Life, etc

Crispr vs HIV: Pitting the weaponry of a bacterium against the cunning of a virus

By Andrea Teagle 5 April 2016

State-of-the-art gene-editing tool Crispr-Cas9 has been widely touted as a means of understanding and eliminating diseases. Recently, scientists used it to successfully and safely delete the HIV genome from human immune cells and to prevent reinfection. But defeating HIV in a culture dish is a little like taking down an opponent in handcuffs. It’s an exciting first step, but there’s still a long way to go. By ANDREA TEAGLE.

Recently, a thriving collection of human immunodeficiency virus (HIV) came to a sudden and satisfying end in a petri dish at Temple University, Philadelphia.

Using a gene-cutting tool, researchers from the Lewis Katz School of Medicine snipped the entire HIV genome out of infected T-cells. They also managed to prevent the reinfection of the white blood cells, and showed that their gene-cutting tool hadn’t in the process accidentally attacked any of the wrong DNA. This equalled a small, early-stage victory for humans in Homo Sapiens v HIV. To put it in perspective, defeating HIV in a petri dish is a little like taking down an opponent in handcuffs. There’s a long way to go before we can do the same on the battlefield of the human body, but at least we’re starting to see how to do it.

The gene-editing tool that the Temple University researchers used is known for its precision, efficiency and versatility. The name is Crispr. Crispr-Cas9. The advent of Crispr in the science world is to a molecular biologist what a telescope is to an astronomer; steroids to an athlete or a wand to Harry Potter. Gene editing was happening before Crispr, but the invention – or, more accurately, the discovery – elevated it to new levels. It’s also cheap and easy to use, as gene-editing tools go.

As Professor Susan Kidson of the department of human biology at the University of Cape Town explained, “The marvellous thing about Crispr is the ability to be very, very specific to fix pieces of DNA. It’s never been possible before, which is why it was such a revolution.”

Long before Crispr popped up in labs the world over, it existed out of the hands of human beings. It was and is part of the immune system of bacteria, which is where we found it ready-made, compliments of evolution. Crispr-Cas9 is a beautiful example not of biomimicry but bioborrowing.

Humble beginnings

All the way back in the 1980s, scientists noted an odd pattern in the genomes of E. coli, later seen in other bacteria and micro-organisms. Like a chorus of a song, the same sequence of DNA was spotted over and over, with unique sequences (dubbed “spaces”) in between. Even more intriguing was that the repeat sequences were palindromes: they read the same backwards and forwards. It was like discovering Stonehenge in a molecular world: scientists didn’t know what the pattern meant, but they figured it probably meant something. Thus “clustered regularly interspaced short palindromic repeat” (Crispr) was coined.

It was only in 2007 that the function of Crispr was confirmed (by Rodolphe Barrangou and his colleagues, who happened to be food scientists working with yoghurt). It turned out that the spaces in the Crispr sequences exactly mirror the DNA of viruses that preyed on bacteria. These, scientists realised, are the bacterium’s records of enemy viruses, which it keeps so it can recognise and defend against them in future. Once an approaching virus is matched to DNA records, the bacterium launches a devastating counter-attack. For this, it sends along RNA-guided enzymes called Cas9 to snip the virus’s double-stranded DNA helix in two. If the viral assailant is unknown, a new identifying spacer is afterwards added to the records. Quite brilliant, really.

Crispr today

The way people use Crispr is essentially the same as the process described above – except that scientists decide on the “spaces”, and the targeted DNA could be that of humans, fruit flies, plants or anything else.

Crispr is used in two ways. Cas9 can be encoded to simply snip away – or “silence” – particular gene sequences, as in the case of eliminating HIV from infected cells. It can also replace the targeted gene sequences with other genetic material. The first application is less contentious because it doesn’t introduce foreign DNA into the organism, but can still have a significant impact on, for example, the way a plant responds to drought. (In some countries, this may mean that it does not classify as GMO, and so might avoid stringent regulation.) The second application means that problematic genes can actually be replaced, and this is the theoretical basis for eliminating genetic diseases such as muscular dystrophy.

On a simpler level, Crispr is extremely useful for understanding human biology and the genetic basis for diseases. This is what South African scientists working with Crispr are occupied with, says Professor Kidson, who is herself working with the tool.

Say you suspect a particular gene is playing a particular role in a disease – you create cells that have that defect, and you study what’s causing that defect. Or, you take some that you know have the defect and you say, what happens if I fix it? How will the cells behave?”

Crispr and HIV

For three decades – about as long as Crispr has been known – the human immunodeficiency virus (HIV) has evaded the efforts of scientists across the world to develop a vaccination or cure. The retrovirus is a master of disguise. Just as the body creates antibodies that can fight it, the virus evolves to take on a new, unrecognisable form, integrating itself into immune cells called CD4 cells – the very cells that should be fighting it. Every time they replicate, they replicate the HIV genome too.

Today, thanks to the development and rollout of antiretroviral therapy (ART), the disease is no longer the death sentence it once was. In South Africa, more than 3-million people are currently on this life-saving treatment. But although it is effective at keeping HIV in check in HIV-1 patients, ART is still unable to root out reservoirs of the HI virus.  

For this reason, lead researcher of the Temple University study, Dr Kamel Khalili said, patients on antiretroviral therapy who stop taking the drugs suffer a rapid rebound in HIV replication”.

Most approaches to curing HIV/Aids focus on trying to reactivate these dormant stores of the virus – bring the virus out of hiding, so to speak – so that the body’s antibodies can attack it. In the Temple University study, Khalili and his team showed that Crispr/Cas9 can isolate and delete the HIV genome directly from latently infected cells – at least in a petri dish. More significantly, they showed that the clear cells were protected from reinfection.

If a new virus comes along, this system stays in the cell. So it keeps working, if you manage to keep it in all the cells,” Prof Kidson explained. “That’s the key element in all this. In a culture dish, the infected cells are in high concentration.”

In a human body, although scientists have an idea of where reservoirs might be – mainly in bone marrow – they don’t know how to find latently infected cells. Sending Crsispr/Cas9 straight in would be futile: like parachuting a task team to Earth with the charge of finding a group of bad guys hiding somewhere on the planet.

What you’d have to do if you were to do it in a human, if you were to do it one day, is take samples of the blood, put them in a dish, correct them, treat the body with total radiation to kill everything that’s there in the bone marrow…” Kidson paused for breath, “and then replace with those that you’ve corrected, making sure that every cell that you replace is corrected. So it’s a huge mountain to climb still.”

The procedure is essentially the same one used to treat patients with leukemia. The reason that it works in that case is that noncancerous cells are more vigorous and will replace the few remaining unhealthy ones that remain after radiation. That’s not true of HIV. Only a few infected cells need to survive for the virus to rebound energetically. This, Kidson says, is why it hasn’t been done – because you can’t eliminate all of the cells.

Not a case of mistaken identity

The other primary hurdle in using Crispr-Cas9 against HIV is one that pertains to the gene-editing technique more generally: the possibility exists that Cas9, as accurate as it is, misidentifies genomes, and attacks the wrong ones. It might mistake the DNA equivalent of “aptitude” for “attitude”, with potentially far-reaching consequences. (There are various checks – which we’re only now discovering – that Crispr uses to guard against this, and which might help scientists to design more accurate Cas9 variants in future.) The University of Temple team’s study was encouraging, as molecular miscommunication did not happen in this case. Cas9 left the human genomes alone.

[The findings] show… that the technology is safe for the cells, with no toxic effects,” said Dr Kalili. Based on the results, his team is confident that they could start clinical trials on humans within three years. However, even then, there’s still a long way to go before a cure becomes a possibility.

There’s a very long road from what we call a proof in principle to clinical translation,” said Kidson. Referring to the current findings, which she noted were positive, she added, “It’s many, many years from the possibility that it becomes therapeutically viable – like 10 years maybe. It’s just one of many, many approaches that people are trying.”

In some ways, it’s an ancient war playing out on a modern battlefield: the weaponry of a bacterium against the cunning of a virus. This time though, that ancient weapon is in human hands, and we’re leading the charge. DM

Photo: A patient lies in bed at the Hillcrest Aids Center in Durban, South Africa, 01 December 2011. EPA/NIC BOTHMA

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