Cardiff Crock of Gold?

 

One of the oddities of science is that we are aware that we know little and understand less and yet manage to be surprised at frequent intervals when some bright spark discovers something new. So, surprised most of us indeed were by a paper from Andrew Sewell and colleagues at Cardiff University who have tracked down a hitherto unknown sub-population of white blood cells that may turn out to be extremely useful.

Before we get to the really exciting bit we need a follow-up word on CRISPR-Cas9, because that was what the Cardiff group used, and a clear picture of how the immune system works in cancer.

CRISPR-Cas in short

This method adapts a bacterial defence system for detecting and destroying invading viruses. It uses RNA guides to locate specific bits of DNA inside a cell, enabling molecular scissors to cut that section of DNA. This can disable a specific gene or allow a new gene to be inserted — described in Sharpening CRISPR and Re-writing the Manual of Life.

However, as well as being able to knock out genes or insert new ones, CRISPR has another feature. By using designer guide RNAs, CRISPR can scan the entire range of the genome. This DNA scanning feature can be scaled up to screen many genomic sites in parallel in one experiment. Synthesis of short fragments of nucleic acids (oligonucleotides) is carried out automatically using computer-controlled instruments (oligonucleotide synthesizers). The scale is astonishing: high-throughput DNA synthesis platforms can produce libraries of oligos (millions of them), each encoding a different guide RNA sequence and hence a different DNA target. Oligo libraries can be cloned into a lentiviral (a retrovirus) vector system for delivery to cells. This generates parallel, high-throughput, loss-of-function of specific genes from which their function can be inferred.

The immune system and cancer

The immune system can recognise cancer cells as abnormal and kill them. This happens because cancer cells (and cells infected by pathogens) break down proteins made within the cell and display those fragments on their surface. Thus cancer cells can ‘present’ their own antigens thereby stimulating an immune response that leads to their elimination by the immune system. Antigens on the cell surface bind to killer T cells (aka cytotoxic T cells) via the T-cell receptor (a complex of proteins on the T cell surface). This provokes the release of perforin that makes a pore, or hole, in the membrane of the infected cell. Cytotoxins then pass into the cell through this pore, destroying it. Almost all cell types can present antigens in some way and the loss of ‘antigen presentation’ is a major escape mechanism in cancer. It allows tumour cells to become ‘invisible’ and avoid immune attack by anti-tumour white blood cells.Scheme showing a cytotoxic T cell, (a type of lymphocyte aka a killer T cell, cytolytic T cell or CD8+ T cell), that kills cancer cells, interacting via its TCR with an antigenic peptide attached to an MHC molecule on the surface of a target cell. Granzymes are enzymes that cause apoptosis in targets cells.

What is the major histocompatibility complex?

Antigen-presenting cell (APCs) display antigen on their surface attached to major histocompatibility complexes (MHCs). MHCs are essential for the adaptive immune system to work, i.e. the sub-system of the immune response that eliminates pathogens. Human MHCs are also called the HLA (human leukocyte antigen) complex to distinguish it from other vertebrates. They’re encoded by a group of genes that are highly polymorphic — meaning that there are many different variant forms of the genes (alleles). The upshot of this is that no two individuals have exactly the same set of MHC molecules, with the exception of identical twins. This is the cause of transplant rejection wherein an immune response is switched on against HLA antigens expressed on APCs transferred along with the transplanted organ.

And now for the exciting news

The CRISPR screen used by Andrew Sewell and colleagues turned up a new type of T cell — one that differs from conventional T cells by presenting fragments of tumour proteins attached not to HLA proteins but to a different a receptor called MR1. The difference is critical because MR1 doesn’t vary between humans, unlike the highly variable HLAs. This appears to be why, in laboratory experiments, T cells with the MR1-seeking receptor can mediate killing of most types of human cancer cells without damaging healthy cells.

What they did was to take a sample of peripheral blood and select lymphocytes that proliferated in the presence of a cancer cell line (derived from a human lung cancer). They found that this cell clone kills a wide range of cancer cells in culture — so they used the CRISPR screening method to track down what the clone was targetting on cancer cells. Answer: MR1.

The novel T cell clone kills a broad range of tumour cells but does not kill cancer cell lines lacking MR1 or a range of healthy cells from various tissues. From Crowther et al., 2020.

The Cardiff group were further able to show that T-cells of melanoma patients modified to express this new TCR could destroy not only the patient’s own cancer cells, but also other patients’ cancer cells in the laboratory, regardless of HLA type (see Self Help – Part 2 and Gosh! Wonderful GOSH for how adoptive cell transfer works).

Transfer of the clone carrying the novel T cell receptor redirects patient T cells to recognize their own melanoma cells. Normal cells are unaffected. Black dots: + MR1; Grey dots: – MR1. From Crowther et al., 2020.

The data show (left) two T cell populations from two patients with metastatic melanoma. T cells transduced with the T cell receptor that binds MR1 recognized their own melanoma cells and killed them. Normal cells were unaffected regardless of MR1 expression.

These findings describe a TCR that exhibits pan-cancer cell recognition via the invariant MR1 molecule. Engineering T cells from patients that lacked detectable anti-cancer cell activity rendered them capable of killing the patients’ melanoma cells. However, these cells did not attack healthy cells so this method of genetic engineering, coupled with adoptive cell transfer, offers exciting opportunities for pan-cancer, T cell–mediated immunotherapy.

This discovery is most timely because, although CAR-T therapy is personalised to each patient, it targets only a few types of cancers and thus far has not worked for solid tumours.

CRISPR and related technologies are leading us into a new world in which Chinese scientists have already made the first CRISPR-edited human embryos and the first CRISPR-edited monkeys and, very recently in the first human trial of cells modified with CRISPR gene-editing technology, shown that the treatment is safe and lasting. This work, by You Lu at the West China Hospital in Chengdu, took immune cells from people with aggressive lung cancer and disabled the PD-1 gene. The PD-1 protein normally attenuates the immune system to prevent it attacking its own tissues but, as this reduces its anti-cancer capacity, knocking out PD-1 should overcome that restriction.

These advances are remarkable but we are still at the very beginning of gene therapy for cancer and the promise is almost limitless.

Reference

Crowther, M.D., Sewell, J.D. et al., (2020). Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1. Nature Immunology  21,  178–185.

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.