A New 'Cloaking' System Makes It Possible To Administer AAV Gene Therapies Without The Dangerous Immunological Response

The mechanism our immune system uses to fend off viruses also attacks AAV vectors. Adding telomeric sequences to these vectors helps them to fly under the radar.

By: Dan Robitzski - Mar. 8, 2021
First-author Ying Kai Chan at Harvard University found a way to cloak AAV delivery vectors from...
First-author Ying Kai Chan at Harvard University found a way to cloak AAV delivery vectors from induced unwanted immune responses - a long-standing issue for AAV-delivered gene therapies. Credit: Wyss Institute/Seth Kroll

A team of scientists led by Harvard University researchers Ying Kai Chan and George Church developed what they call a 'cloaking' device that's helping to solve a long-standing issue in adeno-associated virus (AAV) gene therapies.

That problem, specifically, is that our immune systems struggle to differentiate between an AAV vector and a pathogenic virus. From the T cells' point of view, that lack of discrimination keeps us safe from infections i.e. foreign DNA merely is foreign DNA, and it doesn't matter that the AAV is harmless. So the T cells tend to attack AAVs just like they might any other invader.

»The component of the AAV that leads to inflammation is not unlike the components of infectious diseases that leads to inflammation. But it's much less inflammatory than a pathogenic infectious disease. I would summarise it as having similar mechanisms that lead to inflammation as pathogenic viruses,« says Harvard visiting scholar and molecular engineer Ying Kai Chan.

This immune response to AAVs can negate the benefits of a gene therapy and even cause dangerous levels of inflammation, so the research team found a way to sneak AAVs into the body — without alarming the immune system — by adding a telomeric DNA sequence into the AAV's genetic code. Their study, which was recently published in the journal Science Translational Medicine, shows that this alteration helps to mask the AAV, letting it get to work without causing a hazardous immune response. If this approach is validated in clinical research, scientists suspect that it could make human gene therapies safer and more accessible.

»It is a very big issue. Gene therapy, in general, has been plagued by the immune system throughout its history,« says Dr Julie Crudele, an acting assistant professor at the University of Washington's department of neurology, who didn't work on the AAV cloaking study.

Still, she frequently runs into this troublesome immune response during her research on the immunological aspect of gene therapies designed to treat various muscular diseases as well as other conditions.

DNA is a danger signal to the immune system

The immunological response to AAV therapies was first discovered during a gene therapy trial seeking to treat haemophilia, Crudele explains. The treatment seemed to correct the problem at first, but then the condition returned. The patient's T cells had attacked the liver cells that received the treatment, negating its therapeutic effects and bringing the patient back to square one.

However, different gene therapies, especially at higher doses, can provoke more severe immune responses that's annoying at best and dangerous — or even deadly — at worst.

The immune system relies on Toll-like receptor 9 (TLR9) to identify foreign DNA inside the body. The presence of foreign DNA usually means that the body is under attack by some infectious agent, so TLR9 activation prompts the immune system to launch a counterattack to combat the threat.

The so-called cloaking device built by the Harvard scientists antagonises TLR9 activation, essentially making the AAV vector appear as a sequence of natural, human DNA rather than a virus. To do that, they used a sequence of DNA found naturally in telomeres, which are structures made up of repetitive nucleotide patterns found at the ends of our chromosomes.

There is so much hype and interest in engineering the [AAV] capsid that people forget that the DNA is also a danger signal to the immune systemKai Chan

»If you think about it, the [TLR9] system evolved so we can detect pathogenic viruses to fight them off. The problem is that it's detecting incoming DNA, which appears the same as an AAV vector. It can't distinguish that 'hey, this is a friendly virus',« says Chan.

»There is so much hype and interest in engineering the [AAV] capsid that people forget that the DNA is also a danger signal to the immune system,« he adds.

Invisibility by blocking TLR9 dimerisation

So how does the so-called invisibility cloak work? Telomeres have been implicated in aging and regenerative medicine studies, but they are also thought to safeguard our chromosomes from damage by functioning as protective caps. Now, as Chan explains, geneticists suspect that telomeres also bind to TLR9 and play a crucial role in keeping our immune system from targeting our DNA.

TLR9 has two distinct DNA binding sites. Previous research showed that activation of both sites prompts the most efficient immune response. But either site can recognise foreign DNA, which prompts loop-shaped TLR9 molecules to form dimers and activate, triggering an immune response. To keep that from happening, the researchers built on previous studies that used DNA oligonucleotides to block TLR9 dimerisation by physically blocking its binding sites.

The two telomeric sequences used in the study were fairly short, under 100 and 200 base pairs, respectively, Chan explains. That made it simple to add them into the AAV, which has a genetic sequence of about 5,000 base pairs. Chan says he was initially concerned that tinkering with the AAV's genetic sequence might reduce the yield of gene therapy vectors, but neither sequence seemed to reduce vector titers at any point in the experiment. That means that there was no trade-off between blocking the immune system's response and how well the gene therapy worked.

By introducing that sequence into an AAV's genetic code, Chan and his team found that the resulting gene-editing vectors could pass through our bodies safely without incident.

»We have a strategy that we like to say is inspired by nature,« Chan says.

»We essentially emulated this sequence from nature. We took telomeric sequences, put them in AAV DNA, and saw reduced inflammation,« he later added.

In the study's animal trials, the cloaking system mostly worked. The inflammation response still occurred in one specific case, so the next step for Chan's team is to figure out whether fixing it is a matter of refining the cloaking system or if something different than TLR9 detected the AAV.

The researchers also tested the masked vector in liver, muscle, and retinal gene therapies administered to mice, pigs, and macaques. In nearly every case, the AAV managed to deliver its genetic package without provoking inflammation. However, in one experiment, a particularly high dose that was delivered through an intravitreal injection near a macaque's retina resulted in the same level of inflammation as an uncloaked AAV would have — albeit the response was delayed.

»While our strategy can work for many routes of administration, when we applied it to this high-dosage challenge, we were merely delaying the inflammation. But the other side of the coin is that this is precious information. This suggests that this TLR9 DNA-sensing mechanism is not the only pathway. This opens up a path to the field to find the other immune vector,« Chan says.

Human trials may be near

It's not surprising that the intravitreal injection didn't work out — administering a gene therapy into the vitreous humour in the middle of an eyeball generally prompts a stronger immune response than other routes of administration. But a successful trial would have been a big step forward for gene therapies; unlike subretinal injections, which have to be delivered to the back of the retina by a specially-trained surgeon, intravitreal injections can be administered in clinics by nurses, Chan explains.

Dr Julie M Crudele, at the University of Washington says the new approach could quickly be applied...
Dr Julie M Crudele, at the University of Washington says the new approach could quickly be applied also in human trials. Photo: Courtesy of Dr Crudele.

Opening up that specific administration route would likely make any potential treatments both more accessible and affordable for the people who need them.

But that said, there's no significant reason why researchers conducting clinical research wouldn't be able to implement the telomere sequence into their own gene therapies.

Neither Crudele nor Chan conduct clinical research themselves, but both were optimistic that others could and would use this new cloaking technique.

»I don't think [human trials] are a long way off,« says Crudele.

»Including one of these sequences of DNA into your construct seems like something that people could definitely — easily — start doing tomorrow. I don't think that the regulatory agencies would have much of an issue with that. I don't think it would ever be tested separately, « she adds.

»This strategy is very plug-and-play. You don't have to change your promoter, your transgene. This can be advanced by incorporating our strategy into constructs that are destined for the clinic. We think that because of the ease of the plug-and-play aspect, in this case, there is an obvious path for [clinical use],« says Chan.

However, the cloaking strategy won't be enough to solve AAV-induced inflammation on its own — the macaque trial is evidence enough of that.

»The authors acknowledge, correctly, that this tool is probably inadequate alone. It would need to be used in a cocktail to truly and reduce immune responses,« says Crudele.

»We keep adding in tools to try to dampen these [immune system] responses. And if it seems like it's helping — which based on this paper, I suspect it will — it's a good and straightforward thing to include in new gene therapies. There's not much of a downside for including it. None of these things are perfect, so the idea is you just layer all these imperfect-but-helpful things on top of each other and hope you get the immune system under control,« she adds.

Certainly, many questions and mysteries remain about how our immune systems are likely to respond to future gene therapies. But before ending the interview, Chan shared a message of hope — and urgency — for the field.

»There's so much enthusiasm about gene therapy. If we really want to advance it and deploy it for more and more diseases, immunogenicity is a key issue that we as a field need to address to create these next-generation therapies. I think about that broad overview a lot,« he says.

Link to the original article in Science Translational Medicine: Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses

Dan Robitzski is a science journalist and former neuroscientist based in Los Angeles.


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News: A New 'Cloaking' System Makes It Possible To Administer AAV Gene Therapies Without The Dangerous Immunological Response
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