Lab-on-a-Drone and Other Innovative Point-of-Care Devices

  • Lab-in-a-Box…Think Bento
  • Lab-on-a-Robot…Rolls Along 
  • Lab-on-a-Drone…PCR-on-the-Fly

Honey! I shrunk the lab! 

Taken from gene-quantification.de

Researchers have long dreamed of a “lab-on-chip” (LOC) wherein common laboratory procedures have been miniaturized and integrated in various formats using microfluidics—small, interconnected channels resembling electronic circuits on a chip—that provide low-cost assays for “point-of-care” (POC) applications. The cartoon to the right humorously but concisely depicts the general concept of LOC, for which there are virtually an infinite number of specific embodiments made possible by continuing development of many clever fabrication and microfluidic technologies for “shrinking” lab procedures.

Importantly, lab personnel are thus freed-up from slavish, repetitive tasks to instead carry out discovery and development work. Testament to the significance of LOC is evident from the astounding—to me—130,000 items I found in Google Scholar by searching LOC as an exact-word phrase. There is also a LOC Wikipedia site and a journal for LOC specialists named—appropriately—Lab on a Chip, which is already in its 15th year.

What follows is my take on some of the conceptual morphing, so to speak, of LOC-enabled devices that can be packed for portability, driven by remote control, or flown-“in-and-out” for all manner of unconventional, but critically important POC situations needing nucleic acid-based tests.

Lab-in-a-Box 

In archived blogs I’ve previously commented on examples of commercially available portable POC devices that are variations of a lab-in-a-box that can be easily carried in luggage or a back pack. By way of updates, here are some new applications for these systems illustrating wide diversity of use and location:

  • Ubiquitome’s hand-held qPCR system for molecular testing in New Zealand forests aimed at protecting indigenous Kauri trees—the oldest tree species in the world.
  • Amplyus’ miniPCR system for combating Ebola in villages deep in Sierra Leone, Africa.
  • Oxford Nanopore’s thumb-drive size DNA sequencer to identify organisms in the Canadian high Arctic.

Taken from @WhyteLab

RAZOR system by BioFire Defense. Taken from biofiredefence.com

In the above examples, sample prep workflow is still in need of automation with appropriate LOC technology. However, progress in this regard is being made. One example is the RAZOR system developed by BioFire Defense (pictured below) that features a qPCR lab-in-a-box with ready-to-use, freeze-dried reagent pouches for the detection and identification of pathogens and bio-threat agents. While the progress is impressive, there is still work to be done. A dramatized video for RAZOR usage revealed that much manual manipulation and dexterity with syringes are still required, which suggests the need for complete LOC automation in the future.

Another example of facilitating POC sample prep is the Bento Lab, which is named to be word-play on Bento Box—a complete Japanese lunch in a small, partitioned box-like plate. This portable DNA laboratory created by Bethan Wolfenden and Philipp Boeing at University College London is small enough to fit into a laptop bag, weighs only 6.6 pounds, and can now be preordered for ~$1,000 as a “must have” accessory for so-called “citizen scientists,” some of whom have had early access and have posted their personal Bento Lab stories.

The Bento Lab. Taken from Bento Lab

Lab-on-a-Robot

Biohazard accidents happen, as do bio-threat acts of terrorism. In these seriously scary situations, it may be safer or necessary for first-responders to deploy an Autonomous Vehicle as a self-navigating/driving lab-on-a-robot. Sounds far out, but the first example of a mobile lab-on-a-robot was demonstrated in 2008 by Berg et al., and is pictured below.

Taken from Berg et al. (2008)

This particular lab-on-a-robot is able to autonomously navigate by GPS, acquire an air sample, perform multi-step analysis [i.e. injection, capillary electrophoretic separation, and electrochemical (EC) detection], and send data (electropherogram) to a remote station without exposing an analyst to the testing environment. It’s easy to imagine adapting this kind of robot for carrying out qPCR with EC or fluorescence detection, or nanopore sequencing, for rapidly identifying pathogens.

Lab-on-a-Drone

A logical variation of lab-on-a-robot is to attach the lab part to Unmanned Aerial Systems, (more commonly called drones), thus affording a means for “fly-in, fly-out” applications that require speed to and from a location, or for deployment to otherwise inaccessible locations. This biotech version of drone delivery was initially demonstrated for drone pick-up to aerially transport blood samples from patients to central testing labs, as reported by Amukele et al.

Victor Ugaz. Taken from tamu.edu

The much more difficult task of attaching a lab testing module to a drone has been recently demonstrated by Prof. Victor Ugaz and coworkers at Texas A&M University. Their pioneering 2016 publication titled Lab-on-a-drone: toward pinpoint deployment of smartphone-enabled nucleic acid-based diagnostics for mobile health care is loaded with details, and is a “must read” for technophiles. What follows is my extraction of some unique highlights of that work, as well as information I learned by contacting Prof. Ugaz, who incidentally has received numerous awards and honors.

The basic idea investigated by these researchers was to design a drone-compatible system that could perform what I call “qPCR-on-the-fly.” The drone would require low power consumption and use a smartphone for both fluorescence detection—via its camera—and data analysis via radio transmission of results on-the-fly.

To reduce power consumption by conventional PCR using thermal cycling, which uses power for both heating and cooling during each cycle of amplification, the Texas team invented a radically different approach to achieve isothermal PCR. As depicted below, this new method called convective thermocycling operates isothermally at 95oC and involves movement of reactants upward, away from the heater, through progressively cooler regions and then traveling downward to repeat heating, etc. in a cyclic manner.

Taken from Ugaz & coworkers (2016)

They mimicked POC for an Ebola virus epidemic, which required on-site sample prep and then reverse transcription of viral RNA into cDNA prior to hot start qPCR that is incompatible with convective PCR. The sample prep step was very cleverly achieved using centrifuge adapters that connect to the drone in place of propellers. These centrifuges in turn—pun intended—were fabricated using state-of-the-art 3D printing, and are pictured below.

Taken from Ugaz & coworkers (2016)

The in-flight lab-on-a-drone is pictured below. While in-flight, smartphone-enabled qPCR (as depicted above) takes place during the return trip to home base in order to save time for re-equipping the drone to return to another site, thus increasing overall patient analysis throughput per drone.

Taken from Ugaz & coworkers (2016)

I contacted Prof. Ugaz to ask whether the reverse transcription (RT)-PCR could also be carried out in flight to further automate and increase drone throughput. He replied as follows:

“Many thanks for your interest in our work!  For the purposes of these proof of concept studies, we performed the RT and hot start steps off-device in a conventional thermocycler. However, these steps could straightforwardly be embedded in the portable device.  In principle it should be just a matter of either programming the heater to run through these additional steps (in which case we need to consider the thermal transient between steps, since we are trying to keep the device as simple as possible), or possibly have multiple separate heating zones on the device and have the user physically move the reactor from one to another for each step.  There are multiple possibilities to achieve this that can be explored, and the ‘best’ choice is likely related to the specific application that is envisioned.  But to answer your question, yes this is possible as a relatively straightforward extension of the current design…I have a student who will be working on this during the summer.” 

To my surprise and delight, Prof. Ugaz also informed me of his interest in investigating TriLink’s CleanAmp™ technologies for CleanAmp™ hot start PCR and CleanAmp™ hot start RT-PCR. He said that “[w]e are looking forward to testing this soon and will keep you posted!”

This work by Prof. Ugaz will hopefully lead to encouraging results, and provide a great example of how TriLink CleanAmp™ technologies are enabling both scientific advancement as well as an amazingly interesting, new application such as that in this lab-on-a-drone story.

As always, your comments here are welcomed.

Nanopore Sequencing by Synthesis (Seq-by-Syn)

  • Yet Another Notable Achievement Involving George Church, ‘The Most Interesting Scientist in the World’ 
  • Team of 30 Coauthors Reports Seq-by-Syn with DNA Polymerase-Nanopore Protein Construct on an Integrated Chip
  • Challenging Improvements Needed for Commercial Reality

Prof. George M. Church. Taken from evolutionnews.org

Devotees of my blog will know that I’m prone to word play such as calling myself a “huge” fan of “tiny” nanopores for DNA sequencing, about which I’ve previously opined. They will also recall that I’m an admitted scientific admirer of George Church, who I think is The Most Interesting Scientist in the World.

Having said this, it’s not surprising that I closely follow what’s trending in nanopore sequencing, and also make an attempt to read all of Church’s papers as they get published because they are almost invariably quite interesting, involve “big ideas,” and in some new way are very educational, at least for me. Following are my comments about a recently published paper on nanopore sequencing in venerable Proceedings of the National Academy of Sciences of the United States of America (aka PNAS) wherein Church is the designated corresponding author.

Backstory

The seminal origins and early history of nanopore sequencing have been recently chronicled and criticized—then clarified—in Nature Biotech in several “To the Editor” items, which collectively provide enlightening insights into who did what when, so to speak. Those of us who are ‘Nanoporati’—a clever term tweeted by Nick Lowman—should definitely read those Nature Biotech items. For now, however, I’ll set the stage, as it were, by echoing a bit of what I’ve posted in the past for nanopores.

Patented but prophetic (i.e. no data) methods for nanopore sequencing DNA is actually a relatively old (~20 year) idea posited by Church and other creative visionaries. On the other hand, nanopore sequencing was first reduced to practice commercially not too long ago by Oxford Nanopore Technologies (ONT). Many years of delay between concept and commercialization was due to the need for gradual evolution of lots of “nanopore-ology” and sequencing biochemistry, as well as developing highly sophisticated electronics and complex algorithms for data analysis.

Nanopore Sequencing-by-Scanning (Seq-by-Scan)

Taken from rsc.org

As depicted below, and as can be best seen in a video, ONT’s commercially available MinION Seq-by-Scan system essentially involves threading a strand of DNA through a protein-based nanopore and converting resultant ionic current fluctuations into nucleotide base sequence.

While there are issues with base-calling accuracy, the remarkably small and readily portable MinION provides fast, real-time sequencing results for a wide variety of applications. These included unique or otherwise compelling Point-of-Care analyses, such as pathogen surveillance, which has been achieved in remote geographical locations and even in outer space aboard the International Space Station, as I’ve previously posted.

Nanopore Seq-by-Syn

In contrast to DNA Seq-by-Scan using a nanopore, which is challenged by pore-based differentiation of similarly sized A, G, C, and T bases, DNA Seq-by-Syn has no such limitation as it uses the DNA as a template for base-by-base (i.e. stepwise) detection of enzymatic synthesis of complementary DNA. Various Seq-by-Syn methods and challenges have been discussed elsewhere, and currently available commercial systems include those from Illumina and PacBio. The former employs nucleotides that are reversible terminators equipped with cleavable fluorescent “tags” on each base. The latter detects fluorescently labeled tags on polyphosphates released upon nucleotide incorporation.

The presently featured DNA Seq-by-Syn publication by Stranges et al., which builds upon two earlier reports cited therein, differs from the above approaches by using nanopore-based detection of mass tags rather than fluorescent tags. In principle, mass tags could afford higher accuracy compared to DNA Seq-by-Scan. However, as will now be explained, achieving improved accuracy is far easier said than done.

The general approach taken to demonstrate proof-of-concept for mass-tagged nanopore DNA Seq-by-Syn is depicted below in simplified cartoon form, but involves a true tour de force—in my opinion—of three key technologies. The first is design and synthesis of the nucleotides with appropriate mass tags, which involves very sophisticated chemistry that is best appreciated by reading detailed, extensive supporting information (SI) for Stranges et al. and SI for an earlier publication by Fuller et al. In a nutshell, these nucleotides have 5’-hexaphosphates linked to relatively large mass tags comprised of complex oligonucleotide structures.

Taken from Stranges et al. PNAS 2016

The second area of technical innovation involves attachment of a single molecule of ϕ29 DNA polymerase to each α-hemolysin (αHL) nanopore in such a manner as to retain its enzyme activity and be positioned such that every released mass tag transits through (i.e., is “captured” by) the nanopore leading to base identification by its current signature. As depicted below in two related representations, each of these heteroheptameric pores is comprised of one modified αHL subunit to which a peptidyl SpyTag moiety is attached, and six unmodified αHL subunits. This allows attachment of one ϕ29 DNA molecule modified with a cognate peptidyl SpyCatcher moiety at a predetermined, time-average distance from the pore.

Taken from Stranges et al. PNAS 2016.

The third key area of innovation deals with insertion of the enzyme-pore conjugate into a lipid bilayer residing on a silanized array (aka chip) of 256 Ag/AgCl electrodes such that there is one functional pore per electrode. Interested readers are encouraged to consult the publication for details, as well as check out related fabrication and methods patents that I found by searching Google Scholar.

Representative Results

The first image shown above depicts what base tag-specific detection would ideally look like if each of the four different bases would have a characteristic current-blockage intensity and persistence. In addition, all pores would ideally function similarly. Not surprisingly, given the stochastic nature of single-molecule systems in general, Stranges et al. found less than ideal behavior.

For example, out of 70 single pores obtained, 25 captured two or more tags, whereas only six of those pores showed detectable captures of all four tagged nucleotides. Data obtained for the pore with the most transitions between tag capture levels (i.e. the best results) is shown below, while results for the other five are given in the SI.

Taken from Stranges et al. PNAS 2016

To quote the authors:

“All four characteristic current levels for the tags and transitions between them can be readily distinguished…Homopolymer sequences in the template, and repeated, high-frequency tag capture events of the same nucleotide in the raw sequencing reads were considered a single base for sequence alignment. We recognized 12 clear sequence transitions in a 20-s period. Out of the 12 base transitions observed in the data, 85% match the template strand, showing that this method can produce results that closely align to the template sequence.” 

Interested readers need to consult and carefully read the SI for Stranges et al. regarding the interpretation of the “repeated, high frequency capture events,” such as that exhibited by C in the above current vs. time plot.

All of the above snippets in aggregate suggest to me that, while this huge amount of work has made progress toward one approach to Seq-by-Syn, many improvements will need to be made before achieving a robust system to successfully compete in the commercial sector.

Authorship, Affiliations, and Acknowledgments

The relatively large team of 30 coauthors listed for Stranges et al. include the following numbers of investigators and affiliations: 1 at Arizona State Univ., 4 at Harvard, 11 at Columbia University, and 14 at Genia Technologies, which is a Santa Clara, CA company that was acquired by Roche in 2014, and is part of Roche Sequencing.

Acknowledgments in Stranges et al. refer to support by Genia and NIH Grant R01 HG007415, which I found was awarded to coauthors George M. Church (Harvard), Jingyue Ju (Columbia), and James J. Russo (Columbia). The end of the abstract of this grant reads as follows:

“The nanopore chips will be enhanced and expanded from the current 260 nanopores to over 125,000 using advanced nanofabrication techniques. We will conduct real-time single molecule Nano-SBS on DNA templates with known sequences to test and optimize the overall system. These research and development efforts will lay the foundation for the production of a commercial single molecule electronic DNA sequencing platform, which will enable routine use of sequencing for medical diagnostics and personalized medicine.”

The conflict of interest statement in Stranges et al. indicates that the technology described therein (called “Nanopore SBS”) has been exclusively licensed by Genia, and that specified coauthors are entitled to royalties through this license. In addition, Church is a member of the Scientific Advisory Board of Genia.

Parting Comments

Long gone are the days when government-funded academic researchers thumbed their noses, if you will, at commercial development. Nowadays almost all academics parlay their government grants into university patents that get licensed to companies, usually with some type of corporate involvement of said academics.

I hasten to add that I’m not implying that NIH-funded academic research being a “seed” for corporate profitability is negative—especially in view of its Small Business Innovative Research (SBIR) program—but rather view it as a paradigm shift for the better, as it allows academic creativity to be harnessed into applications that can hopefully greatly benefit society.

In conclusion, and coming back to George Church, who I highlighted in the introduction to this blog, I must say that he might very well be the academic researcher with the longest list of technology transfer, advisory roles, and founded companies—13 to date—according to a public list that is truly mind boggling, at least to me.

As usual, your comments are welcomed.

Postscript

After writing this blog, Roche announced on December 15, 2016 that “it has officially notified Pacific Bioscience (PacBio) of its intention to terminate its [2013] agreement and efforts to develop a sequencing instrument for use in the clinical research and clinical market using their Single Molecule, Real-Time (SMRT®) technology,” about which I have commented previously. The announcement went on to say Roche would instead focus on internal development efforts” and “actively pursue multiple technologies and commercial strategies.” A GenomeWeb headline was more specific:  “Roche Will Focus on Genia’s Nanopore Technology for Dx Market After Ending Deal With PacBio.”

On December 30, 2016 it was reported that the University of California (UC) filed a patent suit against the Chief Technology Officer (CTO) at Genia, and Genia Technologies, claiming the CTO produced key inventions during his time at UC that he later assigned to Genia, but which should have automatically been assigned to UC. Stay tuned…

Top 10 Innovations 2016

  • Sequencing with Sequel, but with a Shocking Surprise
  • Endonuclease CRISPR-Cas9 Makes the Cut Twice—Pun Intended
  • My Predicted 2016 Innovation Winners Made Lots of News

Welcome to my first blog of the New Year, 2017! My New Year’s resolution is to continue to do my best in providing interesting and informative content about what’s trending in nucleic acid research. As in the past, this first blog of the year comments on the Top 10 Innovations in 2016 that were selected by a panel of judges and published last month in The Scientist. Also like the past, you can peruse TriLink’s top products by clicking here.

So, with an imaginary loud flourish of trumpets, read on to learn about the 10 winners, starting with 1st place.

  1. Milo from ProteinSimple enables single-cell Western blotting in a benchtop instrument that allows researchers to search for specific proteins in about 1,000 single cells simultaneously. A titered cell-suspension is pipetted on a 1-by-3-inch glass microscope slide covered by a 30-micron gel-layer dotted with 6,400 microwells. Some wells will remain empty, but ~1,000 will collect individual cells for antibody-based protein analysis, following lysis and other steps, all done automatically. Indeed simple!
  2. ExVive Human Kidney Tissue from Organovo is a replica of the kidney proximal tubule created using 3D bioprinting, which is like uber-trending 3D printing with plastic, but instead uses tiny aggregates of cells. This novel product offers drug developers a reliable means of testing for renal toxicity that is more predictive than traditional cell culture, and avoids animal testing.
  3. Sequel System from Pacific Biosystems is not neither small (see below) nor inexpensive ($350,000), but is nevertheless a third the size and weight—and half the cost—of PacBio’s original long-read, single molecule, real-time (SMRT) sequencer, PacBio RS II, about which I’ve favorably blogged several times in the past. Moreover, Sequel has seven-times the throughput of PacBio RS II.

Taken from fiercebiotech.com

Development of what would become Sequel was announced by PacBio in 2013 as part of a potentially $75M deal with Roche Diagnostics aimed at DNA sequencing-based products for clinical diagnostics. Surprisingly—if not shockingly—in December 2016, after Sequel was launched, Roche stated it was terminating this deal with PacBio in order to focus on its internal development efforts.

At this time, I can only speculate that Roche’s internal efforts include single-molecule DNA sequencing using nanopore technology developed by Genia Technologies, Inc., which Roche acquired in 2014 for $125M—plus even greater contingent payments. By remarkable coincidence, I had been drafting a blog about Genia from a purely technical perspective, but will now update that for posting on January 24th as a sequel to Sequel—pun intended.

  1. Lumos from Axion Biosystems is a 48-well light-delivery device allowing researchers to incorporate cutting-edge optical assay techniques, such as optogenetics, into their in vitro research. Lumos delivers user-specified intensity and duration of light with up to four wavelengths simultaneously for assay flexibility.
  2. LentiArray CRISPR Libraries from Thermo Fisher Scientific make applying lentivirus-encoded CRISPR for gene editing more accessible to researchers. Given the continuing explosive-like interest in DNA endonuclease CRISPR-Cas9, which in 2015 also made the Top 10 cut—pun intended—I was expecting to find this endonuclease system among the 2016 Top 10, too. What surprised me, however, was seeing this system split into two parts—pun intended—i.e. CRISPR per se and separately as Cas9, which you’ll find below.
  3. nCounter Vantage 3D Panels from NanoString utilize an automated microscope that counts color-coded barcodes conjugated to target molecules that are mRNAs, DNAs, proteins, and even phosphorylation status of proteins—all at the same time, which I think is quite a technical achievement. The nCounter analysis system ranges from $149,000 to $280,000, and the nCounter Vantage 3D Panels run from $275 per sample and upward.

Taken from nanostring.com

  1. ZipChip from 908 Devices is a cleverly designed microfluidic chip that radically speeds up sample prep for mass spectrometry, requires only a few microliters of sample volumes, and broadens the range of materials that a mass spectrometer can handle. In less than 3 minutes per sample, ZipChip can process cell growth media, cell lysates, blood, plasma, or urine. The 1-by-3-inch chip is in a small box, less than a foot long, which mounts directly onto a mass spec. The device costs $30,000 and an auto sampler adds another $20,000 to the price.
  2. HAP1 Cells from Horizon have earned a spot in The Scientist Top 10 Innovations for the third year in a row—this time as Turbo GFP Tagged HAP1 Cells, which were selected for their ability to tag proteins of interest with green fluorescent protein (GFP) without requiring that the gene be overexpressed. Turbo GFP cells, which are custom-made using CRISPR-Cas9 gene-editing technology, cost $3,400 and take about 16-18 weeks to develop.

Roger Y. Tsien (February 1, 1952 – August 24, 2016).
Taken from wikipedia.org

As a sad side note, Roger Y. Tsien, who was awarded the 2008 Nobel Prize in Chemistry for his discovery and development of GFP, in collaboration with Osamu Shimomura and Martin Chalfie, passed away in 2016 at the age of 64.

  1. Prime sCMOS Camera from Photometrics is a 4.2-megapixel camera that has a built-in algorithm to reduce what is called “shot noise”—the variation inherent in measurements taken using light microscopes—without having to acquire many extra images and then average across them, or increase the light intensity, which can damage samples. The Prime sCMOS camera costs $15,950.
  2. GeneArt Platinum Cas9 Nuclease from Thermo Fisher Scientific is a recombinant Streptococcus pyogenes Cas9 protein purified from E. coli that contains a nuclear localization signal that aids in delivery to target-cell nuclei, where Cas9 works in conjunction with CRISPR. I should add that CRISPR-based gene editing is alternatively achieved by transfection of Cas9 mRNA, which is offered by TriLink, and used as described in a recent exemplary publication by a team of international collaborators.

Revisiting Jerry’s Predictions for 2016 Top 10 Innovations

Devotees of my blog may recall the following predictions I offered in January 2016 as winning innovations-to-be:

  • Direct Genomics in China will resurrect and morph Helicos single-molecule sequencing into a diagnostics instrument.
  • GnuBIO—acquired by Bio-Rad—will offer its long-delayed next-generation sequencing system.

While none of these were selected by The Scientist, they did make news in various ways:

  • Oxford Nanopore’s VolTRAX system for automated sample has very recently been launched. I should also note that its tiny MinION sequencer was rocket-launched—literally—to the International Space Station (ISS) for evaluation in rapid identification of pathogens that might infect astronauts on the ISS, as I’ve commented on in a previous blog. Launch puns intended.
  • Direct Genomics recently announced its GenoCare Analyzer, the world’s first single-molecule genome sequencer that is being engineered exclusively for the clinic, and promises to improve the cost, speed, and quality of clinical genome sequencing by directly reading a patient’s original DNA or RNA molecules without prior amplification.
  • GnuBio now offers a fully integrated sequencing platform which allows users to simply load genomic DNA onto cartridge, place the cartridge into the GnuBIO sequencer, and then press “run.” Within hours, results can be exported directly from the instrument with real-time informatics onboard.

Although my picks weren’t among those in The Scientist list for 2016, I take satisfaction in believing that choosing winning biotechnology products is like art appreciation or judging beauty, both of which are in the eye of the beholder, who can disagree on what their eyes behold.

As usual, your comments are welcomed.

Nanobombs for Light-Activated MicroRNA Drug Delivery

  • Nanoparticles Designed to “Explode” Upon Irradiation with Light
  • Light Used Can Penetrate Skin and Tissue
  • Nanoparticle “Cargo” of MicroRNA Released Locally, Where Needed 

Every once in a while the title of a publication grabs my attention so much that I just have to read it, and that’s what happened when I read this one A Near-Infrared Laser-Activated “Nanobomb” for Breaking the Barriers to MicroRNA Delivery.

Taken from ajamtafireworks.com

My interest was primarily piqued by the word ‘nanobomb,’ which I had never heard of even though I consider myself quite well-read scientifically. Moreover, microRNA (miRNA) and drug delivery are both currently trending hot topics. I couldn’t find an eye catchy image of a nanobomb other than fireworks by this name sold in India, which are oddly touted as being “pollution free”! But I digress…

Delivery of miRNA and other types of nucleic acid-based drugs continues to be perhaps the biggest challenge facing successful clinical development of this broad new class of drugs. In that regard, an expert review titled Nucleic acid delivery: the missing pieces of the puzzle?, written by my good friend and long-time collaborator Prof. Frank Szoka (UCSF), is a “must read” for those of you who are interested in further details and perspectives by an expert.

Prof. Xiaoming He. Taken from bme.osu.edu

Another “hook” in the nanobomb article’s title was its use of lasers, which are also trending in biomedical or biotechnological applications. So, all of this taken together, led me to request a pdf from the corresponding coauthor, Prof. Xiaoming He at Ohio State University, who promptly complied. Since it’s a pay-to-read publication, I’ve tried my best to convey the essence of this work in what follows.

Symphony of Science

Like a symphony, which is an elaborate musical composition for full orchestra, I learned that Prof. His nanobombs for laser-induced drug delivery are also quite elaborate and composed of a full array of scientific principles. To my surprise, a Google Scholar search of the term “nanobomb” gave a large number—more than one hundred—of items that included a wide variety of ways to use nano-size, i.e. sub-micron materials and light to somehow “blow up” or otherwise kill diseased cells, and thus fall under the umbrella-term photodynamic therapy.

Carbon nanotubes (~100 nm diameter) seen through an electron microscope. Taken from item.fraunhofer.de

For example, there’s a report in 2005 coauthored by Eric Wickstrom—who coincidentally is a friend of mine from early antisense oligo days—on the first application of single-wall carbon nanotubes (SWCNT) as potent therapeutic nanobombs for killing breast cancer cells in vitro. This is accomplished by adding water molecules into SWCNTs, which then get adsorbed on target cells for 800-nm (red) laser light irradiation to vaporize the entrained water.

A more elaborate rendition of nanobomb drug delivery was published in 2013 by Lu et al., who reported that intracellular gold nanoparticles under laser irradiation generate “nanobubbles” that can kill cells. To formulate a targeting gold nanoparticle as a cancer cell-specific “nanobomb,” a 31-nt DNA aptamer was conjugated to the gold surface. While biological results from this approach have yet to be published, there’s a good tutorial on the properties and applications of gold nanoparticles that are commercially available in discrete sizes, as seen here:

Taken from simaaldrich.com

Symphonic Variations of Science

This section heading extending my musical metaphor for nanobomb drug delivery is meant to convey the fact that the study by Prof. He and his coworkers featured herein is a symphony-like variation of photodynamic therapy composed of many parts. This work stands out—in my opinion—as being a remarkably comprehensive proof-of-concept involving lots of in vitro data and, more importantly, compelling in vivo animal results. I think you’ll “tune in”—musical pun intended—on this as I now summarize the complex scientific symphony by He and coworkers pictured below.

(A) Three agents are encapsulated inside nanoparticles: microRNA-34a (miR-34a) anti-mRNA drug for gene therapy of prostate cancer stem cells, indocyanine green (ICG) for absorbing laser light, and ammonium bicarbonate for gas generation under heating. (B) Laser light causes the nanoparticles to expand, penetrating the cancer cell’s endosomal/lysosomal barrier (green circles), blowing up cancer cells (yellow), and releasing miR-34a to inhibit protein CD44, which is crucial for cancer stem cell survival. Credit: Hai Wang et al./Advanced Materials. Taken from kurzweilai.net

What’s the Nanobomb?

The top part of the above depiction labeled (A) doesn’t do justice to the elegant methodology used to prepare the water-in-oil-in-water (W-in-O-in-W) double-emulsion nanobomb, so interested readers will have to consult the publication by He and coworkers for details. However, as shown in A, the nanobomb’s active components are indocyanine green (ICG), ammonium bicarbonate (NH4HCO3) and microRNA-34a (miR-34a), the latter of which I found is a single-stranded 22-nt RNA rather than the misleading drawn double-stranded species. Chitosan’s amino groups form a polyplex with miRs like miR-34a.

I also found many references to miR-34a as an anticancer agent, and in this instance it serves to negatively regulate CD44 overexpression, which is associated with survival and growth of prostate cancer stems cells (CSCs) as previously studied by a number of other groups.

Indocyanine green (ICG). Taken from Wikipedia

Having said this, the real novelties of this nanobomb are—in my opinion—its “ignition” and “explosive” materials, namely ICG and NH4HCO3, respectively. Some among you may recognize from the name or structure of ICG that it’s a “chemical cousin,” so to speak, of cyanine-3 and cyanine-5 dyes popular for labeling, which BTW are now available from TriLink as NHS esters upon inquiry.

The ICG dye is used to absorb laser light and convert the light’s energy into heat, which then causes NH4HCO3 to very rapidly generate CO2 and NH3 gases and thus release miR-34a (as depicted in part B of the above cartoon from He and coworkers). This “release” aspect deserves the following further comments.

Why’s a Nanobomb Needed?

This rhetorical question relates to the “D-word,” i.e. delivery of miR (or other classes of oligo agents) into a cell’s cytoplasmic compartment where it is intended to block target mRNA expression after its release from the endosome/lysosome compartment, as depicted in B above. The following electron micrograph shows what these sub-compartments look like.

Electron micrograph of endosomes in human HeLa cells. Early endosomes (E), 5 minutes after internalization; late endosomes/ multivesicular bodies (M) and lysosomes (L) are visible. Bar, 500 nm. Taken from Wikipedia.com

As depicted by He and coworkers, laser light via ICG is used to “ignite” or trigger “explosive” release of miR-34a from these compartments, rather than rely on natural phenomenon or use of pH-sensitive triggering mechanisms. For those of you who are not familiar with the endosome/lysosome compartment, details can be found in the aforementioned review by Prof. Frank Szoka, which was coauthored by Juliane Nguyen.

Is the Nanobomb Effective In Vivo?

Saline (control); HLPP = formulation; miR-34a (R) unformulated plus laser light (L); HLPP with ICG (I) and NH4HCO3 (A) plus L; HLPP with A and R plus L; HLPP with I and A and R plus L. Taken from He and coworkers.

Prof. He and his coworkers carried out extensive controlled experiments in vitro and in vivo to assess efficacy (as well as biodistribution and safety) of their nanobombs, and to me the most compelling data are shown below. Briefly, mice capable of harboring tumors derived from subcutaneously placed human prostate CSCs were treated with various formulations or control, and afterwards the extent of tumor growth was accessed in various ways including by tumor weight. It’s obvious in (C), shown below, that the only statistically significant (*) effect was obtained by the nanobomb formulation.

But is This Nanobomb Practical?

Here’s what He and coworkers state with regard to the scope and practicality of their nanobomb approach:

“Although the tissue penetration of the NIR [near infrared laser] is limited to less than ~1 cm and multiple polymers are needed for preparing the nanosystem, NIR could be delivered into deep tissue using minimally invasive approaches [e.g. endoscopy] and the preparation of the nanosystem using the double-emulsion method is quite straightforward. Therefore, the present study demonstrates the great potential of the NIR laser-activated “nanobomb” for microRNA delivery to achieve augmented cancer therapy.”

Time will tell what aspects of this approach will ultimately be reduced to practice in the clinic and/or lead to an approved photodynamic therapy for cancer. However, in the meantime, I’m betting that it will also “ignite” others to explore variations on this symphony of science.

As usual, your comments are welcome.

Postscript

After this blog was written, Jalani et al. published a paper titled Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery. This work is conceptually related—in my opinion—to the above nanobombs in that low energy red-light-absorbing nanoparticles emit higher energy UV light (i.e. photon upconversion) to simulataneously image and release drugs specially absorbed on these nanoparticles. This NIR light penetration, UV imaging, and drug release are said therein to be effective down to a depth of ~2 cm of tissue, which is twice that reported above by He and coworkers.

Frightening Fungus Among Us

  • Clinical Alert for Candida auris (C. auris) Issued by CDC
  • US Concerned About C. auris Misidentification and Drug Resistance
  • Sequencing C. auris DNA in Clinical Samples is Preferred for Identification
Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

Strain of C. auris cultured in a petri dish at CDC. Credit Shawn Lockhart, CDC. Taken from foxnews.com

When I was a kid and didn’t know better, there was a supposedly funny rhyme that “there’s fungus among us.” While this saying is thankfully passé nowadays, the growing number of infections by a formerly obscure but deadly fungus is frightening. This so-called “superbug” is an antibiotic-resistant fungus called Candida auris (C. auris) that’s worth knowing about, and is the fungal focus of this blog.

First, Some Fungus Facts

Fungi are so distinct from plants and animals that they were allotted a biological ‘kingdom’ of their own in classification of life on earth, although that was only relatively recently, i.e. 1969. There are 99,000 know fungi, which exist in a wide diversity of sizes, shapes and complexity that extends from relatively simple unicellular microorganisms, such as yeasts and molds, to much more complex multicellular fungi, such as mushrooms and truffles.

It was previously thought that genomes of all fungi are derived from the genome of the model fungus Saccharomyces cerevisae, which has been used in winemaking, baking and brewing since ancient times. However, genome sequencing of more than 170 fungal species has revealed that, while the genome size of S. cerevisae is only ~12 Mb, seven species of fungus have genome sizes larger than 100 Mb. This is attributed to various evolutionary pressure-factors generating transposable elements, short sequence repeats, microsatellites, and genome duplication, and noncoding DNA.

Fungal cell walls are made up of intertwined fibers mostly comprised of long chains of chitosan, the same tough compound found in the exoskeletons of animals such as spiders, beetles and lobsters. The chitin in fungal cells is entangled with glucans and other wall components, such as proteins, forming a mass that protects the cell membrane behind it—and posing a formidable barrier against antifungal drugs.

Taken from Wikipedia.org

Taken from Wikipedia.org

In researching whether there are any nucleic acid drugs against fungi, I found one early patent by Isis (now Ionis) Pharmaceuticals for use of antisense phosphorothioate-modified oligonucleotides for the treatment of Candida infections, but virtually no other reports. I suspect that will change in the future as pathogenic fungi and other disease-causing microbes become more resistant to conventional drugs.

Fungal infections of the skin are very common and include athlete’s foot, jock itch, ringworm, and yeast infections. While these can usually be readily treated, infections caused by pathogenic fungi have reportedly risen drastically over the past few decades. Moreover, with the increase in the number of immunocompromised (burn, organ transplant, chemotherapy, HIV) patients, fungal infections have led to alarming mortality rates due to ever increasing phenomenon of multidrug resistance.

Segue to a Serious Situation

Emergence of drug-resistant fungi is, in part, the segue to the serious story of the present blog. The other part being incorrect identification of a certain fungus as being a common candida yeast, which is not only scary but seemingly inexcusable in today’s era of highly accurate PCR-based assays to accurately identify microorganisms. Here’s the situation in a nutshell.

  1. auris infection, which is associated with high mortality and is often resistant to multiple antifungal drugs, was first described in 2009 in Japan but has since been reported in countries throughout the world. Unlike many Candida infections, C auris is a hospital-acquired infection that is contracted from the environment or staff of a healthcare facility, and it can spread very quickly.

To determine whether C. auris is present in the United States and to prepare for the possibility of transmission, the Centers for Disease Control (CDC) and Prevention issued a clinical alert in June 2016 requesting that C. auris cases be reported.

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

(A) MALDI-TOF schematic; (B) mass spectra from three C. parapsilosis; and (C) two C. bracarensis isolates. Taken from researchgate

This official alarm bell, if you will, was triggered by the following facts:

  • Many isolates are resistant to all three major classes of antifungal medications, a feature not found in other clinically relevant Candida
  • auris identification requires specialized methods such as a MALDI-TOF mass spectrometry or sequencing the 28s ribosomal DNA, as pictured below.
  • Using common methods, auris is often misidentified as other yeasts, which could lead to inappropriate treatments.

The CDC subsequently found that seven cases were identified in Illinois, Maryland, New York and New Jersey. Five of seven isolates were either misidentified initially as C. haemulonii or not identified beyond being Candida. Five of seven isolates were resistant to fluconazole; one of these isolates was resistant to amphotericin B, and another isolate was resistant to echinocandins. While no isolate was resistant to all three classes of antifungal medications, emergence of a new strain of C. auris that is would pose a serious public health issue.

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Sequencing 28s ribosomal DNA. Taken from microbiologiaysalud.org

Based on currently available information, the CDC concluded that these cases of C. auris were acquired in the U.S., and several findings suggest that transmission occurred:

  • First, whole-genome sequencing results demonstrate that isolates from patients admitted to the same hospital in New Jersey were nearly identical, as were isolates from patients admitted to the same Illinois hospital.
  • Second, patients were colonized with auris on their skin and other body sites weeks to months after their initial infection, which could present opportunities for contamination of the health care environment.
  • Third, auris was isolated from samples taken from multiple surfaces in one patient’s health care environment, which further suggests that spread within health care settings is possible.

A related Fox News story adds that C. auris was found on a patient’s mattress, bedside table, bed rail, chair, and windowsill. Yikes!

While the above situation in the U.S. might not seem particularly worrisome to you, the potential for emergence of more infectious C. auris strains with higher lethality should be of concern. That has already reportedly occurred in several Asian countries and South Africa. Obviously, deployment of the best available methods for pathogen identification can, in principle, lessen the likelihood of the emergence and/or spread of C. auris in the U.S. and other countries.

Case for Point-of-Care C. auris Nanopore Sequencing?

Taken from extremtech.com 

Taken from extremtech.com

Regular readers of my previous blogs know that I’m an enthusiastic fan of the Oxford Nanopore Technologies minION sequencer, which is proving to be quite useful for characterizing pathogens in very remote regions on Earth—and even on the International Space Station to diagnose astronaut infections! Notwithstanding various current limitations for minION sequencing of microbes, it seems to me that it would be relatively straightforward to generate minION data for many available samples of pathogenic fungi and genetically related microbes to assess the feasibility using minION for faster, cheaper, better unambiguous identification of C. auris minION in centralized or Point-of-Care applications.

Taken from rnaseq.com

Taken from rnaseq.com

If you think this suggestion is farfetched, think again, after checking out these 2016 publications using minION:

The 51.4-Mb genome sequence of Calonectria pseudonaviculata for fungal plant pathogen diagnosis was obtain using minION.

The first report of the ~54 Mb eukaryotic genome sequence of Rhizoctonia solani, an important pathogenic fungal species of maize, was derived using minION.

Sequence data is generated in ~3.5 hours, and bacteria, viruses and fungi present in the sample of marijuana are classified to subspecies and strain level in a quantitative manner, without prior knowledge of the sample composition.

CDC on C. auris Status and FAQs

In the interest of concluding this blog with the most up-to-date and authoritative information, I consulted the CDC website and found statements and replies to FAQs that are well worth reading at this link.

As a scientist, my overriding question concerns the lack of adoption of improved microbiological methods by hospitals and clinics. The above noted misidentifications of C. auris infections resulting from use of flawed lab analyses seems unacceptable. Although I don’t know all the facts or statistics to generalize, I suspect that there are other incorrect lab analyses due to use of outdated methods. On the other hand, I’m hopeful that, with the FDA’s widely touted Strategic Plan for Moving Regulatory Science into the 21st Century, the section entitled Ensure FDA Readiness to Evaluate Innovative Emerging Technologies—think nanopore sequencing—becomes actionable, sooner rather than later.

Changing established—dare I say entrenched—clinical lab tests is not simple or easy, but if it doesn’t begin it won’t happen, about which I’m quite certain. I can only wonder why development of infectious disease analytical methods and treatments seem to require a crisis. Sadly, I think it boils down to the complexities and socio-political dynamics of who pays.

Frankly, it’s my personal opinion that maybe it’s time Thomas Jefferson’s philosophy about hammering guns into plows is directed to health care.

Postscript

After writing this blog, I learned that T2 Biosystems has received FDA approval to market in the U.S. the first direct blood test for detection of five yeast pathogens that cause bloodstream infections: Candida albicans and/or Candida tropicalis, Candida parapsilosis, Candida glabrata and/or Candida krusei.

Yeast bloodstream infections are a type of fungal infection that can lead to severe complications and even death if not treated rapidly. Traditional methods of detecting yeast pathogens in the bloodstream can require up to six days, and even more time to identify the specific type of yeast present. The T2Candida Panel and T2Dx Instrument (T2Candida) can identify these five common yeast pathogens from a single blood specimen within 3-5 hours.

T2Candida incorporates technologies that break the yeast cells apart, releasing the DNA for PCR amplification for detection by greatly simplified, miniaturized nuclear magnetic resonance (NMR) technology, as can be seen in this video.

In my opinion, this fascinating new technology is another example of what could be rapidly deployed toward detecting C. auris.

RNA and DNA Examples of ‘Seek, and Ye Shall Find’

  • New RNA Modification Found in the Epitranscriptomic Library for Mammals
  • Ditto for a DNA Modification in Mammalian Epigenetics
  • What Will ‘Ye’ Find Next with Improved RNA and DNA Analytical Methods?

‘Seek, and ye shall find’ is a biblical quote (Matthew 7:7) that many might say is a self-evident statement indicating that to find something you need to look for it. In any case, I used it as a segue for us scientists to remember that our collective understanding of RNA and DNA is limited, in part, by the analytical tools we use for these nucleic acid molecules. To me this predicament is akin to a person under the lamp post at night being limited to find whatever is illuminated, and miss what is not. The old cartoon below conveys a humorous variant of this. But I digress…

Taken from quoteinvestigator.com

Taken from quoteinvestigator.com

In previous postings here, I’ve commented on new illuminations—pun intended—for RNA modifications, such as pseudouridine, about which understanding of function is just emerging. And likewise for congeners of the 5-methyl group of cytidine in DNA, such as 5-hydroxymethyl or 5’-formyl moieties.

To continue this metaphor—but to get to the point—the lamp post’s light-field has been recently expanded to allow researchers to now find previously unseen modified bases in mammalian RNA and DNA, namely, N1-methyladenosine and N6-methyl-2’-deoxyadenosine, respectively. Briefly, here’s what’s been reported.

Expanding Epitranscriptomics

Just like modification of bases in DNA after its replication leads to what’s called epigenetics—which I’ll get to in the next section—modification of bases in RNA after its transcription leads to epitranscriptomics. This somewhat of a tongue-twister term was first introduced in 2013 by Sibbritt et al. in reference to increasing amounts of data for methylated bases in mRNA in the form of N6-methyladenosine (m6A) and 5-methylcytosine (m5C).

Both m6A and m5C have long been known to exist in both prokaryotic and eukaryotic organisms, but only more recently have a flurry of publications shed light—pun intended—on the precise locations, as well as enzymatic introduction and removal of m6A and m5C. This dynamic “writing” and “erasing” has been expertly reviewed by He and coworkers, who have also contributed to these discoveries.

m1A; taken from genengnews.com

m1A; taken from genengnews.com

He and collaborators at the University of Chicago, together with researchers in Israel, have recently reported in venerable Nature magazine a new mRNA modification, namely N1-methyladenosine (m1A), which occurs on thousands of different gene transcripts in eukaryotic cells spanning genetically simple yeast to much more complex mammals.

Taking advantage of newly developed sequencing approaches—i.e. brighter lamp posts in my metaphor—they showed that m1A is enriched around the start codon upstream of the first splice site. More specifically, they observed that m1A “preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production.”

They conclude by noting that “these unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m1A in promoting translation of methylated mRNA.” So, as I said at the beginning of this blog, seek and ye shall find, and He did—capital H and pun intended.

Incidentally, those of you who are chemists will recognize that the ionic structure for m1A pictured above can undergo deprotonation to form a formally neutral counterpart having an H-N=C-N-CH3 moiety, as shown in TriLink’s catalog for the triphosphate derivative of m1A. Which one, or the proportion of these two structures for m1A exists in an mRNA of interest will depend on sequence context, local pH, etc. In this regard, scientists will need to somehow seek, in order to find, those answers in epitranscriptomics.

Expanding Epigenetics

m1A; taken from genengnews.com

m1A; taken from genengnews.com

Until recently, mammalian epigenetics had been solely focused on 5-methyl-2’-deoxycytidine and sequentially oxidized versions thereof, i.e. having hydroxyl, formyl, and carboxyl moieties, all of which are offered by TriLink as various products as either 5’-triphosphates or oligonucleotides. In less complex, prokaryotic organisms such as bacteria, N6-methyl-2’-deoxyadenosine (m6dA) is prevalent, but whether it is found in mammals has remained unclear until now.

A multi-university collaboration with the single-molecule-sequencing company Pacific Biosciences now report (Wu et al. 2016) the existence of m6dA in mouse stem cells. This landmark discovery was even more exciting because of the identification of an enzyme that removes methyl groups from m6dA, and by finding that m6dA is enriched in certain regulatory DNA sequences. Together these data provide clues to the possible function of m6dA in mammalian genomes.

Akin to my introductory light-from-the-lamp-post metaphor, detection and location of m6dA was enabled by more powerful analytical methods compared to those available in the past. Using state-of-the-art liquid chromatography-tandem mass spectrometry (LC-MS/MS), Wu et al. found that m6dA represented only 6–7 bases per million (!) adenines genome-wide. m6dA was enriched ~4-fold in genomic regions associated with a rare histone protein, H2AX, although the reasons for this association remain unclear.

The authors next determined specific locations of m6dA in sequences of DNA bound to H2AX by using Pacific Biosystems single-molecule real-time (SMRT) sequencing, which detects different kinetics with which a polymerase enzyme replicates modified bases compared with standard ones. Readers interested in this clever—dare I say “SMaRT”—method can check out my previous blog on SMRT.

In my opinion, the most challenging aspect of investigating epigenetics and epitranscriptomics is deciphering the dynamics and functional impact of “writing” and “erasing” methyl groups on bases in DNA and RNA, respectively. Erasure, i.e. removal of methyl groups is carried out by so-called demethylase enzymes. Several demethylases of the AlkB protein family have been shown to remove methyl groups from m6A in RNA as a means of regulating mRNA function. In mammals, this protein family has nine members, among which AlkBH1-deficiency was found by Wu et al. in mouse embryonic stem cells (ESCs) to lead to accumulation of m6dA in the genome, and that AlkBH1 could remove methyl groups from m6dA in DNA in vitro.

Intriguingly, Wu et al. further discovered that AlkBH1 worked most effectively on single-stranded DNA in vitro, thus raising the question of whether the enzyme preferentially operates during transcription or DNA replication in vivo, when DNA is transiently single stranded.

It’s also worth noting that enzymes that “write” (i.e. add) methyl groups to the N6-position of dA in mammalian DNA remain to be defined, as do the “reader” proteins that detect genomic m6dA.

In conclusion, I hope that this brief synopsis of new findings in “epi-marking” RNA and DNA has been illuminating—pun intended—with regard to molecular biology and the need for applying ever more powerful analytical methods to find more of what we are looking for: the molecular basis for living organisms.

On second thought, it seems to me that scientific discovery is perhaps more like moving forward from one lighted area to the next…

As usual, I welcome your comments.

New CRISPR System Reported for Targeting RNA Instead of DNA

  • Current “CRISPR Craze” for DNA Editing is Catalyzing Creativity
  • Early CRISPR Innovator Feng Zhang Now Reports Targeting RNA
  • This New “C2c2” System has Specificity Issues But is Nevertheless Promising

Just when you thought that the “CRISPR craze” would soon transition from the fundamental discovery phase to the improvements phase, something entirely new for CRISPR has come along. That something, recently published by Feng Zhang and others in venerable Science magazine, targets RNA instead of DNA. Consequently, this may lead to transient vs. permanent editing, as well as other RNA- vs. DNA-based applications.

Before further commenting on this exciting new RNA-targeting approach using CRISPR, here are a few snippets about the original DNA version of CRISPR to set the stage, and substantiate my tongue-in-cheek referral to the craze about it.

Backstory

Taken from igtrcn.org

Taken from igtrcn.org

Editing with CRISPR, which is short-form for CRISPR-Cas9, uses sequence-specific guide RNA (gRNA) to target DNA for cutting by Cas9 nuclease, as depicted below. Guide RNA and Cas9 can be introduced into cells either encoded in a vector or as synthetic gRNA and synthetic Cas9 mRNA, which TriLink offers in either wild-type or base-modified forms of Cas9 mRNA. CRISPR for genome editing was publicly described in Science in 2012 by co-corresponding authors Jennifer A. Doudna, a biologist at the University of California, Berkeley, and the French microbiologist Emmanuelle Charpentier. But Feng Zhang, at the Broad Institute, was first to obtain a patent on the technique.

Not surprisingly, given the financial potential for DNA editing by CRISPR, which has been called the ‘biotech discovery of the century,’ there is ownership litigation. This dispute is getting rather ugly, if you will, according to an article in Science titled Accusations of errors and deception fly in CRISPR patent fight.

crispPotential financial gain aside, PubMed stats I found clearly substantiate the craze factor in numeric terms: >4,000 publications to date with a rapidly increasing trajectory, i.e. ~600 in 2014 and ~1,200 in 2015, which is an average of roughly 4 publications every day in that year!

By the way, in the chart above that I made for CRISPR publications in PubMed, there was only one report in 2002, which was the first publication to identify CRISPR. These Dutch investigators used computer analysis to find a novel family of repetitive DNA sequences that is present among both domains of the prokaryotes (Archaea and Bacteria), but absent from eukaryotes or viruses. They noted that “[t]his family is characterized by direct repeats, varying in size from 21 to 37 bp, interspaced by similarly sized non-repetitive sequences. To appreciate their characteristic structure, we will refer to this family as the clustered regularly interspaced short palindromic repeats (CRISPR).” 

Taken from youtube.com

Taken from youtube.com

CRISPR was also selected as 2015 Science Breakthrough of the Year, and is featured in an interesting YouTube video that is definitely worth watching, in my opinion.

Enough said for CRISPR editing of DNA, let’s move on to RNA editing with CRISPR that offers a fundamentally different editing approach: whereas DNA editing makes permanent changes to the genome of a cell, CRISPR-based RNA-targeting approach may allow researchers to make temporary changes. Moreover, this can be adjusted up or down, and may one day provide greater specificity and functionality than existing methods for RNA interference (RNAi) using either siRNA or antisense oligos.

CRISPR Targeting RNA

Feng Zhang. Taken from mit.news.edu

Feng Zhang. Taken from mit.news.edu

At the risk of seeming to be too trendy, this section heading could have read “Feng Zhang 2.0” in that Zhang at the Broad Institute, along with co-corresponding author Eugene V. Koonin at NIH and uber-famous “Broadster” Eric Langer plus others on a large team, have characterized a new CRISPR system that targets RNA—but not DNA. In their recent Science publication they demonstrated that this new system involves a Class 2 type VI-A CRISPR-Cas effector—aptly abbreviated C2c2 (pronounced “see too, see too”)—that has RNA-guided RNase function.

The researchers originally identified C2c2 in the bacterium Leptotrichia shahii (L. shahii) in a systematic search for previously unidentified CRISPR systems within diverse bacterial genomes. They focused on C2c2 because its sequence contained two copies of a domain called higher eukaryotes and prokaryotes nucleotide-binding (HEPN) that has only been found in RNases. Mutating the putative catalytic site within either of C2c2’s HEPN domains demonstrated that none of the mutated enzyme versions could cut RNA in vitro, suggesting that both HEPN domains are necessary for C2c2 to work.

CRISPR-C2c2 from L. shahii reconstituted in E. coli to mediate interference of the RNA phage MS2 via crRNA facilitated by the two HEPN nuclease domains. Taken from Abudayyeh et al.

CRISPR-C2c2 from L. shahii reconstituted in E. coli to mediate interference of the RNA phage MS2 via crRNA facilitated by the two HEPN nuclease domains. Taken from Abudayyeh et al.

But C2c2 Cleaves Collateral RNA—a Case for “Lemons into Lemonade”?

Unlike Cas9, which cuts DNA only within the sequence dictated by the CRISPR gRNA, C2c2 was found to make cuts within the target sequence and adjacent, nonspecific sequences. While this collateral cleavage obviously presents a specificity problem, Zhang and his colleagues were able to create a deactivated C2c2 (dC2c2) variant by alanine substitution of any of the four predicted HEPN domain catalytic residues. To me this clever trick is like converting “lemons into lemonade” in that undesired non-specific cleavage is transformed into a programmable RNA-binding protein having potential utility.

For example, the investigators speculate that the ability of dC2c2 to bind to specified sequences could be used in the following ways:

  • Bring effector modules to specific transcripts in order to modulate their function or translation, which could be used for large-scale screening, construction of synthetic regulatory circuits, and other purposes.
  • Fluorescently tag specific RNAs in order to visualize their trafficking and/or localization.
  • Alter RNA localization through domains with affinity for specific subcellular compartments.
  • Capture specific transcripts through direct pull-down of dC2c2 in order to enrich for proximal molecular partners including RNAs and proteins.

Listen to Zhang’s Grad Students 

While the details of this seminal work published in Science is not easily summarized, its practical implications have been concisely translated, if you will, by first coauthor Omar O. Abudayyeh, and second coauthor Jonathan S. Gootenberg—both graduate student members of the Zhang lab—in three short videos that I encourage you to watch at this link.

Left: Omar Abudayyeh. Taken from zlab.mit.edu Right: Jonathan Gootenberg Taken from zlab.mit.edu

Left: Omar Abudayyeh. Taken from zlab.mit.edu Right: Jonathan Gootenberg Taken from zlab.mit.edu

Publication protocol generally lists coauthors in order of contribution, so in this C2c2 publication that has many coauthors, these fellows know what they’re talking about because they did lots of the lab work. Congrats to them, Feng Zhang (again), and all of the other contributors.

As always, your comments are welcomed and encouraged.

Jerry’s Favs from the Recent OTS Meeting

  • 12th Annual OTS Meeting in Montreal Très Excitant!
  • RNAi Approach to Treating Preeclampsia Spurred by Researchers’ Personal Experiences
  • Ionis Video for Spinal Muscular Atrophy Amazes the Audience
  • 30 years later, PS-Modified Oligonucleotides Continue to be Enabling!
montreal

City line of Montreal. Taken from oligotherapeutics.org

The 12th Annual Meeting of the Oligonucleotide Therapeutics Society (OTS) held on September 25-28, 2016 in French-speaking Montreal, Quebec, Canada brought together hundreds of enthusiastic investigators from around the world. All attendees share a common interest in oligo-based therapeutics, and we gratefully say merci beaucoup to the organizers of this very well run event.

Commenting here on my “favs” is limited by space, and highly subjective—by intent—focusing on only several impressions that struck me as worth sharing. Readers interested in perusing all of the lecture titles and speaker biosketches can do so at this link, which also lists corporate sponsors—including TriLink—and connects with the regularly updated OTS website.

Jerry’s balcony view of OTS 2016 presentations at the Centre Mont-Royal venue

Jerry’s balcony view of OTS 2016 presentations at the Centre Mont-Royal venue

Before getting to my selected topics, I wish to congratulate the OTS Board of Directors and Scientific Advisory Council for their ongoing valuable contributions to this society, and continuing efforts to include participation by the new generation of young investigators, who I’m sure will collectively make exciting advances in the field of oligonucleotide therapeutics. It was pleasure for me to me meet some of these “next gen” scientists, and learn about their current work, which is quite sophisticated by comparison to what I and others did in the early—dare I say primitive—era of antisense oligonucleotide drug discovery.

Toward Treating Preeclampsia: Personalized Drug Developers’ Stories

Preeclampsia (PE) is a disorder that occurs during pregnancy, affects both the mother and the fetus, and is characterized by elevated blood pressure, swelling and protein in the urine. According to a Preeclampsia Foundation fact sheet, every minute somewhere in the world a woman dies in pregnancy or childbirth, which amounts to more than 500,000 deaths each year. In addition, PE causes ~15% of premature births in industrialized countries and is the number one reason doctors decide to deliver a baby early.

Dr. Melissa J. Moore is a professor in the RNA Therapeutics Institute and the Department of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School. Taken from hhmi.org

Dr. Melissa J. Moore is a professor in the RNA Therapeutics Institute and the Department of Biochemistry and Molecular Pharmacology at the University of Massachusetts Medical School. Taken from hhmi.org

In her OTS lecture on clinical development of an RNA interference (RNAi) approach to treat PE, Dr. Melissa J. Moore (pictured below) introduced an attention-grabbing personal element when she revealed her bout with PE. Coincidentally, she discovered that her then attending physician, Dr. S. Ananth Karumanchi, had begun his quest for identifying PE-causality at the molecular level prompted by his daughter’s dangerously premature birth due to PE. This turned out to be a truncated kinase receptor abbreviated sFlt1, which Moore described as Karumanchi’s break-through discovery for possible development of PE therapeutics.

The condensed version of these two remarkably interwoven, PE-related personal stories—that you can hear first-hand from Moore on YouTube—was an agreement between Moore and Karumanchi to collaborate on discovering whether double-stranded short-interfering RNA (siRNA) targeting sFlt1 could be useful as an RNAi-based PE therapeutic agent.

Melissa Moore’s ultrasound sonogram. Taken from her YouTube video

Melissa Moore’s ultrasound sonogram. Taken from her YouTube video

Moore went on to gratefully acknowledge her colleague at the RNA Therapeutics Institute, Prof. Anastasia Khvorova, for setting up “by hook or by crook” the first non-profit academic facility for large-scale production of siRNA suitable for clinical development. This led to designing and producing a nuclease-resistant siRNA comprised of 2’-OMe/2’-F ribonuceosides and phosphorothioates at the ends, with an attached hydrophobic cholesterol moiety for improved delivery.

Even more impressive—at least to me—was Moore’s ability to access pregnant baboons in a non-human primate model of PE. This is inherently difficult due to issues involving non-human primates for any research, and is technically much more challenging compared to, for example, infectious disease models. Dr. Moore showed lots of compelling results from her model studies, and concluded her talk by saying that clinical studies were planned.

In closing this section, it’s worth noting that Moderna—a leading mRNA therapeutics company—has recently announced its appointment of Dr. Moore as Chief Scientific Officer of Moderna’s mRNA Research Platform.

Ionis Pharmaceuticals Drug Video for Spinal Muscular Atrophy Amazes the Audience

Dr. Stanley T. Crooke. Taken from ionispharma.com

Dr. Stanley T. Crooke. Taken from ionispharma.com

This year’s OTS Lifetime Achievement Award address by Dr. Stanley T. Crooke, founder and CEO of Ionis Phamaceuticals (formerly Isis Pharmaceuticals), was presented to an auditorium packed with attendees who, like me, greatly admire Crooke’s many scientific and commercial contributions to the field over the past decades. These currently include more than 450 (!) scientific publications and 38 drugs in the pipeline, with 3 finishing Phase III—very impressive and promising indeed!

Ionis’ impact on providing efficacious oligonucleotide-based therapies to patients was conveyed most powerfully—in my opinion—by a video about Cameron, an infant SMA patient who was born with Spinal Muscular Atrophy (SMA). According to an NIH fact sheet, SMA is a genetic disease that causes weakness and wasting of the voluntary muscles in the arms and legs of infants and children. These disorders are linked to an abnormal or missing gene known as the survival motor neuron gene 1 (SMN1), without which motor neurons in the spinal cord degenerate and die.

As detailed elsewhere by Chiriboga et al., Nusinersen (aka ISIS-SMNRx or ISIS 396443) is a 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified antisense oligonucleotide (ASO) designed to alter splicing of SMN2 mRNA and increase the amount of functional SMN protein produced, thus compensating for the genetic defect in the SMN1 gene. The cartoon shown below depicts how this ASO targets an hnRNP-A1/A2–dependent splicing silencer in intron 7 of the SMN pre-mRNA. Nusinersen displaces hnRNP proteins from this silencer site on the SMN2 pre-mRNA, facilitating accurate splicing of SMN2 transcripts (e.g., increasing the synthesis of transcripts containing exon 7) and resulting in increased production of full-length SMN protein.

Taken from researchgate.org after Chiriboga et al.

Taken from researchgate.org after Chiriboga et al.

Ionis and its commercial partner Biogen announced in August 2016 that Nusinersen met the primary endpoint pre-specified for the interim analysis of ENDEAR, the Phase 3 trial evaluating Nusinersen in infantile-onset (Type 1) SMA. The analysis found that infants receiving Nusinersen experienced a statistically significant improvement in the achievement of motor milestones compared to those who did not receive treatment. In September, Biogen announced that it applied for Priority Review by the FDA that, if granted, would shorten the review period of Nusinersen following the Agency’s acceptance of the NDA filing by Ionis and Biogen.

Cameron at 2+ years enjoying the moment with his mom. Taken from YouTube

Cameron at 2+ years enjoying the moment with his mom. Taken from YouTube

The amazing effect of Nusinersen warranting this Priority Review is best appreciated—in my opinion—by watching a YouTube video showing the progress of Cameron during his treatment. When viewing this video, keep in mind that Cameron’s Type I SMA is typically evident at birth or within the first few months, and that symptoms include floppy limbs and trunk, feeble movements of the arms and legs, swallowing and feeding difficulties, and impaired breathing. Sadly, the prognosis is poor for babies with SMA Type I. Most die within the first two years. But not so for Cameron, shown below at 2+ years, thanks to Nusinersen!

Phosphorothioate-Modified Oligonucleotides Continue to be Enabling—30 Years On!

men

Left: Prof. Fritz Eckstein. Taken from oligotherapeutics.org Right: Prof. Wojciech J. Stec. Taken from cbmm.lodz.pl

That nuclease-resistant phosphorothioate (PS)-modified internucleotide linkages have had an enabling influence on all manner of oligo-based drug development is evident from the first-ever OTS Lifetime Achievement Award in 2015 being given to Prof. Fritz Eckstein for his pioneering and life-long work on PS linkages in DNA and RNA. Following Fritz’s lead, and aided by the availability of ABI’s DNA synthesizer, Prof. Wojciech J. Stec and yours truly published the first completely automated method for synthesis of fully or partially PS-modified DNA oligos in 1984. This opened the door for early antisense experiments with PS-modified oligos, which have continued now for 30 years!

Perhaps surprisingly, the ever evolving field of oligonucleotide therapeutics continues to rely on PS-modifications in a myriad of mechanistically distinct strategies, such as “classic” antisense to mRNA, siRNA, anti-miRNAs, modulators of RNA splicing, etc. In this regard, Dr. Crooke’s concluding remarks on his goal of deciphering the “code” for protein binding to chemically modified ASO, led me to muse about the negatively charged P-Smoiety in such binding of PS-containing ASO.

Taken from zon.trilinkbiotech.com

Taken from zon.trilinkbiotech.com

My initial thought was that P-S likely enhances binding to positively charged amino acid moieties in proteins, due to greater polarizability vs. P-O, and that Sp or Rp P-Sstereochemistry may likely influence binding. Moreover, this spatial aspect of a binding “code” can now be studied using stereospecific synthesis of PS-ASO by either Stec’s OTP method or Wada’s oxazaphospholidine method. Time will tell—stay tuned!
As usual, your comments here are welcomed.

Chinese Scientists to Pioneer First Human CRISPR Clinical Trial

  • Chinese Team Begins First CRISPR-Based Anticancer Immunotherapy Clinical Study
  • US Consortium’s CRISPR Clinical Study OK’d by NIH Panel
  • Survey Indicates More Worry than Enthusiasm for Gene-Editing in Babies
Taken from jeantet.ch

Taken from jeantet.ch

Regular readers of my blog will recall a number of previous postings on gene editing using CRISPR-Cas9 (aka CRISPR), which has rapidly become the hottest trend in nucleic acid-based biotechnology and medical research. That opinion is partly based on the fact that this relatively new technology has already amassed thousands of publications, including 1,250 in 2015 alone! Additional evidence follows from the numerous start-up and established biopharma companies pursuing CRISPR-based therapeutics, which is “the biggest biotech discovery of the century” according to one report.

Now, even more exciting developments for CRISPR are on the horizon. Chinese researchers are poised for the first human clinical trial using CRISPR, and an analogous study in the US awaiting FDA approval. Both of these trials involve forms of cancer immunotherapy, which is a very hot field in itself, and is briefly introduced in the next section.

Cancer Immunotherapy

Cancer immunotherapy is the use of the immune system to treat cancer either actively, passively, or by a combination of these approaches, which exploit the fact that cancer cells often have macromolecules on their surface that can be detected by the immune system (aka tumor-associated antigens, TAAs). Active immunotherapy directs the immune system to attack tumor cells by targeting TAAs, whereas passive immunotherapies enhance existing anti-tumor responses and include the use of monoclonal antibodies, lymphocytes and cytokines.

Numerous antibody therapies have been approved by the FDA and other regulatory authorities worldwide. In contrast, active immunotherapies, which usually involve the removal of immune cells from the blood or from a tumor for reintroduction to the patient, have lagged in such approvals. In fact, the only US-approved cell-based therapy is Dendreon’s Provenge®, for the treatment of prostate cancer. A recent series of NY Times articles highlights some powerful examples of cancer immunotherapy, especially from the perspective of interviews with patients and their physicians.

Taken from howitworksdaily.com

Taken from howitworksdaily.com

For the purpose of this blog, I’ll add that adoptive T-cell therapy is a form of passive immunization by the transfusion of T-cells, which are found in blood and tissue and usually activate when they find “foreign” pathogens. These pathogens can be either infected cells, or antigen presenting cells present in tumor tissue, where they are known as tumor infiltrating lymphocytes. Although these cells can attack the tumor, the environment within the tumor is highly immunosuppressive, preventing immune-mediated tumor death.

As depicted below, T-cells specific to a tumor antigen can be removed from a tumor sample or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the resultant cells being reinfused. Importantly, activation can take place through genetic engineering, such as gene editing by CRISPR.

Cancer specific T-cells can be obtained by fragmentation and isolation of tumor infiltrating lymphocytes, or by genetically engineering cells from peripheral blood. The cells are activated and grown prior to transfusion into the recipient (tumor bearer). Taken from Wikipedia.org

Cancer specific T-cells can be obtained by fragmentation and isolation of tumor infiltrating lymphocytes, or by genetically engineering cells from peripheral blood. The cells are activated and grown prior to transfusion into the recipient (tumor bearer). Taken from Wikipedia.org

CRISPR Goes Clinical in China

As I write this post, Chinese scientists at Sichuan University’s West China Hospital in Chengdu will reportedly become the first in the world to inject people with cells modified using the CRISPR gene-editing method. This landmark trial will involve patients with metastatic non-small cell lung cancer who have not responded to chemotherapy, radiation therapy and/or other treatments.

The team will extract T cells from the blood of the trial participants and then use CRISPR to knock out a gene encoding a protein called PD-1 that normally regulates the T cell’s immune response to prevent it from attacking healthy cells. As depicted below, this so-called checkpoint has been targeted by small molecule inhibitors (anti PD-1), but with somewhat limited success so far—notably including failure of a would-be billion-dollar drug (Opdivo®) recently reported by Bristol-Myers Squibb. Nevertheless, the presently envisaged PD-1 gene-edited cells will be amplified in the lab and re-introduced into the patient’s bloodstream to circulate and, hopefully, attack the cancer.

Taken from smartpatients.com

Taken from smartpatients.com

Potential Concerns of CRISPR Clinical Trials

While the Chinese trial may be groundbreaking, it is not without risk. There is concern that the edited T-cells could attack normal tissue as it’s well documented that CRISPR can result in gene edits at the wrong place in the genome. To help mediate this risk, the T-cells will be validated by biotechnology company MedGenCell—a collaborator on the trial—to ensure that the correct gene is knocked out before the cells are re-introduced into patients.  Since the primary purpose of this phase one trial is to determine if the approach is safe, participants will be closely monitored for side effects, and researchers will pay close attention to biological markers in the blood.

Some may say that this type of trial in humans is premature, but China has always tried to be a leader in CRISPR research. Carl June, a clinical researcher in immunotherapy at the University of Pennsylvania in Philadelphia explains, ‘China places a high priority on biomedical research.’ And Tetsuya Ishii, a bioethicist in Japan, is quoted in Nature magazine as saying that ‘When it comes to gene editing, China goes first.”

Initial CRISPR Clinical Trial in USA Awaiting Review Board and FDA Approvals

While the Chinese team seems positioned to be the first to conduct a CRISPR clinical trial, the US isn’t far behind. As reported earlier this summer, the Recombinant DNA Research Advisory Committee (RAC) at the NIH has approved an analogous—but genetically more complex—proposal to use CRISPR-edited T cells for cancer immunotherapy. The RAC reviews all proposals for human trials involving modified DNA that are conducted in the United States, and the investigators now have to convince review boards at their own institutions, and the FDA, to allow the trial. This will hopefully be done by the end of 2016.

The proposed trial is a collaborative effort involving the University of Pennsylvania, M.D. Anderson Cancer Center in Texas, and the University of California, San Francisco. This trial will be funded by a $250-million immunotherapy foundation formed in April 2016 by former Facebook president Sean Parker. According to a news item in Nature, The University of Pennsylvania will produce the edited cells, and will recruit and treat patients alongside the collaborating medical centers in Texas and California.

In this study, researchers will perform three CRISPR edits with T cells from patients with several types of cancers: 1. a gene for a protein engineered to detect and target cancer cells will be inserted, 2. a natural T-cell protein that could interfere with this process will be removed and 3. the gene for a protein that identifies the T cells as immune cells and prevents the cancer cells from disabling them will be removed. The researchers will then infuse the edited cells back into the patient.

Both of the studies being performed by the Chinese and US teams are very exciting to me. I will be paying close attention to the progress of these studies over the next few months and hope to report back to you with some very interesting and ground breaking results.

Survey Indicates Public Concern in the US About Gene Editing in Babies

A Pew Research Center pole focusing on concerns about gene editing was recently reported in Nature. The poll surveyed more than 4,700 people in the United States and the results indicate that more people are concerned than enthusiastic about gene editing.

The quadrants below (from 0-100%) show responses to the question, “How worried or enthusiastic do you feel about gene editing to reduce disease risk in babies?”

Taken from nature.com

Taken from nature.com

While these findings speak for themselves, I hasten to add that this same pole also asked “Have you heard or read about this topic?” The following replies (in quadrants from 0-100%) indicate that a substantial percentage of respondents who have not at all read about gene editing nevertheless expressed either worry or enthusiasm for gene editing. Ditto for those who read a little about gene editing.

babies

It would seem, then, that we should take this poll with a grain (or two) of salt as it apparently includes a substantial amount of uninformed opinion. This is not too surprising given the complex nature of gene editing and it’s relatively new position in research. We can see from the results of this poll that a significant amount of education may be needed to garner increased public support for gene editing. Hopefully, the results of the studies highlighted above will begin to pave this road.

Hope for the Best but Expect Complications

This section heading, which summarizes my parting comments about the advent of CRISPR-enabled clinical trials, is based upon my having participated in several decades of research on nucleic acid-based concepts for radically different approaches to traditional small-molecule therapeutics. First was the idea of using chemically modified antisense oligonucleotides to block gene expression, which encountered non-specificity issues, low potency, delivery, and a host of other issues that took two decades to sort through. Then there was the idea of using chemically modified short-interfering RNA (siRNA) for modulating gene expression via RNA interference (RNAi). This, too, encountered similar problems in reaching the clinic. Ditto for anti-microRNAs (antagomirs).

I’m hoping that CRISPR-based therapies meet with success far faster—and prove to be affordable to society. I won’t, however, be surprised if progress is slow—and quite expensive.

Are you excited about potential CRISPR-based therapies? Do you have concerns about their safety and efficacy? Do you believe the general public is ready to accept gene editing therapies? Please share your thoughts as your comments are always welcome.

Update on Zika Virus Detection by RT-PCR

  • Various RT-PCR Assays for Zika Virus Have now Received Emergency Use Authorization (EUA) from the FDA
  • Quest Diagnostics’ Assay Approved by FDA for General Use as a Zika Test
  • Troubled Theranos Touts New “miniLab” for Zika EUA
  • Vaccine Development Progressing Albeit Relatively Slowly
Aedes aegypti mosquito. Taken from wcvb.com

Aedes aegypti mosquito. Taken from wcvb.com

In January 2016, I posted a blog about the then emerging public awareness of Zika virus (ZIKV), which is spread by the bite of infected mosquitos—primarily the Aedes aegypti mosquito. Sadly, ZIKV can be passed from a ZIKV-infected pregnant woman to her fetus leading to development of a brain defect (microcephaly) and/or other malformities. Moreover, ZIKV is now associated with sexual transmission and blood transfusion. This is scary news.

This update was prompted by the additional fact that ZIKV is on the rise and there is still no vaccine, according to the Centers for Disease Control and Prevention (CDC)—my “go to” source for loads of authoritative information about this infectious disease. Weekly updated ZIKV-infection “case counts” in US States and US Territories were given as ~1,200 and ~6,500, respectively on Aug 10th, and increasing to ~3,400 and ~20,000 on Sep 21st —only 6 weeks later!

As I pointed out in my previous blog on ZIKV, absent an anti-ZIKV vaccine, there is considerable interest in mosquito abatement as well as early detection, notably by reverse-transcription PCR (RT-PCR) of this RNA Flavivirus. Now that Zika infection by mosquitos in Florida has been found, leading to several CDC warnings, I thought it would be both apropos and technically interesting to provide the following update on ZIKV structure and RT-PCR.

ZIKV Genome and Structure

As of last month, my PubMed search of “Zika [Title/Abstract] AND RT-PCR [Anywhere]” gave 55 publications. Incidentally, there are now a number of Zika genome sequencing publications, such as this lead reference. It’s worth noting that ZIKV genome sequencing enables monitoring the potential evolution of new genomic variants that might foil existing RT-PCR assays and guide the selection of new primers for RT-PCR.

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV genome. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

It’s also worth pointing out that Zika’s overall molecular capsid structure is enveloped, spherical, and has a diameter of ~40 nm in diameter, as depicted below in schematic form, and pictured in the accompanying electron microscopic image. The surface proteins are arranged in an icosahedral-like symmetry.

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

ZIKV capsid structure. Taken from viralzone.expasy.org with permission from SIB Swiss Institute of Bioinformatics, ViralZone

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

This is a transmission electron micrograph (TEM) of ZIKV, which is a member of the family Flaviviridae. Virus particles are ~40 nm in diameter, with an outer envelope, and an inner dense core (see above). The arrow identifies a single virus particle. Taken from cdc.gov

ZIKV RT-PCR

In the interest of giving explicit credit to various investigative groups who have developed RT-PCR assays for ZIKV, here are links and short snippets I’ve selected from publications, in chronological order, found in the aforementioned PubMed search. Interested readers are encouraged to check out the original publication for details.

The first report of an RT-PCR assay for ZIKV appears to be by Faye et al. in 2008 at the Institut Pasteur de Dakar in Senegal, who targeted the envelope protein coding region and tested ZIKV isolates previously collected over a 40-year period from various African countries and hosts. The assay’s detection limit and repeatability were respectively 7.7pfu/reaction and 100% in serum; none of 19 other Flaviviruses tested were detected. Faye et al. in 2013 extended this work to include quantitative RT-PCR detection of ZIKV and evaluation with samples from field-caught mosquitoes.

Kinetics of ZIKV detection in urine compared to serum from 6 patients was described by Gourinat et al. in 2015 at the Institut Pasteur, Noumea, New Caledonia using RT-PCR primers and probes previously reported by others. Urine samples were positive for ZIKV more than 10 days after onset of disease, which was a notably longer period than 2-3 days for serum samples. These researchers concluded that urine samples are useful for diagnosis of ZIKV infections, and are preferred to serum wherein virus titer diminishes more rapidly.

Musso et al. in 2015 working in Tahiti, French Polynesia with 1,067 samples collected from 855 patients presenting symptoms of Zika fever found that analysis of saliva samples increased the rate of detection of ZIKV at the acute phase of the disease compared to serum samples. They noted that saliva was of particular interest when blood was difficult to collect, especially for children and neonates.

Most recently, Xu et al. in 2016 in China reported the development of a SYBR Green (intercalator dye)-based qRT-PCR assay for detection of ZIKV. Although their results indicate that the assay is specific, it’s important to note that SYBR-type detection can be subject to nonspecific artifacts, for which TriLink’s proprietary CleanAmp™ Primers can be investigated to potentially ameliorate such problems, as discussed in this downloadable pdf publication by TriLink researchers.

ZIKV In Vitro Diagnostic Assays

As detailed elsewhere, the Secretary of Health and Human Services (HHS) earlier this year determined that “there is a significant potential for a public health emergency that has a significant potential to affect national security or the health and security of United States citizens living abroad and that involves Zika virus.” The Secretary of HHS further declared that “circumstances exist justifying the authorization of the emergency use of in vitro diagnostics for detection of Zika virus…”.

The following RNA-based assays and suppliers are currently listed by the FDA for this Emergency Use Authorization (EUA):

  • xMAP® MultiFLEX™ Zika RNA Assay (Luminex Corporation)
  • VERSANT® Zika RNA 1.0 Assay (kPCR) Kit (Siemens Healthcare Diagnostics Inc.)
  • Zika Virus Real-time RT-PCR Test (Viracor-IBT Laboratories, Inc.)
  • Aptima® Zika Virus Assay (Hologic, Inc.)
  • RealStar® Zika Virus RT-PCR Kit U.S. (Altona Diagnostics)
  • Zika Virus RNA Qualitative Real-Time RT-PCR (Focus Diagnostics)
  • Trioplex Real-time RT-PCR Assay (CDC)

Among these, two are particularly notable—in my opinion. Trioplex Real-time RT-PCR Assay is a multiplexed laboratory test developed by the CDC to simultaneously detect ZIKA, dengue virus, and chikungunya virus RNA, each of which can be transmitted primarily by Aedes aegypti mosquitos to cause infections with similar symptoms. Consequently, it is useful to have a single test that can detect each of these viruses in the same sample. Full details for the Trioplex assay are provided in a 40-page downloadable pdf from the CDC at this link.

In brief, this CDC-developed test uses virus-specific primer pairs and fluorogenic hydrolysis probes each differentially dual-labeled with fluorescent reporter and quencher dyes for in vitro detection of complementary DNA (cDNA). The detection follows reverse-transcription of RNA isolated from clinical specimens including serum, cerebral spinal fluid, urine, and amniotic fluid. It’s worth pointing out that I subsequently found a publication in 2016 by several collaborating academic groups regarding an analogous triplex RT-PCR assay for the same three viruses.

The other notable assay is Zika Virus RNA Qualitative Real-Time RT-PCR developed by Focus Diagnostics, which in April 2016 was the first of the aforementioned ZIKV tests to receive authorization by the FDA for use by qualified labs to detect ZIKV RNA in blood samples of those meeting CDC clinical criteria or of people who may have lived in or traveled to an affected location or had other exposure to the virus. Quest Diagnostics, the parent company of Focus Diagnostics, announced it would make the test broadly available to physicians, including those in Puerto Rico in May 2016.

ZIKV EUA Sought by Theranos for Its new “miniLab”

Theranos, which has been in the news regarding troubles over its stealthy proprietary system for finger-stick blood tests, appears to be pivoting its strategic plans. It announced at the August 2016 American Association of Clinical Chemistry Meeting its R&D for a new, fully automated miniLab system, including analytical and method comparison results of its ZIKV nucleic acid-amplification-based assay.

According to its press release, the company collected finger-stick samples from subjects, including people in the ZIKV-infested Dominican Republic, and shipped those to Palo Alto, California to run on the miniLab. Although I was unable to obtain these particular results, I did find the following figure at the TechCrunch website showing functional components of the miniLab along with an article by Sarah Buhr that’s worth a quick read, in my opinion.

Taken from techcruch.com

Taken from techcruch.com

According to the aforementioned Theranos press release, the company has submitted assay validation data for this Zika assay to the FDA for an EUA. The company also states that it is unaware of any currently available capillary (i.e. finger-stick) test for ZIKV.

I’ll stay tuned for future general information about the miniLab, as well as information specifically related to ZIKV. If I hear of anything, I’ll add as a comment here or in a new post with technical details about its nucleic acid assays.

ZIKV Vaccine Status

I hope this blog has convinced you that RT-PCR of ZIKV has provided improved molecular diagnostics. I’m guessing, that like me, you find it unfortunate that there isn’t a proven anti-ZIKA vaccine as of yet. This is especially frustrating given the fact that Zika disease has been known for more than 50 years, and that it is evidently on the rise globally, including in the USA according to regularly updated CDC statistics.

In February 2016, the Obama administration requested $1.9 billion in funding for the NIH to develop a ZIKV vaccine. The US Congress continues to be deadlocked by partisan politics despite the fact that Florida state and local officials are scrambling to contain the ZIKV outbreak in Miami Beach. This outbreak poses a serious threat to the health of residents, as well to visitors who drive the region’s $24 billion-a-year tourism industry.

Nevertheless, some progress has been reported for US studies in monkeys, and US-based Inovio says it’s received FDA approval to begin studies in humans. Outside the US, Sanofi Pasteur in France is said to be poised for initiating its trials in humans, and French biotech company Valneva is reported to have succeeded in generating a ‘highly purified inactivated vaccine candidate’ using the same technical approach it used for its encephalitis vaccine that is marketed in the United States and Europe.

Lagging—dare I say “glacially slow”—action against ZIKV by the World Health Organization is quite disappointing to me, and is reminiscent of what I’ve commented on in an earlier blog concerning this bureaucracy’s ineffective response to the Ebola virus. If I had my druthers, TriLink’s previously announced engagement by Battelle in development of Ebola mRNA vaccine would somehow materialize for a Zika mRNA vaccine. In this regard, GlaxoSmithKline is reported to be preparing research studies alongside the NIH’s Vaccine Research Center to test a self-amplifying mRNA vaccine technology for Zika. Interested readers can check out this link to a fascinating PNAS publication by Geall et al. on biosynthetic self-amplifying mRNA vaccines delivered in lipid nanoparticles.

As always, your comments are welcomed.

Postscript

Taken from Fleming et al. (2016) ACS Infectious Diseases

Taken from Fleming et al. (2016) ACS Infectious Diseases

One of my previous posts featured recent elucidation of biologically functional G-quadraplexes in living cells. Consequently, it’s apropos to mention here that Fleming et al. at the University of Utah have just now published the first analysis of potential G-quadruplex sequences (PQS) in the RNA genome of ZIKV. As depicted in this artistic cartoon, several PQS were found, with the most stable located near the end of the 3’ untranslated region (3′-UTR). Importantly, these investigators propose a rationale for screening G-quadruplex-binding compounds as a completely new class of anti-ZIKV drug candidates. In my opinion, this is a great example of how basic biochemical research can lead to new strategies for much needed antiviral drugs.