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.

 

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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.