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 (proteinsnucleic acidssugars 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.

 

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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-pic

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.

 

Self Help – Part 2

In the second type of cancer immunotherapy a sample of a patient’s T lymphocytes is grown in the lab. This permits either expansion of the number of cells that recognize the tumour or genetic engineering to modify the cells so they express receptors on their surface that target them to the tumour cell surface. Infusion of these manipulated cells into the patient enhances tumour cell killing. We’re now in the realms of ‘personalized medicine’.

A little more of a good thing

The first of these methods picks up a weakness in the patient’s immune system whereby it makes lymphocytes that kill tumour cells but can’t make enough – their protective effect is overwhelmed by the growing cancer. By taking small pieces of surgically removed tumours and growing them in the lab, it’s possible to select those T cells that have killing capacity. These are expanded over a few weeks to make enough cells to keep on growing when they’re infused back into the patient. The upshot is a hefty boost for the natural anti-tumour defence system. The pioneer of this method, called adoptive cell therapy, is Steven Rosenberg (National Cancer Institute, Bethesda) and it has been particularly effective for melanomas. Responses are substantially improved by treatment with drugs that reduce the white cell count before samples are taken for T cell selection – probably because the system responds by making growth factors to restore the balance and these drive the expansion of the infused cells.

A wonderful benefit of this method is its efficacy against metastases – i.e. tumour growths that have spread from the primary site – perhaps not surprising as it’s what Rosenberg calls a “living” treatment, in other words it just gives a helping hand to what nature is already trying to do.

93. Fig. 1Selecting naturally occurring T cells with anti-tumour activity

Tumour fragments are grown in the laboratory: lymphocytes that kill tumour cells are selected and expanded in culture.  About 6 weeks growth yields enough cells to infuse into the patient.

Gene therapy

A more sophisticated approach to boosting innate immunity is to introduce new genes into the genetic material (the genome) of T cells to target them to tumour cells with greater efficiency. An ordinary blood sample suffices as a starting point from which T cells are isolated. One way of getting them to take up novel genes uses viruses – essentially just genetic material wrapped in an envelope. The virus is ‘disabled’ so that it has none of its original disease-causing capacity but retains infectivity – it sticks to cells. ‘Disabling’ means taking just enough of the original genome to make the virus – a viral skeleton – and then inserting your favourite gene, so the engineered form is just a handy vehicle for carrying genes. No need to panic, therefore, if you see a press headline of the “HIV cures cancer” variety: it just means that the human immunodeficiency virus – well and truly disabled – has been used as the gene carrier.

93. Fig. 2

Genetic modification of blood lymphocytes

T cells are isolated from a blood sample and novel genes inserted into their DNA. The engineered T cells are expanded and then infused into the patient.

 This method of re-directing T cells to a desired target was pioneered by Gideon Gross and colleagues at The Weizmann Institute of Science in Israel in the late 1980s and it has led to sensational recent results in treating chronic lymphocytic leukemia (CLL), albeit in just a few patients so far. To the fore have been Renier Brentjens and his group from the Memorial Sloan-Kettering Cancer Center, New York. The genetic modification they used made the patient’s T cells express an artificial receptor on their surface (called a chimeric antigen receptor). This T cell receptor was designed to stick specifically to a protein known to be displayed on the surface of CLL cells. The result was that the T cells, originally unable to ‘see’ the leukemic cells, now homed in on them with high efficiency. Astonishingly, and wonderfully, the modified cells divide in the patient so that, in effect, their immune system has been permanently super-charged.

A critical part of the strategy is that CLL cells carry a known molecular target but the absence of such defined markers for most cancers is currently a severe limitation. On the bright side, however, this type of gene therapy has now been attempted in at least three different centres and, despite inevitable minor differences in method, it clearly works.

One of the leading figures in gene therapy is Carl June of the University of Pennsylvania. Some of his colleagues have made a brilliant video explaining how it works whilst June himself has described in wonderfully humble fashion what it means to work in this field.

References

Rosenberg, S.A. and Restifo, N.P. (2015). Adoptive cell transfer as personalized immunotherapy for human cancer. Science 348, 62-68.

Gross, G., et al. (1989). Expression of immunoglobulin-T-cell receptor chimeric molecules as functional receptorswith antibody-type specificity. Proc. Natl. Acad. Sci. U.S.A. 86, 10024–10028.

Brentjens, R.J., et al. (2013). CD19-Targeted T Cells Rapidly Induce Molecular Remissions in Adults with Chemotherapy-Refractory Acute Lymphoblastic Leukemia. Sci Transl Med., 5, 177ra38. DOI:10.1126/scitranslmed.3005930.

Kalos, M., et al. (2011). T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia. Sci. Transl. Med. 3, 95ra73.

Kochenderfer, J.N., et al. (2012). B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor–transduced T cells. Blood 119, 2709–2720.