influence of stereopure Rp or Sp linkages. Consequently, I and everyone else listened very attentively to the talk titled Stereochemical Control of Antisense Oligonucleotides Enhances Target Efficacy given by Chandra Vargeese, PhD, Senior Vice President Drug Discovery at Wave Life Sciences (Cambridge, MA).

Dr. Vargeese introduced the approach being used, and then presented data for antisense oligonucleotides shown here. These oligonucleotides were comprised of stereorandom or stereopure Rp (red ˄) or Sp ( black ˅) linkages at specific positions in a 20-base gapmer antisense nucleotide (gray filled ο) of unspecified sequence that was used to measure the kinetics of RNase H-mediated cleavage of complementary RNA as a model target.

Vargeese and coworkers Poster 30 titled Optimized, Stereopure Antisense Oligonucleotides Achieve Broad Tissue Distribution and Excellent Exposure, Enabling Potent and Durable Knockdown of Nuclear Malat1 in Mice and Nonhuman Primates. Photo by Jerry Zon.

Under the reported conditions, this construct gave initial kinetics that were 2-fold faster than the corresponding stereorandom antisense sequence. While this difference in efficiency was not very impressive, in my opinion, it was followed by in vitro results for free-uptake of stereopure and stereorandom antisense oligonucleotides against Malat1. In these experiments, the inhibitory concentration (IC50) values indicated 20-fold increased potency for the stereopure compound, which is quite impressive. Moreover, knockdown of Malat1 mRNA by the stereopure compound was about 10- to 100-times more effective than the stereorandom compound in mouse eye at 1 week.

Dr. Vargeese presented very intriguing data on uptake and distribution, which is not easily summarized here, but will hopefully be published with all of the above data in the near future alongside efficacy results for disease models in animals.

OTS Lifetime Achievement Award

Prof. Marv Caruthers award lecture. Taken from @OTSociety with its permission

The aforementioned synthesis of stereopure PS-oligonucleotides, which is enabled by phosphoramidite coupling followed by P3 → P5 conversion with a sulfurizing agent, is my segue into the OTS Lifetime Achievement Award. These year’s honoree was Prof. Marvin (“Marv”) Caruthers, whose talk was titled Chemical Synthesis of DNA/RNA and Biological Activity of Selected Analogues.

Marv began with his recollections of his lab’s development of the now universally employed phosphoramidite synthesis method, which he said was aimed at the younger members of the audience, jokingly adding that “oligos aren’t magically made by Federal Express.” Marv ended his talk with a description of how he has morphed his method to provide a wide variety of novel analogues. My blog about Marv’s marvelous contribution can be read here.

Delivery of Oligonucleotides to the Brain

At the risk of oversimplification, the human brain is sort of a “sanctuary” organ by virtue of a biochemical/vascular barrier (aka the blood brain barrier) that prevents or mutes continuous assaults, if you will, by chemicals or biochemicals that are harmful to the brain and its central nervous system (CNS). This makes delivering small drugs to the brain difficult, let alone relatively large single-stranded oligonucleotides. Consequently, current approaches for delivery of oligonucleotides to the brain have been largely restricted to using invasive intrathecal administration.

Blood brain barrier. Taken from Shutterstock. //  Intrathecal lumbar administration. Taken from Chung et al. (2016) PLOS One. Credit decade3D

Dr. Suzan Hammond. Photo by Jerry Zon.

By striking contrast to intrathecal administration of oligonucleotides, Dr. Suzan Hammond, pictured here, reported use of conventional systemic administration. Dr. Hammond is a postdoc with Prof. Matthew Wood in the Department of Physiology, Anatomy, and Genetics at the University of Oxford in the UK. She presented a poster titled Targeting the Brain and Spinal Cord with Antisense Oligonucleotides.

This work, which was carried out in collaboration with my long-time friend and oligonucleotide synthesis guru Prof. Michael Gait, employs a rather remarkable peptide-“oligonucleotide” conjugate. The “oligonucleotide” portion is a neutral-backbone, 6-membered ring morpholino (PMO) structure, which is quite unlike a deoxyribonucleotide, and has always been somewhat of an oddity in the oligonucleotide field. In any case, the PMO is conjugated to a PMO-internalizing peptide (Pip).

Motor neuron controls muscle movement. Taken from Shutterstock by Alila Medical Media

Interested readers can consult full details in a publication by Hammond et al. in the prestigious Journal of the Proceedings of the National Academy of Sciences. Dr. Hammond’s poster reports preclinical data that demonstrates potent efficacy in both the central nervous system (CNS) and peripheral tissues in severe spinal muscular atrophy (SMA), a leading genetic cause of infant mortality primarily due to lower motor neuron degeneration and progressive muscle weakness, which results from loss of the ubiquitous survival motor neuron 1 gene (SMN1).

Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate a functional SMN protein. Peptide-PMOs yield SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs.

As a follow-up to my question about future plans, Prof. Gait kindly provided the following reply by email:

“There is ongoing work in progress in Oxford, including a small spin off
company, to take the concept of peptide-PMO drugs towards clinical
trials in the next 2 or 3 years for one or more of the well-known
neuromuscular diseases, that include SMA. However, in the case of SMA, a
further grant-funded study is shortly to be underway to select a peptide
that might have a favourable toxicology profile, whilst maintaining
strong activity than the current peptides reported here, in the
expectation of reaching a suitable clinical candidate as soon as possible.”  

Concluding Impressions

Mid-morning and mid-afternoon coffee break networking was very enthusiastic. Photo by Jerry Zon

As a researcher who shared the initial vision of oligonucleotides as new therapeutics in the late 1970s—which faced far more naysayers than believers—I was more than gratified to be part of OTS 2018, and feel the positive “vibes” among nearly 600 participants, especially since this group included many young next-generation investigators, who represent the critical mass needed for future successes in this now firmly established field.

And speaking of the future, please note that in 2019 the 15th Annual OTS Meeting will be held in Munich, Germany.

Ich hoffe dich dort zu sehen!

Taken from oligotherapeutics.org


Given the difficulty of obtaining grant money for research, some of you will be pleased to know that the Ono Pharma Foundation sponsors the Breakthrough Science Initiative Awards Program. At OTS 2018, a representative of Ono Pharma gave the following information:

This program targets “groundbreaking work using synthetic oligonucleotides to gain valuable insights into molecular mechanisms, delivery strategies, or physiologic targets.” The grant is for $900,000 plus up to 15% indirect costs for 3 years of research, and there is no IP (intellectual property) obligation. Eligibility is for institutions in the US and Canada. Previous awardees are Jonathan Watts, PhD (Univ. of Mass.) in 2017, and William Mobley, MD, PhD (Univ. of Calif., San Diego).






3’-End Labeling of RNA or DNA by a Polymerase Ribozyme

  • Revisiting a Polymerase Ribozyme for 3’-End Labeling Oligos
  • Using a Wide Variety of Modified Nucleotide Triphosphates from TriLink to Demonstrate Versatility of Labeling

Gerald Joyce. Taken from scripps.edu

In September 2016, I wrote a blog featuring a remarkable publication by the Gerald Joyce lab at Scripps Research Institute in La Jolla, CA. The researchers wrote about the in vitro evolution of an RNA catalyst (i.e. ribozyme) that had RNA polymerase activity and could amplify RNA. This purely RNA-based synthetic chemistry, in the complete absence of any proteins, provided further evidence for the feasibility of “RNA world,” a phenomenon first discussed by Walter Gilbert in 1986, who hypothesized the existence of prebiotic era billions of years ago during which life began without DNA or proteins.

This blog post once again spotlights the Joyce lab, but in the context of applying this novel polymerase ribozyme as a means to carry out 3’-end labeling of RNA or DNA with 50 modified nucleotides. I’m pleased to add that many of the requisite modified nucleotide triphosphates were obtained from TriLink! Interested readers can consult this June 2018 publication in Nucleic Acids Research for more details if they wish to supplement the brief overview that will be given here.


RNA polymerase ribozymes are in vitro evolved RNA molecules that extend an RNA primer on a complementary RNA template using NTP substrates. Currently, the most advanced RNA polymerase ribozyme is the ‘24-3’ polymerase, which was reported in 2016 by Horning & Joyce to have an extension rate of ~1 nucleotide (nt) per minute, and can operate on most template sequences. Using specially designed templates, the 24-3 polymerase can generally be limited to the addition of only a single modified nucleotide, thus enabling efficient 3’-end labeling of a target RNA or DNA using various NTP and dNTP analogs shown here in red.

Taken from Joyce and coworkers Nucleic Acids Research 2018

The highly structured 24-3 polymerase ribozyme, which is depicted here in 2D, contains 180 nt. The ribozyme also has a short “tag” sequence (5’ GUCAUUG 3’) at the 5’ end of the polymerase that is complementary to a sequence (3’ CAGUAAC 5’) at the 5’ end of the template. Besides this feature, the template sequence is not constrained. The primer, which corresponds to the template nucleic acid, binds to the template through Watson–Crick pairing and is extended by the polymerase to achieve 3’-end labeling.

Although the 24-3 polymerase ribozyme can add multiple successive NTPs to the 3’ end of a template-bound primer, the reaction can mostly be restricted to the addition of a single residue by choosing an appropriate template and providing only one of the four nucleobase substrates. By way of example and as shown here, four templates were constructed, each with a different templating nucleotide (red) at the first position of primer extension, followed by several non-complementary nucleotides. Together, this set of templates enables the testing of triphosphate analogs containing each of the four nucleobases.

Taken from Joyce and coworkers Nucleic Acids Research 2018

Exemplary Results

Although a great variety of functionalized nucleotides can be prepared by chemical synthesis, this study by Joyce and coworkers focuses on commercially available nucleotide triphosphate analogs, such as sugar, nucleobase, and backbone modifications, in order to demonstrate the general utility of the approach. Fifty different analogs were tested in a reaction employing a 0.8 μM RNA (or DNA) primer with the following sequence: 5’ UUGCUACUACACGAC 3’ (or corresponding DNA sequence), together with 1 μM ribozyme and 1 μM RNA template. The reactions were carried out in the presence of 200 mM MgCl2 and 0.5 mM NTP analog at pH 8.3 at 17C for 1 h. Yields by PAGE ranged from 11% to 89% overall, with 84% to 89% yield for five of the analogs.

The exemplary results tabulated here highlight the versatility of 24-3 polymerase ribozyme toward incorporation of NTP analogs with very diverse molecular structures that provide different types of functionality.

Exemplary NTP Analogs (TriLink) and Incorporation Yield by the 24-3 Polymerase Ribozyme

NTP analog Yield % NTP analog Yield %
N6-methyl-2-amino-ATP 49 2’-amino-dATP 85
7-propargylamino-dGTP 89 2’-amino-dGTP 70
biotin-16-aminoallyl-dUTP 28 2’-amino-dCTP 80
Pseudo-UTP 66 2’-amino-dUTP 50
α-thio-ATP 70 5-aminoallyl-CTP 67
α-thio-GTP 86 Cy5-aminoallyl-CTP 47
α-thio-CTP 84 5-formyl-CTP 85
α-thio-UTP 11 5-formyl-UTP 58
thieno-GTP 50 1-borano-dGTP 37
thieno-UTP 25 1-borano-dCTP 12

For instance, N6-methyl-2-amino-ATP is a member of the diaminopurines that are discussed elsewhere, while pseudo-UTP shown below is an isomer of UTP that is now widely used in modified mRNAs.

Pseudo-UTP. Taken from TriLink BioTechnologies

In a TriLink white paper by Paul and Yee titled PCR incorporation of modified dNTPs: the substrate properties of biotinylated dNTPs, it is noted that the high affinity of streptavidin for the biotin ligand is one of the strongest and most widely utilized interactions in biology. The strength and specificity of this interaction has been exploited in many biological applications, including secondary label introduction and affinity isolation. While there are various length linkers that have been employed for attachment of biotin to the nucleotide, the relatively long biotin-16-aminoallyl-2′-dUTP used for incorporation by 24-3 polymerase ribozyme is often preferred.

Modification of the 3’-end by incorporation of 7-propargylamino-dGTP, 2’-amino dNTPs or 5-aminoallyl-CTP provides a reactive primary amine group as a versatile “chemical handle” to attach virtually any type of moiety that is needed for an application, whether that be a detectable label or synthetic peptide. The α-thio-NTPs (aka 1-thio-NTPs) and 1-borano-dNTPs demonstrate that these phosphate modifications are compatible with the 23-3 polymerase ribozyme.

α-Thio-ATP (1-Thio-ATP). Taken from Trilink BioTechnologies // 1-Borano-dCTP. Taken from TriLink BioTechnologies

Thieno-UTP. Taken from TriLink BioTechnologies

5-Formyl-nucleotides provide a reactive formyl (i.e. -CHO) group for conjugation reactions with, for example, hydroxylamine-functionalized labels of the type reported elsewhere. The incorporation of thieno-NTPs is interesting because of the inherent fluorescent properties of this relatively new class of analogs offered by TriLink, which can be read about at this link.

Concluding Comments

According to the aforementioned Joyce publication, the simple, one-step installation of a fluorophore or affinity probe using the 24-3 polymerase ribozyme is likely to have broad application, offering an attractive alternative to 3’-end labeling using a polymerase protein such as poly(A) polymerase or terminal transferase. These polymerase proteins operate in the template-independent manner, and thus result in multiple successive additions, unless the NTP analog itself is a chain terminator.

As usual, your comments are welcomed.

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 wikipedia.org

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 zon.trilinkbiotech.com  //  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.



Spotlight on TriLink Product Applications

  • Nearly 500 Publications in 2017 Cite Use of TriLink Products
  • Jerry Spotlights 20 Citing Oligos, Nucleotides, mRNA and Aptamers
  • 10 of These 20 Spotlighted Items Show Global Reach of TriLink Products

While thinking about possible topics to blog about, it occurred to me that researching recent publications on the applications of TriLink products would likely lead to many options. Using Google Scholar to do just that, I was given nearly 500 items, which is indeed plenty. However, choosing which to feature was neither an easy nor objective task. Having said that, and with sincere apologies to publications not spotlighted here, my “faves” and comments are given below, listed arbitrarily (not ranked) in four product categories: oligonucleotides, nucleotides, mRNA, and aptamers.

Taken from depositphotos.com

For convenience, each publication title can be clicked on to access the original article. Links to the cited TriLink products are also provided, alongside links to other adjunct information. Several trending “hot topics” and previous blogs are also noted.


Taken from researchgate.net


8-oxo-dGTP; taken from TriLink BioTechnologies // dPTP; taken from TriLink BioTechnologies


Modified mRNA for new therapeutic approaches continues to be an amazingly hot area of R&D, which I have previous dubbed “modified mRNA mania” in a previous blog. Interested readers can peruse this link to ~300 items found in my Google Scholar search for TriLink and mRNA publications in 2017.

pseudo-UTP; taken from TriLink Biotechnologies // 2-thio-UTP; taken from TriLink Biotechnologies


2’-F-dCTP; taken from TriLink BioTechnologies // 2’-F-dUTP; taken from TriLink BioTechnologies

Global Reach

A pleasantly surprising aspect of the selected-product search results given above is the worldwide distribution of researchers using TriLink products. This global reach, if you will, is evident from the following countries outside of the USA, which I made point of mentioning:

The Netherlands, India, Austria, Switzerland, Turkey, Germany, Italy, Belgium, Republic of Korea, and Denmark.

All of the publications listed above were selected solely on the type of TriLink product used. Given the relatively small “sample size” of these selected publications, which are only 20-of-500, finding investigators in 10 countries outside of the USA is a compelling testimonial for the TriLink global reach.

World Science Day

Truth be told, when I was searching for a fitting image to visually convey the concept of “global science,” I came across the fact that the United Nations Educational, Scientific, and Cultural Organization (UNESCO) has designated November 10 as World Science Day, with an emphasis on peace and development. The stated intention is to highlight “the important role of science in society and the need to engage the wider public in debates on emerging scientific issues. It also underlines the importance and relevance of science in our daily lives.”

Taken from monitor.co.ug

According to UNESCO, “[t]he theme for 2018 is ‘Science, a Human Right’, in celebration of the 70th anniversary of the Universal Declaration of Human Rights (art. 27), and of the Recommendation on Science and Scientific Researchers. Recalling that everyone has a right to participate in and benefit from science, it will serve to spark a global discussion on ways to improve access to science and to the benefits of science for sustainable development.”

To me, this is a long-term objective which is indeed critical for betterment of future generations.

As usual, your comments are welcomed.




Clinical Efficacy Found: Two Oligonucleotide Drugs for Transthyretin (TTR)-Related Amyloidosis (ATTR)

  • ATTR Is a Class of Life-Threatening and Progressive Genetic Diseases    
  • ATTR Results from Misfolding of TTR, a Blood Protein Carrier of Retinol (Vitamin A), and Thyroxine (T4)
  • TTR mRNA Knockdown by an Antisense Oligonucleotide (ASO) or a Short-Interfering RNA (siRNA) Leads to Clinical Efficacy for ATTR

A recent blog here lauded the beginning of American and European clinical studies of gene-editing by CRISPR, which is enabled by sequence-specific interaction of synthetic guide RNAs with genomic DNA targets. At the end of that post, I mentioned that future clinical successes with CRISPR may encounter the challenge of unforeseen drug development issues, as has been the case for antisense oligonucleotide (ASO) and short-interfering RNA (siRNA) approaches. It took a couple of decades for FDA-approved ASO drugs to progress from “bench to bedside,” and the first siRNA drug has yet to be approved.

After opining the above just a few weeks ago, I am pleased to say that another ASO drug and the first siRNA drug have been reported to both be on the brink of FDA approval, based on back-to-back publications of the results of Phase 3 clinical studies in the highly respected Journal of New England Medicine. Interestingly, both of these mechanistically different oligonucleotide drugs are aimed at the same clinical indication, namely, transthyretin (TTR)-related amyloidosis (ATTR). This blog will serve as an introduction to ATTR, as well as a brief summary of both of these very promising clinical studies.

Introduction to ATTR

As detailed elsewhere, ATTR represents a class of life-threatening and progressing diseases that is associated with the misfolding of TTR, a major blood protein that is a carrier of retinol (vitamin A) and thyroxine (thyroid hormone T4). TTR is primarily (>95%) produced in the liver as a tetramer, as depicted below. Amyloid aggregation is believed to be a result of decreased tetramer stability, resulting in dissociation of TTR into monomers. These monomers are prone to unfolding, and can self-assemble into oligomers and amyloid fibrils found in peripheral neurons, the gastrointestinal tract, and heart.

Taken from link.springer.com

The three major forms of ATTR disease are described in the NIH Genetic Home Reference (GHR), which also provides information about their underlying genetics and diagnosis by, for example, DNA sequencing. In hereditary ATTR with polyneuropathy [aka familial amyloid polyneuropathy (FAP)], the peripheral nerves are primarily affected, while in cardiomyopathy-related ATTR [aka familial amyloid cardiomyopathy (FAC)], neuropathy is usually less prominent.

According to the GHR, ATTR among Americans of European descent is estimated to affect one in 100,000 people, while FAC is far more frequent among people with African ancestry. For example, it is estimated that this form affects between three to four in 100 African Americans. Although the reasons for this disproportionately higher incidence are unknown, suggestions on how to raise awareness on this issue are discussed in a review I found while researching FAC among African Americans.

For the most part, patients develop a severe disease and die within 5 to 15 years after onset. While these forms of ATTR can be ascribed to a dominant expression of the TTR gene variants, (their number is ~100, as depicted here in an artistic rendering), only wild-type TTR is expressed in senile systemic amyloidosis (SSA), a type of amyloidosis frequently found in elderly people.

Taken from pharmaceuticalintelligence.com

The ATTR pathway above depicts the possibility of using oligonucleotides that interfere with mRNA function to block the synthesis of TTR. While blocking synthesis of a specific mutant of TTR is possible, biochemical and clinical evidence suggests that the wild-type TTR can also significantly contribute to the disease. In 2016, results obtained from an in vitro model system were jointly published by Ionis Pharmaceuticals and Alnylam Pharmaceuticals. The study supports the notion that ASOs or siRNAs can block TTR synthesis, and can therefore represent a possible therapy for ATTR. These findings paved the way for each company to independently carry out individual clinical development programs for oligonucleotide drug candidates, as outlined below.

ASO Drug Inotersen

Ionis’ inotersen (formerly IONIS-TTRRx/ISIS 420915), which is a 2′-O-methoxyethyl (MOE)– and phosphorothioate (PS)-modified RNA (orange)-DNA (black) “gapmer” ASO shown here, is an inhibitor of the hepatic production of TTR. Readers interested in ASO chemistry and mechanistic details for gapmers can consult an excellent review by Shen and Corey.

Taken from Shen and Corey Nucleic Acids Research (2018)

Taken from Buxbaum Journal of New England Medicine (2018)

As shown here, the genome of patients with hereditary amyloidotic polyneuropathy contains one copy of wild-type TTR and one copy with a base substitution (mutation), resulting in a change in the amino acid sequence. In hepatocytes, both copies are equally transcribed and translated. Inotersen binds to wild-type and mutant TTR mRNA transcripts, resulting in their degradation by ribonuclease H.

In healthy volunteers, inotersen showed dose-dependent and sustained reductions of circulating TTR levels. Ionis then conducted a randomized, double-blind, placebo-controlled, Phase 3 trial (NEURO-TTR) to determine the efficacy and safety of inotersen treatment in patients with hereditary transthyretin amyloidosis with polyneuropathy, in the presence or absence of cardiomyopathy. According to Benson et al., eligible patients were randomly assigned (in a 2:1 ratio) to receive 300 mg of inotersen or placebo. Patients 18 to 82 years of age received three subcutaneous injections during the first week to achieve near steady-state drug levels, followed by a once-weekly subcutaneous injection for the next 64 weeks.

A total of 172 patients (112 in the inotersen group and 60 in the placebo group) received at least one dose of a trial regimen, and 139 (81%) completed the intervention period. In the inotersen group, reductions in circulating TTR reached steady-state levels by week 13 and were sustained through the end of the intervention period. From week 13 to week 65, decreases in serum TTR from baseline levels in the inotersen group reached a median nadir of 79%.

Primary efficacy assessments favored inotersen. These improvements were independent of disease stage, mutation type, or the presence of cardiomyopathy. The most frequent serious adverse events in the inotersen group were glomerulonephritis [in 3 patients (3%)] and thrombocytopenia [in 3 patients (3%)]. It was concluded that “inotersen improved the course of neurologic disease and quality of life in patients with hereditary transthyretin amyloidosis” and that “[t]hrombocytopenia and glomerulonephritis were managed with enhanced monitoring.”

siRNA Drug Patisiran

According to the above cited review by Shen and Corey, and as shown here, Alnylam’s patisiran is a double-stranded siRNA comprised of 3’-end DNA (black), RNA (pink), and 2’-O-MOE RNA (blue) moieties. Partisiran is delivered in complex with a lipid nanoparticle (LNP), (also depicted here), and is described in detail elsewhere by Acuitas Therapeutics, an LNP drug-delivery company that partnered with Alnylam.

Taken from Shen and Corey Nucleic Acids Research (2018)

Taken from acuitatx.com

The mechanism of action depicted here for the siRNA patisiran involves the formation of an RNA-induced silencing complex (RISC) with both wild-type and mutant TTR mRNA transcripts and subsequent mRNA degradation.

Taken from Buxbaum Journal of New England Medicine (2018)

Adams et al. have now reported results from a Phase 3 clinical trial by Alnylam wherein patients were randomly assigned (in a 2:1 ratio) to receive patisiran (0.3 mg per kilogram of body weight) or placebo intravenously over a period of approximately 80 minutes, once every 3 weeks for 18 months. A total of 225 patients 24 to 83 years of age were randomly assigned to receive patisiran (148 patients) or placebo (77). In the patisiran group, the reduction in serum TTR levels was rapid and sustained over a period of 18 months, with a median reduction of 81% that remained similar across age, sex, or genotype.

Primary efficacy endpoints favored patisiran and approximately 20% of the patients who received patisiran and 10% of those who received placebo had mild or moderate infusion-related reactions; the overall incidence and types of adverse events were similar in the two groups. It was concluded that “patisiran improved multiple clinical manifestations of hereditary transthyretin amyloidosis.”

One Expert’s Opinion

Joel Buxbaum, an MD and Professor Emeritus in the Department of Molecular Medicine at the Scripps Research Institute, accompanied these back-to-back publications about inotersen and patisiran in the Journal of New England Medicine with an Editorial. Based on his publications in this field, Buxbaum is an expert on the molecular basis of acquired and hereditary human disease related to human amyloidosis. After summarizing the results of each clinical trial, Buxbaum posed the following scientifically intriguing questions, which had not occurred to me after reading these two reports.

“Although each of the trials unequivocally shows a therapeutic effect, there remain questions. Would a single patient have a response to each of the therapeutics to the same degree? If so, it is in the patient’s interest to use the least expensive therapy. If not, N-of-1 trials of sequential treatments, although cumbersome, would be required to identify the best treatment for each patient, conditional on the availability of a rapidly responsive, validated surrogate marker of disease.” In regard to the currently trending concept of N-of-1 trials, Buxbaum refers the reader to a commentary in Nature by Nicholas Schork titled Personalized medicine: Time for one-person trials, from which I borrowed this illustration that visually coveys Schork’s point.

Taken from Schork Nature (2015). Illustration by Greg Clarke

Buxbaum continues his expert commentary as follows:

“It is also possible that a combination of interventions would elicit a more pronounced, durable therapeutic effect. Although there remains much work to be done, the trials by Adams et al. and Benson et al. represent a landmark: together, they show that the rate of progression of a peripheral neurologic disease can be slowed, and perhaps ameliorated, through the use of oligonucleotide drugs that are administered systemically.”

In my humble opinion, the promising outcomes of these two trials are indeed great news for people afflicted with ATTR, who can be future recipients of this oligonucleotide therapy. The results are likewise great news for families and friends. The many researchers, who collectively contributed—directly or indirectly—to achieving this milestone, are also undoubtedly pleased by these results.

As usual, your comments are welcomed.

Reported: First Visualization of I-Motif DNA in Human Cells

  • Although Akin to Well-Known G-Quadruplexes, I-Motif DNA In Vivo Has Been Debatable
  • Now, Researchers Have Reported the First Visualization of I-Motifs in Human Cells Using an Antibody
  • These Researchers Raise Questions About Antibody Specificity for Visualizing G-Quadruplex vs. I-Motif Structures

In April 2013, in my second ever blog, DNA G-quadruplexes (GQs) were featured as a then trending hot topic in nucleic acid research, following a report in Nature Chemistry of the first visualization of these DNA structures in human cells. That published work, led by University of Cambridge Prof. Shankar Balasubramanian, has been cited nearly 2,000 times. This puts it in the 99th percentile (ranked 160th) of the 309,600 tracked articles of a similar age in all journals, according to an Altmetric score.

Sir Shankar (©Caroline Hancox). Taken from tri.com.ac.uk

The aforementioned 2013 blog includes an interview with Prof. Balasubramanian, who was knighted in January 2017 in part for that pioneering work. Although somewhat akin to GQs, iM DNA has eluded detection in vivo until now. Aussie researchers have visualized iM DNA for the first time, as described in an April 2018 publication in Nature Chemistry. Following my brief synopsis of these new findings, Sir Shankar was kind enough to share some additional perspectives on iMs with us for discussion.

Introduction to GQ and iM DNA Structures

DNA is well-known to adopt alternative non-B-form conformations in vitro, including GQ (aka G4) and iM structures, which are depicted here in a figure taken from the aforementioned Nature Chemistry report in 2018 by the Aussie team of Zeraati et al. Of the two structures, GQ DNA formed within guanine (G)-rich regions of the genome is by far the more studied. In spite of extensive bioinformatic and in vitro characterizations of GQs, the in vivo existence of GQs in human cells remained speculative until the visualization of these structures by Sir Shankar’s team. The team used an antibody fragment that recognizes GQs (shown in red) in a structure-specific manner.

Taken from Zeraati et al. Nature Chemistry (2018)

In marked contrast, insights into the biological role of iM DNA are limited. This structure is formed via a stack of intercalating hemi-protonated C—neutral C base pairs (shown in green), which are stabilized at a slightly acidic pH. As C-rich regions are common within the human genome and can occur opposite G-rich regions (hence the interconversion arrows in the figure), iMs have been characterized in vitro using a range of biophysical techniques. These analyses demonstrated the formation of both intramolecular (as depicted in the figure) and intermolecular structures, with the overall stability of the structure dependent on the number of Cs in the iM core, as well as the length and composition of the loops.

However, the in vivo existence of this four-stranded iM DNA structure in the human genome has been a matter of scientific debate. In particular, observations that in vitro formation of the iM structure is dependent on acidic conditions has led to questions concerning biological relevance. But now, as you’ll read in what follows, such doubt about the biological relevance of iM DNA has been countered by compelling evidence in support of the in vivo existence of iM DNA, and suggestions of regulatory roles for iM DNA in the genome.

An Antibody Fragment Specific for iM Structures

The recent publication by Zeraati et al. should be read in its entirety to fully appreciate the methodology used to isolate an iM-specific antibody fragment for the study of iM structures in vitro and in vivo. Briefly, three rounds of phage-display selections were performed to isolate iM DNA-binders. The binders were then initially characterized using soluble fragment enzyme-linked immunosorbent assay (ELISA). This was followed by evaluation of the bio-layer interferometry (BLI) off-rate ranking, resulting in the identification of a lead candidate, which was termed iMab.

Diagram of BLI activity with immobilized binding partner attached. Taken from fortebio.com

Further ELISA analyses revealed highly specific binding, as indicated by the absence of iMab binding to a wide range of control proteins and nucleic acids, including double-stranded DNA, hairpin DNA, and microRNA. To investigate whether iMab is capable of binding to partially folded iM structures, Zeraati et al. tested truncated constructs with either three or two tandem runs of Cs. The two truncated constructs displayed either considerably reduced binding (three C-runs) or no detectable binding (two C-runs), indicating that recognition by iMab is highly dependent on the folded structural framework of C-containing DNA.

The pH-dependence of iM stability and iMab binding was investigated because iM structures are considered most stable at acidic pH, reflecting the requirement for the formation of hemi-protonated C—neutral C base pairs (see above). Zeraati et al. thus hypothesized that pH and the binding of iMab should be inversely correlated, assuming that the antibody predominantly recognizes folded iM rather than unfolded species. Using BLI and a constant concentration of iMab binding to an iM structure, it was found that the iMab-iM complex antibody formed at pH 6, and could be readily dissociated when switching to pH 9 buffer conditions.

iMab Differentiates iM Structures from GQ Structures

Zeraati et al. reasoned that iMs and GQs could display common epitopes, i.e. a specific piece of DNA to which iMab binds. Moreover, in genomic DNA, iMs are often accompanied by complementary G-rich sequences, which can adopt GQ structures as depicted above. For applications such as visualization by immunostaining, a high level of specificity and the absence of binding to GQs is therefore important. To evaluate iMab cross-reactivity, Zeraati et al. examined binding to six GQs of known structures, which were selected to represent a diverse range of conformations. ELISA results showed that iMab does not display any detectable binding to these molecules, suggesting that it is highly capable of differentiating between GQ and iM structures.

The studied presumptive negative controls included commercially available GQ antibody BG4, which was initially described by Sir Shankar and coworkers in Nature Chemistry as noted above. BG4 showed substantial binding to all analyzed GQ structures, as expected; however, BG4 also displayed detectable cross-reactivity to iMs and competed with iMab for binding to several iMs. Given the potential complications that could result from BG4 cross-reactivity with iMs, independent confirmatory investigations are needed to address the nature and scope of this putative cross-reactivity reported by Zeraati et al.

iM Structures are Formed in the Nuclei of Human Cells

Zeraati et al. used iMab for indirect immunofluorescent staining of three different human cell lines. This staining revealed punctate foci in the nuclei of MCF7, U2OS and HeLa cell lines that were dependent on the presence of iMab, as shown here for MCF7 cells. In addition, DNase I treatment significantly decreased the number of foci in the nuclei, presumably due to nuclease-resistant DNA structures or shielding by proteins. Zeraati et al. speculated that another possibility was the formation of iM RNA structures that are not removed by DNase I digestion. A small decrease in the number of iMab foci after RNase A treatment supported this speculation.

Imaging of iMab foci in the nuclei of MCF7 cells using confocal microscopy. Nuclei were counterstained with DAPI. Yellow dashed lines in the DNase I treated samples indicate the borders of nuclei. Scale bars, 5 μ m. Taken from Zeraati et al. Nature Chemistry (2018)

pH-Dependent Formation of iM Structures

Zeraati et al. examined how the formation of iM structures is affected by pH in the following way. As depicted here, H2O and CO2 react to form carbonic acid, H2CO3. Consequently, intracellular pH is inversely correlated with the concentration of CO2 in a cell culture system.

Taken from wateronline.com

These investigators hypothesized that they could study the effect of pH on iM formation by changing the CO2 concentration. MCF7 cell cultures were incubated at 5% CO2 until they reached ~70% confluency and then placed in 2%, 5% or 8% CO2 incubators for a further 2.5 h. From the box-and-whiskers plot shown here, staining with iMab led to observation of a relatively small but statistically significant increase in the numbers of foci with increasing CO2 concentration. As expected, measurements of pH in the MCF7 culture medium indicated an inverse correlation with the CO2 concentration. In view of the relatively small lowering of pH, it seems to me that iM structures are quite sensitive to pH changes in cell culture solution.

iMab foci of 200–300 nuclei were counted per condition. Green boxes represent 25th to 75th percentiles. Horizontal lines and “+” indicate medians and means, respectively. Whiskers indicate highest and lowest values of the results. Statistical significance **P < 0.01, ****P < 0.0001. Taken from Zeraati et al. Nature Chemistry (2018)

Cell-Cycle-Dependent Formation of iM Structures

As depicted here, the human cell cycle consists of four distinct phases: G1 phase (growth; cells increase in size), S phase (synthesis; DNA replication), G2 phase (growth and preparation for mitosis, i.e. cell division) and M phase (mitosis). M phase is composed of two tightly coupled processes: karyokinesis, in which the cell’s chromosomes are divided, and cytokinesis, in which the cell’s cytoplasm divides forming two daughter cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called G0 phase.

Taken from commons.wikimedia.org                                     Taken from difference.wiki

To investigate whether iM formation is affected by cell-cycle progression, Zerati et al. used the iMab antibody to stain HeLa cells synchronized at G0/G1, G1/S boundary or early S phase. As shown here, they observed the minimum number of foci at the G0/G1 phase and the highest number of foci at G1/S boundary phase. When the cells synchronously began (early) S phase, the number of iMab foci was reduced compared to the G1/S boundary phase. Similar trends of iM formation during cell-cycle progression were observed in the MCF7 cell line.

Taken from Zeraati et al. Nature Chemistry (2018)

iMab foci of 200–300 nuclei were counted per condition. Green boxes represent 25th to 75th percentiles. Horizontal lines and “+” indicate medians and means, respectively. Whiskers indicate highest and lowest values of the results. Statistical significance **P < 0.01, ****P < 0.0001.


Rather than paraphrase Zeraati et al., here are some salient statements taken from the discussion section:

  • “[W]e were able to detect iMs within a pH range of 6–8. Our results thus indicate that iM DNA structures can indeed form under physiological conditions….”
  • “[T]he formation of iM structures is highly cell-cycle specific…we observed that the highest level of iM formation occurs in late G1 phase, which is characterized by high levels of transcription and cellular growth…iMs may act as scaffolds for the binding of transcription factors during transcription.” 
  • “This behavior is markedly different to GQ formation, which occurs predominately during the S phase. In contrast, we observed a reduction in the number of iMab stained foci in early S phase, indicating that iM structures are resolved during replication.” 
  • “The difference between iM and GQ formation during cell-cycle progression agrees with recent findings demonstrating that these structures are often mutually exclusive and play opposite roles in the regulation of gene expression.”

As mentioned in the introduction, Sir Shankar kindly provided these perspectives on the report by Zeraati et al.:

“[It] is a nice study that adds to the emerging view that DNA secondary structure is more interesting and dynamic than was thought a few decades back.  The results of the study would seem to counter the view that the pH-dependence iMs makes them unlikely to form.  Previous studies have suggested iMs may play roles that are distinct from those proposed for GQs and there is a subtle indication of this from the differences observed in the cell-cycle dynamics of iM, which seem to be most abundant at M1/S and not S-phase, as compared with GQs that max out during S-phase.  As next steps it will interesting to further probe when and where iMs form in the genome and the potential links between iM and function.”  

I thank Sir Shankar for his willingness to share his thoughts here and, as usual, I welcome your comments.


While researching literature for this blog, I became curious about evidence for coexistence of GQ and iM structures in the same region of DNA. One such publication by Chen et al. reports the following:

A diagram of equilibrium between two forms of NHE III1 (nuclease hypersensitivity element III). The left state represents a transcriptionally active form, which can regulate 80-90% of c-Myc transcription, and the right state represents a silenced form, with both GQ and iM structures being shown, which represses the transcription of c-Myc. CNBP: cellular nucleic acid binding protein; hnRNP: heterogeneous nuclear ribonucleoprotein; TBP: TATA-box-binding protein; RNA Pol II: RNA polymerase II. Taken from Chen et al. Int J Biol Sci (2014)

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 quantamagazine.org

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 socratic.org

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 statnews.com 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 highwaysupply.net

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.”




ATP Synthase and ATPase: In Memory of Paul D. Boyer and Jens C. Skou

  • ATP Is the “Molecular Unit of Currency” of Intracellular Energy Transfer
  • 1997 Nobelists Boyer and Skou Elucidated Fundamental ATP Biochemistry
  • Both Laureates Have Recently Passed Away at Age 99

In 1977, the Nobel Prize in Chemistry was divided among three scientists for their fundamental contributions related to adenosine triphosphate (ATP), which is often referred to as the “molecular unit of currency” of intracellular energy transfer. One half of this prize was jointly awarded to Paul D. Boyer and John E. Walker for their elucidation of the enzymatic mechanism underlying the synthesis of ATP, and the other half was awarded to Jens C. Skou for the first discovery of an ATP-driven ion-transporting enzyme: sodium/potassium-ATPase.

Taken from pinterest.com

ATP sodium. Taken from TriLink BioTechnologies

The recent passing of Boyer (June 2, 2018) and Skou (May 28, 2018) is doubly saddening, as well as striking in that both Nobelists died at age 99, and within only five days of each other. In memory of their fundamentally important contributions related to ATP, which is arguably the most important nucleotide for life, this blog provides a brief synopsis of the amazing mechanism by which ATP is produced by ATP synthase, and the equally amazing mechanism by which ATP pumps sodium and potassium ions across cell membranes.

ATP Synthase

I highly recommend reading Boyer’s historical account and personal perspective on how, alongside his graduate students and postdoctoral fellows, over a span of many years, he concluded that ATP is made by an unusual rotational catalysis of ATP synthase. Boyer’s reflections also explain how an x-ray structure reported by Walker—now an emeritus director of the MRC Mitochondrial Biology Unit in Cambridge—came to play a critical role in establishing this novel mechanism.

Taken from Boyer J Biol Chem (2002)

There are three major conformations of catalytic sites (1, 2 and 3) for synthesis of ATP or hydrolysis by ATP. These three sites are depicted here with asymmetric interactions to the central subunit. During catalysis, sites are converted sequentially into three different states, accompanying rotation of the central subunit. The sequence for synthesis is 1 → 2 → 3, while for hydrolysis it is 3 → 2 → 1. Site 1 binds adenosine diphosphate (ADP) better than ATP and is the site at which ADP and inorganic phosphate (Pi) must be present for rapid synthesis to occur. Site 2 has the ability to catalyze chemical transformation and to be present as a form with ADP and Pi present or with ATP present. ATP can be released from Site 3 during synthesis and must be present at this site for rapid hydrolysis.

The rotation of the central subunit is powered, so to speak, by protons. To fully appreciate the wonderous complexity of this “molecular machine”, it’s necessary to consider all of the structural components and the functions ascribed to each. The colorized depiction of membrane-imbedded ATP synthase from E. coli shown below is a useful visual aid for such mechanistic considerations.

Taken from Boyer J Biol Chem (2002)

This ATP synthase consists of two regions called F1 and F0. The F1 portion subunits are designated as α3β3γδε and the F0 portion subunits are designated as ab2c9-12. The passage of protons (H+), at the interface of subunit a and the ring of c subunits, causes a rotation of the c subunits and attached ε and γ subunits relative to the rest of the enzyme. The asymmetric γ subunit (yellow and light green) extends through the center of the α3β3 cluster. The b2 and δ subunits serve as a stator, i.e. the stationary part of a rotary system. The rotation of the γ subunit results in sequential conformational changes of the catalytic sites that promote ADP and Pi binding, ATP formation, and ATP release. You can access and read a concise recap of this process written by Boyer at this link in a Nature article titled What makes ATP synthase spin?

Amazing ATP Facts

I was truly amazed—and totally surprised—by the following facts on ATP, especially the last bullet point:

  • Cellular ATP concentration is normally maintained in the range of 1 to 10 mmol/L, with a normal ratio of ATP/ADP of approximately 1000.
  • The totally quantity of ATP in an adult is approximately 0.10 mol/L.
  • Approximately 100 to 150 mol/L of ATP are required daily, which means that each ATP molecule is recycled some 1000 to 1500 times per day.
  • Transmembrane proton flux through the mitochondrial ATPase synthase complex occurs at an estimated rate of 3 × 1021 protons per second.
  • This corresponds to ATP reformed at a rate of 9 × 1020 molecules/sec, or approximately 65 kg ATP recycled per day in a normal resting adult.
  • Basically, the human body recycles its weight in ATP daily!

Sodium/Potassium-ATPase (Na/K-ATPase)

Taken from cellularscale.blogspot.com

To fully appreciate the scientific story regarding the first discovery of Na/K-ATPase, I recommend reading Skou’s historical account of the circumstances that led him to this achievement. Skou recalls reading a paper that mentioned the presence of an ATP hydrolyzing enzyme in the sheath part of the giant axon of a squid: an ATPase. Knowing then that ATP is the energy source within cells, Skou was intrigued, but he had no access to giant axons and therefore no way of investigating this putative nerve membrane ATPase. He reasoned that crab nerves might be a suitable alternative, so in 1954, he made a deal with a local fisherman to provide him with crabs. He isolated the sciatic nerve from the legs, starting what would become his pioneering discovery.

By 1965, Skou had accumulated enough data to publish a review, summarizing the following properties for the enzyme he named “Na+, K+-ATPase”:

  • It is located in the cell membrane.
  • It has a higher affinity for Na+ than for K+ on the cytoplasmic side; and a higher affinity for K+ than Na+ on the extracellular side.
  • It has enzymatic activity and catalyzes ATP hydrolysis; the rate of which depends on cytoplasmic Na+ and extracellular K+.
  • It is found in all cells that have coupled active transport of Na+ and K+.

The simplified series of depictions shown below represent the current view of the mechanistic steps for how Na/K-ATPase functions as a sodium-potassium pump. (1) The Na/K pump binds three sodium ions and a molecule of ATP. (2) The splitting of ATP into bound Pi and ADP provides energy to change the shape of the channel, which leads to sodium ions being driven through the channel. (3) The sodium ions are released to the outside of the membrane, and the new shape of the channel allows two potassium ions to bind. (4) Release of Pi allows the channel to revert to its original form, releasing the potassium ions on the inside of the membrane.

Taken from socratic.org

Legacy of Literature

In my humble opinion, one of the most gratifying aspects of being a scientist is recognizing that your published investigations will be available for others to learn from after you are no longer among the living. This immortalization, if you will, is what we can collectively think of as our “legacy of literature” that can benefit, in some way, future generations of scientists and other members of society.

The Nobel-worthy contributions by Boyer and Skou on the elucidation of ATP biochemistry are obviously very special examples of this legacy, as reflected in the huge numbers of citations to their publications. I used Google Scholar to find several of the most highly cited articles by each of these Nobelists that you can peruse by clicking the link to each:

Citations          Title

2513               The influence of some cations on an adenosine triphosphatase from peripheral nerves

2378                 Enzymatic Basis for Active Transport of Na+ and K+ Across Cell Membrane

1823                 The ATP synthase—a splendid molecular machine

1157                 The binding change mechanism for ATP synthase—some probabilities and possibilities

873                    Further investigations on a Mg+++ Na+-activated adenosine triphosphatase…

700                     Oxidative phosphorylation and photophosphorylation

By the same token, Walker’s ATP-related legacy of literature can be perused via this link to all of his publications in PubMed. In researching the literature for background and details, I learned a lot about ATP synthase and ATPase biochemistry. Hopefully, you have also found some of this information new and interesting.

Finally, I came across this quote by Paul Boyer expressing his thoughts about being awarded the Nobel Prize after many years of slogging through seemingly endless enzymology; it resonated with me and may also resonate with you:

“The experience reminds me of a favorite saying: Most of the yield from research efforts comes from the coal that is mined while looking for diamonds.”

Taken from pinsdaddy.com

As usual, your comments are welcomed.

June Is Acute Myeloid Leukemia (AML) Awareness Month

  • N6 Methylation of Adenosine in mRNA by METTL3 Linked to AML
  • Linking METTL3 to AML by Gene Knockdown with RNA Interference
  • Targeting METTL3 May Lead to a New Drug for AML

Designation of June as Acute Myeloid Leukemia (AML) Month was announced in 2016 by CancerCare®, which provides free emotional and practical support for people with cancer, caregivers, loved ones and the bereaved. The stated goal of the campaign is to put a spotlight on this rare and difficult-to-treat blood cancer that typically affects older adults. In support of that goal, the present blog focuses on newly reported mechanistic findings related to AML.

Taken from Wikipedia.org

By way of an introduction, previous blogs here on RNA epigenetics dealt with discovery of post-transcriptionally modified bases in RNA and enzymes that add (“write”) and remove (“erase”) these modifications, and the effects on RNA function. Among these base-modified RNAs, N6-methyladenosine (m6A; shown here), which is present in bacterial and eukaryotic cells, has been found to have a regulatory role in RNA processing.

According to Wei et al., m6A occurs mainly in the 3′ untranslated regions (UTRs) and near the stop codons of mRNA. Dynamic regulation (“editing”) of m6A is found in metabolism, embryogenesis, and developmental processes, indicating an epigenetic regulatory role. For example, it is known that m6A editing is involved in nuclear RNA export, mRNA degradation, protein translation, and RNA splicing. More recently, as summarized below, m6A editing has been linked to human cancer.

m6A Formation in mRNA Mediated by METTL3 Linked to AML

Although m6A is required for differentiation of mouse embryonic stem cells, it has been unknown whether m6A controls differentiation of normal and/or malignant myeloid hematopoietic cells. Compelling evidence in support of such control by m6A was recently published by Vu et al. in Nature Medicine based on collaborative work by a large team of investigators at Memorial Sloan Kettering Cancer Center (NY), Weill-Cornell Medical College (NY), and elsewhere. Readers interested in details should consult this lengthy paper, but for now here’s my short synopsis of what was done.

By way of background, N6-adenosine-methyltransferase 70-kDa subunit (METTL3) is an enzyme that in humans is encoded by the METTL3 gene and, in complex with an analogous subunit (METTL14), is involved in post-transcriptional methylation of the N6 position of adenosine residues in eukaryotic mRNAs to form m6A. The molecular structure and mechanism of how this happens are published and are schematically depicted in the drawing shown below, taken from that work by Śledź and Jinek.

S-adenosyl methionine (SAM); active site loops (ASLs) 1 and 2. Taken from Śledź & Jinek eLife (2016)

Taken from seer.cancer.gov

Since myeloid differentiation (see below) is frequently dysregulated in leukemia, Vu et al. determined if METTL3 expression is altered in leukemia. METTL3 mRNA expression in human AML samples is significantly higher than in other cancer types. To further assess the relative abundance of METTL3 in myeloid leukemia, they examined METTL3 mRNA and protein levels in 11 multiple leukemia cell lines and compared these to primary human cord blood-derived CD34+ cells. METTL3 mRNA was more abundant in AML cell lines (8/11), as was METTL3 protein (11/11). They found no significant difference in METTL3 expression across multiple subtypes of AML.

Taken from dreamtimes.com

After finding increased METTL3 in leukemic cells versus normal cells, they next measured m6A abundance in mRNA and found a significant increase in an AML cell line (MOLM13) compared to CD34+ control cells. These data suggested that elevated m6A might be critical to maintaining an undifferentiated state in myeloid leukemia. To directly address the role of m6A in human myeloid leukemia cells, they examined the effect of METTL3 mRNA knockdown in MOLM13 cells using short hairpin RNA (shRNA). METTL3 knockdown significantly decreased m6A levels, blocked cell growth, induced differentiation and resulted in an increase in apoptosis (aka “programmed cell death”). Similar effects of shRNA-mediated METTL3 depletion were seen in two other AML cell lines.

Vu et al. next investigated whether METTL3 was required in vivo for induction of leukemia. MOLM13 cells were transduced with METTL3-shRNA and transplanted into immunodeficient recipient mice. These cells exhibited delayed leukemia development compared to MOLM13 cells transduced with the scrambled-shRNA. To determine how METTL3 depletion affects m6A -containing transcripts, they performed RNA-Seq on MOLM13 cells following METTL3 knockdown. Consistent with the idea that m6A destabilizes mRNA, transcripts with at least one m6A site showed increased abundance following METTL3 depletion compared to transcripts with no called m6A sites. Moreover, the change in abundance was directly correlated with the number of m6A sites per transcript.

Clinical Relevance

To assess clinical relevance of the MOLM13 m6A profile, Vu et al. mapped m6A in two AML patient samples and compared these transcripts to m6A-enriched transcripts found in MOLM13 cells. They found that the mRNAs with the highest levels of m6A (top 300 transcripts) from the patient samples were enriched with the m6A transcripts from the MOLM13 cells.

Vu et al. stated that this study is the first to demonstrate that m6A is critical for maintaining the differentiation program in the hematopoietic system and that this process is dysregulated in myeloid leukemia. Rather than rephrasing the final conclusion by these researchers, here’s the direct quote:

Our data suggest that inhibition of METTL3 could be exploited as a therapeutic strategy for myeloid malignancies. It is notable that leukemia cells show elevated abundance of METTL3 compared to normal hematopoietic cells. Furthermore, we find that METTL3 depletion shows markedly increased levels of apoptosis in leukemia cells compared to normal cells. These findings suggest a possible therapeutic index. Future studies that target METTL3, potentially in combination with current and emerging therapeutic agents, should be explored.

Parting Comments

Given the increasing interest in elucidation of how mRNAs bearing various types of base-modifications are related to cancer, it seems likely that more discoveries will lead to future therapies of the sort envisaged by Vu et al. for m6A in AML.

Finally, in recognition of June as AML Awareness Month, I thought it would be worth sharing the following information about this disease taken from an authoritative website maintained by the National Cancer Institute.

  • The number of new cases of AML was 4.3 per 100,000 people per year, while the number of deaths was 2.8 per 100,000 people per year.
  • Lifetime risk of developing AML is ~5% of people will be diagnosed with AML at some point during their lifetime, based on 2013-2015 data.
  • Less than 30% of people survive5 years or more after being diagnosed with AML.
  • AML is most frequently diagnosed among older people but it effects all age groups.
  • Leukemia is cancer that starts in the tissue that forms blood. Most blood cells develop from cells in the bone marrow called stem cells. In a person with leukemia, the bone marrow makes abnormal white blood cells. The abnormal cells are leukemia cells. Unlike normal blood cells, leukemia cells don’t die when they should. They may crowd out normal white blood cells, red blood cells, and platelets. This makes it hard for normal blood cells to do their work.
  • There are four main types of leukemia: AML, acute lymphoblastic leukemia, chronic lymphocytic leukemia (CLL), and chronic myelogenous leukemia (CML).

Common symptoms for leukemia are as follows:

Taken from pinterest.com

As usual, your comments are welcomed.