Now You See It

 

In the pages of this blog we’ve often highlighted the power of fluorescent tags to track molecules and see what they’re up to. It’s a method largely pioneered by the late Roger Tsien and it has revolutionized cell biology over the last 20 years.

In parallel with molecular tagging has come genetic engineering that permits novel genes, usually carried by viruses, to be introduced to cells and animals. As we saw in Gosh! Wonderful GOSH and Blowing Up Cancer, various ‘virotherapy’ approaches have been used with some success to treat leukemias and skin cancers and a trial is underway in China treating metastatic non-small cell lung cancer.

A major aim of genetic engineering is to be able to control the expression of novel genes (i.e. protein production from the encoding DNA sequence) that have been introduced into an animal — in the jargon, to ‘switch’ on or off at will. That can be done but only by administering a drug or some other regulator, either in drinking water, by injection or squirting directly into the lungs. An ideal would be something that’s more controlled and less invasive. How about shining a light on the relevant spot?!

Wacky or what?

That may sound as though we’re veering towards science fiction but reflect for a moment that every animal with vision, however rudimentary, sees by transforming light entering the eyes into electrical signals that the brain turns into a picture of the world around them. This relies on photoreceptor proteins that span the membranes of retinal cells.

How vision works. Light passes through the lens and falls on the retina at the back of the eye. The photoreceptor cells it activates are rod cells (that respond to low light levels — there’s about 100 million of them) and cone cells (stimulated by bright light). Sitting across the membranes of these cells are photoreceptor proteins — rhodopsin in rods and photopsin in cones. Photoreceptor proteins change shape when light falls on them — the driver for this being a small chemical attached to the proteins called retinal, one of the many forms of vitamin A. This shape change allows the proteins to ‘talk’ to the inside of the cell, i.e. to interact with other proteins to switch on enzymes and change the level of ions (sodium and calcium). The upshot is that the signal is passed through neural cells in the optic nerve to the brain where the incoming light signals are processed into the images that we perceive. © Arizona Board of Regents / ASU Ask A Biologist.

The seemingly far-fetched notion of controlling genes by light was floated by Francis Crick in 1999. The field was launched in 2002 by Boris Zemelman and Gero Miesenböck who engineered neurons to express one form of rhodopsin. This gave birth to the subject of optogenetics — using light to control cells in living tissues that have been genetically modified to express light-sensitive ion channels such as rhodopsin. By 2010 optogenetics had advanced to being the ‘Method of the Year’ according to the research journal Nature Methods.

Dropping like flies

One of the most dramatic demonstrations of the power of optogenetics has come from Robert Kittel and colleagues in Würzburg and Göttingen who made a mutant form of a protein called channelrhodopsin-1 (found in green algae) and expressed it in fruit flies (Drosophila melanogaster). The mutant protein (ChR2-XXL) carries very large photocurrents of ions (critically sodium and calcium) with the result that photostimulation can drastically change the behaviour of freely moving flies.

Light-induced stimulation of motor neurons in adult flies expressing a mutant form of rhodopsin ChR2-XXL. Click to run movie.

Left hand tube: Activation of ChR2-XXL in motor neurons with white light LEDs caused reversible immobilization of adult flies. In contrast (right hand tube) flies expressing normal (wild-type) channelrhodopsin-2 showed no response. From Dawydow et al., 2014.

Other optogenetic experiments on flies can be viewed on You Tube, e.g., the TED talk of Gero Miesenböck and the Manchester Fly Facility video of fly maggots, engineered to have a channel protein (channelrhodopsin) in their neurons, responding to blue light.

Of flies … and mice … and men

This is stunning science and it’s opened a new vista in neurobiology. But what about the things we’re concerned with in these pages — treating diseases like diabetes and cancer?

Scheme showing how genetic engineering can make the release of insulin from cells controllable by light. Normally cells of the pancreas (beta cells) take up glucose when its level in the circulation rises (via a glucose transporter protein). The rise in glucose triggers ATP production in the cell. This in turn causes potassium channels in the membrane to close (called depolarization) and this opens calcium channels. The increase in calcium in the cell drives insulin secretion. From Kushibiki et al., 2015.

The left-hand scheme above shows how glucose triggers the pancreas to produce the hormone insulin. Diabetes occurs when either the pancreas doesn’t make enough insulin or when cells of the body don’t respond properly to insulin by taking up glucose.

As a first step to see whether optogenetic regulation of calcium levels in pancreatic cells could trigger insulin release, Toshihiro Kushibiki and colleagues at the National Defense Medical College in Saitama, Japan engineered the channelrhodopsin-1 protein into mouse cells and hit them with laser light of the appropriate frequency. An hour after a short burst of light (a few seconds) the insulin levels had doubled.

The photo below shows a clump of these cells: the nuclei are blue and the channel protein (yellow) can be seen sitting across the cell membranes.

 

Cells expressing a fluorescently tagged channelrhodopsin protein (yellow). Nuclei are blue. From Kushibiki et al., 2015.

 

 

To show that this could work in animals they suspended the engineered cells in a gel and inoculated blobs of the goo under the skin of diabetic mice. Laser burst again: blood glucose levels fell and they showed this was due to the irradiated, implanted cells producing insulin.

Fast forward three years

Those brilliant results highlighted the potential of optogenetic technology as a completely novel approach to a disease that afflicts over 300 million people worldwide.

Scheme showing a Smartphone can be used to regulate the release of insulin from engineered cells implanted in a mouse with diabetes. The key events in the cell are that the light-activated receptor turns on an enzyme (BphS) that in turn controls a transcription regulator (FRTA) that binds to a DNA construct to switch on the Gene Of Interest (GOI) — in this case encoding insulin. (shGLP1, short human glucagon-like peptide 1, is a hormone that has the opposite effect to insulin). From Shao et al., 2017.

In a remarkable confluence of technologies Jiawei Shao and colleagues from a number of institutes in Shanghai, including the Shanghai Academy of Spaceflight Technology, and from ETH Zürich have recently published work that takes the application of optogenetics well and truly into the twenty-first century.

They figured that, as these days nearly everyone lives with their smartphone, the world could use a diabetes app. Essentially they designed a home server SmartController to process wireless signals so that a smartphone could control insulin production by cells in gel capsules implanted in mice. There are differences in the genetic engineering of these cells from those used by Kushibiki’s group but the critical point is unchanged: laser light stimulates insulin release. The capsules carry wirelessly powered LEDs.

The only other thing needed is to know glucose levels. Because mice are only little and they’ve already got their gel capsule, rather than implanting a monitor they took a drop of blood from the tail and used a glucometer. However, looking ahead to human applications, continuous glucose monitors are now available that, placed under the skin, can transmit a radio signal to the controller and, ultimately, it will be possible for the gel capsules to have a built-in battery plus glucose sensor and the whole thing could work automatically.

Any chance of illuminating cancer?

This science is so breathtaking it seems cheeky to ask but, well, I’d say ‘yes but not just yet.’ So long as the ‘drug’ you wish to use can be made biologically (i.e. from DNA by the machinery of the cell), rather than by chemical synthesis, Shao’s Smartphone set-up can readily be adapted to deliver anti-cancer drugs. This might be hugely preferable to the procedures currently in use and would offer an additional advantage by administering drugs in short bursts of lower concentration — a regimen that in some mouse cancer models at least is more effective.

References

Dawydow, A., Kittel, R.J. et al., 2014. Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications. PNAS 111, 13972-13977.

Kushibiki et al., (2015). Optogenetic control of insulin secretion by pancreatic beta-cells in vitro and in vivo. Gene Therapy 22, 553-559.

Shao, J. et al., 2017. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Science Translational Medicine 9, Issue 387, eaal2298.

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Blowing Up Cancer

To adapt the saying of the sometime British Prime Minister Harold Wilson, a month is a long time in cancer research. {I know, you’ve forgotten – as well you might. He was PM from 1964 to 1970 and again from 1974 to 1976. His actual words were “A week is a long time in politics”}. When I started to write the foregoing Self Helps (Parts 1 & 2) I had absolutely no intention of mentioning the subject of today’s sermon – viral immunotherapy. But how times change and a recent report has hit the headlines – so here goes.

The reason for my reticence is that this is not a new field – far from it. Folk have been trying to target tumour cells with active viruses for twenty years but efforts have foundered to the extent that the new report is the first time in the western world that a phase III trial (when a drug or treatment is first tested on large groups of people) of cancer “virotherapy” has definitively shown benefit for patients with cancer, although a virus (H101) made by the Shanghai Sunway Biotech Co. was licensed in China in 2005 for the treatment of a range of cancers.

Hard bit already done

I appreciate that getting the hang of immunotherapy in the two Self Helps wasn’t a total doddle – but it was worth it, wasn’t it, bearing in mind we’re dealing with life and death here. My friend and correspondent Rachel Bown had to resort to her GCSE biology notes (since she met me I think she keeps them on the coffee table) but is now up to speed.

Fortunately this bit is pretty easy to follow – it’s just an extension of the viral jiggery-pokery we met in Self Help Part 2. There we saw that using ‘disabled’ viruses is a neat way of getting new genetic material into cells. The viruses have key bits of their genome (genetic material) knocked out – so they don’t have any nasty effects and don’t replicate (make more of themselves) once inside cells. Inserting new bits of DNA carrying a therapeutic gene turns them into a molecular delivery service.

Going viral

In virotherapy there’s one extra wrinkle: the viruses, though ‘disabled’, still retain the capacity to replicate – and this has two effects. First, more and more virus particles (virions) are made in an infected cell until eventually it can hold no more and it bursts. The cell is done for – but a secondary effect is that the newly-made virions spill out and drift off to infect other cells. This amplifies the effect of the initial injection of virus and, in principle, will continue as long as there are cells to infect.

A new tool

The virus used is herpes simplex (HSV-1) of the relatively harmless type that causes cold sores and, increasingly frequently, genital herpes. The reason for this choice is that sometimes, not very often, science gets lucky and Mother Nature comes up with a helping hand. For HSV-1 it was the completely unexpected discovery that when you knock out one of its genes the virus becomes much more effective at replicating in tumour cells than in normal cells. That’s a megagalactic plus because, in effect, it means the virus targets tumour cells, thereby overcoming one of the great barriers to cancer therapy. In this study another viral gene was also deleted, which increases the immune response against infected tumour cells.

All this cutting and pasting (aka genetic engineering) is explained in entertaining detail in Betrayed by Nature but for now all that matters is that you end up with a virus that:

  1. Gets into tumour cells much more efficiently than into normal cells,
  2. Makes the protein encoded by the therapeutic gene, and
  3. Replicates in the cells that take it up until eventually they are so full of new viruses they go pop.

The finished product, if you can get your tongue round it, goes by the name of talimogene laherparepvec, mercifully shortened by the authors to T-VEC (made by Amgen). So T-VEC mounts a two-pronged attack – what the military would call a pincer movement. Infected tumour cells are killed (they’re ‘lysed’ by viral overload) and the inserted gene makes a protein that soups up the immune response – called GM-CSF (granulocyte macrophage colony-stimulating factor). The name doesn’t matter: what’s important is that it’s a human signaling molecule that stimulates the immune system, the overall result being production of tumour-specific T cells.

Fig. 1 Viral Therapy

Virotherapy. Model of a virus (top). The knobs represent proteins that enable the virus to stick to cells. Below: sequence of injecting viruses that are taken up by tumour cells that eventually burst to release new virions that diffuse to infect other tumour cells.

And the results?

The phase III trial, led by Robert Andtbacka, Howard Kaufman and colleagues from Rutgers Cancer Institute of New Jersey, involved 64 research centres worldwide and 436 patients with aggressive, inoperable malignant melanoma who received either an injection of T-VEC or a control immunotherapy. Just over 16% of the T-VEC group showed a durable response of more than six months, compared with 2% given the control treatment. About 10% of the patients treated had “complete remission”, with no detectable cancer remaining – considered a cure if the patient is still cancer-free five years after diagnosis.

Maybe this time?

We started with Harold Wilson and it was in between his two spells in Number 10 that President Nixon declared his celebrated ‘War on Cancer’, aimed at bringing the major forms of the disease under control within a decade or two. It didn’t happen, as we might have guessed. Back in 1957 in The Black Cloud the astrophysicist Sir Fred Hoyle has the line ‘I cannot understand what makes scientists tick. They are always wrong and they always go on.’ To be fair, it was a science fiction novel and the statement clearly is only partly true. But it’s not far off and in cancer there’s been rather few of the media’s beloved ‘breakthroughs’ and a great deal of random shuffling together with, overall, some progress in specific areas. Along the bumpy highway there have, of course, been moments of high excitement when some development or other has briefly looked like the answer to a maiden’s prayer. But with time all of these have fallen, if not by the wayside, at least into their due place as yet another small step for man. The nearest to a “giant leap for mankind” has probably been coming up with the means to sequence DNA on an industrial scale that is now having a massive impact on the cancer game.

When Liza Minnelli (as Sally Bowles in Cabaret) sings Maybe this time your heart goes out to the poor thing, though your head knows it’ll all end in tears. But this time, maybe, just maybe, the advent of cancer immunotherapy in its various forms will turn out to be a new era. Let us fervently hope so but, even if it does, the results of this Phase III trial show that a long struggle lies ahead before treatments arrive that have most patients responding.

We began Self Help – Part 1 with the wonderful William Coley and there’s no better way to pause in this story than with his words – reminding us of a bygone age when the scientist’s hand could brandish an artistic pen and space-saving editors hadn’t been invented:

“While the results have not been as satisfactory as one who is seeking perfection could wish, … when it comes to the consideration of a new method of treatment for malignant tumours, we must not wonder that a profession with memories overburdened with a thousand and one much-vaunted remedies that have been tried and failed takes little interest in any new method and shows less inclination to examine into its merits. Cold indifference is all it can expect, and rightly too, until it has something beside novelty to offer in its favour.”

References

Mohr, I. and Gluzman, Y. (1996). A herpesvirus genetic element which affects translation in the absence of the viral GADD34 function. The EMBO Journal 15, 4759–66.

Andtbacka, R.H.I. et al. (2015). Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma. 10.1200/JCO.2014.58.3377