DIY Chromosomes

  • This DIY (do-it-yourself) is Actually DIT (do-it-together)!
  • A Global Network of Undergraduates are Collectively Assembling Designer Chromosomes for Yeast
  • The Inspirational Jolt for this Harnessing of “Student Power” Came in a Caffeinated Coffee Conversation

At a conference held over a decade ago, visionary geneticist Ronald Davis suggested that researchers should synthesize artificial yeast chromosomes and insert them in a living cell. Many of the attendees, including Jef Boeke, didn’t think much of the idea. According to a News Focus article in Science by Elizabeth Pennisi, Davis made the bold prediction that artificial yeast would be the next significant milestone in the then-emerging field of synthetic biology. Boeke didn’t share Davis’s vision, mostly due to the seeming insurmountable task of designing and synthesizing a 12.5-million-base genome comprising 16 chromosomes. According to Pennisi, as Boeke listened to Davis’s talk, he says, ‘I remember thinking “Why on Earth would you want to do that?’“

However, Boeke’s opinion about his own rhetorical question completely flip flopped, and has led to an inspiring “collective can do” attitude among scientists in this field. Led by Boeke, hundreds of researchers around the global are now working together as a gigantic team to successfully achieve a goal befitting a “grand challenge,” which would otherwise not be possible.

Here’s the interesting story of what has happened and how it came about.

Boeke (a geneticist who recently moved from Johns Hopkins University to New York University Langone Medical Center) and his colleagues have just finished the first complete synthetic yeast chromosome. They are well on their way to putting together several more—thanks to technological advances in manufacturing DNA oligonucleotides and—importantly—a global army of collaborators who are—amazingly to me—mainly undergraduate students!

computo

It’s only a cartoon, but definitely not a joke…“designer chromosomes” are now a reality (credit: Dave Simonds, taken from economist.com).

This post, which borrows from Pennisi’s article, briefly touches on only a bit of the truly impressive science—which can be read in detail in the original publication by Boeke and his army—and instead emphasizes the enthusiastic undergraduate student participation, along with an equally interesting—I think—odyssey as to how this project progressed to where it is now.

Why Build Completely Customized Yeast?

Good question. For centuries, Saccharomyces cerevisiae (S. cerevisiae), also know as brewer’s, baker’s, or budding yeast, has played a pivotal role in our food and drinks, from baked goods to beer and wine. It’s also a “workhorse” for cellular and molecular biologists. In 1996, it became the first eukaryote to have its genome fully sequenced. Since then yeast geneticists have studied it every which way—facilitated in part by being able to incorporate foreign DNA through a process called homologous recombination. There is even a comprehensive website dedicated to all things yeast.

In contrast, the “ultimate modification” of yeast is designed completely in silico (i.e. using computer programs), and assembled in the lab piece by piece using chemically synthesized DNA oligonucleotides. Each of the ~6,000 genes can thus be pondered with regard to being kept, modified or removed. The same applies to other genetic elements. Moreover, exogenous genes (i.e. not found in natural yeast) or other exogenous functional genetic elements can be inserted to obtain “custom content” genetic variants intended for specific applications, such as production of commercially valuable products.

Caffeinated Coffee Conversation Jolts Inspiration

Why tinker with yeast when we could ‘synthesize the whole thing.’ Jef Boeke, New York University (credit: Jef Boeke/New York University; taken from sciencemag.org).

Why tinker with yeast when we could ‘synthesize the whole thing.’ Jef Boeke, New York University (credit: Jef Boeke/New York University; taken from sciencemag.org).

I’m sure it was more than the caffeine, but its jolt probably helped a little based on Pennisi’s story about Boeke’s “aha moment.” The following anecdote is taken from Pennisi’s article. Boeke traces his change of heart about the synthetic yeast project to 2006, while over coffee, his Johns Hopkins colleague Srinivasan Chandrasegaran was trying to convince him to make a large number of zinc-finger nucleases. These DNA-modifying enzymes are Chandrasegaran’s specialty, and a toolkit full of them would make the yeast genome easier to manipulate. Boeke wasn’t that interested and, almost as a joke, suggested a more drastic way to take control of the yeast genome: synthesize the whole thing. To his surprise, Chandrasegaran jumped on the idea, and the pair brought Joel Bader, a computer scientist at Johns Hopkins, into the discussion.

Despite Boeke’s dismissal of Davis’s proposal just 2 years earlier, the trio at Hopkins quickly concluded that making an artificial yeast genome would be worthwhile if it could be a testbed for learning about the genome itself. They decided to start with the 90,000-base “R” arm of chromosome 9—the shortest in the yeast’s genome—and spent a year arguing about how the synthetic sequence should differ from the natural one. The trio considered just including the genes they wanted, ‘but we quickly realized it would be very risky to eliminate whole bunches of genes’ without really knowing in advance what the effect of that loss on the whole system would be,’ Boeke recalls.

So the team used the natural sequence of the chromosome 9 arm as the basic building block, and incorporated DNA that would enable them to ‘induce changes at will’. Basically, when activated by a chemical added to cells, it kicks off a chromosomal version of ‘musical chairs.’ By using this process, they were able to generate new yeast strains with different properties. Over time, they have been able to add stability to the yeast genome, helping to ensure integrity of the strain. Readers interested in technical aspects of how they did this—and other genetic edits such as removal destabilizing transposons and non-coding RNA—are referred to the original publication.

From Buy It to DIT—More Inspiration

After months of work and with the help of Bader, Boeke designed a version of the chromosome 9 arm using a software program. He then contracted with a biotech company to synthesize the chromosomal arm. The company was having difficulty synthesizing such a long piece of DNA and they didn’t hear back regarding the project for almost a year. During that time, Boeke began to wonder if he could speed things up himself. He came up the idea of offering a course dedicated to building a synthetic yeast genome. In 2007, the course was offered for the first time as a summer school class, which to me is reminiscent of the innovative strategy used by Mark Twain’s fictional character, Tom Sawyer, to achieve painting a seemingly endless and impossibly high fence. In this case, it’s building a big genome by making it educational for many student workers. sawyer

When tasked with painting a fence in front of his house, Tom Sawyer convinced others that doing so is more fun than sitting in the shade watching other people paint. As other children congregate to watch Tom Sawyer paint, he extols the fun of painting, and eventually has the other children clamoring to get to paint the fence (taken from postcrossing.com via Bing Images).

The course proved to be extremely popular and 6 years later it’s one of the most in-demand classes that Hopkins offers. Every section is packed —even on Friday nights! ‘We were overwhelmed with interest’ from biology, engineering, computer science, public health, and other majors,’ said Boeke, who is working with his colleagues to keep the course going at Hopkins despite his move to NYU.

Student-Power Drives Yeast Project Assembly

Genome builder, Katrina Caravelli (credit: Marty Taylor/Johns Hopkins University; taken from sciencemag.org).

Genome builder, Katrina Caravelli (credit: Marty Taylor/Johns Hopkins University; taken from sciencemag.org).

As a former professor and still active researcher, I have nothing but high praise for Boeke’s Tom Sawyer-like innovation to harness interest and enthusiasm of students through an educational course that achieves a truly grand challenge: a totally synthetic designer-yeast genome. More importantly, here’s what Pennisi reported about a couple of representative undergraduate student genome-builders, James Chuang and Katrina Caravelli, whose “Build A Genome” coursework consisted of sequences of DNA comprised of just four letters: A, C, G, and T in DNA:

For students, the course offers intensive training in synthetic biology and the thrill of taking part in frontline research. ‘I was really fascinated by the potential and by my ability to have an impact,’ recalled Caravelli, who signed up for the course in 2008 and is now Boeke’s senior lab coordinator at New York University. During the semester-long course, students learned basic molecular biology procedures such as performing PCR and cloning DNA in bacteria.

Each student committed to completing 10,000 DNA bases during the course. ‘My building blocks went to chromosome 8,’ Chuang, now a graduate student in biomedical engineering at Boston University, proudly told Pennisi. After being supplied with the DNA sequence he needed to synthesize, he started out with 16 DNA oligonucleotides, each about 75 bases long, ordered from a commercial DNA synthesis company. The ends of some pieces overlapped, so when he mixed the pieces together with enzymes, they self-assembled into 750-base spans dubbed building blocks. After making sure the building blocks had the correct sequence, he put them into bacteria to generate many copies of the newly assembled DNA. Now, Johns Hopkins graduate student Jingchuan Luo is putting those bigger DNA sections into yeast and making “minichunks” about 3,000 bases long. If the yeast stays healthy, then she adds the next chunk in line to it, and so on. Her goal: to make a yeast strain with a totally new chromosome 8.

Caravelli recalls that her own bacteria sometimes wouldn’t grow with the yeast segment in their midst. She enjoyed figuring out why. ‘The trial-and-error process challenges you to think properly like a scientist.’

Boeke’s course has proved such a success that three other U.S. universities are hosting or will soon host their own. Because it’s now cheaper to buy 750-base segments than to have students make them, current students start with such DNA blocks and turn them into 3000-base spans, and, ultimately, 10,000-base chunks. Class productivity has soared. Each student’s target is now 30,000 to 50,000 bases.

The students have high hopes for their work. ‘I want to see synthetic biology do really useful things for society,’ Chuang told Pennisi. ‘The synthetic yeast genome provides a template for doing [that].’

synthesis

Initial assembly steps carried out by undergraduate students (taken from sciencemag.org).

Global Momentum and Sc2.0

After the students in Boeke’s course completed chromosome 9, they moved on to chromosome 3. It took 49 students over 18 months to successfully assemble the 272,871 bases. Their work was detailed in a publication in Science in March of this year. The publication showed that ‘yeast carrying the shortened, modified chromosome grew normally and looked little different from their natural counterparts under almost all of the growing conditions tested.’

You can watch and listen to Jef Boeke talk about this achievement in a 2 minute video.

As they neared completion of chromosome 3, the team continued work on other chromosomes as word was spreading about the novel work coming out of Hopkins. Ying-Jin Yuan of Tianjin University in China heard about the project and was eager to participate. He convinced Huanming Yang from BGI to get involved and Yang helped organize the first synthetic yeast genome meeting, held in Beijing in 2012. Yuan didn’t want to stop there. He forged an international collaboration with Boeke and through his own 2012 “Build-A-Genome” course, he enlisted the help of 60 Chinese students and assembled all the bases needed for chromosome 5.

Popularity of the project continued to grow in other labs around the world. Tom Ellis, a synthetic biologist at Imperial College London, had attended Yang’s meeting in Beijing and was also intrigued by Boeke’s work. He helped organize a second synthetic yeast meeting in July 2013. Soon after, the British government committed £1 million to the project.

The momentum has continued. These like-minded yeast chromosome-builders have created a great website for their project that is called Sc2.0. This site is definitely worth visiting, especially to read the FAQ and learn more about the international Team members. I’ve included a great visual from the website that depicts where in the world each of the 16 chromosomes is being built.

xvi

The goal of the yeast genome project is to complete each of the yeast’s chromosomes within 2 years. This will be done by various partners around the global. Once the individual chromosomes are complete, Boeke will lead a team through the largest challenge of all—putting all of the chromosomes together into one organism. ‘It’s a little surprising that we can have this grand idea with so many moving parts and multiple organizations making different chromosomes,’ Boeke’s senior lab coordinator, Katrina Caravelli told Pennisi. But so far, ‘it’s working well.’

The Vision for Custom Yeast

Many yeast biologists are waiting in anticipation for the synthetic yeast genome project to be completed as they are eager to begin testing it themselves. Aside from studying how induced mutations influence function of genes, the way will be open for various applications, such as a new model for human diseases. Boeke, for example, told Pennisi that he ‘would like to install in yeast all of the genes of the molecular pathway that, in humans, is defective in Lesch-Nyhan syndrome, a rare disorder characterized by gout and kidney and neurological problems, including self-mutilating behavior. By modeling the molecular defect in yeast, he hopes to figure out how the mutation affects eukaryotic cells in general.’

On the other side of the equation, Kirsten Benjamin, a synthetic biologist at Amyris Inc., is looking to study what’s wrong with the synthetic strain. ‘We’re going to find a bunch of ways where it doesn’t work,’ she says. This, she says, ‘will allow us as a scientific community to discover all these unappreciated phenomena.’

While the commercial applications for synthetic genomes are still to be determined due to uncertainty around financial feasibility, it’s not the long term viability that Boeke is after. ‘In a way, you can put it as a kind of milestone, like the first human genome was a milestone for genomics,’ he says. ‘When we finish it, we can really plant a flag in it.’

My opinions on this are that the cost for synthesizing DNA oligonucleotides has continually decreased, while innovative methods for up-scaled mass production and stitching together oligos have continually improved. In concert, these will nullify the aforementioned ‘too costly’ argument and enable custom synthesized genomes.

What do you think?

As always, your comments are welcomed.

Children at Risk from Deadly Respiratory Virus EV-D68

  • Frightening Statistics From CDC
  • CDC Updated U.S. Map of Outbreak & Advice of What to be Aware
  • CDC Develops Rapid Real-Time RT-PCR Test for Detection
  • Some Speculate on Linking Outbreak to the “Southern Border Invasion”

Ebola virus is dominating news reports lately, and perhaps rightly so considering the worldwide impact. Turning our attention, however, to actual incidents of infection and death in the U.S., enterovirus (EV) D68 poses a much greater threat and warrants our attention—especially if you or your friends have young children.

On September 24, Eli Waller’s parents were worried that their 4 year-old son had pink eye and kept him home from school so that he wouldn’t infect other children. He seemed otherwise healthy. What happened next was shocking.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

‘He was asymptomatic and fine, and the next morning he had passed,’ said Jeffrey Plunkett, the township’s health officer. ‘The onset was very rapid and very sudden,’ quoted the NY Times.

A week later the Centers for Disease Control and Prevention (CDC) confirmed that Eli had been infected with EV-D68.

EV-D68 was seen as early as August of this year as hospitals in Missouri and Illinois reported increased visits from children with respiratory illness. Soon, the virus was identified in 43 states and detected in 594 patients, 5 of which died.

After reading this very sad—if not frightening—story, I decided to research EV-D68 for this “hot topic” blog, which I’m dedicating to little Eli Waller.

EV-D68 Facts in the U.S. for 2014

Following are facts and related information I’ve selected from the CDC website for EV-D68 that is well worth consulting if you or your friends have any young children.

  • The United States is currently experiencing a nationwide outbreak of EV-D68 associated with severe respiratory illness. From mid-August to October 30, 2014, CDC or state public health laboratories have confirmed a total of 1105 people in 47 states and the District of Columbia with respiratory illness caused by EV-D68.
Activity of enterovirus-D68-like illness in reporting states. Taken from CDC website for EV-D68.

Activity of enterovirus-D68-like illness in reporting states. Taken from CDC website for EV-D68.

  • Every year, various species of EV and rhinovirus (RV) cause millions of respiratory illnesses in children—see my Postscript below for details. This year, EV-D68 has been the most common type of enterovirus identified, leading to increases in illnesses among children and affecting those with asthma most severely.
  • CDC has prioritized testing of specimens from children with severe respiratory illness. There are likely many children affected with milder forms of illness. Of the more than 1,400 specimens tested by the CDC lab, about half have tested positive for EV-D68. About one third have tested positive for an enterovirus or rhinovirus other than EV-D68.
  • As of October 22, EV-D68 has been detected in specimens from seven patients who died and had samples submitted for testing. Investigations are ongoing; CDC reviews and updates available data every Thursday.

Lakeia Lockwood, mother of D’Mari Lockwood receiving treatment, said he was “Struggling to breathe, coughing. [Doctors] said the airways were so tight they actually, in Gary, said I almost lost him.” Taken from wgntv.com via Bing Images.

Lakeia Lockwood, mother of D’Mari Lockwood receiving treatment, said he was “Struggling to breathe, coughing. [Doctors] said the airways were so tight they actually, in Gary, said I almost lost him.” Taken from wgntv.com via Bing Images.

What CDC Is Doing about EV-D68

According to a CDC website for EV-D68, the following are now operative:

  • CDC is continuing to collect information from states and assess the situation to better understand EV-D68, the illness caused by this virus, and how widespread EV-D68 infections may be within each state and the populations affected.
  • CDC is helping states with diagnostic and molecular typing for EV-D68. CDC has obtained one complete genomic sequence and six nearly complete genomic sequences from viruses representing the three known strains of EV-D68 that are causing infection at this time.
  • Comparison of these sequences to sequences from previous years shows they are genetically related to strains of EV-D68 that were detected in previous years in the United States, Europe, and Asia. CDC has submitted the sequences to GenBank to make them available to the scientific community for further testing and analysis.
cold

Researchers at The Genome Institute at Washington University have recently sequenced the genome of EV-D68, which is similar to the germ that causes the common cold, rhinovirus, shown above. This picture, taken from a Bioscience Technology interview with senior author Gregory Storch, who is quoted as saying that ‘Having the DNA sequence of this virus enables additional research. It can be used to create better diagnostic tests. It also may help us understand why this epidemic seems to be producing severe and unusual disease, and why it’s spreading more extensively than in the past.’

CDC Developed a New, Rapid Test for EV-D68 Detection

In an important breakthrough that once again demonstrates the “power of PCR,” CDC has developed a new PCR-based test to quickly detect EV-D68, enabling CDC to process over 2000 specimens. As a result, the number of confirmed EV-D68 cases increased substantially in the past few weeks as CDC began using the test on October 14. About 40% of the specimens have tested positive for EV-D68.

Because EV-D68 is an RNA virus, CDC’s new lab test utilizes reverse transcription polymerase chain reaction (RT-PCR), which offers fast, “real-time” detection. More information about RT-PCR in general is available at TriLink’s webpage for CleanAmp™ One-Step RT-PCR 2X Master Mix.

M. Steve Oberste, chief of the polio and picornavirus laboratory branch at CDC, recently told PCR Insider that until the new CDC test was released on October 14, the agency was detecting the virus using a 10 year-old semi-nested PCR protocol from J. Clin. Microbiol followed by sequencing. CDC’s new test is specific for the VP1 gene of EV-D68, and ‘is a straightforward TaqMan® rRT-PCR test’, Oberste said. The sample prep step remains standard RNA or total nucleic acid extraction.

The lab is currently working on publishing the new method, as well as putting the basic protocol on the CDC website. ‘That way, states and clinical labs can adopt it as [a Laboratory Developed Test (LTD)],’ Oberste said.

Is CDC Hiding EV-D68 Link to Illegal Alien Kids?

At the risk of my being accused of posting politically incorrect comments, the above question is the headline of an online editorial in Investor’s Business Daily on October 17 that, within five days—when I wrote this blog—prompted over 500 comments and more that 7,500 tweets.

I won’t comment on the editorial’s assertion of CDC’s ‘bungling…about the Ebola outbreak’ but I am of the opinion—at least for now—that CDC is probably not ‘hiding’ anything about EV-68. On the other hand, I strongly agree with the editorial’s comment that ‘dispersal of …unaccompanied minors, throughout the U.S. without proper medical screening is an appalling dereliction of duty by…an administration sworn to protect the health and safety of American citizens.’

Let’s hope that the EV-D68 outbreak—regardless of its origin–ends soon, without further loss of children like little Eli Waller.

Postscript

For readers interested in details of microbiology, here is selected information taken from Blomqvist et al. published in J. Clin. Microbiol. (2002).

The family Picornaviridae contains two large and important genera of common human pathogens, Enterovirus and Rhinovirus. In structural and genetic properties, these two genera are very much alike. However, rhinoviruses, which multiply mainly in the nasal epithelium, differ from enteroviruses, infectious agents of the alimentary tract, by their sensitivity to acid and by their growth at a lower optimal temperature. The genus Enterovirus contains 64 serotypes pathogenic to humans, which have been distinguished by the neutralizing antibodies against them. There may still be uncharacterized serotypes, as some clinical enterovirus isolates are not typeable by existing antisera and show genetic segregation indicative of an independent serotype. Nucleotide analysis of the RNA genomes of different human enterovirus (HEV) serotypes has provided new insight into the classification of enteroviruses, resulting in the division of these viruses into four main genetic clusters, designated HEV species A to D. Poliovirus serotypes 1 to 3 are genetically related to HEV-C but are classified as a species of their own. The genus Rhinovirus contains 102 serotypes, which are numbered from 1 to 100. Serotype 1 contains two subtypes, 1A and 1B. More recently, a strain referred to as the Hanks strain has been proposed to represent a new serotype.

Pseudouridine: 2014 Modified Nucleobase of the Year

  • A Minor Modified Nucleobase Playing a Major Role in RNA Therapeutics
  • Nucleobase is “Acrobatically” Formed in RNA by an Enzyme
  • Its Original Identification is Linked to the Atomic Bomb! 

The Molecule of the Year was started in 1989 by Science magazine and in 1996 was changed to ‘Breakthrough of the Year’. After a brief hiatus, the Molecule of the Year designation was revived in 2002 by the International Society for Molecular and Cell Biology and Biotechnology Protocols and Research. If you peruse the list of these lauded molecules, you’ll find that DNA has received this coveted award twice: “PCR and DNA polymerase” (1989) and “DNA repair enzyme” (1994).

While the Molecule of the Year is fascinating and well deserved, I think we should show some love for individual nucleobases given that this blog focuses on “what’s trending in nucleic acid research”—and since TriLink scientists are “The Modified Nucleic Acid Experts”. So, I thought it would be apropos (and fun) to start an annual post on Modified Nucleobase of the Year.

After mulling over which modified nucleobase merits this accolade for 2014, I decided on pseudouridine (aka 5-ribosyluracil)—an unusual isomer of uridine—for several reasons that are given below, followed by some history of its discovery that I found to be quite interesting.

pseudo-u-1st

Pseudouridine-Modified RNA Therapeutics

An earlier post entitled “Modified mRNA Mania” commented on relatively recent research demonstrating an exciting new paradigm in therapeutics based upon in vivo delivery of modified mRNAs to express target proteins. These proteins encoded by modified mRNAs have been applied to vaccines, cellular reprogramming, and other therapeutic modalities. In that post, I go on to indicate how such proof-of-concept publications have quickly spawned several new start-ups and mega-million dollar investments by Big Pharma companies. Although it’s still early days, the future for modified mRNA therapeutics is very promising.

Pseudouridine-5′-triphosphate (Pseudo-UTP) is used to impart desirable mRNA characteristics such as increased nuclease stability, increased translation or altered interaction of innate immune receptors. Pseudo-UTP, along with 5-methylcytidine-5′-triphosphate (5-methyl-CTP) has shown innate immune suppression in cell culture and in vivo, while actually enhancing translation.

For example, Warren et al. determined an efficient means of reprogramming multiple human cell types using modified mRNA that can express the four primary reprogramming proteins. These cells are referred to as induced pluripotent stem cells (iPSCs). Warren et al. found that modified mRNA substituted with Pseudo-UTP, 5-Methyl-CTP and ARCA effectively evaded the cell’s innate immune response, a crucial component in their success. Reduced toxicity due to substitution with Pseudo-UTP, 5-Methyl-CTP was critical since it allowed repeated transfection with modified mRNA over several weeks.

Readers interested in these approaches can access two posters here by TriLink scientists and collaborators.

“Acrobatic” Enzymatic Synthesis of Pseudouridine from Uridine

From the structures of uridine and pseudouridine shown above, you’ll notice that the red-colored nitrogen (N) and a double-bonded carbon in uridine have “switched places” in pseudouridine. Whether and how nature does this switch via one or more enzymes are intriguing questions that have been extensively investigated for many years.

The non-controversial answer to the first question about “whether” this switch occurs naturally is that nature has indeed evolved an enzyme for isomerizing uridine to pseudouridine that is named—appropriately—pseudouridine synthase. The second question about “how” is less clear, if not controversial, to some. Here’s why:

Pseudouridine synthase achieves this “acrobatic” positional switch on uridine that is already incorporated in RNA, as opposed to during biosynthesis of monomeric uridine prior to RNA synthesis. Since only a small number of uridines in RNA are converted to pseudouridine, understanding which ones are isomerized has been investigated. Readers interested in this can consult a recent report by Sibert & Patton entitled “Pseudouridine synthase 1: a site-specific synthase without strict sequence recognition requirements.”

The controversial aspect of “how” deals not with sequence context but mechanistic details, which Santi and coworkers in 1999 proposed as follows, based on elegant substitution of hydrogen (H) with fluorine (F) at the C-5 ring position—BTW, my apologies for the organic chemistry “arrow pushing” but it’s a necessary evil to put up with to show the “how”. 

onthe

On the other hand, subsequent work by Spedaliere et al. has led to questioning whether a different mechanism is operative. This is explored in detail  in a publication entitled The pseudouridine synthases: revisiting a mechanism that seemed settled.” Readers interested in this alternative view are encouraged to read that publication.

Pseudouridine-Modified Oligonucleotides

I hope this brief overview has spurred you to learn about the amazing things you can do with pseudouridine. Companies like TriLink offer custom chemical synthesis of pseudouridine-modified oligonucleotides that can be used for a wide variety of physicochemical, biochemical, or biological studies. Among biological applications, RNA interference (RNAi) using short, double-stranded RNA (aka siRNA) is especially important and widely employed. RNAi has been expertly reviewed by Beal & Burrows et al. with regard to incorporation of modified bases to probe and enhance RNAi.

Highlighted in the Beal & Burows review are comprehensive investigations by Nawrot and coworkers, who incorporated pseudouridine, as well as other modified nucleobases, at various positions to evaluate thermodynamic stability and gene silencing activity. As detailed in those results, certain locations for pseudouridine provided potent gene-silencing activity. I think you’ll see that these publications and reviews support my claim that pseudouridine is truly deserving of Modified Nucleobase of the Year status.

Fractionating Fractions and Chasing Spots

While I believe I’ve provided significant justification for my obesssion with pseudouridine, I understand that some of you may not be convinced. If the current range of potential uses and pivotal roles of this facscinating nucleobase haven’t been compelling enough, perhaps I’ll make you a convert when you learn more about this nucleobase’s origin.

Several groups of investigators in the 1950s were engaged in identifying minor constituents of RNA by developing then state-of-the-art chromatographic methods to separate hydrolyzed RNA into its components. Those of you who may not be familiar with column chromatography or paper chromatography simply need to know that eluted (aka separated) fractions are collected and further fractionated, or paper spots are cut out, followed by analyses to determine purity and identity. Sounds easy, but it’s not—especially in the 1950s.

Column chromatography in the 1950s (taken from Wikipedia)

Column chromatography in the 1950s (taken from Wikipedia)

What these early investigators actually did for “fractionating fractions and chasing spots” of minor RNA constituents is way too complicated to get into here. So, I’ve taken the liberty to focus on the following work of Waldo E. Cohn, who has the distinction of being the first to definitively identify the very unusual structure of pseudouridine, and is associated with its naming.

Waldo E. Cohn:  From the Atomic Bomb to Pseudouridine

In researching Cohn’s work leading up to identifying and naming pseudouridine, I learned about how his earlier involvement with the ultra-secret Manhattan Project to develop the first atomic bomb led to pioneering investigations of radioactive 32P biodistribution in rats! Here’s a brief excerpt on that taken from Simoni et al., which I’ll tie back to Cohn’s pseudouridine story.

Dr. Ralph Overman uses a probe counter to check the shoes of Dr. Waldo E. Cohn for radioactive contamination on June 14, 1946 in Oak Ridge, Tennessee (AP Photo/Clinton Labs; taken from snippits-and-slappits.blogspot.com via Bing Images).

Dr. Ralph Overman uses a probe counter to check the shoes of Dr. Waldo E. Cohn for radioactive contamination on June 14, 1946 in Oak Ridge, Tennessee (AP Photo/Clinton Labs; taken from snippits-and-slappits.blogspot.com via Bing Images).

By the mid-1930s, the cyclotron had been invented and developed at the University of California at Berkeley by Ernest O. Lawrence. With it came the capability of creating artificial radioactive isotopes. One of the first radioactive isotopes produced was 32P, which was immediately put to use in seeking solutions to biological problems.

Waldo E. Cohn was a graduate student at University of California Berkeley working with his thesis advisor Prof. David M. Greenberg. The 32P was prepared in the cyclotron of the Radiation Laboratory at Berkeley by bombardment of red phosphorus with deuterons. After purification and oxidation to H3PO4, the radioactive material was administered to rats, and the distribution of radioactivity in various tissues was followed with time, which you can read about here and is considered a “JBC Classic”.

Cohn’s experiences with radioactive isotopes as a graduate student shaped his career. After receiving his Ph.D. degree in Biochemistry from Berkeley in 1938, and a postdoc at Harvard, he was recruited to the Metallurgical Laboratory of the University of Chicago in 1942 as a member of the Manhattan Project. His role was to study the biological effects of fission products on biological systems. Cohn moved to the Oak Ridge National Laboratory (ORNL) where he became Senior Chemist and Group Leader of the Oak Ridge Biology Division, a position that he held until his retirement in 1975.

Cohn was involved in the formation of the Isotope Distribution Committee, which was assigned the responsibility of making isotopes available to qualified researchers. In 1946, Cohn and Paul Aebersold published a landmark paper in Science that established the administration of isotope distribution and preparation of the catalog of isotopes that were available for research. It was one of the first post-war efforts toward the “peaceful use” of atomic energy. Cohn became well known for his efforts to establish and enforce standardized biochemical nomenclature. From 1965 to 1976, he was Director of the Office of Biochemical Nomenclature of the National Academy of Sciences.

Now back to pseudouridine!

The Right Scientist at the Right Place

Cohn’s experience with radioisotopes coupled with his expertise in nucleic acid-related chemistry and the unique resources offered by ORNL added up—in my opinion—to him being the right scientist at the right place to doggedly pursue identification of minor constituents of RNA.

Here are actual excerpts from his key publications on this hunt.

note note2

In concluding this post, I’d like to add one more historical “factoid” that I thought was interesting. I tracked down the identity of “Dr. A Michelson.” He is actually A. M. Michelson who coauthored with über-famous Sir Alexander Todd the first synthesis of ATP published in Nature in 1948. My thanks to Drs. Cohn and Michelson for their respective roles in giving “birth” to pseudouridine, which in turn has enabled development of new molecular tools for nucleic acid-based applications.

As we look at the historical significance as well as modern applications of pseudouridine, I hope I’ve convinced you of the amazing work this nucleobase is capable of. Let me know if you support my nomination for Modified Nucleobase of the Year, or share your nominations for other nucleobases below.

Reflections on Advances in Medicinal Oligonucleotides

  • Oligos Are Not “Magic Bullets”
  • Oligos Have, Nevertheless, Enabled New Drug Paradigms
  • Oligos Continue to Attract Significant Corporate Investments

Reflections

The 10th Annual Meeting of the Oligonucelotide Therapeutics Society (OTS) is in full swing today in San Diego, CA where it began on Oct 12 and concludes on Oct 15. Having worked on antisense oligos since the early days (~30 years ago) participating in this meeting led me to several thoughts that I wish to share with you in this post.

First of all, contrary to many sceptics in those early days, the concept of using synthetic oligonucleotides as an entirely new class of medicinal agents has not only survived but also greatly expanded in terms of the biological target/mechanism of action and types of oligo constructs used—each with a seemingly endless array of chemical modifications to evaluate. In approximate chronological order of discovery, these targets and the types of oligos they have now come to include are listed below coincidentally, most of these are represented in the 2014 OTS agenda.

Target
mRNA
dsDNA
transcription factors
immunostimulation
proteins
splice junctions
RNA interference
Oligo Type
antisense
triplex
dsDNA decoys
CpG
aptamers
blockers
siRNA

 

 

 

 

 

Secondly, while it has been possible for oligo chemists to design and synthesize a plethora of modified oligos to achieve optimized nuclease stability, binding to target, etc., efficient delivery has remained the single most challenging problem to deal with. In talks on medicinal oligos, this situation is oftentimes eluded to as something to the effect of “there are only three remaining problems to solve: delivery, delivery, and delivery.”

Lastly, contrary to early hopes of being Dr. Ehrlich’s “magic bullets” (see caption below), oligos didn’t quite prove to be the new paradigm for a speedy concept-to-clinic solution. As all of us in the oligo world know, oligo-based therapeutics have encountered long and costly R&D timelines and clinical development paths typical of all other classes of therapeutic compounds.

magic

Dr. Ehrlich’s Magic Bullet is a 1940 biographical film starring Edward G. Robinson, based on the true story of the German doctor and scientist Dr. Paul Ehrlich (14 March 1854 – 20 August 1915), who worked in the fields of hematology, immunology, and chemotherapy. His laboratory discovered arsphenamine, a synthetic arsenic-containing compound that provided the first effective medicinal treatment for syphilis, thereby initiating and also naming the concept of chemotherapy. Ehrlich popularized the concept of a “magic bullet”. In 1908, he received the Nobel Prize in Physiology or Medicine for his contributions to immunology.

Notable Advances

Given the aforementioned breadth of therapeutic targets being pursued with oligos, selecting notable advances for comment here is necessarily limited to only a few, and is obviously highly subjective. Having said that—and with apologies for not highlighting other important advances—here are my favorites.

Ebola Drug – As reported in a Scientific American story, Thomas W. Geisbert, now at the University of Texas Medical Branch at Galveston, and his many collaborators have devised a highly promising treatment that has saved the lives of six monkeys infected with Ebola virus—the causative agent of the current Ebola disease outbreak recently commented on in a previous post. One of Geisbert’s collaborators, Ian MacLachlan of Burnaby, British Columbia–based Tekmira Pharmaceuticals, and his team have received a $140-million grant from the U.S. Department of Defense to develop the therapy further.

Working together, the scientists engineered an siRNA to prevent the Ebola virus from making a particular protein, without which it cannot replicate itself. “If you knock out that one, in theory you knock out everything,” Geisbert says. The researchers also designed another siRNA to thwart manufacture of a second protein that the virus uses to weaken an infected individual’s immune system. There is no danger of the siRNAs interfering with typical cellular duties because the targeted viral proteins do not exist in the cells of humans or other mammals.

MacLachlan and his colleagues encapsulated the lab-made siRNAs in Tekmira’s proprietary lipid nanoparticle (LNP) formulation to enable vascular delivery and transport across cellular membranes.

Simplified cartoon of Tekmira’s lipid nanoparticles (LNPs) formulations of siRNA (taken from an interview with Ian MacLachlan in GEN via Bing Images).

Simplified cartoon of Tekmira’s lipid nanoparticles (LNPs) formulations of siRNA (taken from an interview with Ian MacLachlan in GEN via Bing Images).

Then they injected the preparation into several rhesus macaques, which had been infected with Ebola virus less than an hour earlier. In one study, two of three monkeys given a total of four doses of the treatment in the first week after exposure survived. In a second study designed to test the effectiveness of a higher dose, all four monkeys that received seven siRNA injections lived. Tests revealed that the treated monkeys had far fewer virus molecules in their blood than is typical for an infected animal. The macaques tolerated the siRNA injections well, and those that survived were still healthy 30 days later.

The study was a “milestone,” says Gary Kobinger of the University of Manitoba, who is working on a different Ebola treatment based on antibodies. He believes Geisbert and his team “are leading the effort toward clinical development.”

Exon Skipping – This novel strategy is used to restore the reading frame within a gene. Exons are the sections of DNA that contain the instruction set for generating a protein; they are interspersed with non-coding regions called introns. The introns are later removed before the protein is made, leaving only the coding exon regions.

Splicing naturally occurs in pre-mRNA when introns are being removed to form mature-mRNA that consists solely of exons. The mechanism behind exon skipping is a mutation specific antisense oligonucleotide (AON). The AON binds to the mutated exon, so that when the gene is then translated from the mature mRNA, it is “skipped” over, thus restoring the disrupted reading frame. This allows for the generation of an internally deleted, but largely functional protein.

Shown below is a schematic diagram of how this applies to the AON named Drisapersen, developed by Prosensa and currently in late stage development for treating Duchenne Muscular Dystrophy (DMD) patients. Drisapersen induces exon 51 skipping in the dystrophin gene and is intended for ~13% of all DMA patients. On June 3, 2014, Prosensa announced that the United States Food and Drug Administration (FDA) outlined a regulatory path forward for drisapersen, under an accelerated approval pathway, based upon existing data.

Drisapersen binds to exon 51 in pre-mRNA leading to an in-frame mRNA transcript that produces a shortened but functional protein like that in Becker muscular dystrophy (BMD), which is a less form of muscular dystrophy.

Drisapersen binds to exon 51 in pre-mRNA leading to an in-frame mRNA transcript that produces a shortened but functional protein like that in Becker muscular dystrophy (BMD), which is a less severe form of Duchenne muscular dystrophy.

Notable Corporate Investments

Santaris Pharma – According to an Aug 4, 2014 press release, Roche announced that it has agreed to acquire Santaris Pharma based near Copenhagen, Denmark. Santaris Pharma has pioneered its proprietary Locked Nucleic Acid (LNA) platform that has contributed to an emerging era of RNA-targeting therapeutics.

Roche plans to maintain Santaris Pharma’s operations in Denmark, where the existing site will be renamed Roche Innovation Center Copenhagen. Under the terms of the agreement, Roche will make an upfront cash payment of $250 million to Santaris Pharma shareholders and make additional contingent payments of up to $200 million based on the achievement of certain predetermined milestones.

Isis Pharmaceuticals – As detailed in multiple press reports here, Isis Pharmaceuticals has announced that it has earned three $1 million payments for advancement of each of three programs with GlaxoSmithKline (GSK), a $10 million milestone payment from Biogen-Idec, and a $15 million milestone payment from AstraZeneca.

Isarna Therapeutics – It was announced earlier this year that Isarna Therapeutics GmbH raised $7.6 million from its current investor groups to further its TGF-ß therapeutics using oligos.

In closing, my reflections turned to thinking that, while it has taken more time and money than originally envisaged by early proponents of medicinal oligos, the pipelines are full and investment is strong.

All of us in the oligo therapeutics arena are greatly interested in hearing about what you are doing with medicinal oligos, so please feel free to share in the comments section below. Also, if you’re at OTS stop by the TriLink booth or look for me during the talks, I’d love to meet you face-to-face.

#OligoMeeting2014

October is Down Syndrome Awareness Month

Down syndrome remains the most commonly diagnosed chromosomal condition with approximately 6,000 afflicted babies born in the U.S. each year. This means that Down syndrome occurs in about 1 out of every 700 babies.

In recognition of Down Syndrome Awareness Month, this post provides information about Down syndrome taken from the U.S. Centers for Disease Control (CDC) and Prevention website. It also provides a personal story about John Nguyen, a coworker at TriLink, whose first born son, Jordan, has Down syndrome. I’d like to first introduce the Nguyen’s story and then continue on with an overview of Down syndrome causes, risk factors and diagnosis.

The Nguyen Family

When John and his wife, Jessica heard, “Your child may have Down syndrome,” they, like most parents who receive this news, were saddened and confused. What did this mean? Will he or she be healthy? What do we do next? So many questions and emotions shook up their world in a way they never expected.

baby boyWhen they welcomed Jordan into the world, his condition was still unknown and his heart needed to be monitored so they were only able to hold him for a few seconds before he was whisked away. But that’s all they needed to know they were a family and they were in this together. Jordan spent 10 days in the NICU. In addition to having Down syndrome, he was also diagnosed with a congenital heart condition called Ebstein’s anomaly.

Jessica said, “Although it was difficult in the beginning, we quickly realized how lucky we were and how amazing he is. His heart condition remains stable. Throughout this journey we have remained positive and refuse to let his diagnoses damper or put a limit to what Jordan can accomplish and achieve… The route to parenting we thought we were going to take was detoured and I consider it a blessing. We now get to appreciate things we never previously considered. We have met people we never would have met otherwise. And now know a love so deep that we never could have imagined. Love not only for and from our son but also from the love and support of our family, friends, and community. Those questions and emotions in the beginning really shook up our world….but in the best way possible.”

boyJohn and Jessica actively fundraise for the Down Syndrome Association of San Diego, whose mission is to enhance the quality of life for people with Down syndrome and their families through education, social and support programs. They participate in the Buddy Walk® each year.

TriLink is donating $5* to the Nguyen’s Buddy Walk® team for each share this post receives. Help us spread awareness about this common and often misunderstood condition.

Types of Down Syndrome  

Down syndrome, which was first characterized by English physician John Langdon Down in 1862, is a condition in which a person has an extra chromosome. Typically, a baby is born with 46 chromosomes—23 from both mother and father. Babies with Down syndrome have an extra copy of one of these chromosomes, chromosome 21. A medical term for having an extra copy of a chromosome is ‘trisomy.’ Down syndrome is also referred to as Trisomy 21. This extra copy changes how the baby’s body and brain develop, which can cause both mental and physical challenges for the baby.

Taken from http://theoneinathousand.blogspot.com/2012/10/october-is-down-syndrome-awareness-month.html

Taken from http://theoneinathousand.blogspot.com/2012/10/october-is-down-syndrome-awareness-month.html

There are three types of Down syndrome. People often can’t tell the difference between each type without looking at the chromosomes because the physical features and behaviors are similar.

  • Trisomy 21: ~95% of people with Down syndrome have Trisomy 21, wherein each cell in the body has 3 separate copies of chromosome 21 instead of the usual 2 copies.
  • Translocation Down syndrome: This type accounts for ~3% of people with Down syndrome. This occurs when an extra part or a whole extra chromosome 21 is present, but it is attached or “trans-located” to a different chromosome rather than being a separate chromosome 21. 
  • Mosaic Down syndrome: This type affects ~2% of the people with Down syndrome, wherein some of their cells have 3 copies of chromosome 21, but other cells have the typical two copies of chromosome 21.

Causes and Risk Factors

Researchers know that Down syndrome is caused by an extra chromosome, but no one knows for sure why Down syndrome occurs or how many different factors play a role. One factor that increases the risk for having a baby with Down syndrome is the mother’s age. Women who are 35 years or older when they become pregnant are more likely to have a pregnancy affected by Down syndrome than women who become pregnant at a younger age. However, the majority of babies with Down syndrome are born to mothers less than 35 years old, because there are many more births among younger women.

Diagnosis

Doctors can diagnose Down syndrome during pregnancy or after the baby is born. Some families want to know during pregnancy whether their baby has Down syndrome. Diagnosis of Down syndrome during pregnancy can allow parents and families to prepare for their baby’s special needs. 

Screening for Down syndrome

Screening tests often include a combination of a blood test, which measures the amount of various substances in the mother’s blood (e.g., MS-AFP, Triple Screen, Quad-screen), and an ultrasound, which creates a picture of the baby. During an ultrasound, one of the things the technician looks at is the fluid behind the baby’s neck. Extra fluid in this region could indicate a genetic problem. These screening tests can help determine the baby’s risk of Down syndrome. Rarely, screening tests can give an abnormal result even when there is nothing wrong with the baby. Sometimes, the test results are normal and yet they miss a problem that does exist.

A relatively new test used since 2010 to detect certain chromosome problems including Down syndrome, screens for small pieces of the developing baby’s DNA that are circulating in the mother’s blood. This test is recommended for women who are more likely to have a pregnancy affected by Down syndrome, especially mothers over the age of 35. The test is typically completed during the first 3 months of pregnancy, and it is becoming more widely available from Sequenom, Natera, and other companies.

Following a positive screening test, diagnostic tests are usually performed in order to confirm a Down syndrome diagnosis. Types of diagnostic tests include:

  • Chorionic villus sampling (CVS)—examines material from the placenta
  • Amniocentesis—examines the amniotic fluid (the fluid from the sac surrounding the baby)
  • Percutaneous umbilical blood sampling (PUBS)—examines blood from the umbilical cord

These tests look for changes in the chromosomes that would indicate a Down syndrome diagnosis.

What to expect after birth

Most pregnancies involving Down syndrome are uncomplicated and proceed normally. Once a baby with down syndrome is born, however, he/she should receive an echocardiogram (an ultrasound picture of the heart) because almost half of all babies born with Down syndrome have a heart defect. The doctor will usually order tests of the baby’s blood to confirm whether there is an extra chromosome (chromosome 21) in the baby’s cells and make an official diagnosis.

Living with Down syndrome

Down syndrome is a lifelong condition. Services early in life will often help babies with Down syndrome improve their physical and intellectual abilities. Most of these services focus on helping children with Down syndrome develop to their full potential and include speech, occupational, and physical therapy. These services are offered through early intervention programs in each state. Children with Down syndrome may also need extra help or attention in school, although many children are included in regular classes.

In addition to state-run programs, families of Down syndrome children often find support and resources by connecting with one another and joining national organizations such as National Down Syndrome Society and National Association for Down Syndrome. Several campaigns including Buddy Walk® and Down Syndrome Awareness Month have taken shape over the past few decades to raise awareness of and acceptance for those with Down syndrome. It is important for everyone to understand that people with Down syndrome can lead productive and fulfilling lives well into adulthood. They hold jobs, live independently and contribute to their communities.

Share this blog and raise money for Down syndrome research

In support of Down Syndrome Awareness Month, I hope you’ll take a moment to find out more about Down syndrome, or better yet to support a fundraising or awareness effort in your community. I personally invite you to share this blog to help raise money* for Down syndrome.

Thanks to all of our loyal readers, we reached our initial goal and raised $1,500 for the Buddy Walk® in less than 8 hours! But we’re not stopping there, we’ll donate an additional $1* for every new share we receive.

* up to $2,500.

Hot Topic in Oligonucleotide Therapeutics: Nanomedicine

  • What is it?
  • Why is it Hot?
  • How do Nucleic Acids Fit in?

The upcoming Oligonucleotide Therapeutics Society (OTS) annual meeting on Oct 12-15 in San Diego has an agenda with a number of presentations that could each qualify as a hot topic to highlight in this blog post. While mulling over which to pick, the talk by Prof. Weihong Tan—an extraordinarily prolific researcher at the University of Florida—entitled DNA-based molecular medicine and nanomedicine triggered my decision to comment on nanomedicine because it’s at the nexus of multiple scientific disciplines and various aspects of medicine.

What is Nanomedicine?

Nanomedicine, like almost everything these days, has a wiki site; however, it simply defines nanomedicine as “the medical application of nanotechnology,” which begs questions of what’s nanotechnology and what are some of these applications? So, I dug deeper and found—to my surprise—that there is no widely agreed upon definition of nanomedicine!

Instead, several related but different definitions of nanomedicine have been adopted by groups of expert researchers that actually practice nanomedicine. There’s a scholarly editorial in the International Journal of Nanomedicine (IJM) that addresses this curious circumstance. At the risk of “losing you” by being pedantic, here are several snippets that were my key takeaways from this article:

  • “Although defining a term such as nanomedicine may sound simple, by comparing several main funding agencies from around the world, one quickly realizes that a uniform international definition of nanomedicine does not currently exist. This is typical of a new field, but can be problematic to those trying to understand the field, make significant contributions to it, and especially in how the public views nanomedicine.”
  • “[In NIH’s 2006] Roadmap for Medical Research in Nanomedicine, [it] is defined as ‘an offshoot of nanotechnology [that] refers to highly specific medical interventions at the molecular scale for curing disease or repairing damaged tissues, such as bone, muscle, or nerve’.”
  • “[N]anomedicine emerged from nanotechnology which is generally defined by the creation and use of materials at the level of molecules and atoms (sometimes specifically less than 100 nm, other times this dimension is more diffuse and confusing).”

The last point resonated with me, and prompted a search for a picture that puts nanometer dimensions into perspective relative to familiar objects. The scale shown below nicely achieves that and highlights the 10-100 nm range applicable to representative nanodevices used in nanomedicine.

Nanometer dimensions of common objects (taken from piterest.com via Bing Images).

Nanometer dimensions of common objects (taken from piterest.com via Bing Images).

In closing this section, it’s worth pointing to two authoritative sources of up-to-date information on nanomedicine. First is the NIH website dedicated to nanomedicine, and second is the American Society for Nanomedicine (ASN) website. BTW, the current president of ASN is Georgetown University Prof. Esther Chang, whose cell-targeted nanoparticle delivery investigations were featured here earlier this year in a post entitled Nanomedicine: Using ‘Tiny Little FedEx Trucks’ to Target Breast Cancer Tumors.

Why is Nanomedicine Hot?

Before offering my personal perspectives on why nanomedicine is hot, perhaps I should first convince you that it is hot. For instance, entering a general search of PubMed using the word nanomedicine found over 8,000 publications since 1999, with the number of nanomedicine-related publications growing exponentially since 2006—coinciding with the aforementioned NIH Roadmap’s funding. These articles reached an impressive rate of ~4 publications per day in 2012.

This intense interest in nanomedicine over that short period is underscored by a similar search of Google Scholar that found over 97,000 items, while that for Google Patents found nearly 24,000 items.

As for why nanomedicine is hot, let me first echo what I said in the introduction above, i.e., nanomedicine is at the nexus of multiple scientific disciplines and various aspects of medicine. Given this circumstance, nanomedicine as an application is exceptionally diverse and, hence, powerful. This potential power, which can enable major advances having medical significance, is emphasized in the concluding statement taken from the IJM editorial mentioned above:

“IJN takes a firm stance…and emphasizes nanomedicine research in which significantly changed medical events are elucidated only by concentrating on nanoscale events. In this respect, our attempt to separate nanomedicine from other traditional medical research fields is a focus on significantly changed medically related events that result by concentrating solely on the nanoscale.”

How do Nucleic Acids Fit in?

I’m neither clairvoyant nor privy to Prof. Tan’s upcoming lecture at OTS, but I’ve used the illustration below to point out a prime example of the kind of application his talk will likely refer to: tumor-specific, nanoparticle-encapsulated, drug delivery enabled by nucleic acid aptamers. This therapeutic application of aptamers—and its “chemical cousin” wherein imaging agents are delivered—can be read about in two earlier posts from October and November of last year. In brief, nucleic acids provide 3D-structure for aptamers to allow specific bonding to “molecular markers” on target cells or tissue.

Targeted nanoparticles for imaging and therapy (taken from medicalnewstoday.com via Bing Images).

Targeted nanoparticles for imaging and therapy (taken from medicalnewstoday.com via Bing Images).

More futuristic applications of DNA in nanomedicine are predicated on predictable “self-assembly” of nano-scale structures via base pairing to achieve a functional state. This remarkably powerful attribute is unique to nucleic acids. This is elaborated on in the following abstract of a review by Chhabra et al. entitled DNA Self-assembly for Nanomedicine:

“Self-assembling DNA nanostructures based on rationally designed DNA branch junction molecules have recently led to the construction of patterned supramolecular structures with increased complexities. An intrinsic value of DNA tiles and patterns lies in their utility as molecular pegboard for deterministic positioning of molecules or particles with accurate distance and architectural control. This review will discuss the state-of-art developments in self-assembled DNA nanostructural system. Biomedical aspects of information guided DNA nanostructures will also be summarized. We illustrate both the use of simple DNA artworks for sensing, computation, drug delivery and the application of more complex DNA architectures as scaffolds for the construction of protein and nanoparticle arrays.” 

The Future is Now!

You may be thinking that achieving self-assembling DNA nanostructures for practicing nanomedicine is decades away and, even if doable then, it will be limited to only a handful of experts. Parabon NanoLabs, however, is proving that may not be true.

Parabon NanoLabs, with the support of an NSF Technology Enhancement for Commercial Partnerships grant, is using a simple “drag-and-drop” computer interface and DNA self-assembly techniques, to develop a new automated method of drug development that could reduce the time required to create and test medications.

“We can now ‘print,’ molecule by molecule, exactly the compound that we want,” says Steven Armentrout, the principal investigator on the NSF grants and co-developer of Parabon’s technology, according to an online interview. “What differentiates our nanotechnology from others is our ability to rapidly, and precisely, specify the placement of every atom in a compound that we design.”

The Parabon Essemblix Drug Development Platform combines computer-aided design (CAD) software with nanoscale fabrication technology, developed in partnership with Janssen Research & Development, LLC, part of the Janssen Pharmaceutical Companies of Johnson & Johnson.

Scientists can use the CAD software to design molecular pieces with specific, functional components for development of a new drug-delivery agent, as depicted below. The software then optimizes the design using a cloud supercomputing platform that uses proprietary algorithms to search for specific sets of DNA sequences that can self-assemble those components.

Parabon Essemblix process (credit: Parabon NanoLabs)

Parabon Essemblix process (credit: Parabon NanoLabs)

With the resulting sequences, the scientists chemically synthesize trillions of identical copies of the designed molecules. The process, from conception to production, can be performed in weeks, or even days — much faster than traditional drug discovery techniques that rely on trial and error for screening potentially useful compounds, the company says.

Parabon R&D scientists also claim that in vivo experiments, funded by an NSF SBIR award, have validated this approach to faster, more targeted rational drug design, which—as the saying goes—is a good thing.

Frankly, I’m amazed and truly excited about this advance in nanomedicine made possible by oligo base-pairing rules! I also look forward to hearing Prof. Tan’s talk at OTS and finding out more about the latest developments in nanotechnology. If you’re attending OTS, please look for me at the talks as I’d really like to meet you!

As always, your comments are welcomed.

Practical Applications of DNA Testing

  • Italy is Keeping Cities Beautiful by DNA Testing Dog-Droppings
  • Fines Issued to Owners who Don’t Pick Up
  • Smart Idea or Story-plot for an Italian Comic Opera?

As you know, I like to explore all things related to nucleic acids. In this post, I take a little respite from scientific analysis and instead explore an interesting real-life application for DNA testing. As summer comes to an end and we’ve all returned from our recent holidays, this post provides a tie-in to Naples, which is both a great vacation destination and a place where dog-droppings are a controversial topic. Sound intriguing? Read on.

Naples’ historic city center, which is listed by UNESCO as a World Heritage Site, is the largest in Europe, covering 4,200 acres and enclosing 27 centuries (!) of history. This historic city is leading the way in employing modern technology to keep its streets clean. Naples is the first metropolitan city to use “CSI-style” DNA forensics to rid its sidewalks of dog poop and impose stiff fines to those who let Fido leave his DNA behind. It’s a story that strangely juxtaposes quaint European old-city charm with an interesting mix of science and societal topics to evoke a wide spectrum of opinions.

By the way, in researching Naples, I learned that it has long been a major cultural center with a global sphere of influence, particularly during the Renaissance and Enlightenment eras. In the immediate vicinity of Naples are numerous culturally and historically significant sites, including the Palace of Caserta and the Roman ruins of Pompeii and Herculaneum. Naples is synonymous with pizza, which originated in the city as Neapolitan flatbread and migrated to America with the Italians. Neapolitan music has furthermore been highly influential, credited with the invention of the romantic guitar and the mandolin, as well as notable contributions to opera.

Good Idea or Waste of a City’s Resources?

Despite its glorious past, the city is not without it’s slew of current problems. Tommaso Sodano, the vice mayor of Naples, recently acknowledged in an interview that the city is facing many challenges related to huge debt, unfunded service agencies, wide-spread organized crime and general filth related to illegal dumps and dog droppings…yes, poop.

The city administration is positioning Naples ‘at the cutting edge of dog-waste eradication’. The initiative takes DNA samples of dogs to create a database used to identify and fine owners who leave their dog’s poop on city streets.

“I know some people find it funny,” Mr. Sodano is quoted as saying, smiling, “that with all the problems the city has, we would focus on dog poop. I know that.”

Via Toledo is Naples’s principal shopping street and a “must do” for tourists (taken from Wikipedia)

Via Toledo is Naples’s principal shopping street and a “must do” for tourists (taken from Wikipedia)

While it may sound a bit humerous at first, it’s an issue that is wide-spread and difficult to control as there are some 80,000 dogs in Naples. While other cities have tried everything from mailing dog poop back to its owner to publicly shaming offenders, Naples is pursuing a more modern approach. Blood samples will be taken from every dog in the city and used to create a database matched with contact information for the dog’s owner. ‘When an offending pile is discovered, it will be scraped up and subjected to DNA testing. If a match is made in the database, the owner will face a fine of up to 500 euros, or about $685.’ The program is in its infancy, but officials say it’s already making an impact on Via Toledo and surrounding areas where it’s being piloted.

Veterinary workers in Naples drew blood from Fiona, a pit bull, for the DNA database. Credit Gianni Cipriano for the NY Times.

Veterinary workers in Naples drew blood from Fiona, a pit bull, for the DNA database. Credit Gianni Cipriano for the NY Times.

Many residents and officials are skeptical that the program will work given the city’s existing challenges with basic services like garbage collection and sewage. Others disagree with the program’s expenditures in light of the city’s mounting debt. Sodano remains committed to the program saying “The main goal is respect for the rules.” He’s quick to add that other mounting issues shouldn’t prevent city administration from keeping Naples beautiful. “Governing Naples,” he said, “certainly requires a sparkle of madness.”

What’s the DNA-Based ID Used?

I tried, unsuccessfully, to find out what DNA-based identity testing is being used in Naples. In researching this, however, I did find the abstract of a relatively recent publication that suggests to me that Naples may be analyzing mitochondrial DNA (mtDNA), analogous to TriLink’s forensic products (mitoPrimers™) for human identity testing using PCR-sequencing.

According to this abstract, the researchers sequenced the entire ∼16 kb canine mtDNA genome of 100 unrelated domestic dogs (Canis lupus familiaris) and compared these to 246 published sequences to assess hypervariable region I (HVI) haplotype frequencies. They then used all available sequences to identify informative single nucleotide polymorphisms (SNPs) outside of the control region sequence—identical in all dogs—for use in further resolving mtDNA haplotypes corresponding to common HVI haplotypes. They identified a total of 71 informative SNPs that they concluded are “useful forensic tools to further resolve the identity of individual dogs from mitochondrial DNA (mtDNA).”

Even though we don’t know the exact details of the testing being used in Naples, I wrote this post because I find the practical applications of DNA-based testing to be fascinating. If you know of other real-life applications, please share them in the comments section below.

ALS Ice Bucket Challenge: What You May Not Know but Should

  • The Story of How this Challenge Began
  • How Baseball and the “Big Bang” are Connected to this Challenge
  • How a Dearth of Genetic Understanding Led to Crowdfunding ALS Research
  • The First Drugs May be on the Horizon

How It Began

For those very few of you who have for some reason been disconnected from mainstream and social media, the ALS Ice Bucket Challenge involves dumping a bucket of ice water on someone’s head to promote awareness of the disease amyotrophic lateral sclerosis (ALS) and encourage donations—typically $100—to research ALS. The challenge dares nominated participants to be recorded having a bucket of ice water poured on their heads, and challenging others to do the same. A common stipulation is that nominated people have 24 hours to comply or forfeit by way of a charitable financial donation.

Those of you already familiar with the ALS Bucket Challenge may, however, not know that it was first issued by golfer Chris Kennedy to his cousin Jeanette Senerchia of Pelham, New York, whose husband, Anthony, has had ALS for 11 years.

Anthony and Jeanette Senerchia of Pelham and their daughter Taya, 6, watch as local politicians participate in the ice bucket challenge, July 25, 2014 outside Pelham (NY) Town Hall. Photo: Tania Savayan; taken from lohud.com via Bing Images.

Anthony and Jeanette Senerchia of Pelham and their daughter Taya, 6, watch as local politicians participate in the ice bucket challenge, July 25, 2014 outside Pelham (NY) Town Hall. Photo: Tania Savayan; taken from lohud.com via Bing Images.

According to a local newspaper story, Jeanette Senerchia, 40, said “I was going to donate,” but they were really relentless with their texts counting down the time.” When she finally poured icy water on herself, having her 6-year-old daughter Taya record it, she posted the video on Facebook and “it went crazy,” she said. In a little more than a week, 1,000 people across the country, in Canada, Europe and Japan had joined in.

Soon the challenge circled back to Pelham, where Town Supervisor Pete DiPaola took a dousing, along with Town Councilman Timothy Case and Court Clerk Fran Ardisi. State Assemblywoman Amy Paulin and Westchester County Legislator Jim Maisano also got in on the act.

Girl Scouts from Troop 1662 did the honors in front of Town Hall, pouring the water from giant, 5 gallon Home Depot buckets baring the words “Let’s DO this.”

Photo taken from lohud.com.

Photo taken from lohud.com.

In drought-stricken regions a cool—pun intended—variant has been proposed as seen in this video wherein San Luis Obispo, California photographer Brittany App—yes, it’s really her last name—has a compromise: instead of dumping a bucket of ice water on one’s head, she is challenging people to live on only 5 gallons of water for a day.

BTW, lest we get caught up in all the fun associated the ALS Ice Bucket Challenge, keep in mind that ALS currently has no cure and affects an estimated 350,000 individuals around the globe, killing more than 100,000 annually. The disease can impact anyone, anywhere, regardless of age, ethnicity, or socioeconomic background.

Famous Persons with ALS

While I will echo the last sentences again, it’s apparent that society in general has a fascination—for lack of a better word—in knowing which famous persons have reached a certain age, or married, or passed away, or become afflicted with a disease. In the case of ALS, a website lists a number of such persons among which the most famous are American baseball great Lou Gehrig and British theoretical physicist Stephen Hawking. Captions for their photos below provide further information about each of these two “professional polar opposites” who nevertheless have ALS in common.

lou

Henry Louis “Lou” Gehrig (June 19, 1903 – June 2, 1941), born Ludwig Heinrich Gehrig, was an American baseball player in the 1920s and 1930s, and set several Major League records and was popularly called the “The Iron Horse” for his durability. His record for most career grand slam home runs (23) still stands today. At the midpoint of the 1938 season, Gehrig’s performance began to diminish. After six days of extensive testing at Mayo Clinic in Rochester, Minnesota, the diagnosis of ALS was confirmed on June 19, Gehrig’s 36th birthday. ALS is commonly referred to as “Lou Gehrig’s disease” in the U.S. Photo taken from exequy.wordpress.com via Bing Images.

 

 

hawking

Professor Stephen Hawking—born Jan 8, 1942 in Oxford, England—has conducted work concerning the basic laws that govern the universe itself. Along with Roger Penrose, he has shown that Einstein’s General Theory of Relativity implied space and time would have a beginning in the, “Big Bang,” and end in “black holes.” In regards to the disability Stephen experiences, he has some things to say: “I am quite often asked: How do you feel about having ALS? The answer is, not a lot. I try to lead as normal a life as possible, and not think about my condition, or regret the things it prevents me from doing, which are not that many.” Photo taken from picfind.bloguez.com via Bing Images.

 

 

 

ALS Genetics in Brief

Given that this blog deals with “all things nucleic acids,” it’s apropos to mention a bit about the genetics, but quickly add that they are quite complex, as can be read about here.

There is a known hereditary factor in familial ALS. A defect on chromosome 21, which codes for superoxide dismutase, is associated with ~20% of familial cases of ALS, or about 2% of ALS cases overall. This enzyme is a powerful antioxidant that protects the body from damage caused by superoxide, a toxic free radical generated in the mitochondria. Free radicals are highly reactive molecules produced by cells during normal metabolism. Free radicals can accumulate and cause damage to DNA and proteins within cells.

This mutation has over 100 different genotypes. The most common ALS-causing mutation is a mutant SOD1 gene, seen in North American patients; this is characterized by an exceptionally rapid progression from onset to death. The most common mutation found in Scandinavian countries, D90A-SOD1, is more slowly progressive than typical ALS and patients with this form of the disorder survive for an average of 11 years.

In 2011, a genetic abnormality known as a hexanucleotide repeat was found in a region called C9orf72, which is associated with ALS combined with frontotemporal dementia ALS-FTD, and accounts for some 6% of cases of ALS among white Europeans. The gene is also found in people of Filipino descent.

I found it rather surprising that, where no family history of the disease is present—i.e., in a whopping ~90% of cases—there is no known cause for ALS! How stressful it must be to be afflicted with a disease having no known cause and no drug. Consequently, it’s easy to understand the importance of raising more research funding through the ALS Ice Bucket Challenge, and other money raising endeavors such as the following.

Crowdfunded ALS— Project MinE

I’ve previously commented here on the rapidly growing popularity of crowdfunding as a new, socio-web-based mechanism for “reaching out” to obtain money for conducting scientific research—including ALS. Project MinE is an independent, large-scale, whole-genome research project that has been initiated by two Dutch individuals with ALS and started on World ALS day (June 21) last year. These individuals provide their personal views on Project MinE at their Treeway website, which provides several modes of “communal contributing” that include jumping into Amsterdam canals (!!) as well as the now “classic” Ice Bucket Challenge.

Project MinE is a research project aimed at systematically interrogating the human genome for common and rare genetic variation in ALS—aka genetic “data mining.” The project will involve obtaining donated-DNA sequence information for 15,000 ALS samples and 20,000 healthy controls to obtain a large number of single-nucleotide polymorphisms (SNPs). This and additional sequencing will be performed on a sample size large enough to reliably analyze whole genome sequencing data outside of a family context.

The long-term benefit of the approach taken for project MinE is a catalogue of many non-ALS whole genomes that can be used to investigate other human diseases, including Diabetes Mellitus, some types of cancer, and other neurological disorders. Project MinE is the largest genetic study worldwide for ALS and was started in the second quarter of 2013. Complete information—and details on how to donate may be found at the Project MinE website, which I encourage you to visit.

Harvard Team Reports Possible ALS Drug Target

I thought it would be best to end this post on a positive note—specifically a possible drug target for ALS that is the subject of an online account by Cynthia Fox in Drug Discovery & Developement. Snippets of this exciting story are as follows.

A Harvard University team reported they may have found an ALS therapy—or two! When they blocked a gene for prostanoid receptor DP1 in ALS brain glia cells in a dish, neurons made from human embryonic stem (ES) cells were “completely protected” from death.

When they created ALS mice with that same gene deleted, the mice lived 6.7 percent longer.

This helps validate the idea that neurons made from human stem cells—in a dish—can be drug screens, team leader Kevin Eggan told a press conference. The 6.7 percent survival increase may rise even more, he said, if/when their DP1 antagonist is given with a drug his team earlier found has anti-ALS properties. And as both drugs are FDA-approved for other indications, clinical trials could move fast.

“We think this is a significant advance—both in terms of the use of stem cells for understanding disease, and with respect to understanding the degenerative processes of ALS and how we might inhibit them,” said Eggan.

Let’s all hope that this all proves to be true, and soon!

As always, your comments are welcome.

Could the Current Ebola Outbreak Have Been Prevented?

  • Deadliest Outbreak Yet Shows no Sign of Abating
  • Lack of Funds Hampered Clinical Development of Drugs and Vaccines
  • Treatments Exist so Why are Doctors Left with no Cure to Offer the Infected?

You’ve undoubtedly seen or read ongoing—seemingly continuous—news stories about a serious outbreak of Ebola virus disease (EVD) in Africa, and the two infected American health workers who were brought back to the US for treatment. The title of this blog post is adapted from Sara Reardon’s article in venerable Nature magazine, which I draw from following a brief overview of EVD.

The current Ebola outbreak involving the most dangerous Zaire species has killed more than 1,000 and infected an estimated 1,975 people in West Africa. Sierra Leone, Guinea and Liberia have been hit the hardest, with Nigeria experiencing a handful of confirmed cases and 3 deaths. Tom Frieden, director of the U.S. Centers for Disease Control and Prevention, estimated the outbreak will take three to six months to contain under the best of circumstances. Although the outbreak is the deadliest to date, the chances of infection in the US is remote, albeit theoretically possible that one of the 10,000+ travelers to and from the region over the next three-months could carry the virus back to the US. “Ebola poses little risk to the U.S. general population,” Frieden is quoted as saying. Let’s all hope he’s right.

The current outbreak of the Ebola virus in West Africa has killed more people than any previous outbreak. According to a spokesman from the organization “Doctors Without Borders,” the disease is now “out of control” (taken from inquisitr.com via Bing Images).

The current outbreak of the Ebola virus in West Africa has killed more people than any previous outbreak. According to a spokesman from the organization “Doctors Without Borders,” the disease is now “out of control” (taken from inquisitr.com via Bing Images).

EVD – Key Facts

Electron micrograph of an Ebola virus virion (taken from readtiger.com via Bing Images).

Electron micrograph of an Ebola virus virion (taken from readtiger.com via Bing Images).

The following selected statements about EVD were taken from a World Health Organization (WHO) website that was updated on April 2014, and should be consulted for further details.

    • EVD, formerly known as Ebola hemorrhagic fever, is a severe, often fatal illness in humans.
    • EVD outbreaks have a case fatality rate of up to 90%.
    • EVD outbreaks occur primarily in remote villages in Central and West Africa, near tropical rainforests.
    • The virus is transmitted to people from wild animals and spreads in the human population through human-to-human transmission, with infection resulting from direct contact (through broken skin or mucous membranes) with the blood, secretions, organs or other bodily fluids of infected people, and indirect contact with environments contaminated with such fluids.
    • EVD is a severe acute viral illness often characterized by the sudden onset of fever, intense weakness, muscle pain, headache and sore throat. This is followed by vomiting, diarrhea, rash, impaired kidney and liver function, and in some cases, both internal and external bleeding. Laboratory findings include low white blood cell and platelet counts and elevated liver enzymes.
    • Severely ill patients require intensive supportive care. No licensed specific treatment or vaccine is available for use in people or animals.

Only 18,959 Nucleotides Encode Much Human Suffering

Simplified schematic drawing of key molecular components of Ebola virus (taken from primehealthchannel.com via Bing Images).

Simplified schematic drawing of key molecular components of Ebola virus (taken from primehealthchannel.com via Bing Images).

Like HIV and other RNA viruses, Ebola is encoded in a relatively tiny genome that nevertheless leads to huge problems for society through complex life cycle/human host molecular biology. As detailed elsewhere, the genome of the Zaire Africa Ebola virus—the most deadly species and the one involved in the current outbreak—is only 18,959 nucleotides in length and contains seven transcriptional units that direct synthesis of at least nine distinct primary translation products: the nucleoprotein (NP), virion protein (VP) 35, VP40, glycoprotein (GP), soluble glycoprotein (sGP), small soluble glycoprotein (ssGP), VP30, VP24 and the large (L) protein. L is the catalytic subunit of the polymerase complex. Ebola virus encodes a multi-protein complex to carry out replication and transcription. Ebola viral RNA synthesis requires the viral NP, VP35, VP30 and L proteins. Each Ebola virus mRNA is presumed to be efficiently modified with a 5′-7-methylguanosine (m7G) cap and a 3′ p(A) tail.

RT-PCR Enables Effective Diagnostics for Ebola Viral RNA

Ebola virus infections can be diagnosed definitively in a laboratory through several types of tests, such as antibody-capture enzyme-linked immunosorbent assay (ELISA), serum neutralization test, and virus isolation by cell culture. Not surprisingly, however, RT-PCR has been demonstrated to be highly specific and sensitive, as outlined in this abstract published by a collaborative team lead by the Diagnostic Systems and Virology Divisions at the United States Army Medical Research Institute of Infectious Diseases:

Viral hemorrhagic fever is caused by a diverse group of single-stranded, negative-sense or positive-sense RNA viruses belonging to the families Filoviridae (Ebola and Marburg), Arenaviridae (Lassa, Junin, Machupo, Sabia, and Guanarito), and Bunyaviridae (hantavirus). Disease characteristics in these families mark each with the potential to be used as a biological threat agent. Because other diseases have similar clinical symptoms, specific laboratory diagnostic tests are necessary to provide the differential diagnosis during outbreaks and for instituting acceptable quarantine procedures. We designed 48 TaqMan™-based polymerase chain reaction (PCR) assays for specific and absolute quantitative detection of multiple hemorrhagic fever viruses. Forty-six assays were determined to be virus-specific, and two were designated as pan assays for Marburg virus. The limit of detection for the assays ranged from 10 to 0.001 plaque-forming units (PFU)/PCR. Although these real-time hemorrhagic fever virus assays are qualitative (presence of target), they are also quantitative (measure a single DNA/RNA target sequence in an unknown sample and express the final results as an absolute value (e.g., viral load, PFUs, or copies/mL) on the basis of concentration of standard samples and can be used in viral load, vaccine, and antiviral drug studies.

According to WHO, Ebola was first detected in 1976 and strides have been made in developing sophisticated tests that can accurately diagnosis the virus, as demonstrated above. There is currently no FDA approved vaccine or treatment for Ebola, but that doesn’t mean one doesn’t exist.

Could Outbreak Have Been Avoided?

Although several vaccines and treatments for Ebola do exist, they are stalled in various stages of testing owing to a lack of funding and of international demand. So, for now, doctors have no cure to offer those with EVD and understaffed clinics must make do with isolating infected people, finding and quarantining their families, and educating the public on how to avoid spreading the disease.

A doctor in Sierra Leone enters the high-risk area of the Ebola treatment center. Credit: Sylvain Cherkaoui/Cosmos/eyevine (taken from Nature).

A doctor in Sierra Leone enters the high-risk area of the Ebola treatment center. Credit: Sylvain Cherkaoui/Cosmos/eyevine (taken from Nature).

The fact that the Ebola virus was identified almost 40 years ago and there’s been ongoing research ever since begs the question, “Was the current Ebola outbreak preventable?” According to Reardon, researchers such as Heinz Feldmann, a virologist at the US National Institute of Allergy and Infectious Disease (NIAID) in Hamilton, Montana, think that the situation could have been avoided. In 2005, Feldmann published a vaccine approach based on vesicular stomatitis virus (VSV) that has since yielded an Ebola vaccine that is effective in macaques. But money is not available to take the next step—testing the vaccine’s safety in healthy humans. Compared with malaria or HIV, “Ebola is just not that much of a public-health problem worldwide”, he told Reardon, and consequently draws little interest from public or private funders.

“What works for Ebola is good old-fashioned public health,” says Thomas Frieden, director of the US Centers for Disease Control and Prevention in Atlanta, Georgia, according to Reardon. “It would be great to have a vaccine, but it’s not easy to do and not clear who you’d test it on.”

There are other possible vaccines as well. The NIAID Vaccine Research Center in Bethesda, Maryland, has developed a vaccine that is carried by a chimpanzee adenovirus, similar to the virus that causes the common cold. The institute hopes to begin testing in healthy people as early as September. Barney Graham, deputy director of the research center, told Reardon that the institute is talking with the Food and Drug Administration (FDA) to speed up the approval process, a position that is strengthened by the outbreak in West Africa.

Biotechnology companies are also developing treatments at a pace that could now be accelerated, as we’ve seen with the ZMapp™ vaccine (discussed in detail below) that arrived in Liberia a few days ago. ZMapp™ was developed by Mapp Biopharmaceutical in San Diego, California. The potenital treament uses monoclonal antibodies (mAbs) that target the virus.

Another potential therapeutic backed by US$140 million from the US Department of Defense, is being tested by Tekmira in Burnaby, Canada. The treatment, called TKM-Ebola, uses chemically synthesized small RNA (siRNA) molecules to bind the virus and target it for destruction. The company began testing TKM-Ebola in humans in January, but in July of this year, the FDA put the study on hold until the company could provide more data on how the treatment works. According to an article in Streetsider.com, here’s what the CEO said in response to the FDA news:

“We have completed the single ascending dose portion of this study in healthy volunteers without the use of steroid pre-medication. The FDA has requested additional data related to the mechanism of cytokine release, observed at higher doses, which we believe is well understood, and a protocol modification designed to ensure the safety of healthy volunteer subjects, before we proceed with the multiple ascending dose portion of our TKM-Ebola Phase I trial,” said Dr. Mark Murray, President and CEO of Tekmira Pharmaceuticals. “We will continue our dialogue with the FDA, provided for under our Fast Track status, in order to advance the development of this important therapeutic agent.”

A treatment could be approved by the FDA on a ‘compassionate use’ basis, but that process would have to mesh with a host country’s rules. “A country has to request these things; it’s not something we can force on them,” says Gene Olinger, a virologist at the contract research organization MRIGlobal in Frederick, Maryland. “We have to follow their internal policies for drug development and for testing.” It appears that Liberia, at least, has made such a request and it has been honored.

Coincidentally (or maybe not so much), on August 7, the FDA reduced the full clinical hold on Tekmira’s TKM-Ebola drug to a partial hold, potentially enabling use of the compound in patients. It remains to be seen if the drug will be sent to West Africa to be administered, but such a move leaves some to wonder why the FDA can act so swiftly now but refused to do so back in July, prior to the outbreak.

Mapp’s ‘Mystery’ Ebola Virus Drug Said to be ‘Miraculous’

Major media outlets frequently employ attention-grabbing words—such as ‘mystery’ and ‘miraculous’—so it’s not surprising that these descriptors have been used in recent news reports about two American health workers in Liberia infected with Ebola virus. The Los Angeles Times reported that Mapp Biopharmaceutical’s experimental drug, ZMapp™, was given to Dr. Kent Brantly and Nancy Writebol under circumstances described by the LA Times as “a mysterious treatment.”

With all involved wearing full protective gear, a man believed to be Ebola patient Dr. Kent Brantly is helped from an ambulance at Emory University Hospital in Atlanta on Saturday.

With all involved wearing full protective gear, a man believed to be an Ebola patient, Dr. Kent Brantly is helped from an ambulance at Emory University Hospital in Atlanta. Credit Associated press/WSB-TV Atlanta (taken from LA Times).

Intrigued by the ‘mystery,’ I did some quick research and found ZMapp™ had not been previously evaluated for safety in humans, and “very little of the drug is currently available,” according to LeafBio (San Diego, California), which is Mapp’s commercialization partner.  In fact, the available supply of ZMapp™ is said to have been exhausted, according to a statement posted August 12, 2014 on Mapp’s website. The statement also notes that ZMapp™ is the result of a collaboration by Mapp, LeafBio, Defyrus Inc. (Toronto, Canada)—a biodefense company—and both the U.S. government and the Public Health Agency of Canada.

I went on to read that “ZMapp™ is composed of three ‘humanized’ mAbs manufactured in plants, specifically Nicotiana” (aka tobacco plant—and origin of the word nicotine). In other words, tobacco plants are cleverly repurposed by genetic engineering to produce mAbs suitable for use in humans, as detailed here in a review by Mapp that describes this approach as a “revolutionary advance” in antibody manufacturing.

The tobacco plant: Nicotiana tabacum. Credit Joachim Mullerchem (taken from Science via Bing Images).

The tobacco plant: Nicotiana tabacum. Credit Joachim Mullerchem (taken from Science via Bing Images).

The LA Times also said that CNN reported that the drug had prompted a ‘miraculous’ recovery and that Brantly’s condition improved within an hour after treatment, but that this was greeted with skepticism by longtime Ebola virus researchers.

This skepticism is based on the following series of quotes in the LA Times story:

‘I would be ecstatic if Larry’s product helped save these people, but I also need to be extremely cautious,’ said Thomas Geisbert, a professor of microbiology and immunology at the University of Texas Medical Branch at Galveston.

‘To say the whole thing cleared up in an hour, that doesn’t happen in reality,’ Geisbert said. ‘That’s like something that happens in a movie.’

Dr. Anthony Fauci, head of the National Institute of Allergy and Infectious Disease, said the company had manufactured only three ‘courses’ of the drug, and that two of them were provided to the American patients.

‘This was the first time it was put into humans, because all the previous work was done on animals and the results had been encouraging,’ Fauci said.

In closing, I’ll sadly add that it’s unfortunate—to say the least—that funding for timely development of an Ebola vaccine had not been forthcoming from some agencies that knew full well that it was only a matter of time for the next outbreak to occur in Africa. Corporations, however, seem to be stepping in where these agencies may have failed. On Monday, August 11, World Bank announced it will give $200M to help fund the fight against Ebola. Let’s all hope that the current crisis provides the necessary catalyst for that development so as to preclude yet another outbreak and more unnecessary deaths.

As always, your comments are welcomed.

mtDNA Replacement: Eliminating Disease or Creating Designer Babies?

  • Mitochondrial DNA Replacement to Preclude Mitochondrial Disease Shown Feasible in Monkeys
  • US FDA Advisors Ponder Pros and Cons for Allowing Human Clinical Trials Amidst “Designer Baby” Concerns
  • UK Government Decision “Up in the Air”

Prologue

Before jumping into the headline and bylines of this post, I thought it would be worth sharing TriLink’s connection with mitochondrial DNA (mtDNA), as well as provide a bit of background about mtDNA and diseases related to it.

TriLink recently introduced its mitochondrial DNA (mtDNA) PCR-sequencing primers (mitoPrimers™) and mtDNA master mix for forensic science and casework. These products have been used by several well-known experts in mtDNA forensics, including Prof. Rhonda Roby, a well-known expert in mtDNA forensics. Prof. Roby’s expertise in mtDNA sequencing extends beyond forensic applications to academic interests in disease-related mtDNA dysfunction, as described in her recent publication in Nature Scientific Reports.

To better understand those interests, I researched mtDNA diseases at an NIH.gov website and found that, while mtDNA is only a “tiny” (~16,569 base-pairs) genetic component compared to genomic DNA (~2 billion base-pair), and encodes only 37 genes compared to ~20,000 genes for genomic DNA, the list of mtDNA related diseases is lengthy.

The vast majority (90%) of the energy needs of the human body are provided by mitochondrial oxidative phosphorylation that takes place entirely in mitochondria, and is a highly efficient system for producing the energy required to maintain the structure and function of the body. Consequently, according to the NIH.gov website, mutated mtDNA disrupts the mitochondria’s ability to efficiently generate energy for the cell, leading to organ-related health conditions. These conditions can be serious and are most pronounced in organs and tissues with high energy requirements, such as the heart, brain, and muscles. Frequently observed symptoms include muscle weakness and wasting, problems with movement, diabetes, kidney failure, heart disease, loss of intellectual functions (dementia), hearing loss, and abnormalities involving the eyes and vision.

mito

Taken from “Powerhouse Rules: The Role of Mitochondria in Human Diseases,” online coursework at MIT (ocw.mit.edu) via Bing Images.

The image (right) depicts multiple copies of circular mtDNA that reside in mitochondrial organelles external to a cell’s nucleus (in blue), which contains genomic DNA. Whether or not these organelles evolved by free-living bacteria that were taken inside a human cell-to-be (aka endosymbiotic theory) is an evolving—pun intended—debate worth reading about if you’re intrigued—as I am—by pondering how our cells came to be what they are.

Interestingly, women’s egg cells have many copies of mtDNA while men’s sperm cells have just enough to enable it to swim to the egg and fertilize it. The mtDNA of the sperm usually disappears once fertilization occurs. On the other hand, the mtDNA of the egg pass on to all of a woman’s children (male and female), but only women pass on their mtDNA from generation to generation. This maternal aspect of genetics is depicted in the cartoon below, posted by genetic-ancestry company 23andMe to celebrate Mother’s Day.

Taken from blog.23andme.com via Bing Images.

Taken from blog.23andme.com via Bing Images.

Unfortunately, we might not always be thanking Grandma for her mtDNA. In her daily blog, Mighty Mito Mom, Amy Boyd chronicles the challenges and experiences as a Mom whose daughter—affectionately called Little Miss Mollypop—has a mitrochondrial disease. I also found numerous YouTube videos that give a real sense of how families struggle with mitochondrial disease, such as this poignant example involving three generations affected by mitochondrial-related conditions. These examples raise the question, can something be done to preclude mtDNA disease by substituting “good mtDNA” for “bad mtDNA” during in vitro fertilization (IVF)?

The answer is yes, but the process involves what some view as three parents—two females and one male. As you may imagine, the procedure prompts considerable concern and controversy.

Mitalipov’s Mitochrondrial Manipulations

I’ve “borrowed” this catchy alliterative heading from the title of a March 2014 NY Times story. The article is by Sabrina Tavernise about Oregon Health and Science University Dr. Shoukhrat Mitalipov, who has developed a procedure to help women conceive children without passing on their mtDNA genetic defects. Salient snippets of this article are as follows, including this self-charactrization by Mitalipov: “my colleagues, they say I’m a ‘mitochondriac,’ that I only see this one thing. Maybe they are right.”

Dr. Mitalipov, 52, has shaken the field of genetics by perfecting a version of the world’s tiniest surgery: removing the nucleus from a human egg and placing it into another. In doing so, this Soviet-born scientist has drawn the ire of bioethicists and the scrutiny of federal regulators. Credit Leah Nash, NY Times.

Dr. Mitalipov, 52, has shaken the field of genetics by perfecting a version of the world’s tiniest surgery: removing the nucleus from a human egg and placing it into another. In doing so, this Soviet-born scientist has drawn the ire of bioethicists and the scrutiny of federal regulators. Credit Leah Nash, NY Times.

About one in 4,000 babies in the US is born with an inherited mitochondrial disease. These diseases are terminal, there is no known treatment, and few of these afflicted children live into adulthood. Women who carry mtDNA mutations are understandably eager not to pass them to their children. Remember, although sperm carry mitochondria, they are usually degraded shortly after fertilization, so mitochondrial diseases are passed down through the mother. Dr. Mitalipov’s procedure (shown below) would allow these women to bear children by placing the nucleus from the mother’s egg into a donor egg whose nucleus has been removed. The defective mitochondria, which float outside the nucleus in the egg’s cytoplasm, are left behind, thus eliminating the possibility of passing along defective mitochondria.

Those of you interested in Mitalipov’s published feasibility results with human oocytes can read a 2013 report in Nature, and a 2014 review of promises and challenges in Fertility and Sterility entitled “Three-parent in vitro fertilization: gene replacement for the prevention of inherited mitochondrial diseases.”

Mitochondrial DNA replacement could help carriers of severe disease have healthy children. Credit K. Sutliff, Science.

Mitochondrial DNA replacement could help carriers of severe disease have healthy children. Credit K. Sutliff, Science.

Are “Designer Babies” on the Horizon?

While some advocates of this procedure say it’s a “major breakthrough,”others aren’t as enthusiastic since the resulting baby would carry genetic material from three parents—the mother, the host egg’s donor and the father—an outcome that ethicists have deplored.

That specter drew critics from all over the country to a hotel in suburban Maryland in February 2014. It was here that Dr. Mitalipov tried to persuade a panel of experts convened by the FDA that the procedure, which he has pioneered in monkeys, was ready to test in people.

Some experts voiced concerns about unintended consequences, such as introducing new genetic mutations into the human gene pool. Others warned that it could be used later for something ethically murkier—perhaps, said Marcy Darnovsky, executive director of the Center for Genetics and Society, “to engineer children with specific character traits.”

Darnovsky’s assertion of a slippery slope situation possibly leading to “designer babies” is not new, and can be read about in a lengthy Science perspective entitled Stirring the Simmering “Designer Baby” Pot written by Thomas H. Murray. The metaphorical “pot” that he refers to ties Mitalipov’s proposed procedure to a 2013 patent granted to 23andMe—a controversial want-to-be genetic testing company currently foiled by the FDA. This “hot button” patent covers genetic calculations that enable would-be parents “to select a donor and view other possible phenotype of the hypothetical child resulting from the recipient’s and the donor’s gametes.”

Dr. Mitalipov waved off those warnings, according to Tavernise, by noting that mtDNA comprises just 37 genes, which direct the production of enzymes and molecules that the cell needs for energy. Those genes, he says, have nothing to do with traits like eye and hair color, which are encoded in the nucleus. She quotes Mitalipov as saying that “there are always people trying to stir things up. Many of them made their careers by criticizing me.”

“Suspended Animation”

In his Science perspective, Murray observes that “it is unrealistic to expect the US policy lacuna [gap or missing piece] over assisted reproductive technologies to be filled any time soon. Regulation of preimplantation genetic diagnosis remains in a ‘state of suspended animation’—according to a cited publication.

He adds that “professional self-regulation likely works well when the interested professions reflect a well-established public consensus. But when it comes to empowering parents to decide what sort of children they have beyond questions of serious childhood diseases, professional organizations cannot even agree on the appropriate ethical framework.”

Murray concludes by stating that “discussion of the ethics of mitochondrial manipulation cannot be postponed indefinitely. With little prospect of sensible legislation in the near term, and conflicting guidance from professional organizations, a national conversation about current and emerging technologies shaping the choices that parents will have is urgent. The UK conducted a similar exercise a decade ago that combined polling, focus groups, and the Internet.”

Status of mtDNA Replacement in the UK

In Britain, the government has issued draft regulations that would govern clinical trials of mtDNA replacement in people. If accepted into law by Parliament, such trials (which are now banned), would be allowed to go forward, although regulators would have to license any clinical application.

Polly Toynbee of The Guardian reported in February 2014 that, while a year has passed since the public was consulted at extraordinary length on the ethics of mtDNA replacement to prevent the birth of children with incurable genetic diseases—with most of the public saying yes, go ahead—the government has “dragged its feet.”

Inquiring about its progress with the Department of Health a couple of weeks before drafting her report, the answer was, “it’s up in the air.” Pressed on the question the day before publishing, she was told that draft regulations may appear next month. But the public will have to be consulted again, for a further three-month period. Polly opined that, since this procedure “arouses deep passions, plentiful responses are expected” and it will take yet more months to consider them. Doubts remain about the government’s eagerness to push this through parliament, rousing a controversy close to the general election, she added.

My comment is that difficult policy decisions and political party concerns for reelection seem to be similar on “both sides of the pond,” as the UK and the US are referred to.

Newcastle University neurologist Prof. Doug Turnbull, who is co-leading investigations with embryologist Dr. Mary Herbert, has been putting pressure on the government to prepare legislation that will allow experts in Newcastle to use human embryos containing DNA from three people to be used in clinical treatment, according to an earlier report.

Ms. Toynbee points out that the Human Fertilization and Embryology Authority (HFEA) conducted its scientific review of the procedure’s safety and efficacy and concluded back in 2011 that it would be unethical for the government not to press ahead, to prevent any more needless suffering. Toynbee was a member of the HFEA’s oversight group that supervised a massive public consultation, ensuring these complex issues were fairly aired and comprehensible to all.

Overall, the public was in favor, she says, and adds that when randomly selected people looked at the evidence they didn’t think this was a slippery slope that would lead to “designer babies,” or that it amounted to “three-parent IVF,” as there is no genetic effect on identity. The HFEA recommends that mitochondria are treated like tissue, a kidney for example—so donors would not be considered ‘parents.’

She adds “parliament is often less rational than the public. Stuffed with the religious and rabble-rousers who stir up fears of Frankenstein babies, many in both houses will make noisy speeches, ignoring the science.” That despite an estimated 73 people dying each year from mitochondrial disease, many of them children. The impact is even greater when you consider that some mitochondrial diseases go undiagnosed and that over 2,500 women of child-bearing age carry faulty genes – putting their children at risk.

Ms. Toynbee concluded her story by saying that “just as I reach the end of writing this, a call comes from the Health Department: it wants me to know that it is still absolutely committed, and regulations will be out soon. Why the year’s delay? Ah, um. When will the regulations go to parliament? ‘By the end of the year’ was the reply. Left so close to the election, let’s hope No. 10 strategists don’t veto it.”

As always, your comments are welcomed.

Postscript

After writing this blog, The New York Times Magazine featured a lengthy cover story by Kim Tingley entitled One Child, Three Parents. Also, a lengthy and freely available article by Ewen Callaway was published in venerable Nature magazine. Overall, the same pro and con issues are presented by Callaway, along with simplified diagrams showing methodological differences between pronuclear transfer and maternal spindle transfer techniques for genome transfer to prevent children from inheriting their mother’s mutant mitochondria. I found this article’s lead-in graphic (credited to Vasava) show below to be symbolically riveting and worth sharing here.

3parent