Lorenzo’s Oil for Nervous Breakdowns

 

A Happy New Year to all our readers – and indeed to anyone who isn’t a member of that merry band!

What better way to start than with a salute to the miracles of modern science by talking about how the lives of a group of young boys have been saved by one such miracle.

However, as is almost always the way in science, this miraculous moment is merely the latest step in a long journey. In retracing those steps we first meet a wonderful Belgian – so, when ‘name a famous Belgian’ comes up in your next pub quiz, you can triumphantly produce him as a variant on dear old Eddy Merckx (of bicycle fame) and César Franck (albeit born before Belgium was invented). As it happened, our star was born in Thames Ditton (in 1917: his parents were among the one quarter of a million Belgians who fled to Britain at the beginning of the First World War) but he grew up in Antwerp and the start of World War II found him on the point of becoming qualified as a doctor at the Catholic University of Leuven. Nonetheless, he joined the Belgian Army, was captured by the Germans, escaped, helped by his language skills, and completed his medical degree.

Not entirely down to luck

This set him off on a long scientific career in which he worked in major institutes in both Europe and America. He began by studying insulin (he was the first to suggest that insulin lowered blood sugar levels by prompting the liver to take up glucose), which led him to the wider problems of how cells are organized to carry out the myriad tasks of molecular breaking and making that keep us alive.

The notion of the cell as a kind of sac with an outer membrane that protects the inside from the world dates from Robert Hooke’s efforts with a microscope in the 1660s. By the end of the nineteenth century it had become clear that there were cells-within-cells: sub-compartments, also enclosed by membranes, where special events took place. Notably these included the nucleus (containing DNA of course) and mitochondria (sites of cellular respiration where the final stages of nutrient breakdown occurs and the energy released is transformed into adenosine triphosphate (ATP) with the consumption of oxygen).

In the light of that history it might seem a bit surprising that two more sub-compartments (‘organelles’) remained hidden until the 1950s. However, if you’re thinking that such a delay could only be down to boffins taking massive coffee breaks and long vacations, you’ve never tried purifying cell components and getting them to work in test-tubes. It’s a process called ‘cell fractionation’ and, even with today’s methods, it’s a nightmare (sub-text: if you have to do it, give it to a Ph.D. student!).

By this point our famous Belgian had gathered a research group around him and they were trying to dissect how insulin worked in liver cells. To this end they (the Ph.D. students?!) were using cell fractionation and measuring the activity of an enzyme called acid phosphatase. Finding a very low level of activity one Friday afternoon, they stuck the samples in the fridge and went home. A few days later some dedicated soul pulled them out and re-measured the activity discovering, doubtless to their amazement, that it was now much higher!

In science you get odd results all the time – the thing is: can you repeat them? In this case they found the effect to be absolutely reproducible. Leave the samples a few days and you get more activity. Explanation: most of the enzyme they were measuring was contained within a membrane-like barrier that prevented the substrate (the chemical that the enzyme reacts with) getting to the enzyme. Over a few days the enzyme leaked through the barrier and, lo and behold, now when you measured activity there was more of it!

Thus was discovered the ‘lysosome’ – a cell-within-a cell that we now know is home to an array of some 40-odd enzymes that break down a range of biomolecules (proteins, nucleic acids, sugars and lipids). Our self-effacing hero said it was down to ‘chance’ but in science, as in other fields of life, you make your own luck – often, as in this case, by spotting something abnormal, nailing it down and then coming up with an explanation.

In the last few years lysosomes have emerged as a major player in cancer because they help cells to escape death pathways. Furthermore, they can take up anti-cancer drugs, thereby reducing potency. For these reasons they are the focus of great interest as a therapeutic target.

Lysosomes in cells revealed by immunofluorescence.

Antibody molecules that stick to specific proteins are tagged with fluorescent labels. In these two cells protein filaments of F-actin that outline cell shape are labelled red. The green dots are lysosomes (picked out by an antibody that sticks to a lysosome protein, RAB9). Nuclei are blue (image: ThermoFisher Scientific).

Play it again Prof!

In something of a re-run of the lysosome story, the research team then found itself struggling with several other enzymes that also seemed to be shielded from the bulk of the cell – but the organelle these lived in wasn’t a lysosome – nor were they in mitochondria or anything else then known. Some 10 years after the lysosome the answer emerged as the ‘peroxisome’ – so called because some of their enzymes produce hydrogen peroxide. They’re also known as ‘microbodies’ – little sacs, present in virtually all cells, containing enzymatic goodies that break down molecules into smaller units. In short, they’re a variation on the lysosome theme and among their targets for catabolism are very long-chain fatty acids (for mitochondriacs the reaction is β-oxidation but by a different pathway to that in mitochondria).

Peroxisomes revealed by immunofluorescence.

As in the lysosome image, F-actin is red. The green spots here are from an antibody that binds to a peroxisome protein (PMP70). Nuclei are blue (image: Novus Biologicals)

Cell biology fans will by now have worked out that our first hero in this saga of heroes is Christian de Duve who shared the 1974 Nobel Prize in Physiology or Medicine with Albert Claude and George Palade.

A wonderful Belgian. Christian de Duve: physician and Nobel laureate.

Hooray!

Fascinating and important stuff – but nonetheless background to our main story which, as they used to say in The Goon Show, really starts here. It’s so exciting that, in 1992, they made a film about it! Who’d have believed it?! A movie about a fatty acid!! Cinema buffs may recall that in Lorenzo’s Oil Susan Sarandon and Nick Nolte played the parents of a little boy who’d been born with a desperate disease called adrenoleukodystrophy (ALD). There are several forms of ALD but in the childhood disease there is progression to a vegetative state and death occurs within 10 years. The severity of ALD arises from the destruction of myelin, the protective sheath that surrounds nerve fibres and is essential for transmission of messages between brain cells and the rest of the body. It occurs in about 1 in 20,000 people.

Electrical impulses (called action potentials) are transmitted along nerve and muscle fibres. Action potentials travel much faster (about 200 times) in myelinated nerve cells (right) than in (left) unmyelinated neurons (because of Saltatory conduction). Neurons (or nerve cells) transmit information using electrical and chemical signals.

The film traces the extraordinary effort and devotion of Lorenzo’s parents in seeking some form of treatment for their little boy and how, eventually, they lighted on a fatty acid found in lots of green plants – particularly in the oils from rapeseed and olives. It’s one of the dreaded omega mono-unsaturated fatty acids (if you’re interested, it can be denoted as 22:1ω9, meaning a chain of 22 carbon atoms with one double bond 9 carbons from the end – so it’s ‘unsaturated’). In a dietary combination with oleic acid  (another unsaturated fatty acid: 18:1ω9) it normalizes the accumulation of very long chain fatty acids in the brain and slows the progression of ALD. It did not reverse the neurological damage that had already been done to Lorenzo’s brain but, even so, he lived to the age of 30, some 22 years longer than predicted when he was diagnosed.

What’s going on?

It’s pretty obvious from the story of Lorenzo’s Oil that ALD is a genetic disease and you will have guessed that we wouldn’t have summarized the wonderful career of Christian de Duve had it not turned out that the fault lies in peroxisomes.

The culprit is a gene (called ABCD1) on the X chromosome (so ALD is an X-linked genetic disease). ABCD1 encodes part of the protein channel that carries very long chain fatty acids into peroxisomes. Mutations in ABCD1 (over 500 have been found) cause defective import of fatty acids, resulting in the accumulation of very long chain fatty acids in various tissues. This can lead to irreversible brain damage. In children the myelin sheath of neurons is damaged, causing neurological defects including impaired vision and speech disorders.

And the miracle?

It’s gene therapy of course and, helpfully, we’ve already seen it in action. Self Help – Part 2 described how novel genes can be inserted into the DNA of cells taken from a blood sample. The genetically modified cells (T lymphocytes) are grown in the laboratory and then infused into the patient – in that example the engineered cells carried an artificial T cell receptor that enabled them to target a leukemia.

In Gosh! Wonderful GOSH we saw how the folk at Great Ormond Street Hospital adapted that approach to treat a leukemia in a little girl.

Now David Williams, Florian Eichler, and colleagues from Harvard and many other centres around the world, including GOSH, have adapted these methods to tackle ALD. Again, from a blood sample they selected one type of cell (stem cells that give rise to all blood cell types) and then used genetic engineering to insert a complete, normal copy of the DNA that encodes ABCD1. These cells were then infused into patients. As in the earlier studies, they used a virus (or rather part of a viral genome) to get the new genetic material into cells. They choose a lentivirus for the job – these are a family of retroviruses (i.e. they have RNA genomes) that includes HIV. Specifically they used a commercial vector called Lenti-D. During the life cycle of RNA viruses their genomes are converted to DNA that becomes a permanent part of the host DNA. What’s more, lentiviruses can infect both non-dividing and actively dividing cells, so they’re ideal for the job.

In the first phase of this ongoing, multi-centre trial a total of 17 boys with ALD received Lenti-D gene therapy. After about 30 months, in results reported in October 2017, 15 of the 17 patients were alive and free of major functional disability, with minimal clinical symptoms. Two of the boys with advanced symptoms had died. The achievement of such high remission rates is a real triumph, albeit in a study that will continue for many years.

In tracing this extraordinary galaxy, one further hero merits special mention for he played a critical role in the story. In 1999 Jesse Gelsinger, a teenager, became the first person to receive viral gene therapy. This was for a metabolic defect and modified adenovirus was used as the gene carrier. Despite this method having been extensively tested in a range of animals (and the fact that most humans, without knowing it, are infected with some form of adenovirus), Gelsinger died after his body mounted a massive immune response to the viral vector that caused multiple organ failure and brain death.

This was, of course, a huge set-back for gene therapy. Despite this, the field has advanced significantly in the new century, both in methods of gene delivery (including over 400 adenovirus-based gene therapy trials) and in understanding how to deal with unexpected immune reactions. Even so, to this day the Jesse Gelsinger disaster weighs heavily with those involved in gene therapy for it reminds us all that the field is still in its infancy and that each new step is a venture into the unknown requiring skill, perseverance and bravery from all involved – scientists, doctors and patients. But what better encouragement could there be than the ALD story of young lives restored.

It’s taken us a while to piece together the main threads of this wonderful tale but it’s emerged as a brilliant example of how science proceeds: in tiny steps, usually with no sense of direction. And yet, despite setbacks, over much time, fragments of knowledge come together to find a place in the grand jigsaw of life.

In setting out to probe the recesses of metabolism, Christian de Duve cannot have had any inkling that he would build a foundation on which twenty-first century technology could devise a means of saving youngsters from a truly terrible fate but, my goodness, what a legacy!!!

References

Eichler, F. et al. (2017). Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy. The New England Journal of Medicine 377, 1630-1638.

 

Re-writing the Manual of Life

A little while ago we talked about a fantastic triumph by a team at Great Ormond Street Hospital (Gosh! Wonderful GOSH) in using a form of immunotherapy to save a little girl. What they did was to take the T cells from a sample of her blood and use gene editing – molecular cutting and pasting – to remove some genes and add others before growing more of the cells and then putting them back into the patient.

Gene editing – genetic engineering that removes or inserts sections of DNA – uses engineered nucleases, enzymes that snip DNA but do so in a controlled way by homing in on a specific site (i.e. a defined sequence of As, Cs, Gs and Ts).

We mentioned that there are four main ways of doing this kind of engineering – the GOSH group used ‘transcription activator-like effectors’ (TALEs). However, the method that has made the biggest headlines is called CRISPR/Cas, and it has been very much in the news because a legal battle is underway to determine who did what in its development and who, therefore, will be first in line for a Nobel Prize.

Fortunately we can ignore such base pursuits and look instead at where this technology might be taking us.

What is CRISPR/Cas?

CRISPRs (pronounced crispers) are bits of DNA that contain short repetitions of base sequence, each next to a ‘spacer’ sequence. The spacers have accumulated in bacteria as a defence mechanism – they’re part of the bacterial immune system – and they’re identical to sequences found in viruses that infect microbes. In other words, the cunning bugs pick up bits of dangerous viruses to make a rogues gallery so they can recognize and attack those viruses next time they pop in.

Close to CRISPR sit genes encoding Cas proteins (enzymes that cut DNA, so they’re ‘nucleases’). When the CRISPR-spacer DNA is read by the machinery of the cell to make RNA, the spacer regions stick to Cas proteins and the whole complex, including the viral sequences, can roam the cell seeking a virus with genetic material that matches the CRISPR RNA. The CRISPR RNA sticks to the virus and Cas chops its DNA – end of virus. So Cas, by binding to CRISPR RNA, becomes an RNA-guided DNA cutter.

CRISPR-CAS: Bug defence against invaders. Viruses can attack bacteria just as they can human cells. Over time bugs have evolved a cunning defence strategy: they insert short bits of viral DNA into their own genome (above). These contain repeated sequences of bases and each is followed by short segments of ‘spacer DNA’ (above). This happens next to DNA that encodes Cas proteins so that both are ‘read’ to make RNA (transcription). Cas proteins bind to spacer RNA, leaving the adjacent viral RNA free to attach to any complementary viral DNA it encounters. The Cas enzyme is thus guided to DNA that it can cleave. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.

Why is CRISPR/Cas in the headlines?

We saw in Gosh! Wonderful GOSH how the Great Ormond Street Hospital team tinkered with DNA and in Self Help – Part 2 we summarized another way of doing this using viruses (notably a disabled form of the human immunodeficiency virus) to carry novel genes into cells.

A further arm of immunotherapy attempts to reverse an effect called checkpoint blockade whereby the immune system response to tumours is damped down – e.g. by using antibodies that target a protein called PD-1 (Self Help – Part 1).

Now comes news of a Chinese trial which will be the first time cells modified using CRISPR–Cas9 gene editing have been injected into people. The chap in charge is Lu You from Sichuan University’s West China Hospital in Chengdu and the plan is to take T cells from the blood patients with metastatic non-small cell lung cancer for whom chemotherapy, radiation therapy and other treatments have failed.

The target will be the PD-1 gene, the idea being that, if you want to stop PD-1 doing its stuff, far better than mucking about with antibodies is to just knock out its gene: no gene no protein! What could possibly go wrong?

Well, wonderful though CRISPR is, it doesn’t always hit the right target but in this trial the cells can be tested to make sure it’s the PD-1 gene that’s been zonked – so that shouldn’t be a problem. However, it’s a blockbuster in that all the multiplied T cells put back into the patient will be active – i.e. will have lost the PD-1 brake. Whilst that may be good for zonking tumours, goodness knows what it might do elsewhere.

The initial trial is on a small scale – just 10 people. If there are problems one possibility is to try to take the T cells from the site of the tumour, which might select those specifically targeting the tumour – not straightforward as lung cancers are difficult to get at.

Anyone for a DNA upgrade?

It’s hard to say where all this is leading. However, as Chinese scientists have already made the first CRISPR-edited human embryos and the first CRISPR-edited monkeys, the only safe bet is that China will be to the fore.

 

Gosh! Wonderful GOSH

Anyone who reads these pages will long ago, I trust, have been persuaded that the molecular biology of cells is fascinating, beautiful and utterly absorbing – and all that is still true even when something goes wrong and cancers make their unwelcome appearance. Which makes cancer a brilliant topic to talk and write about – you know your audience will be captivated (well, unless you’re utterly hopeless). There’s only one snag, namely that – perhaps because of the unwelcome nature of cancers – it’s tough to make jokes. One of the best reviews I had for Betrayed by Nature was terrifically nice about it but at the end, presumably feeling that he had to balance things up, the reviewer commented that it: “..is perhaps a little too light-hearted at times…” Thank you so much anonymous critic! Crikey! If I’d been trying to do slap-stick I’d have bunged in a few of those lewd chemicals – a touch of erectone, a bit of PORN, etc. (btw, the former is used in traditional Chinese medicine to treat arthritis and the latter is poly-ornithinine, so calm down).

I guess my serious referee may have spotted that I included a poem – well, two actually, one written by the great JBS Haldane in 1964 when he discovered he had bowel cancer which begins:

I wish I had the voice of Homer

To sing of rectal carcinoma,

Which kills a lot more chaps, in fact,
Than were bumped off when Troy was sacked.

Those couplets may reflect much of JBS with whom I can’t compete but, nevertheless, in Betrayed by Nature I took a deep breath and had a go at an update that began:

Long gone are the days of Homer
But not so those of carcinoma,
Of sarcoma and leukemia

And other cancers familia.
But nowadays we meet pre-school
That great and wondrous Molecule.
We know now from the knee of Mater
That DNA’s the great creator.

and went on:

But DNA makes cancer too

Time enough—it’ll happen to you.
“No worries sport” as some would say,
These days it’s “omics” all the way.

So heed the words of JBS

Who years ago, though in distress,
Gave this advice on what to do

When something odd happens to you:
“Take blood and bumps to your physician
Whose only aim is your remission.”

I’d rather forgotten my poem until in just the last week there hit the press a story illustrating that although cancer mayn’t be particularly fertile ground for funnies it does gloriously uplifting like nothing else. It was an account of how science and medicine had come together at Great Ormond Street Hospital to save a life and it was even more thrilling because the life was that of a little girl just two years old. The saga brought my poem to mind and it seemed, though I say it myself, rather spot on.

The little girl, Layla, was three months old when she was diagnosed with acute lymphoblastic leukemia (ALL) caused by a piece of her DNA misbehaving by upping sticks and moving to a new home on another chromosome – one way in which genetic damage can lead to cancer. By her first birthday chemotherapy and a bone marrow transplant had failed and the only remaining option appeared to be palliative care. At this point the GOSH team obtained special dispensation to try a novel immunotherapy using what are being called “designer immune cells“. Over a few months Layla recovered and is now free of cancer. However, there are no reports of Waseem Qasim and his colleagues at GOSH and at University College London dancing and singing the Trafalgar Square fountains – they’re such a reserved lot these scientists and doctors.

How did they do it?

In principle they used the gene therapy approach that, helpfully, we described recently (Self Help Part 2). T cells isolated from a blood sample have novel genes inserted into their DNA and are grown in the lab before infusing into the patient. The idea is to improve the efficiency with which the T cells target a particular protein (CD19) present on the surface of the leukemia cells by giving them artificial T cell receptors (also known as chimeric T cell receptors or chimeric antigen receptors (CARs) – because they’re made by fusing several bits together to make something that sticks to the target ‘antigen’ – CD19). The engineered receptors thereby boost the immune response against the leukemia. The new genetic material is inserted into a virus that carries it into the cells. So established is this method that you can buy such modified cells from the French biotech company Cellectis.

Adoptive cell transfer immunotherapy. T cells are isolated from a blood sample and novel genes inserted into their DNA. The GOSH treatment also uses gene editing by TALENs to delete two genes. The engineered T cells are expanded, selected and then infused into the patient.

Is that all?

Not quite. To give themselves a better chance the team added a couple of extra tricks. First they included in the virus a second gene, RQR8, that encodes two proteins – this helps with identifying and selecting the modified cells. The second ploy is, perhaps, the most exciting of all: they used gene editing – a rapidly developing field that permits DNA in cells to be modified directly: it really amounts to molecular cutting and pasting. Also called ‘genome editing’ or ‘genome editing with engineered nucleases’ (GEEN), this form of genetic engineering removes or inserts sections of DNA, thereby modifying the genome.

The ‘cutting’ is done by proteins (enzymes called nucleases) that snip both strands of DNA – creating double-strand breaks. So nucleases are ‘molecular scissors.’ Once a double-strand break has been made the built-in systems of cells swing into action to repair the damage (i.e. stick the DNA back together as best it can without worrying about any snipped bits – these natural processes are homologous recombination and non-homologous end-joining, though we don’t need to bother about them here).

To be of any use the nucleases need to be targeted – made to home in on a specific site (DNA sequence) – and for this the GOSH group used ‘transcription activator-like effectors’ (TALEs). The origins of these proteins could hardly be further away from cancer – they come from a family of bacteria that attacks hundreds of different types of plants from cotton to fruit and nut trees, giving rise to things like citrus canker and black rot. About six years ago Jens Boch of the Martin-Luther-University in Halle and Adam Bogdanove at Iowa State University with their colleagues showed that these bugs did their dirty deeds by binding to regulatory regions of DNA thereby changing the expression of genes, hence affecting cell behavior. It turned out that their specificity came from a remarkably simple code formed by the amino acids of TALE proteins. From that it’s a relatively simple step to make artificial TALE proteins to target precise stretches of DNA and to couple them to a nuclease to do the cutting. The whole thing makes a TALEN (transcription activator-like effector nuclease). TALE proteins work in pairs (i.e. they bind as dimers on a target DNA site) so an artificial TALEN is like using both your hands to grip a piece of wood either side of the point where, using your third hand, you make the cut. The DNA that encodes the whole thing is inserted into plasmids that are transfected into the target cells; the expressed gene products then enter the nucleus to work on the host cell’s genome. There are currently three other approaches to nuclease engineering (zinc finger nucleases, the CRISPR/Cas system and meganucleases) but we can leave them for another time.

The TALENs made by the GOSH group knocked out the T cell receptor (to eliminate the risk of an immune reaction against the engineered T cells (called graft-versus-host disease) and CD52 (encodes a protein on the surface of mature lymphocytes that is the target of the monoclonal antibody alemtuzumab – so this drug can be used to prevent rejection by the host without affecting the engineered T cells).

What next?

This wonderful result is not a permanent cure for Layla but it appears to be working to stave off the disease whilst she awaits a matched T cell donor. It’s worth noting that a rather similar approach has been used with some success in treating HIV patients but it should be born in mind that, brilliant though these advances are, they are not without risks – for example, it’s possible that the vector (virus) that delivers DNA might have long-term effects – only time will tell.

Almost the most important thing in this story is what the GOSH group didn’t do. They used the TALENs gene editing method to knock out genes but it’s also a way of inserting new DNA. All you need to do is add double-stranded DNA fragments in the correct form at the same time and the cell’s repair system will incorporate them into the genome. That offers the possibility of being able to repair DNA damage that has caused loss of gene function – a major factor in almost all cancers. Although there is still no way of tackling the associated problem of how to target gene editing to tumour cells, it may be that Layla’s triumph is a really significant step for cancer therapy.

Reference

Smith, J. et al. (2015). UCART19, an allogeneic “off-the-shelf” adoptive T-cell immunotherapy against CD19⁺ B-cell leukemias. Journal of Clinical Oncology 33, 2015 (suppl; abstr 3069).