Sunday, January 25, 2015

Up-Coming: Computational Genetics and the Evolution of Ebola

Image: NIAID Ebola Virus

"Zaire Ebola virus (EBOV) is an emerging zoonotic pathogen that causes hemorrhagic fever in humans. Fatality rates in some human outbreaks have approached 90% (reviewed in reference. Because of its lethality, the lack of FDA-approved therapeutics, and its potential use as a bioweapon, EBOV is classified as a category A pathogen and is studied under biosafety level 4 containment."(Source: 

The current outbreak of Ebola virus across West Africa involves a strain known as EBOV/Mak. "Viruses like Ebola are notoriously sloppy in replicating, meaning the virus entering one person may be genetically different from the virus entering the next. The current Ebola virus’s hyper-evolution is unprecedented; there has been more human-to-human transmission in the past four months than most likely occurred in the last 500 to 1,000 years. ( "Researchers at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) published their findings in American Society for Microbiology journal mBio. In it, they tracked the genetic mutations of the virus over the last 40 years to identify changes in the current strain that could interfere with experimental, sequence-based therapies. The most promising drugs being developed bind to and target part of  Ebola's genetic sequence or protein sequence. However, if this sequence changes, the drugs will not work as effectively." (Source: "As of 8 January 2015, the mean lethality in this outbreak, caused by Ebola virus (EBOV), reached 39.4% (Source:

Emerging Technologies: Lowering the threshold for ISIS (Islamic State of Iraq and Syria) Mass Casualty Terrorism

Emerging technologies are those technologies that are innovative and competitive both in terms of function and accessibility. When we consider emerging technologies, one of the primary novel technologies is 3D printing and its application to weapon and weapon systems development. What sets 3D printing apart however, from other emerging technologies is the lowering of a technical threshold which makes it user friendly to a much wider population. In so doing, it lowers the technical and economic threshold of weapon production.

3D printing has already been used in drone construction and the potential that terrorist organizations either supported by states (Hezbollah/Iran) or with vast financial resources such as the Islamic Stata in Syria and Iraq, could acquire the means to mass produce disposable weapons at a low cost point is a counter-terror concern. Not only would 3D printing of weapons increase the potential for mass casualty terrorism but standoff weapon systems could well increase this threat replacing the need to even arm terrorists.  3D printed disposable drones could deliver conventional and unconventional payloads. The ability to swiftly replace captured or destroyed drones would significantly impact methods we currently use in counter-terrorism and warfare.  In an article by Yochi Dreazen entitled: The Next Israeli-Arab War will be Fought with Drones, Mr. Dreazen contends, “In October, near the West Bank city of Hebron, Palestinian  security personnel arrested a team of operatives preparing to launch a drone packed with explosives. The events have set off alarms within the Israeli Defence Forces, which last April released a statement declaring unmanned aerial vehicles (UAV’s) to be a serious threat to the country. Hezbollah’s drones represent the next evolution of warfare by remote control, when weaponized robotic planes give terrorist groups de facto air forces.” See:

As if this were not concerning enough, the 3D printing of drone technology will likely rapidly increase the ability of terrorist organizations, such as Hamas, Hezbollah and ISIS specifically, to deploy disposable drones, reprint them and rapidly replace lost drones. Moreover suicide drones could easily be employed to take out infrastructures and used in mass casualty attacks against civilians. 3D mass production disposable drones would be a game changer for weapons of mass destruction and future terrorist methods and tactics allowing incredible versatility. Smaller 3D produced drones and those designed with swarming technology are likely to evade current counter measures. In a scenario where mixed drones are used, some with conventional payloads, some with un-conventional payloads, multiple strikes would be possible and while the conventional attack would be considered immediate, there could well be long term casualties either from loading the payloads with low level radiological material (small aerial dirty bombs) or biological and chemical weaponized agents.  Such agents could well create multiple rolling outbreaks of pandemic disease or be used as stealthy force reducers/force multipliers. 4D technology, developed at MIT, could mean that printed payloads using biological agents could be weaponized based on target specific data. This would obscure identification and remove some of the barriers which previously served to make this type of weaponization process the domain of state military labs. Essentially making it user friendly to terrorists.

While Israel is one of the best placed nation, both technically and in terms of experience, in countering potential future terrorist weapons, the use of 3D printed technology in a European scenario, would offer ISIS real advantages. With the possible exception of France, most European governments are not quite as well prepared to counter this threat, nor do they believe it is remotely on the horizon. This gap in assessment and experience advantages ISIS and other terrorists, even lone terrorists who might access 3D printing technology within Europe. To understand how real and how close this technology is and it's accessibility to terrorists, we need only look at a recent article by Adam Clark Estes wherein he notes: “A team from the Advanced Manufacturing Research Center at the University of Sheffield, built their disposable drone, a five foot wide guy made of just nine parts that looks like a tiny stealth bomber, using a technique called fused deposition modeling. This additive manufacturing technique has been around since the 1980’s but has recently become faster and cheaper thanks to improved design processes. The ultimate vision, as UAS describes it, is for ‘cheap and potentially disposable UAV’s that could be built and deployed in remote situations potentially within as little as 24 hours. Forward operating teams equipped with 3D printers could thus generate their own semi-autonomous micro air force squadrons or airborne surveillance swarms, a kind of first strike desktop printing team hurling disposable drones into the sky.” (

Hezbollah and Hamas both have sophisticated intelligence collection capabilities, what we will likely see with 3D printable drones is the ability to drop disposable surveillance equipment into theaters where previously they would not have access. If the surveillance equipment can be mass produced, and cost effective (disposable) we are likely to see its use by terrorist organizations; a risk assessment of this emerging technology and terrorist applications, could not be undertaken too soon.

Turning our attention to ISIS, recent reports note that ISIS has increased its use of chemical weapons (see: Several biological agents which are within the purview of ISIS could be well suited for drone deployment. ISIS taking of Palmyra, an area known for its phosphates, is worrying but more so because ISIS possess the technical sophistication to arms drones with CW and BW. Lacking effective in threatre counter measures advantages them in ways which are probably best left to the imagination.  

As breathtaking as this seems it is now reality and one few European governments are planning effectively to counter. Unfortunately there is a technological gap between disposable 3D printed drones and counter technologies to identify, evade and destroy such technology in civilian situations. An advantage our enemies are likely to exploit and in the very near term, not two years or five years down the road.  

Thursday, January 15, 2015

Revolutionizing Drug Discovery and Delivery: How Ebola Vaccine Research changed Bio-Pharma

Source: CDC

Recently, a paper entitled: Rethinking the development of Ebola treatments by Rajesh Gupta, was published in The Lancet. The paper stipulates:
"Therapeutic Ebola research is heavily funded by the US government under the auspices of threats to national security,11and international activities are limited to a few research groups. To allow for greater participation of researchers globally, real-time accessibility of crucial data is necessary.In silico methods are still in development and rapidly evolving, but have been successful in identifying potential candidates for various diseases and the risk of using such methods are very low. Their ability to affect, at scale, drug development processes, costs, and timelines is unknown but likely to be considerable given the private sector's strong interest and investment in this area. Equally likely is that these approaches will be able to affect a wide range of diseases. Although these approaches are currently directed towards diseases with clear revenue streams (eg, inflammatory bowel disease and cancer), such approaches could be used for unprofitable diseases that affect the most underserved populations of the world." Source:
"The inequities already posed by a disease of poverty such as Ebola become further exacerbated when novel technologies are used first to explore diseases that are viable commercial opportunities. This does not have to be the pattern moving forward, and Ebola might provide the opportunity to apply new technological approaches to drug development (such as in silico methods) for traditional “market failure” diseases. If the global community is truly committed to rapidly developing a new drug for Ebola, multiple novel approaches, methods, and technologies will need to be used to beat the inherent hurdles of drug development." Source:
Diseases of poverty and their orphan drug development counterparts are often considered so highly limited that the costs of research and  investment are prohibitive. Most Category A and some Category B diseases (defined under CDC), considered a national security priority are funded by a multitude of US government programs. Such funding was put in place following the US anthrax attacks. What has changed since then is our approach from one bug one drug to development of a range of technologies which foresee one drug covering a wide range of infectious disease. This is particularly true of Ebola vaccines and therapeutics. 
We need only look to the annual WHO smallpox retention debate at the World Health Congress, held each May to understand how research into even eradicated disease (smallpox being the only one to date), offers a host of novel drug research, development and manufacturing techniques which would not perhaps have been generated quite so swiftly without the crisis aspect defining the current Ebola outbreak in West Africa.  We must also consider how emergency investigational new drug (IND's) applications are evaluated and approved by FDA; although this may be considered a downstream issue. Drug development for Ebola will likely convey significant benefit to other diseases, particularly influenza and HIV, which may not have been the case in the time frames we are now witnessing. 

In order to take full advantage of this opportunity we need protocols and evaluations in step with the pace of accelerated discovery. We need to rethink evaluation processes in light of far more agile drug and drug platform discovery and development. Not only will drug research and development on Ebola vaccines and therapeutics prove advantageous to other emerging disease but will likely revolutionize fundamental aspects of this. One example is of course DARPA and Medicago's one month challenge to produce millions of vaccines. Immuno-therapies are likely to gain a strong foothold due to application to Ebola research in markets they held lesser positions. Additionally, advances in delivery such as nano vaccines and VLP's (virus like particles) as a delivery platform for gene therapy and other therapeutics is likely to become a serious technology a range of applications perhaps not considered for emergency use. The most recent discovery announced by MIT "In a paper published July 27 in the journal PLoS One, the researchers tested their drug against 15 viruses, and found it was effective against all of them — including rhinoviruses that cause the common cold, H1N1 influenza, a stomach virus, a polio virus, dengue fever and several other types of hemorrhagic fever. The drug works by targeting a type of RNA produced only in cells that have been infected by viruses. “In theory, it should work against all viruses,” says Todd Rider, a senior staff scientist in Lincoln Laboratory’s Chemical, Biological, and Nanoscale Technologies Group who invented the new technology." (Source: These types of discoveries which move far away from one bug one drug, an issue which has haunted bio-defence could well be an obsolete approach given the revolution in drug discovery we are currently witnessing. 
While the current Ebola outbreak has devastated West African and I don't think this can be understated, if there is a positive to be drawn, it is the explosion of drug discovery and bio-technology applications which are likely to effect the future of how we control epidemic and pandemic disease. I believe significant strides in broad spectrum drug development will greatly increase our ability to eradicate several diseases entirely, polio being first on the list. 

Stopping a Hemorrhagic Killer: BCX4430

Colorized transmission electron micrograph (TEM) revealing some of the ultrastructural morphology displayed by an Ebola virus virion.
Ebola Image: CDC

"Filoviruses, such as Ebola and Marburg virus, constitute serious threats to our national defense," said Colonel Erin P. Edgar, commander of USAMRIID. "Development of cost-effective and versatile treatment options to combat these agents remains an unmet medical need and a high biodefense priority for the U.S. Government." See:

Case fatality rates associated with filovirus disease outbreaks are the highest reported for any infection, exceeding 90 percent. These pathogens are classified as Category A bioterrorism Agents by the Centers for Disease Control and Prevention. BCX4430 completely protected cynomolgus macaques from Marburg virus infection and Ebola virus infections. In addition, BCX4430 was shown to be active in vitro against a broad range of other RNA viruses, including the emerging viral pathogen Middle East Respiratory Syndrome Coronavirus (MERS-CoV)." See: BCX4430-in-a-Non-Human-Primate-Model-of-Filovirus-Infection#sthash.6b2rLOfA.dpuf

Although Crucell, a Dutch based pharmaceutical firm has been working on an Ebola vaccine candidate for several years and notes on their site that "In experiments conducted in 2004 by the VRC together with the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), our vaccine candidate confirms single-dose protection of monkeys against Ebola. Our results are distinct from the earlier trials in that our vaccine is based on PER.C6® cells, makind it suitable for large scale manufacturing." (See: One is not exclusive to the other and indeed a viable vaccine candidate would be a valuable addition to any national strategic stockpile. However, BCX4430 offers a novel approach with wide applications. 

BioCryst Pharmaceuticals, Inc. announced on March 3, 2014 in the journalNature extensive laboratory and nonclinical characterizations of BCX4430, "including efficacy results in animal models of infection with Marburg virus and Ebola virus, two highly virulent pathogens responsible for viral hemorrhagic fever diseases. The Nature publication entitled,"Protection against filovirus diseases by a novel broad-spectrum nucleoside analogue BCX4430," represents the first report of protection of non-human primates from filovirus disease by a small molecule drug, and describes efficacy results generated from an ongoing collaboration between scientists at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) and BioCryst." See: "BCX4430, resembles the famous "A" found in DNA: adenosine. (Recall that DNA is made of Adenosine, Thymidine, Cytidine and Guanosine.) The RNA-based filoviruses also use "A" in their genomes. BCX4430, because it resembles "A", can be accidently used by the virus when it is trying to grow inside of our cells. For the virus, this is a fatal mistake. BCX4430 blocks further growth and reproduction." See: 

While both Crucell and BioCryst offer potentially novel ways of combating filovirus outbreaks, BCX4430 has the advantage of greater application. This is an exciting discovery, not only as a counter-measure for Ebola and other VHF, but the potential application to other highly pathogenic diseases for which we currently have no treatment or preventive medical countermeasures i.e. vaccines. For years, bio-defence drug discovery has endured a one bug one drug approach, but advanced drug discovery means our ability to counter emerging and re-emerging diseases for which their previously was low to no investment incentives, may mean research and development for orphan diseases will become more enticing. The recent outbreak of Ebola in Guinea and Liberia, serves to remind us that emerging and re-emerging diseases continue to pose a global public health threat. The emergence of MERS-CoV and the increasing mortality associated with it mean it  too will likely remain a health concern for years to come. 

Charlie Hebdo: Should Europe worry about Mass Casualty Bio-Terrorism?

“Al Qaeda in the Maghreb is probably the most operationally capable affiliate in the organization right now.” 

--Roger Cressey, a former senior counterterrorism official at the National Security Council under Presidents Bill Clinton and George W. Bush Source: 
Virus - Public Domain
Source: CDC
While it may be a bit early to reflect on the Paris massacre of media staff at Charlie Hebdo and the Jewish market, the tactics are textbook conventional terrorist tactics used against innocent civilians. As analysts pour over data to discover cells and develop profiles of potential loan wolf perpetrators is Europe at increased risk of chemical or biological agents being used in conventional attacks? 

In 2009 the Washington Post published an article entitled: "Al Qaeda bungles Arms Experiment." The article posited that a biological weapon accident had occurred noting: "the accident killed at least 40 al Qaeda operatives, but [ ] the mishap led the militant group to shut down a base in the mountains of Tizi Ouzou province in eastern Algeria." "AQIM, according to U.S. intelligence estimates, maintains about a dozen bases in Algeria, where the group has waged a terrorist campaign against government forces and civilians. In 2006, the group claimed responsibility for an attack on foreign contractors. In 2007, the group said it bombed U.N. headquarters in Algiers, an attack that killed 41 people." Source:  It is difficult to surmise from public domain reports if this particular case was one of accidental exposure or work on plague, which is endemic in the region. What remains concerning about AQLIM is their repeated stated intent to develop and use biological weapons in Europe. While some speculate that his is the purview of state actors and would not likely be used in a terrorist attack, there is an increasing sense of concern that this might well be on the horizon. 

"Plague ( ) is a naturally occurring disease caused by the bacterium Yersinia pestis. This bacterium is found in rodents and fleas that infest them and exists in many parts of the world including the western United States. According to the U.S. CDC, there are 1000 to 3000 cases of plague diagnosed in humans every year; between five and fifteen of those cases occur in the United States. Y. pestis can infect humans in three ways. The bacteria cause pneumonic  plague when inhaled, though pneumonic plague can also occur when plague bacteria from another form of transmission infect the lungs. Bubonic  plague results when the bacteria enter through a break in the skin (such as a flea bite), and septicemic plague occurs when the bacteria multiply in the victim's blood (usually after being infected by one of the other types). In general, a flea bite is the primary form of infection, and if 
the infection is left untreated, it can evolve into a case of pneumonic or septicemic plague." Source:

Bubonic and septicemic plagues are not normally spread from person to person. Pneumonic plague can be contagious if a person inhales respiratory droplets containing the bacteria from an infected person, which usually requires close contact with the infected individual. Y. pestis is a fragile bacterium and does not last long in sunlight or after it is dried. Plague is treatable with antibiotics, which are especially effective if administered early. Wearing a simple surgical mask can protect a person from pneumonic plague infection. Like many biological agents, there are great challenges associated with producing and employing large quantities of a virulent biological agent. Certainly, plague can be obtained from the environment in a place where it occurs naturally, such as Algeria, but taking that bacterium and producing a large quantity of it in a virulent form and then disbursing it in an efficient manner is another matter entirely." Source:

Three things stand out  when we conider the likelihood of AQ using in particular plague as a bioweapon in the statements above. First, it is unlikely that AQ would use plague for a number of reasons not least of which that their highly educated microbiologists certainly are aware that yersinia pestis is not highly transmissible and weaponizing it to increase virulence or at the edges of their scientific capability modifying to make it so is extremely remote. Secondly, AQ microbiologists would certainly select a pathogenic agent for which there was no treatment available and finally, while terrorists may have the capability to manufacture quantities of biological warfare agents Category A agents, manufacturing large quantities is considered an obsolete past time by most bio-defence experts as BW can be deployed in small quantities where the quality not the quantity is important. Manufacturing large quantities of BW agents is generally considered a state activity prohibited since 1976 under the BTWC. 


So how likely is AQ to posses and deploy a biological warfare agent in a terrorist attack in Europe? My answer would be that while the risk remains relatively low, it is not zero and one attack with BW is akin to an airline going down, it kills a lot of people at once. We should better ask why Al Qaeda has highly trained microbiologists, where did they receive their training and how do we interdict this both with AQ as well as state offensive weapon programs which might be transferred to terror cells. Our current understanding of BW and synthetic biology may well mean that the technical thresholds we thought were a safeguard against the use of weaponized biological agents by terrorists, may be a false sense of security. AQ's microbiologists are likely to select agents for which there is no known treatment, highly transmissible and infective and which can be deployed in small highly concentrated quantities in areas such as transportation infrastructures where exposure to sunlight and UV is limited.  As shocking at the terrorist attack in Paris was we must remain vigilant that AQ will continue to up the ante and BW may well be on the horizon. 

Wednesday, January 14, 2015

Emerging Technologies: Bio-hacking and the future of bio-terrorism

For some time concern has been raised over 3D and 4D technologies, (with synthetic biology the emerging technological forerunner of these concerns and the NSABB playing watchdog), with regard to how inherent de-skilling may reduce the technical threshold which inhibits most would-be weaponeers from developing and deploying a weaponized biological agent capable of mass destruction. At the somewhat more extreme end, bio-hacking could reduce barriers which are perhaps better left in place.  Bio-hacking  was put on the map in 2013 when molecular biologist Ellen Jorgensen delivered a TED talk about Genespace, the DIY science lab she opened in New York in late 2010. See:  

(Image: Mac Cowell/FutureLabCamp)
The lab Jorgensen oversees is one of approximately 45 DIY international science groups,of  more than twenty in the US. While some of these labs are rather extreme in their goals, emerging technology such as 3D bioprinting could theoretically reduce the knowledge needed to develop synthetic weapons. So far several of the bio-hacking groups seem to be content with using themselves in experiments and implanting magnets but coding life for the masses and or the non-scientific community, could become a lot easier in a relatively short period of time.
"But we don't smuggle plutonium. We don't supply chemical weapons. We don't build rockets. Instead we have a hobby that the FBI believes could be so dangerous that they have come up with a special programme to make sense of it. That hobby is to play with genes, proteins and bacteria in our spare time in a homemade lab we constructed from scratch. We are part of a rapidly growing community of amateur geneticists, who are often labelled biopunks, or outlaw biologists. Or, better still, in an analogy to the computer programming enthusiasts of a generation ago, some call us bio-hackers. But instead of software code, we try to tinker with DNA, the code of life. The FBI has set up the Biological Countermeasures Unit ( ) one of their goals in preventing acts of terrorism is to reach out to leading names in the field to quiz them about what they do." See:

This surely must be cutting edge bio-security, however, how close are bio-hackers to actually crossing what was considered the technological threshold to creating what might even be considered synthetic biological weapons? After 911 and the US anthrax attacks, I advised governments that mass casualty bio-terrorism was not as simple as it was being touted. In fact I, and several other scientists, focused on state warfare laboratories, considering bio-terrorism not of real world interest. Emerging technology which results in de-skilling however, may make the life of the would be bioweaponeer far easier and reduce what was always considered to be rather insurmountable technical barriers, certainly in the deployment of a mass casualty weapon. 

What is the current view of life sciences deskilling, given the increase in DIY science? Johnathan B.Tucker, a former long time colleague, presented an excellent analysis of the issue in his paper, "Could Terrorists Exploit Synthetic Biology? published in The New Atlantis, see:, although notably before bio-hacker movement emerged more openly into the media with a cohesive defined goal and group structure. Tucker, in his analysis states: 

"Member of this second school point to a contradictory trend in biotechnological development that they claim will ultimately prove stronger. They note that the evolution of many emerging technologies involves a process of de-skilling that, over time, reduces the amount of tacit knowledge required for their use. Chris Chyba of Princeton, for example, contends that as whole-genome synthesis is automated, commercialized, and 'black-boxed,' it will become more accessible to individuals with only basic scientific skills, including terrorists and other malicious actors (16).De-skilling has already occurred in several genetic-engineering techniques that have been around for more than twenty years, including gene cloning (copying foreign genes in bacteria), transfection (introducing foreign genetic material into a cell), ligation (stitching fragments of DNA together), and the polymerase chain reaction, or PCR (which makes it possible to copy any particular DNA sequence several million fold). Although one must have access to natural genetic material to use these techniques, the associated skill sets have diffused widely across the international scientific community. In fact, a few standard genetic-engineering techniques have been de-skilled to the point that they are now accessible to undergraduates and even advanced high school students, and could therefore be appropriated fairly easily by terrorist groups." See:   

Gerald Epstein, of the Center for Science, Technology and Security Policy, write that whole-genome synthesis 'appears to be following a trajectory familiar to other useful techniques: Originally accessible only to a handful of top research groups working at state of the art facilities, synthesis techniques are becoming more widely available as they are refined, simplified, and improved by skilled technicians and craftsmen. Indeed, they are increasingly becoming 'commoditized,' as kits, processes, reagents, and services become available for individuals with basic lab training." (17). In 2007 Epstein and three co-authors predicted that 'ten years from now, it may be easier to synthesize almost any pathogenic virus than to obtain it through other means," although they did not imply that individuals with only basic scientific training will be among the first to acquire this capability.(18)" See: 

"To date, the de-skilling of synthetic genomics has affected only a few elements of what is actually a complex, multi-step process. Practitioners of de novo viral synthesis note that the most challenging steps do not involve the synthesis of DNA fragments, which can be ordered from commercial suppliers, but the assembly of these fragments into a functional genome and the expression of the viral proteins. According to a report by the U.S. National Science Advisory Board for Biosecurity, a federal advisory committee, "The technology for synthesizing DNA is rapidly accessible, straightforward and a fundamental tool used in current biological research. In contrast, the science of constructing and expressing viruses in the laboratory is more complex and somewhat of an art. It is the laboratory procedures downstream from the actual synthesis of DNA that are the limiting steps in recovering viruses from genetic material." (19)" See: 

As technology emerges which contributes to deskilling and with the advent of DIY science, we may witness rather rapid advancements which overcome the long time presumed threshold. The bio-hacking community has emerged because techniques used in molecular biology have been de-skilled and the cost has dropped. 

"A couple of decades ago, it took three years to learn how to clone and sequence a gene, and you earned a PhD in the process. Now, thanks to ready made kits you can do the same in less than three days. Specialized materials and second hand equipment are much more affordable, not to mention more available. Machines for amplifying DNA can now be purchased online, whilst enzymes and chemicals for creating, manipulating and sticking together DNA an be ordered off the shelf. The cost of sequencing DNA has plummeted , from about 100,000 for reading a million letters or base pairs, of DNA code in 2001, to around 10 cents today. See: full review: Warfare Technology Analytics

Sunday, January 11, 2015

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)

CRISPR (Clustered Regularly Inter-spaced Short Palindromic Repeats), is a basic acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. Each repetition contains a series of base pairs followed by the same or a similar series in reverse and then by thirty or so base pairs known as 'spacer DNA.' The spacers are short segments of DNA from a virus and serve as a 'memory' of past exposures. CRISPRs are found in the genomes of approximately 40% of sequenced eubacteria and 90% of sequenced archaea. CRISPR's are often associated with cas genes which code for proteins that perform various functions related to CRISPRs. The CRISPR-Cas system functions as a prokaryotic immune system, in that it confers resistance to exogenous genetic elements such as plasmids and phages and provides a form of acquired immunity. CRISPR spacers recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms. Since 2012, the CRISPR-Cas system has been used for a novel technique of gene editing (silencing, enhancing or changing specific genes) which even works in eukaryotes. By inserting a plasmid containing cas genes and specifically designed CRISPRs, the organisms genome can be cut at any desired location. (2) See:
Microinjection of embryos with TALEN mRNA 
The Biochemical Society noted the following in an announcement of a focused meeting entitled: CRISPR: Evolution, Mechanisms and Infection:

"The extreme evolutionary pressure exerted on cells by viruses, and vice verse, is a key driving force in evolution. Perhaps the most exciting development in this area in the past ten years is the discovery of the CRISPR system for antiviral defence. These clusters of regularly inter-spaced palindromic repeats are genomically encoded by many prokaryotes and carry a record of past viral infections. Transcription and processing of the CRISPR RNA leads to RNA-directed cleavages of the nucleic acid of invading mobile elements, mediated by large, complex molecular machines assembled from CRISPR associated (Cas) proteins. The process of viral DNA capture and incorporation int CRISPR loci is still enigmatic and is an interesting example of pure Lamarckian evolution. The dynamic interplay between viruses and hosts also has clear parallels to the predator: prey relationship studied more widely in biology. CRISPRs are the most rapidly evolving parts of most prokaryotic genomes, providing opportunities for the study of evolution in the field in real time." See:

The applications of CRISPR will likely revolutionize genetic engineering, allowing scientists to engineer any part of the human genome with exceptionally accurate precision. Caribou Biosciences, a Berkeley based company, specializes in utilizing the Cas9 enzym, an extremely efficient genome editing platform. Dr. Haurwitz, of Caribou noted: "I would say that Cas9, like other site-specific genome engineering technologies such as TALENs (transcription activator-like effector nucleases) or ZFNs (zinc finer nucleases), does have the capability of being deployed appropriately in a therapeutic context to modify patients cells at the genomic level in order to either repair disease causing genes or otherwise prevent infections. For example Sangamo Biosciences, which is a company here in the Bay Area, has clinical trials ongoing where they are using ZFN technology to modify T-cells or stem cells in order to knock out the receptor that is necessary for HIV infections. So they are using site specific genome engineering technology with the ultimate goal of curing HIV. In terms of Cas9 specific therapeutic applications, I think there is tremendous opportunity. I also think the trickiest part is not actually editing the cells. There are a number of technologies for editing the cells; it's appropriately and safely delivering the editing technology that is the tricky part." See:

It will be interesting to follow Caribou Biosciences as a forerunner in Cas9 and I will update this as things progress.

Saturday, January 10, 2015

Nanoparticle Vaccines: Next-Generation Vaccine Platforms

In Vaccine, authors Liang Zhao, Arjun Seth, Nani Wibowo, Chun-Xia Zhoa, Neena Mitter, Chengzhong Yu and Anton P.J. Middelberg contend: 
Vaccine development is historically based on Louis Pasteur's “isolate, inactivate, inject” paradigm. As vaccine development moves increasingly to draw on modern concepts of rational design, the number of candidate vaccines is increasing  [1] and [2]. Most candidate vaccines represent “minimalist” compositions[3], which typically exhibit lower immunogenicity. Adjuvants and novel delivery systems that boost immunogenicity are increasingly needed as we move toward the era of modern vaccines.
Nanotechnology offers the opportunity to design nanoparticles varying in composition, size, shape, and surface properties, for application in the field of medicine [4] and [5]. Nanoparticles, because of their size similarity to cellular components, can enter living cells using the cellular endocytosis mechanism, in particular pinocytosis [6]. These cutting-edge innovations underpinned a market worth US $6.8 billion in 2006 [7] and predicted to reach US $160 billion by 2015 [8]. Source: "Nanoparticle Vaccines",Vaccine, Vol.32, Issue 3, 9 January 2014,

Anil Mahapatro and Dinesh K. Singh describe the advantages of nanaparticle drug delivery in their research paper entitled, " Biodegradable nanoparticles are excellent vehicle for site directed in-vivo delivery of drugs and vaccines," which appeared in the Journal of Nanobiotechnology, (Source: Mahapatro and Singh provide the following analysis "

Polymer-based nanoparticles are submicron-sized polymeric colloidal particles in which a therapeutic agent of interest can be embedded or encapsulated within their polymeric matrix or adsorbed or conjugated onto the surface [6]. These nanoparticles serve as an excellent vehicle for delivery of a number of biomolecules, drugs, genes and vaccines to the site of interest in-vivo. During the 1980's and 1990's several drug delivery systems were developed to improve the efficiency of drugs and minimize toxic side effects [7]. The early nanoparticles (NPs) and microparticles were mainly formulated from poly-alkyl-cyanoacrylate. The initial enthusiasm for the use of microparticles in medicine was later on dampened due to the size of the microparticles. There is a size limit for the particles to be able to cross the intestinal mucosal barrier of the gastrointestinal (GI) tract after the drug has been delivered orally. Most often, macroparticles could not cross mucosal barrier due to their bigger sizes resulting in failed delivery of drugs. Nanoparticles on the other hand have an advantage over microparticles due their nano-sizes. They are also better suited for intravenous (i.v.) delivery [8] compared to microparticles. Nanoparticles, however, had a different set of problems of their own. They had a very short circulating life span within the body after intravenous administration. The nanoparticles administered intravenously were rapidly cleared from the body by phagocytic cells. The therapeutic effect of drugs delivered via nanoparticles was thus minimized and could not be sustained. In recent years the problem of phagocytic removal of nanoparticles has been solved by surface modification of nanoparticles [7]. The surface modification protected nanoparticles from being phagocytosed and removed from the blood vascular system after intravenous injections. Now, a wide variety of biomolecules, vaccines and drugs can be delivered into the body using nanoparticulate carriers and a number of routes of delivery. NPs can be used to safely and reliably deliver hydrophilic drugs, hydrophobic drugs, proteins, vaccines, and other biological macromolecules in the body. They can be specifically designed for targeted drug delivery to the brain, arterial walls, lungs, tumor cells, liver, and spleen. They can also be designed for long-term systemic circulation within the body." Source:

If we consider applications for the current outbreak of Ebola across West Africa the advantages are quite dramatic. One of the forerunners of nanovaccine technology applications for Ebola is Novovax. According to Emily Mullin, "In rodents and monkeys, Novavax recombinant glycoprotein (GP) nanoparticle vaccine was highly effective, generating antibodies against the virus in the blood of animals challenged with the 2014 Guinea Ebola strain, which is responsible for the current Ebola epidemic in West Africa." (Source: 
Yet, nanotechnology applications for future vaccines is not the only application which could benefit the current outbreak of Ebola. Ceres, a biotech company has developed a nanoparticle technology 'Nanotrap' which provides biofluid sample processing capabilities for a wide array of diagnostic applications and sample handling needs. (Source:

During the current outbreak of Ebola raging through parts of West Africa several Ebola Treatment Units and Centers (ETU's and ETC's) shifted their policy to reduce the use of IV's particularly in late stage cases. The advantages of nanoparticle delivery platforms, particularly in treating VHF's should not be underestimated.  "Only a handful of mucosal vaccines have been approved for human use; the best-known example is the Sabin polio vaccine, which is given orally and absorbed in the digestive tract. There is also a flu vaccine delivered by nasal spray, and mucosal vaccines against cholera, rotavirus and typhoid fever." (Source; Darrell Irvine, an MIT professor of materials science and engineering and biological engineering and the leader of the research team at MIT notes: "Mice vaccinated with nanoparticles were able to quickly contain the virus and prevent it from escaping the lungs. Vaccinia virus usually spreads to the ovaries soon after infection, but the researchers found that the vaccinia virus in the ovaries of mice vaccinated with nanoparticles was undetectable, while substantial viral concentrations were found in mice that received other forms of the vaccine. Mice that received the nanoparticle vaccine lost a small amount of weight after infection but then fully recovered, whereas the viral challenge was 100 percent lethal to mice who received the non-nanoparticle vaccine.
To create better ways of delivering such vaccines, Irvine and his colleagues built upon a nanoparticle they developed two years ago. The protein fragments that make up the vaccine are encased in a sphere made of several layers of lipids that are chemically “stapled” to one another, making the particles more durable inside the body." Source:
Pulmonary drug delivery with nanoparticle vaccines will be the drug delivery platform for for future Ebola and VHF outbreaks, in addition to its applications across a wide spectrum of infectious disease. 

Sunday, January 4, 2015

DARPA's 7-Day Bio-Defence and the Future of Synthetic Vaccines

US military personnel don special biohazard gear during a training exercise designed to simulate a biological weapon attack. The Department of Defense and other agencies routinely hold training sessions throughout the country as part of a domestic bioterrorism preparedness program. 'The challenge is to integrate these forces to mount an effective response under various attack scenarios," says Prof. Steven Block. Courtsey: US Navy 

Author's note: In 2007, I attended a bio-defence briefing in Washington D.C., delivered by a DARPA scientist. It was as one would expect from DARPA, an incredible presentation on pipeline technologies. Since then I have always thought of DARPA as the 'bugs on the wall folks' and indeed they have successfully produced robotic bugs, but more significantly, in the briefing they discussed advanced bio-defence technologies which we were not allowed to take notes on or photograph the slides of; it was extremely exciting and I left with a sense of awe which DARPA tends to inspire.

Six years later, they have successfully manufactured 10 million doses of H1NI (flu) vaccine within a month. For pharmaceutical companies who invest up too and over a billion per drug and not forgetting the length of time one vaccine can take to bring to market, about ten years, the DARPA/Medicago manufacturing of 10 million doses of H1N1 in one month was a phenomenal feat. Here we are on the threshold of major breakthroughs in vaccine research, development and production, even manufacturing technologies and the future of bio-defence couldn't look brighter. While industry of course continues to work on live attenuated vaccine production is the future a synthetic one?

Current vaccine production based on inactivated  viruses (live attenuated vaccines), has been successful in reducing significant disease burden associated with major epidemics of the 19th and 20th centuries. However, a major draw back has been the lengthy research and development phase, the significant investment costs and the inability to respond rapidly to changing strains. Synthetic vaccines may overcome many of the more traditionally based production obstacles. In 2012 DARPA announced it's Blue Angel Program. On their site they lay out the problem quite concisely with regard to responding to pandemics of the future. Such pandemics may or may not be natural and may or may not be caused by natural pathogens, viruses and toxins. Consider fighting a synthetically produced outbreak. The DARPA site states:

"The 2009 Army Posture Statement, cites a World Health Organization estimate of between 20 annd 50 percent of the world's population being effected if a pandemic were to emerge. WHO forecasts 'it may be six to nine months before a vaccine for a pandemic virus strain becomes available." In a separate report on pandemic influenza, the WHO described several challenges to producing sufficient volumes of vaccine using current, egg based protein-production technology, including the likelihood that two doses per person could be required due to the absence of pre-existing immunity. In short, the potential for a pandemic exists and current technological limitations on defensive measures put the health and readiness of U.S. military forces at risk. A technological solution to increase the speed and adaptability of vaccine production is urgently needed to match the broad biological threat. DARPA's Blue Angel Program seeks to demonstrate a flexible and agile capability for the Department of Defence to rapidly react to and neutralize any natural or intentional pandemic disease. Building on a previous DARPA program, Accelerated Manufacture of Pharmaceuticals, Blue Angel targets new ways of producing large amounts of high quality, vaccine grade protein in less than three months in response to emerging and novel biological threats. One of the research avenues explores plant made proteins for candidate vaccine production. "Vaccinating susceptible populations during the initial stage of a pandemic is critical to containment," said Dr. Alan Magill, DARPA program manager. "We're looking at plant based solutions to vaccine production as a more rapid and efficient alternative to the standard egg-based technologies, and the research is very promising." In a recent milestone development under Blue Angel, researchers at Medicago Inc. produced more than 10 million doses (as defined in an animal model) of an H1N1 influenza vaccine candidate based on virus-like particles (VLP) in one month. Production adhered to Phase 1 appropriate current good manufacturing practices. The work was part of a 'rapid fire' test that ran from March 25, 2012 to April 24, 2012, at a facility in Durham, NC. A third party laboratory tested the production lots to confirm the immunogenicity of the vaccine candidate. Testing confirmed that a single dose of the H1N1 VLP influenza vaccine candidate induced protective levels of hemagglutinin antibodies in an animal model when combined with a standard aluminum adjuvant. The equivalent dose required to protect humans from natural disease can only be determined by future, prospective clinical trials. 

DARPA's 7 Day Bio-Defence 

Photo: DARPA
In May, 2013 Medicago Inc. announced it had successfully produced a VLP vaccine candidate for the H7N9 virus responsible for an influenza outbreak in China. Medicago's future in VLP vaccines couldn't look brighter especially given their success with rapid vaccine production and their work with DARPA. 
An additional, although slightly different project which I believe will make significant strides in bio-defence counter-measures is DARPA's 7-Day Bio-Defence project, again, worth considering in terms of how future vaccines will be manufactured and how investment in this technology could well shift. As stated on their site:

"Military readiness and national security depends on the health and well being of military service members. The Department of Defence's (DoD) cumulative investment in personnel comprises the second largest share of the total defence budget. As such DoD seeks advances in health care to ensure war-fighters can operate at peak performance. Research into natural and synthetic pathogens, and treatments against them is one plank of ensuring military readiness in the face of accidental and offensive biological threats to both war-fighters and the supply chain supporting them. In this context, the 7-Day Biodefence program will seek to develop novel technologies focused on preventing infection by any emerging pathogen, sustaining survival once infected, and building immunity. In recent years, global surveillance networks have determined an increase in the frequency and diversity with which new infectious micro-organisms are emerging. While this increase is due in part to improved reporting, multiple examples demonstrate it is also promulgated by changes in natural systems and possibly human activity. The potential biological threat breaks down into two primary categories: 

1. Exposure to natural pathogens that are the result of: increased human-animal interface; increased population densities and co-location of vulnerable species with pathogen reservoirs; climate change, particularly affecting migration and spread of vectors; and narrowing of genetic diversity among food-animal stocks. 

2. Exposure to synthetic and highly diverse pathogens that have become easier to produce as bio medical and genetic-engineering technologies proliferate internationally; such that pathogens could be used by adversaries for offensive purposes in a direct attack on war-fighters for covert sabotage of the agricultural industry that supports war-fighters. 

Together, these emerging threat agents challenge current medical countermeasures. Today's research and development cycle for countermeasures is ill-equipped for rapid response to emerging biological threats. In response to the unspecified potential threat from emerging pathogens, the goal of the 7-Day Biodefence program is to develop innovative approaches to counter pathogens without regard to their exact nature. The methods being explored do not require prior knowledge of the pathogen and are broadly applicable to multiple, unrelated infectious agents. The program consists of four technical areas investigating novel technologies to: 1. prevent infection; 2. sustain survival; 3. provide transient immunity; and 4. create persistent immunity. See:

Advances in synthetic vaccine manufacturing, VLP's and even 3D bio-printing will significantly change our concept of bio-defence and the manufacturing process of medical counter-measures. The incorporation of these counter-measures will additionally change how we approach threat reduction and possibly remove many of the traditional concerns at the technical level. This could increase interest in areas which have typically been an after thought.