What would you do with 900 million dollars of start-up funding?

America, a land of plenty – plenty of land, plenty of food, plenty of crazy politicians and plenty of start-up funding.

Grail, a company formed by sequencing giant Illumina in Jan 2016, recently obtained a hefty $900 million in Series B financing, after already obtaining $100 million in Series A. Grail aims to screen for cancer mutations in circulating tumour DNA (ctDNA) from blood samples via next-generation sequencing (learn more about this booming field in Sensitive Detection of ctDNA). The money came from several large pharmaceutical companies, Johnson & Johnson purportedly with the largest investment followed by others such as Bristol-Myers Squibb, Celgene and Merck. Interestingly, Bill Gates and Jeff Bezos from Amazon has also invested in Grail, together with the venture arm of medical distributor McKesson, China-based Tencent Holdings, and Varian Medical Systems, a radiation oncology treatment and software maker from Palo Alto.

This is the biggest start-up financing deal in biotech by a long-shot, the largest deal in 2016 went to Human Longevity, Craig Venter’s company that raised $220 million in series B. Another one that came somewhat close was RNA company Moderna Therapeutics, which raised $450 million in 2015.

Grail plans to carry out “high-intensity sequencing” on blood samples from vast numbers of people to detect circulating tumour DNA at early stages, essentially in people not showing any signs cancer, as a means of early detection to enable better treatment. This is an especially challenging feat, given that ctDNA makes up < 1% of circulating DNA found in the blood. But there are some hints that Grail is sitting on promising data sets that have turned skeptics into believers.

There are concerns that testing healthy people for cancer might yield false positives that could subject people to unnecessary and potentially dangerous testing procedures and treatments. This was the case for the Prostate-specific antigen (PSA) test used to screen men at risk for prostate cancer. It turned out that PSA testing did not significantly reduce mortality of men with prostrate cancer but did increase the harms associated with the accompanying treatments and tests, some of which are pretty nasty such as urinary incontinence and erectile dysfunction.

So Grail had better be sure the sensitivity and accuracy of their predictions are full-proof as cancer treatments are not exactly pleasant. They seem to be taking it seriously, judging from their embarkation on an ambitious trial called “The circulating cell-free genome atlas study” where they will recruit more than 10, 000 participants – 7000 newly-diagnosed cancer patients of multiple solid tumour types who have not undergone treatment, and 3000 healthy volunteers.  The trial is already recruiting and is projected to be completed within 5 years by Aug 2022, with a primary outcome measure available by Sept this year. Grail hopes to detect shifts in cancer stage severity as they perform their tests over time. How accurately their tests reflect other clinical readouts would give appropriate proof of its reliability. Likely, more trials involving more patients would be necessary to determine if this form of testing is full-proof and whether it may even replace tissue biopsies as a gold standard in cancer diagnosis.

Grail has even drafted plans to make their form of testing available to the medical community by 2019, subject to experimental results. An incredibly ambitious timeline, so its no wonder they need the big amounts of cash to drive it through. Jeff Huber, a former Google Exec, is Grail’s new CEO. His wife Laura died of colorectal cancer, so his new job also fulfils a personal mission. Other members of the team include other former employees from Illumina and Google, including Verily CSO/founder Vikram Bajaj. The Google Life Science company Verily have recently received a similarly outstanding investment of $800 million from who would have guessed, Singapore Temasek Holdings.

The scale of investment in America seriously dwarfs that found in the European biotech scene. Despite conservatives highlighting a potential bubble in US biotech and Trump’s anti-pharma sentiment that may signal a potential decline in available funding, one cannot deny that the lofty research goals being currently undertaken, can only yield an incredible expansion of scientific knowledge. In my opinion, science is expensive, and the more money you have, the more science you can do. They key thing though, is to make sure its good science!

 

 

An exciting time for epitranscriptomics

Epigenetics is a well-established means of gene regulatory control where chemical modifications of DNA bases or their associated histone proteins affect the expression of genes (as opposed to DNA sequence). These epigenetic marks may take the form of methylation (usually on cytosine residues) or histone acetylation/phosphorylation/ubiquitination/sumoylation etc. and can be passed down to daughter cells. Epigenetic changes are induced by the environment and provide a biological mechanism by which nurture as opposed to nature, plays a significant role in shaping our behaviours and characteristics.

Recently, scientists are uncovering the functional significance of epitranscriptomics – or the chemical modification of RNA. The first report of mRNA modification, specifically,  methylation of the N6 position of adenosine (m6A), occurred in 1974 by Fritz Rottman et al.. The m6A modification is the most common eukaryotic mRNA modification however its functional significance remained unclear until the 2010s. In 2012, Dominissini et al. using m6A-antibody enriched RNA-seq, discovered 12000 methylated sites in 7000 coding genes and 250 non-coding genes. Typically highly concentrated around stop codons, within long internal exons and at transcription start sites, it became evident that genes without these modifications tend to be more highly expressed. He writes about it in a Science essay here.

There are several players involved in epitranscriptomics, and they are often referred to as writers, readers and erasers. A methyltransferase METTL3 (“writer“) produces the m6A modification, the YT521-B homology (YTH) domain family of proteins are “readers” that bind to m6A modified RNA and regulate functions that affect protein expression such as RNA degradation, translation and splicing. Finally the “erasers” such as enzyme FTO, implicated in various diseases such as cancer and obesity, removed these m6A marks. Thereby completing the set of actors required for establishing epitranscriptomics as a regulatory mechanism for RNA expression.

Samie Jaffery’s group recently published a controversial paper that identified another epitranscriptomic modification N6,2O-dimethyladenosine (m6Am), located near the 5′ caps of mRNA, that is the main substrate of eraser FTO instead of m6A. m6Am is correlated with increased mRNA stability as it makes 5′ caps harder to remove, which thereby increases protein expression. This is in contrast with m6A modification, which is associated with suppression of protein expression. This highlights distinct functional roles of these RNA methylation marks.

There are already studies demonstrating m6A regulation is utilized also by non-coding RNA. The simple self-made schema below shows how m6A modification was used by Xist to carry out its transcriptional repression of the X chromosome, demonstrated by Patil et al. in a recent Nature publication.

xistm6a

Epitranscriptomics is a relatively young field and opens up new possibilities to study how RNAs are regulated and in particular, how non-coding RNAs may carry out their functions. This signals yet more exciting times ahead for RNA researchers!

Two man-made nucleotide bases produces never before seen semi-synthetic organism

As if altering the genetic code was not enough, now there might be a possibility to completely change the way it was written. Two new nucleotide bases have been created that were stably replicated in bacteria, expanding the possible number of base-pairs to 3 and increasing the coding capacity of DNA exponentially.

In his initial study published in Nature, Dr. Floyd E. Romesberg, a Chemistry Professor at the Scripps Research Institute in California, produced two nucleoside triphosphates called dNaMTP and d5SICSTP, which basepair via hydrophobic interactions instead of hydrogen bonding. A nucleotide transporter had to be expressed in the bacteria in order for their uptake, but once inside, DNA polymerases had no problem incorporating and amplifying the synthetic nucleotides, making copies of an introduced plasmid containing a single base-pair of the unnatural bases.

There were concerns initially that DNA repair mechanisms may work to remove these foreign bases, but that did not seem to be the case. The unnatural bases persisted for more than 3 days after he stopped supplying the bacteria with nucleotides at a retention of 45% (at 0 days, retention was in excess of 95% as suggested by termination of reads by Sanger sequencing) and 6 days at 15%. This led him to guess that their decay over time was due to replication-mediated mispriming instead of DNA repair.

In his most recent study in PNAS, Dr. Romesberg and his graduate student Yorke Zhang made improvements to the nucleotide transporter used, which previously induced some toxicity. They also further optimized one of the unnatural bases to be better incorporated by DNA polymerases, increasing copying efficiency. Finally, to prevent the decay of the unnatural bases over time, they used CRISPR to eliminate the DNA that had lost the unnatural bases. The resulting bacteria was able to indefinitely retain the unnatural bases under various sequence context.

romesberg

Image taken from News Medical Life Sciences‘ interview with Professor Romesberg

The so-called semi-synthetic organism produced therefore uses 3 base-pairs and has 6 nucleotide types instead of 4. It remains to be seen whether its DNA can be transcribed into RNA and then more importantly into protein. But it holds the potential to greatly change the way things are currently done – imagine what we could do with 172 amino acids instead of 20!

 

Role models in science – Dr Susan Lindquist

plos_lindquist

It has been a month since renown researcher of protein folding, Dr Susan Lindquist, passed away to cancer at the age of 67. I remember watching her video on prion biology while working on my PhD project looking at mechanisms involved in neurodegenerative disease. She seemed pretty cool, I thought.

She grew up in Chicago to a Swedish father and Italian mother who never expected her to come so far in her career. Being a woman, their hopes for her was to marry someone decent and successful. Coming home from a party the night before New Year’s Eve and seeing their daughter hard at work on a paper, their comments were “Are you still working? When are you going to settle down?”  I’m not so sure that parental thinking has changed significantly since then…

She found inspiration in a book detailing the life of Elizabeth Blackwell, the first woman who obtained a medical degree in the US, and various teachers who stimulated her interest in science. Under the guidance of her microbiology professor Jan Drake, she applied successfully for a National Science Foundation scholarship to do research in his lab. With his encouragement, she applied to graduate school in Harvard, and got in, something she had never dreamed would happen. She worked in the lab of Matthew Meselsen but failed to get any data for her first project. After talking to a colleague down the hall who noted particular phenomena to heat-exposed fruitflies however, she decided to test if any similar responses would happen in cells. That was the turning point in her career, as she found and characterized the upregulation of specific proteins induced by heat, a mechanism termed the heat shock response that would be found to be highly conserved across many organisms.

She continued to work on the heat shock response during her post-doc rather independently in the lab of Hewson Smith at the University of Chicago. She characterized how the expression of these heat shock proteins were regulated via transcription, translation, splicing or degradation. Realising that these proteins were so highly conserved across different species and were being in expressed in every cell in response to stress that occurs frequently in life and disease, Lindquist was driven to find out exactly what these proteins were doing. Her research brought her into broad and vastly different fields, as it was found that these proteins played essential roles from enhancing malignancy in cancer to managing protein aggregates so often found in neurodegenerative diseases.

She had surpassed her initial dream of writing grants under the supervision of a male superior, to managing her own lab at the Whitehead Institute at MIT. She even co-founded a company – FoldRx Pharmaceuticals – which utilized her favourite model, yeast, in a high-throughput functional assay to search for drugs that could alleviate protein aggregation in protein misfolding diseases. This was later bought by Pfizer as they sought to obtain the rights to the drug Tamafidis, which was approved for the treatment of early stage transthyretin-related hereditary amyloidosis or familial amyloid polyneuropathy or FAP.

Susan Lindquist is definitely a role model to look up to, especially for women in science. There are still far lesser women compared to men in leadership positions in science and beyond. I gather this is attributable to the demands of family rearing, the discrimination that comes hand-in-hand with being a woman attempting to lead, and the internal fight women go through to overcome natural feelings of inadequacy. But Susan shows us it can be done. And I think we would probably do a better job than men sometimes as Sandi Toksvig would agree in her hilarious TED Talk.

Read and watch more about Dr Susan Lindquist here:

Fearless about Folding | The Scientist Magazine® http://www.the-scientist.com/?articles.view%2FarticleNo%2F44769%2Ftitle%2FFearless-about-Folding%2

Gitschier, Jane. “A Flurry of Folding Problems: An Interview with Susan Lindquist”. PLoS Genetics. 7 (5): e1002076. doi:10.1371/journal.pgen.1002076. PMC 3093363Freely accessible. PMID 21589898.

Short video Q&A with Susan Lindquist http://www.moleclues.org/interviews/they-can-make-mess-hurry 

CRISPR trial in humans – a look at checkpoint inhibitors

Scientists in China led by oncologist Lu You at Sichuan University in Chengdu have started treating patients in the first ever CRISPR human trial. They have beaten out the US team from U Penn who had previously obtained approval from a bioethics committee in June but were still waiting on FDA approval.

Carried out at the West China Hospital in Chengdu, Lu You is isolating T cells from 10 patients with metastatic non-small cell lung cancer that have failed to respond to chemotherapy, radiation therapy and other treatments. To these T cells, he is introducing CRISPR-mediated genetic modifications to knock-out the Programmed Cell Death Protein 1 (PDCD1) gene, expanding them in culture, and introducing them back into the patients at escalating doses. The key aim of the trial is establishing safety and researchers will be monitoring adverse events, progression free survival, circulating tumour DNA  and other immune system activity readouts such as interferon gamma levels.

T cell therapy against cancer has amassed huge research funding and support, read the CAR T phenomenon in an old blogpost here. And checkpoint inhibition is the latest and most popular strategy to help T cells identify and kill cancer cells. See how it works in this nifty video by the Dana Farber Institute:

Although there has been a lot of success with checkpoint inhibitor drugs (see here for a list), there has also been a recent report in the New England Journal of Medicine that a combination of two check-point inhibitors (Yervoy and Opdivo, against CTLA4 and PD1 respectively) produced severe heart inflammation and damage (myocarditis) that was driven by T cells, causing death in two patients.

The patients in the China trial will not be given any checkpoint inhibitor drugs, but its still uncertain what these modified T cells are capable of once inside the body. Previous trials have however been performed with reported “unprecedented” success rates ranging around 90%. And it was very successful for this little girl named Layla. I have yet to come across the published results but am keeping my fingers crossed!

How your oligos are made

As scientists, we use oligos everyday – primers for PCR, labelled hybridization probes in rtqPCR detection or expression microarrays, biotinylated pulldown probes, sequencing adaptors etc. Oligos, defined as short stretches of chemically-synthesized nucleic acids, can be chemically made, amplified, assembled and ligated together to form synthetic genes. They can be put into cells and animals (including humans) to perform all sorts of functions, not just via their translation into proteins, but also by interacting with the cell’s molecular machinery to block the expression of RNA, trigger immune responses, mediate gene editing and so on. So how much do you know about how they are actually made?

The history of oligo synthesis goes back a long way. The discoveries made in the 1940s and 1950s on the structure and composition of DNA and how they make up the code for life triggered attempts to chemically synthesize oligos. The first report of oligo synthesis was in 1955 by Todd and Michelson in Cambridge who linked two thymine nucleotides together using phenylphosphoryl dichloride. The reaction however worked slowly and was compromised by the instability of the phosphoryl chloride intermediate (leftmost molecule) which was susceptible to hydrolysis.

todd

Then came along the exceptional scientist, Har Gobind Khorana, born in Raipur and based at the University of Wisconsin-Madison, who transformed the way oligos were made by making two significant developments in the 1950s-60s:

  1. Instead of using phenylphosphoryl dichloride to activate the 3′ phosphate, he used condensing agents – sulphonic acid chlorides or carbodi-imides – that allowed the reaction to proceed faster and more efficiently.
  2. He found suitable protective groups to protect the 5′ hydroxyl, 3′ hydroxyl, amino group on the heterocyclic ring, and the phosphate group itself to prevent any non-specific reactions and ensure the correct product was made. These groups could be selectively removed (e.g. with mild acids) at the appropriate time to allow the oligo strand to grow. These protective groups are still used till today.
    • For the 5′ hydroxyl (OH) group: dimethoxytrityl (DMT)
    • For the 3′ hydroxyl (OH) group: acetyl group which was later replaced by diisopropylamino with a 2-cyanoethyl to block branching at the phosphate
    • The amine group: a benzoyl or isobutyryl group

oligo-protective-groups

A phosphoramidite – common building block for oligo synthesis used today

Khorana’s phosphodiester method still had limitations though, producing various by-products that slowed the reaction down and required further purification steps to isolate the target oligo.

Subsequently, an American biochemist, Robert Letsinger protected the 3′ phosphate with a 2-cyanoethyl group which prevented these by-products from forming and significantly sped up the reaction. Letsinger also started using Phosphorous (III) rather than Phosphorous (V), which increased the reactivity of the nucleosides, increasing nucleotide coupling efficiency. This was termed the phosphite-triester approach. Letsinger with the help of Khorana’s former student, Marvin Caruthers, were also the first to initiate synthesis of oligos on solid supports. The link to a solid support or resins, usually made up of polystyrene or controlled pore glass (CPG), is made via the 3′ hydroxyl group thereby initiating chemical oligo synthesis from the 3′ to 5′ direction.

Caruthers continued to further improve oligo synthesis and by substituting the chloride group of the highly unstable phosphomonochloridite intermediate with an amide, he created a much more stable intermediate – phosphoramidite – that could be made and stored and activated with tetrazole when required. So here’s the whole process of oligo synthesis:

synthesiscycle

  1. The protective DMT group is removed from the 5′ hydroxyl in the detritylation step with mild acid. The solid support is then washed with acetonitrile to remove the acids and dehydrate the reaction.
  2. The two nucleosides are coupled with addition only taking place on the 5′ of the unprotected nucleoside, activated by tetrazole.
  3. The oxidation step is required to convert the phosphite to phosphate, which is more stable and also represents the natural form seen in DNA.
  4. Capping is performed to block nucleotides that were unsuccessfully coupled. An acetyl group is generally added to prevent the hydroxyl group from reacting.

The oligos then have to be cleaved from the solid support and eluted. These oligos grow in pores of the solid supports that are typically on columns or in wells of a plate. It is important to note that as the oligo strand grows, the synthesis yield drops as the growing oligo blocks the pore and reduces diffusion of the reagents through the matrix. Larger pores (1000 angstroms) are thus used for longer oligos but can only support oligo lengths up to 100 bases. Added to these physical constraints, varying inefficiencies in the individual reaction steps ensure coupling efficiency is never 100%. Furthermore, depurination can sometimes occur in the detritylation and capping step, where the glycosidic bond between the purine and sugar is cleaved, producing more truncated products. Hence, the yield of full-length product drops as length of oligo increases and truncated products not of the correct length are often present in most ordered oligos unless HPLC purification is employed.

oligo-yield-chart1_158847_284_13928

% expected yield of full-length product as oligo length increases

In recent years however, modern advances have seen oligo synthesis taking place on 2D planar surfaces with higher efficiency and throughput. Agilent for example, employs inkjet printing that takes place in an anhydrous chamber to deposit and couple phosphoramidites on a 1 x 3 inch microarray chip that can hold up to a million growing oligo spots. Oxidation and detritylation are carried out with a flowcell that floods all the oligo features with the necessary reagents. The highly controlled fluidics of the flowcell and the anhydrous property of the printing chamber provides a better coupling efficiency and better control of depurination, allowing oligo lengths of up to 150 nucleotides to be achieved.

Twist Bioscience have scaled it up a notch employing silicon wafers containing 9600 wells, each well being capable of synthesizing 121 different oligos. Furthermore, these oligos can be assembled into longer sequences (synthetic genes) within the well itself, avoiding the fuss that comes with handling small volumes of liquid.

Oligo synthesis has come a long way since the 50s indeed, we now can make a lot of oligos in parallel and can even stitch them together in the process. Ensuring they are error-free and pure however is another problem, but it can only get better from here.

Sources:

Khorana, H. G. (1968). Synthesis in the study of nucleic acids. The Fourth Jubilee Lecture. Biochemical Journal, 109(5), 709–725. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1187021/

Some images adapted and modified from A Short History of Oligonucleotide Synthesis
By Richard Hogrefe, Ph.D.; TriLink BioTechnologies (contains more detailed description of the chemical processes, recommended reading)

Image of % oligo synthesis yield from FAQs > Oligo Synthesis Resource | W.M. Keck Foundation | Yale School of Medicine

LeProust, E. M., Peck, B. J., Spirin, K., McCuen, H. B., Moore, B., Namsaraev, E., & Caruthers, M. H. (2010). Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Research, 38(8), 2522–2540. http://doi.org/10.1093/nar/gkq163

Banner image “Digital DNA” by Wonderlane

 

Unity Biotechnology’s science behind their cure for aging

Can we reverse aging? Unity Biotechnology certainly thinks so and it has managed to convince investors to give them $116 million in series B funding to carry out the necessary steps. The company is led by an impressive team of experienced start-up founders including Nathaniel David and Keith Leonard who together had previously started Kythera Biopharmaceuticals which was recently sold to Amgen for $2.1 billion.

Their scientific team is just as strong, composing of Jan Van Deursen, a Professor of Biochemistry and Molecular Biology at the Mayo Clinic and Judy Campisi, Professor at the Buck Institute for Research on Aging. They recently published a paper in Science describing  that removing senescent cells in cell and animal models of atherosclerotic disease inhibited growth of atherosclerotic plaque, reduced inflammation, and improved the structural risk profile of plaques.

Their previous paper in Nature also demonstrated better aging outcomes when senescent cells were removed. They did this by using a transgenic mouse expressing green fluorescent protein (GFP) and FK506-binding protein-caspase 8 (FKBP-Casp8) fusion protein under control of the promoter for Ink4a (a gene active in senescent cells). They then added a drug, AP20187, which dimerizes and activates FKBP-Casp8, triggering cell death specifically in senescent cells (see diagram below for mechanism, taken from an even earlier publication: Baker et al., Nature, 2011).

senescenttgmice

The transgenic mice treated with AP20187 showed a loss of senescent cells in a tissue-dependent manner, with only the colon and liver not showing expected losses of senescence-associated transcripts. Overall, the loss of senescent cells carried out for a period of 6 months when mice were about 12 months of age:

  • prevented age-dependent fat loss
  • extended median life-span by 17-35%
  • increased maximum life-span in mice with mixed genetic background (only when both sexes combined) but not in C57BL/6 mice
  • did not change incidence of tumor formation
  • delayed cataract formation
  • prevented age-dependent loss of spontaneous motor activity and exploratory behavior but did not prevent loss in memory, muscle strength, or coordination and balance.
  • reduced age-related impairment of kidney function
  • prevented age-associated increase in cardiac myocytes
  • increased tolerance of mice to cardiac stress

Oh and it also made the mice look younger:

apmice

Just look at the luscious fur coats of those AP20187-treated mice!

The only challenge with this approach is it requires genetic engineering. However it does identify the role of senescent cells in age-related diseases, and finding a drug/molecule to get rid of them may work just as well. Previous attempts to stall ageing using Resveratrol has not shown such dramatic effects though in a recent trial it does seem to improve outcomes in Alzheimer’s disease patients. Diabetes drug, Metformin, is also being touted as the best hope as an anti-aging medicine and recently won FDA-approval for a clinical trial to be used for that purpose called Targeting Aging with Metformin (TAME).

Aging was thought of as a mysterious process but it appears we are getting a little closer to understanding it. Let’s hope we can prevent hair-fall and cataracts in our lifetime!