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

PCR Better than Pap Test for Preventing Cervical Cancer

“Power of PCR” as a Transformative Diagnostic Method

  • FDA Approves Roche PCR Test for Cervical Cancer Screening
  • Automated Test Replaces Pap Test as First-line Cervical Cancer Screening
  • Demonstrates the “Power of PCR” as a Transformative Diagnostic Method

Pap Test

The Papanicolaou test—aka Pap test, Pap smear, cervical smear, or smear test—is a method of cervical screening used to detect potentially pre-cancerous and cancerous processes in the endocervical canal of the female reproductive system. Unusual findings are often followed up by more sensitive diagnostic procedures, and, if warranted, interventions that aim to prevent progression to cervical cancer.

Proper interpretation of microscopic results requires a “trained eye.” This is evident from the representative example shown below, which I found in an online educational textbook and, quite frankly, had trouble discerning the visual keys described in the verbatim caption. Notwithstanding this issue, Pap tests were—until now—the accepted “gold standard.”

pap

Taken from homepage.smc.edu via Bing Images.

The source document for this Pap smear reads as follows. “The cytologic features of normal squamous epithelial cells can be seen at the center top and bottom, with orange to pale blue plate-like squamous cells that have small pyknotic nuclei. The dysplastic cells in the center extending to upper right are smaller overall with darker, more irregular nuclei.”

The eponymous test was pioneered by Georgios Papanikolao, a prominent Greek doctor, who in 1928 was the first to report that uterine cancer could be diagnosed by means of a vaginal smear. However, the importance of this work was not widely recognized until his 1943 publication of Diagnosis of Uterine Cancer by the Vaginal Smear, coauthored by Herbert F. Traut, both at Cornell University Medical College.

ResearchGeorgios Papanicolaou moved to Miami, Florida in 1961 to establish the Papanicolaou Cancer Research Institute at the University of Miami, but died in 1962 prior to its opening. Papanicolaou was the recipient of the Albert Lasker Award for Clinical Medical Research in 1950—this award is sometimes referred to as “America’s Nobels,” as eighty-six Lasker laureates have received the Nobel Prize. Papanikolaou’s portrait appeared on the Greek 10,000-drachma banknote of 1995-2001, prior to its replacement by the Euro.

Cervical Cancer Statistics

Cervical cancer is the second most common cancer in women worldwide, according to an NIH publication in 2007. Country-by-country data for cervical cancer reveal a striking geographical distribution. According to currently available U.S. Centers for Disease Control (CDC) FastStats, cervical cancer mortality in the U.S. in 2010 was ~4,000 or ~2.5 deaths per 100,000 females.

The global statistics provided by Cancer Research U.K. are far more saddening. Worldwide there were more than ~275,000 deaths from cervical cancer in 2010 that accounted for ~10% of female cancer deaths.

Remarkably, mortality rates are reported to vary seventeen-fold between the different regions of the world. By estimating the years-of-life-lost (YLL) by young and middle-aged women (25-64 years old) in different regions of the world, YLL attributed to cervical cancer is the most important cause of YLL for all cancers in Latin America, the Caribbean, and populous regions of Sub-Saharan Africa and South-Central Asia. The overall picture is not very sensitive to the age-weighting function used. The report notes that, since this loss of life is preventable using existing technologies, more health-resource allocation in low income settings is needed.

Pap Test Statistics

Currently available CDC FastStats for Pap test use in the U.S. in 2010 (the most recent year available) are as follows:

  • Percent of women 18 years of age and over who had a Pap test within the past 3 years: 73.2%
  • Number of physician office visits during which Pap tests were ordered or provided: 29.4 million
  • Number of hospital outpatient department visits during which Pap tests were ordered or provided: 2.4 million

Pap Test Recommendations as of 2013

To put today’s blog-post headline about switching from Pap to PCR in perspective, here are snippets from the most recent CDC guidelines and comments made available in a January 2013 press release headlined with “more women getting Pap tests as recommended [but] some women get Pap tests without need.”

  • In 2012, the U.S. Preventive Services Task Force, American College of Obstetricians and Gynecologists and American Cancer Society recommended that women, beginning at age 21, should start Pap test screening every three years.
  • The same groups agree that screening is unnecessary for most women who have had a total hysterectomy (removal of the uterus and uterine cervix) for non-cancerous reasons, or for women aged 65 years and older with several years of normal test results.
  • Studies analyzed Pap test survey data from CDC’s Behavior Risk Factor Surveillance System found the following:
    • The percentage of women aged 18-21 years who reported never being screened increased from 23.6% in 2000 to 47.5% in 2010; however, screening is not recommended for women under the age of 21.
    • In 2010, 58.7% of women aged 30 years and older who had a hysterectomy were still given a Pap test.
    • Because of the Affordable Care Act (aka Obamacare), many private health plans and Medicare now cover certain preventive services, including cervical cancer screening, with no copays or other out-of-pocket costs.

HPV: The Cervical Cancer-Causing Agent and Key to Early Detection

In a landmark publication in 1999 entitled Human papillomavirus is a necessary cause of invasive cervical cancer worldwide, Dutch investigators used PCR data to establish that the worldwide HPV prevalence in cervical carcinomas is 99.7 per cent. They noted that “the presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer.” More importantly, they presciently concluded that “the extreme rarity of HPV-negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening.”

Due in part to technical challenges posed by numerous genotypes of HPV with varying cancer causality detailed elsewhere, and unavoidable time-consuming clinical studies required for FDA approval, it has taken ~15 years for a PCR test to now be poised to displace the Pap test as the primary diagnostic approach for early detection of cervical cancer.

Those of you who are interested in the technical underpinnings of Roche’s investigations to this end are referred to this 2013 publication by Roche and collaborators entitled Development and characterization of the cobas human papillomavirus test. In contrast to the tedious Pap test protocol and its “visually challenging” manual microscopic analysis, this “cobas”-based PCR test provided by Roche is fully automated.  The test process involves two instruments: one that completes sample preparation (COBAS® AmpliPrep) and another that performs the PCR process and detection of the pathogen DNA in real time (COBAS® TaqMan® Analyzer).

Incidentally, I traced-back the term “cobas” to late 1970’s Roche instrumentation named the “cobas-bio” analyzer, but could not decipher what “cobas” stands for! If any of you know the answer, please let us know by a comment at the end of this post.

FDA Panel Recommends Replacement for the Pap Test

This attention-grabbing headline of a March 2014 NY Times article by Andrew Pollock was the catalyst for my decision to research and write this blog exemplifying the “power of PCR” as a transformative diagnostic method. While this and numerous other popular news media all made reference to an FDA panel’s report, it took some digging to find the actual source-report, which is an 80-page pdf that can be accessed here to peruse in detail, if you wish. However, a much shorter but essential-fact-laden article by Joyce Frieden, News Editor of MedPage Today provided the following excerpts.

The FDA’s Medical Devices Advisory Committee Microbiology Panel agreed by a vote of 13-0 in each of three successive votes that the cobas® viral DNA test for HPV—made by Roche Molecular Systems—was safe and effective for cervical cancer screening, and that the benefits of the tests outweighed the risks. The Panel recommended that this Roche HPV test replace the Pap smear as the first-line standard of care for cancer screening.

The Roche test is seen as better than Pap tests in finding precancerous lesions (taken from the NY Times).

The Roche test is seen as better than Pap tests in finding precancerous lesions (taken from the NY Times)

The cobas® test currently has approval as a follow-up assessment for women 21 and older who have abnormal Pap tests, and as a co-test with the Pap smear to screen for the high-risk p16 and p18 HPV strains in women 30 to 65. The test comprises genotyping for HPV16 and 18 and pooled assessment of 12 additional high-risk HPV strains.

According to the proposal submitted by Roche, women 25 and older who test positive for HPV16 or 18 would proceed directly to colposcopy for further assessment.

Patients who test negative for HPV16 or 18 but positive for the other high-risk strains would have a Pap test to determine the need for colposcopy. Women who have a completely negative test would be followed at their physician’s discretion.

Panelists did express some concerns about dropping the age at which women should have the test from 30 to 25. The ATHENA study of over 47,000 patients with long-term follow-up used as the basis for the application found that about 11% of women ages 25 to 29 tested positive for HPV16 or 18 with the cobas test, compared with 7.28% among women 25 to 29 who had cytology alone as their first-line screening. Panel member Paula Hillard, MD, of Stanford University in California, was quoted as saying that would mean more patients in that age group “will be anxious about potentially having cancer.”

In addition, Hillard is quoted as expressing concern about off-label use. “I’m concerned that all those women potentially with other high-risk positivity won’t go to Paps next but go [straight] to colposcopy. That’s not what’s proposed here, but what control does FDA have once it’s out there?”

Panelist Kenneth Noller, MD, of the American Board of Obstetrics and Gynecology, in Dallas, agreed that real-world use could differ from the protocol proposed by Roche. He’s quoted as saying that “I’ve been watching how people practice; if you’re high-risk HPV positive you’re going to get colposcopy.” Furthermore, he said “that doesn’t necessarily mean it’s bad—it’s what you do with the colposcopy.”

Noller added that although he was “somewhat biased against dropping the age to 25 before I came here … I find the data presented today somewhat compelling to drop it to 25.”

Agreeing with this was panel member Kimberly Hanson, MD, MHS, of the University of Utah and ARUP Laboratories, both in Salt Lake City: “now we have the opportunity to identify women earlier, and to me that’s compelling,” adding that “although colposcopy is invasive and can be anxiety-provoking, it’s really very safe, so I think I’m leaning toward earlier screening.”

According to the summary submitted by FDA staff members, “The data show that the proposed primary screening indication for the cobas HPV test detects more women with disease and requires fewer women without disease to go to colposcopy than cytology alone.”

Benefit-risk analyses favored the HPV DNA test whether expressed in terms of number of cases of high-grade cervical disease per 10,000 women screened or per 100 colposcopy procedures.

The FDA is not bound to follow its advisory committees’ recommendations, but does so in most cases. On April 25—coincidentally DNA Day 2014—the FDA formally approved Roche’s HPV test as the First-Line Cervical Cancer Screening Method.

The “Entrenchment Factor”

At the risk of “throwing cold water” on the aforementioned PCR test benefits, I feel compelled to quote from Pollak’s NY Times story that ended with the following caveat.

“The Pap test, which is well entrenched and has been highly successful, will not go away quickly, if at all, however.

Assuming the FDA itself agrees with its advisory committee and approves the new use of Roche’s test, it would become just another option, not a replacement for the older testing regimens. And many doctors will not adopt the new test unless professional societies recommend it in guidelines, which could take years.”

Let’s all hope that these professional societies—and any other persuasive factors—lead to relatively rapid adoption by doctors.

As always, your comments are welcomed.

Venter’s Latest Venture: Increasing Human Longevity

  • The Quest to Make 100 the New 60
  • Aiming to Sequence 100,000 Human Genomes Per Year (Wow!)
  • Adding Genomes, Microbiomes, and Metabolomes to Health Records May Lead to Better Health and Longevity

Prologue

Although this post is mainly about a new start-up called Human Longevity, whose mission is to apply genomics to guide increased longevity, the fact that this company was founded by J. Craig Venter has certainly created a “buzz.” The very name Venter—to me—is synonymous with scientifically unorthodox ideas that are big and bold. If you’re familiar with Venter’s accomplishments and “genomics rock star” status, go to the next section; if not, here are some highlights of his rise to fame.

As an investigator at NIH, Venter gained notoriety when he caused a brouhaha with his intentions to patent genes he discovered using expressed sequence tags (ESTs). The controversy was so extensive that it precipitated resignation of Nobel Laureate James D. Watson in 1992, who was then Director of NIH’s Human Genome Office. This caught the eye of a venture capitalist, Wallace Steinberg, who wanted to start a gene-finding company—with Venter as its head. Venter, however, insisted on a nonprofit venture, so Steinberg set him up in a nonprofit entity called The Institute for Genomic Research (TIGR) supported by a new company, Human Genome Sciences. (A NY Times story by Nicholas Wade entitled A Maverick Making Waves provides a nice overview of Venter’s career path through 2000).

Venter generated thousands of EST’s to the human genome that became the intellectual property of Human Genome Sciences and enabled the company to develop far-reaching claims to many medical genes of interest. In partnership with Nobel Laureate Hamilton O. Smith, Venter next used shotgun sequencing—then unproven, and controversial—to completely sequence Haemophilus influenzae. This gave scientists their first glimpse into the set of genes necessary for life. Moreover, this achievement set off a revolution in medical microbiology, inspiring efforts to decode every major pathogen and learn the microbes’ entire playbook for attacking human cells.

These early sequencing successes in turn led Michael W. Hunkapiller—then President of PE Biosystems, which made the leading brand of DNA sequencing instrument—to recruit Venter to run Celera—a new private company. Venter boldly declared to the media that Celera would decode the human genome using shotgun sequencing by 2001—ahead of the public consortium, sparking a contentious “race for the human genome.”

Fast forwarding from his role in decoding the human genome—described as the single most important scientific breakthrough of modern times—Venter is Founder, Chairman, and CEO of the J. Craig Venter Institute (JCVI), a non-profit research organization with approximately 300 scientists and staff dedicated to human, microbial, plant, synthetic and environmental genomic research, and the exploration of social and ethical issues in genomics.

Venter is also Founder and CEO of Synthetic Genomics Inc. (SGI), a privately held company dedicated to commercializing genomic-driven solutions to address global needs such as new sources of energy, new food and nutritional products, and next generation vaccines. Recently Venter announced his latest venture, Human Longevity Inc. (HLI), “a genomics and cell therapy-based diagnostic and therapeutic company focused on extending the healthy, high performance human life span.”

Venter relaxing on his 95-foot sailboat/research vessel named Sorcerer II.

Venter relaxing on his 95-foot sailboat/research vessel named Sorcerer II. Photograph: Rick Friedman/Corbis (taken from theguardian.com via Bing Images).

Since 2003, scientists at the J. Craig Venter Institute have been on a quest to unlock the secrets of the oceans by sampling, shotgun sequencing and analyzing the DNA of the microorganisms living in these waters. In February 2014, the vessel embarked on a sampling expedition of the Amazon River and its tributaries, which contains 1/5th of the Earth’s river flow.

Human Longevity: Genomics-Based Fountain of Youth?

The Fountain of Youth is a spring that supposedly restores the youth of anyone who drinks or bathes in its waters. Tales of such a fountain have been recounted across the world for thousands of years, beginning with writings by the Greek historian Herodotus. The tale was particularly prominent in the 16th century, when it became attached to the Spanish explorer Juan Ponce de León, who was searching for the Fountain of Youth when, in 1513, he traveled to what is now Florida.

Artistic rendering of Ponce de León accepting water from the Fountain of Youth (taken from tabuherbalsmoke.com via Bing Images).

Artistic rendering of Ponce de León accepting water from the Fountain of Youth (taken from tabuherbalsmoke.com via Bing Images).

Given this legendary history and our collective wish for healthy, long lives, it’s not surprising that Venter’s announcement earlier this year attracted widespread media attention and significant funding—to the tune of $70 million. A story in the NY Times refers to Venter as saying that the largest of the investors is K. T. Lim, a Malaysian billionaire who runs Genting Berhad, a gambling conglomerate. Venter adds that a ‘not insignificant’ part of the funding comes from Illumina—for reasons that will be appreciated by reading further.

The press release goes on to say that HLI’s funding is being used “to build the largest human sequencing operation in the world to compile the most comprehensive and complete human genotype, microbiome, and phenotype database available to tackle the diseases associated with aging-related human biological decline.” HLI is also “leading the development of cell-based therapeutics to address age-related decline in endogenous stem cell function.”

In addition, HLI’s “revenue streams will be derived from database licensing to pharmaceutical, biotechnology and academic organizations, sequencing, and development of advanced diagnostics and therapeutics.”

Venter is quoted as saying that “using the combined power of our core areas of expertise—genomics, informatics, and stem cell therapies, we are tackling one of the greatest medical/scientific and societal challenges—aging and aging related diseases,” and that “HLI is going to change the way medicine is practiced by helping to shift to a more preventive, genomic-based medicine model which we believe will lower healthcare costs. Our goal is not necessarily lengthening life, but extending a healthier, high performing, more productive life span.”

HLI cofounder Peter H. Diamandis, M.D. puts this another and trendier way, according to the NY Times, which quotes Diamandis as saying that the goal was not to make people live forever, but rather to make “100 years old the next 60.” The NY Times goes on to say that “Venter, who is 67, sounds as if he might not need the company to succeed. Quoting Venter, “I feel like I have at least 20 or 30 years left in my career.”

HLI’s humongous database-to-be will surely be, in my opinion, a prime example of Big Data—itself a “hot trend.” It aims to have genomic sequences from “a variety of humans—children, adults and super centenarians [i.e. people who have attained the age of at least 110 years] and those with disease and those that are healthy,” according to the press release.

Illumina Looms Large in Longevity’s Plans

HLI has initially purchased two Illumina HiSeq X Ten Sequencing Systems (with the option to acquire three additional systems) to sequence up to 40,000 human genomes per year, with plans to rapidly scale to 100,000 human genomes per year.

Let me repeat this to be sure you don’t think these are typos.

40,000 and then 100,000 human genomes per year!

As pictured below, each of these newly introduced Sequencing Systems is comprised of ten—count them—instruments, which I’ve previously written about as enabling the long-elusive $1,000 genome cost target. Ironically, this goal was set by Venter as a technical challenge in 2002 at a now monumental TIGR conference.

Each Illumina HiSeq X Ten Sequencing System has a list price of $10 million.

Each Illumina HiSeq X Ten Sequencing System has a list price of $10 million.

Microbiome and Metabolome Data

Relative proportion of sequences determined at the taxonomic phylum level at eight anatomical sites. High-throughput sequencing has revealed substantial intra-individual microbiome variation at different anatomical sites, and inter-individual variation at the same anatomical sites. Such site-specific differences and the observed conservation between human hosts provide an important framework to determine the biological and pathological significance of a particular microbiome composition (taken from nature.com via Bing Images).

Relative proportion of sequences determined at the taxonomic phylum level at eight anatomical sites. High-throughput sequencing has revealed substantial intra-individual microbiome variation at different anatomical sites, and inter-individual variation at the same anatomical sites. Such site-specific differences and the observed conservation between human hosts provide an important framework to determine the biological and pathological significance of a particular microbiome composition (taken from nature.com via Bing Images).

Along with the genomic data gleaned from the sequencing of complete human genomes, HLI will also be generating microbiome data for many of these individuals through its Biome Healthcare division. The division is lead by Karen Nelson, who at TIGR led the first human microbiome study on the human gut published in Science in 2006.

The microbiome— a very “hot” trend in genomics research that I wrote about last year—consists of all the microbes that live in and on the human body that contribute to health and disease status of an individual. By better understanding a person’s microbiome—from gut, oral, skin, lung, and other body sites—the company said that it “anticipates developing improved probiotics and other advanced diagnostic and therapeutic approaches to improve health and wellness.”

HLI will also capture and analyze each individual’s metabolomic data. The metabolome is the full complement of metabolites, biochemicals and lipids circulating throughout the human body. HLI has signed an agreement with Metabolon Inc., a diagnostic products and services company offering a biochemical profiling platform, to capture this information from each of the genomic samples that HLI is collecting. “Metabolomics is important because quantifying and understanding the full picture of circulating chemicals in the body can help researchers get a clearer picture of that individual’s health status, and provide markers and pathways associated with drug action,” according to HLI.

Schematic of the 'omic hierarchy: genomics, transcriptomics, proteomics, and metabolomics—yes, the figure leaves out a few others, e.g. epigenomics and phenomics (taken from schaechter.asmblog.org via Bing Images).

Schematic of the ‘omic hierarchy: genomics, transcriptomics, proteomics, and metabolomics—yes, the figure leaves out a few others, e.g. epigenomics and phenomics (taken from schaechter.asmblog.org via Bing Images).

Stem Cell Therapies

This part of the company’s multi-pronged strategy utilizing stem cell therapy advances is said to be “premised on the theory that as the human body ages many biological changes occur, including substantial changes and degradation to the genome of the differentiated, specialized cells found in all body tissues. There is also a depletion and degradation of healthy regenerative stem cell populations in the body over time. HLI will monitor the genomic changes which occur during stem cell differentiation, normal aging, and in association with the onset of disease.”

In this regard, it’s worth mentioning that TriLink BioTechnologies is a leading provider of biosynthetic modified mRNAs that encode factors used for cellular reprograming and regenerative medicine. Further information about these catalog products and custom services is available here.

Commercial Potential

Robert Hariri, M.D., Ph.D., who cofounded HLI with Venter and Diamandis, is quoted in HLI’s press release as saying that “the global market for healthy human longevity is enormous with total healthcare expenditures in those 65 and older running well over $7 trillion.” He adds, “we believe that HLI’s unique science and technology, along with our business leadership, will positively impact the healthcare market with novel diagnostics and therapeutics.”

Time will tell.

Personally, over the many years since Mike Hunkapiller introduced me to then “NIHer” Craig Venter, I’ve learned not to bet against him.

In closing, I should mention that Venter et al. are not the first to eye the commercial potential of longevity. Last September, Google’s chief executive, Larry Page, announced that his company was creating an anti-aging company, Calico, which is being run by Arthur D. Levinson, the former chief executive of Genentech. Even earlier, Oracle’s chief executive, Lawrence J. Ellison, had financed anti-aging research through his foundation. However, last December, Ellison announced this research would end due to a funding crunch.

Your comments are welcomed.

Crowdfunding: Science & Startups Go Social

  • Websites Proliferate for “Reaching out to Engage” for Needed Dollars
  • Academics, Universities and Startups are Now Crowdfunding
  • Some Projects Attract Big Buck But Most Get Little

Wikipedia defines crowdfunding as “the collective effort of individuals who network and pool their money, usually via the internet, to support efforts initiated by other people or organizations.” In the context of science, it’s tempting to think that this is just a trendy way to characterize what has been done for a long time as charitable giving. However, I hope the following will convince you that crowdfunding is quite different and offers a number of unique advantages.

Faced with research goals that, for various reasons, are not fundable by traditional mechanisms, researchers are turning to crowdfunding to get the dollars they need. “Scientists are beginning to use crowdfunding to support their work, but don’t stop filling out those grant applications just yet”—this according to Jessica Marshall writing in the venerable Proceedings of the National Academy of Sciences.

While crowdfunding is a generic process (in the present context it involves asking members of the public to chip in money for projects that interest them), a number of crowdfunding web sites now offer researchers a page to pitch their idea, typically including a short video whereby potential donors can “meet” would-be recipients. Importantly, crowdfunding is also being used to raise money for startup companies in lieu of traditional venture capital (VC) financing.

Crowdfunding research through Web-based social media bypasses traditional grant reviews by peers or other experts (taken from conteudoegeek.blogspot.com via Bing Images).

Crowdfunding research through Web-based social media bypasses traditional grant reviews by peers or other experts (taken from conteudoegeek.blogspot.com via Bing Images).

Intrigued by this radical trend in internet-enabled, social media-catalyzed funding of scientific research, I did some homework. In this post I’ll touch on some similarities and differences among the main crowdfunding websites, explore a few representative “case studies”, and touch on some concerns that have been expressed thus far.

Crowdfunding Websites for Research

Indiegogo (“Fund what matters to you”) is an international crowdfunding site founded in 2008 and headquartered in San Francisco, CA. The site’s structure allows users to create a page for their funding campaign, set up an account with PayPal, make a list of “perks” for different levels of investment—think TV fund raisers—then create a social media-based publicity effort. Users promote the projects themselves through Facebook, Twitter and other social media platforms. The site levies a 4% fee for successful campaigns. For campaigns that fail to raise their target amount, users have the option of either refunding all money to their contributors at no charge or keeping all money raised but with a 9% fee. I assume this “penalty” encourages users to keep trying to meet their target.

RocketHub (“The world’s crowdfunding machine”) was launched in 2010, and shares similarities to Kickstarter, which was started a year earlier and focuses on helping “bring creative projects to life.” If the selected funding target is not reached by the deadline, the project leader is still able to keep the collected funds. RocketHub charges 4% of funds collected, plus 4% payment processing fees, if the project is fully funded, and 8% plus 4% payment processing fees if the project does not reach its goal. Seems like another “early withdrawal penalty” feature. SciFund Challenge has recently been founded to leverage RocketHub’s platform for science projects.

Experiment is a U.S. web site dating back to 2012 that works on the “all-or-nothing” funding model:  5% for Experiment and 3% for payment processing, but only if the campaign is successful. If the campaign does not reach the funding goal, no one is charged. In February 2014, the site changed its name from Microryza to Experiment.com. The former name Microryza was inspired by Mycorrhizae, a symbiotic fungi that live in the roots of plants. Unlike Indiegogo, backers of Experiment projects do not get tangible rewards for giving money. However, researchers share the scientific process directly with the backers and become a part of the project—think symbiosis.

Crowdfunding Numbers

Ethan O. Perlstein, who you’ll read about in the next section, posted this plot of total number of donors (x-axis) vs. the total amount raised (y-axis) for 115 science projects across four different crowdfunding platforms—as of October 20, 2013—with the comment “115 science projects have been crowdfunded, but crowdfunding can’t compete with grants — yet.”

crowdfunding

He added that Kickstarter claims that over 50,000 projects had been successfully funded to the tune of $836 million, with an increasing “pie slice” going to science projects that traditionally would have been funded almost exclusively by the government. Perlstein then offers the following series of comments that I think are quite interesting—including a provocative “universal fundraising statistic.”

“The majority of these 115 science projects fit the academic profile: professionally trained, university-employed, grant-dependent researchers asking focused research questions. But there are also examples of unconventional projects led by self-taught (aka citizen) scientists, student-led, e.g., iGEM teams, and pedagogical research.

Cumulatively, 115 science projects raised $5,082,028 from 47,958 donors, with two megaprojects comprising over half of these totals. Since the average is thrown off by those whoppers, the median is more useful. The median project goal is $3,029, and the median number of project donors is 39. 

The above plot reveals two interesting facts about the distribution of science projects. First, the ratio between dollars and donors is roughly 100-to-1 across three orders of magnitude, from $1,000 goals to $1,000,000 goals. In other words, the average donation for science projects is $100. Technically, the average donation falls within a range of $100-$60 per donor. This range is consistent with non-science projects and is probably a universal fundraising statistic that reflects economic and psychological drivers of charitable giving.

Second, there is a ceiling between $25,000 and $35,000 above which Microryza and RocketHub science projects don’t go, but above which Kickstarter and Indiegogo science projects do boldly go. What’s causing this separation? The answer appears to boil down to incentives, i.e., whether science projects offer products or not. There is also a role for the size and engagement of each crowdfunding platform’s donor community, especially repeat donors. Briefly put: tangible rewards shift the average donation size higher than people might spontaneously donate, and built-in communities on Kickstarter, and to a less extent on Indiegogo, means a larger captive audience.”

Crowdfunding Case Studies

If you’re seriously interested in pursuing crowdfunding for your research—or participating as a contributor to research by others—more information can be found by using keyword (e.g. DNA, genes, etc.) search engines provided at the aforementioned websites. In any case, the following exemplary case studies taken from Jessica Marshall’s PNAS article and elsewhere are intended to provide a sense of scope, scale, and success, or lack thereof.

Lauren Kuehne, University of Washington, Seattle, WA (taken from PNAS).

Lauren Kuehne, University of Washington, Seattle, WA (taken from PNAS).

Crowdfunding Platform: SciFund Challenge
Funding Goal / Raised: $2,000 / $2,048
Project: Soundscape characterization of freshwater lakes
Reward Offered: Sound chart of donor’s name being spoken
Take-home Message: “It was a lot of work for $2,000, I’m not going to lie.”

According to Marshall, while a campaign is live, success hinges on staying engaged, responding to questions and glitches, and continuing to spread the word on social media and among potentially interested networks, including relevant businesses and the press. After the campaign, teams need to update backers on the project and distribute the perks, which can include t-shirts, tokens related to the research, or lab tours for larger donors. “I would recommend that people not do this if their sole purpose is fundraising,” says Kuehne.

So why do it if not for money? “I immediately had this network of people who were aware of the research. People would send me papers and ideas for extending the research, and ideas for data management,” she adds. Also, “I felt like I was this little hub for people who were interested in freshwater, in noise, in data management, in new technologies.” Marshall adds that about one-third of Kuehne’s backers were her friends and family, a similar fraction were people she knew but would not have thought to ask for support, and one-third were strangers.

Rob Knight, American Gut project, University of Colorado, Boulder, CO (taken from PNAS).

Rob Knight, American Gut project, University of Colorado, Boulder, CO (taken from PNAS).

Crowdfunding Platform: Indiegogo
Funding Goal / Raised: $400,000 / $339,960
Project: Global survey of people’s microbiomes
Reward Offered: Profiles of individuals’ microbiomes
Take-home Message: “One thing that’s been very much a learning process is figuring out ways to make the results accessible to the general public.”

With some projects, connecting with the public is an essential part of the research. The American Gut project, and similar project uBiome, invited backers to donate money and samples from their own microbial populations in exchange for a printout detailing the bacteria in their bodies—a continuing “hot topic” of research that I wrote about here last year. The teams counted on growing public awareness about the importance of the microbiome and people’s innate desire to know more about their own microbiome to drive involvement.

Together, the two projects raised almost $700,000. ‘One curious aspect of this crowdfunding approach is that certain groups—like people who ascribe to caloric restriction, or those interested in following paleolithic diets—have shown strong interest’, says Rob Knight, who is the lead scientist on the American Gut project. Marshall added, “this skews the sample population, but it should allow the researchers to make some interesting comparisons, and they plan to contact participants for follow-up studies.”

uBiome is interested in taking crowdsourcing one step further by allowing backers to submit research questions as the database grows. “We wanted to bring the public in in a bigger way, let them ask questions of the data, and really harness the scientific understanding,” uBiome cofounder Jessica Richman told Marshall.

Ethan O. Perlstein, founder of Perlstein Lab, B Corp (taken from PNAS).

Ethan O. Perlstein, founder of Perlstein Lab, B Corp (taken from PNAS).

Crowdfunding Platform: RocketHub
Funding Goal / Raised: $25,000 / $25,460
Project: Interactions of methamphetamine with brain cells
Reward Offered: Model of methamphetamine molecule made with a 3D printer
Take-home Message: “The formula is straightforward: social networks + external media + time and commitment.”

Marshall opines that crowdfunding may be more likely to succeed for a project with broad public appeal. Perlstein’s project capitalized on the popularity of the television program “Breaking Bad”, about a chemistry teacher who starts making methamphetamine. Perlstein and a colleague created a short video explaining the concept and posted it on RocketHub. The scientists offered prizes, including the chance to talk science over beer for people who donated $100 or more. The men quickly raised $25,000 from family, friends and strangers.

This past February, Amy Dockser Marcus reported in The Wall Street Journal that Perlstein plans to use crowdfunding to raise $1.5 million for his research at the eponymous Perlstein Lab, B Corp. But why do this? In a nutshell, Perlstein had earned a Ph.D. in molecular biology from Harvard, spent five years doing postdoctoral research at Princeton and there led a team that published two papers on pharmacology, which all sounds very promising as a springboard to become an independent academic researcher. But he was turned down by 27 universities when he sought a tenure-track position, and decided to instead set up his own lab as a Benefit Corporation (aka B Corp) and raise money through crowdfunding.

Dr. Perlstein’s specialty is something he calls “evolutionary pharmacology.” He plans to study lysosomal storage diseases in yeast, fish, flies and worms, trying to find drugs that slow or stop the disorders, then test the drug candidates in patient cells collected by researchers. He anticipates making money by licensing or auctioning the findings to drug companies or others that can run trials in humans.

Needless to say, there are probably hundreds of researchers who will be eagerly watching to see if this successfully plays out.

Crowdfunding in Academia

Universities are also exploring how to incorporate crowdfunding into their operations, and view the campaigns as potentially valuable outreach—this according to Jessica Marshall—and quite predictable to me. She adds that The University of California, San Francisco (UCSF), has forged a partnership with Indiegogo that, among other things, allows backers to donate under tax-exempt status. Tuhin Sinha, a UCSF administrator who is coordinating the efforts is quoted by Marshall as saying that “we’re totally in a pilot mode. We’re having mixed reviews with this.”

Taking a different approach is Michael Greenberg, Director of Innovation and Strategic Initiatives for the Office of Research at the University of California, Los Angeles, and the cofounder of ScaleFunder, a company that has developed a crowdfunding platform tailored to universities. ScaleFunder lists UCLA, UCSF, and UCSC among its clients. Since all sorts of new trends, like the weather, tend to “blow from West to East” it’s likely that Harvard and MIT might also try this route.

According to Greenberg, when universities coordinate crowdfunding campaigns, researchers can access a much larger network of potential backers. The universities can also provide support as the researchers prepare their text and videos, and ensure that no university regulations are breached. “Institutions have the ability not only to bring in major donors but also major corporations,” Greenberg is quoted as saying.

Crowdfunding Startups

Stephanie M. Lee is a San Francisco Chronicle staff writer who entitled her February 15th 2014 story “Biotech startup turns to crowdfunding,” from which I’ve taken these selected excerpts.

“When Exogen Biotechnology needed to raise money, the startup turned not to venture capitalists but to crowdfunding site Indiegogo. In just 10 days this year, nearly 300 donors from all over the world chipped in $50,000.

jon

Jonathan Tang (left) and Sylvain Costes developed an agent-based model that is the first multi-scale model of the development of a mammary gland from the onset of puberty all the way to adulthood (photo by Roy Kaltschmidt; taken from technology.org via Bing Images).

The two biophysicists at Lawrence Berkeley National Laboratory are developing a technology that measures DNA damage, which could determine if a person might develop cancer, neurodegenerative disorders and immunological diseases.

‘If you find you have a high level of DNA damage, you can try to find out what’s causing it to reduce it,’ Tang said.

At a time when venture capital firms are investing fewer dollars in young biotech companies, a growing number of entrepreneurial scientists are raising money through online crowdsourcing communities like Indiegogo, Medstartr, VentureHealth, RocketHub and AngelList.

Since the recession in 2008, investors have been increasingly reluctant to pour cash into risky biotech startups that face a long and uncertain road to winning approval from the U.S. Food and Drug Administration. First-time venture deals in biotechnology hit a 17-year low in the first three quarters of 2013, according to a Burrill Report analysis of the U.S. life sciences industry. As a result, startups are looking to alternative sources like angel investors, venture philanthropists and now crowdfunding sites.

‘Early-stage life science is just in poor shape in terms of having a variety of funding sources,” said Stephanie Marrus, director of UCSF’s Entrepreneurship Center. “Everyone should keep their eyes on (crowdfunding). Looking at the global trends, it’s definitely become a source of capital. If it becomes appropriated adaptively to health care, maybe we have a new avenue to go down.’

Exogen’s founders say they turned to Indiegogo in part because they want to treat the campaign as a citizen science project. Two preliminary trials with 100 people last year gave the scientists confidence in their idea when they found that the older the subjects were, the higher their level of DNA damage. In addition, four former cancer patients had among the highest levels of DNA damage for their age group.

Donors who chip in $99 or more receive a kit to extract blood samples. The samples are analyzed by Costes, a biophysicist who studies the effects of low-dose radiation on cellular processes, and Tang, a postdoctoral fellow in Costes’ lab.

Traditionally, if a scientist wanted to measure the level of a person’s DNA damage, they would examine cells in a microscope and manually count the number of DNA breaks. Costes, however, said he has invented a way to automate the task with a machine that can scan and objectively score the damaged DNA.

Exogen plans to allow users to check their scores privately online. To prevent the onset of diseases like cancer, people with high levels of DNA damage could try to modify their diet and exercise or limit exposure to the sun. The founders are careful to note that for now, the company won’t dish out medical advice or sequence people’s genomes. But the company plans to seek FDA approval for its technology as a diagnostic tool.”

Crowdfunding Concerns and Criticisms

While crowdfunding has proven to be an efficient and effective way to raise funding, there are obvious concerns around fraud and the quality of the research being conducted. I won’t comment on fraud, since this is a concern that is common to any online—or old fashion mail—solicitation. On the other hand, quality of science is an issue peculiar to crowdfunding. After cogitating about various ways to address this concern, I concluded that it’s not easy or practical to deal with given that the vast majority of donors are non-scientists. Perhaps posting “letters of support” from bone fide academics would be an effective way to handle this.

Lee quotes Arthur Caplan, a bioethics professor at New York University’s Langone Medical Center, as saying that “you can say anything and everything when you’re crowdfunding, so the person donating money has to be vigilant. They could say, ‘I’m going to find a cure for your child’s brain cancer—if you fund it, I’ll do it.”

Erika Check Hayden, writing in Nature this past February, said that a crowd-funded HIV vaccine project called the Immunity Project has sparked debate, as scientists question the tactics for the public campaign for this project. “It is dreaming big,” she quotes co-founder Reid Rubsamen as saying, adding that his enthusiasm makes him an effective pitch man for the Immunity Project, based in Oakland, CA, which has raised more than $400,000.

But missing from Rubsamen’s promotional campaign are any HIV researchers or data supporting the effort’s scientific strategy. The unorthodox approach raises the question of whether crowdfunding, which tends to be more impressed with technology and marketing than peer-reviewed data, is compatible with medical research. This is an increasingly pertinent issue as scientists appeal to the public to fund more projects aimed at developing therapies.

I’m obviously intrigued by the rising popularity and future potential of crowdfunding. I welcome your thoughts, opinions and comments regarding this topic.