World's-first clinical trial for a CRISPR-Cas3 phage therapy. Interview: Joseph Nixon, Locus Biosciences.

In a world's first, Locus Biosciences has enrolled the first patients in a clinical trial using bacteriophage boosted with CRISPR to precision-kill bacteria. Inside the phages is a DNA-shredder - CRISPR-Cas3 - that enhance the bacteriophage killing efficiency by orders of magnitude. The completely new antibacterial principle comes as multi-drug resistance is on the rise.

By: Rasmus Kragh Jakobsen - Mar. 19, 2020
Joseph Nixon, Senior Vice President of Business Development at Locus Biosciences, explains how they...
Joseph Nixon, Senior Vice President of Business Development at Locus Biosciences, explains how they are using bacteriophages loaded with CRISPR-Cas3 'DNA-shredder' to cure disease. Photo: Courtesy of Locus Biosciences.

- The world's first clinical trial for a CRISPR enhanced bacteriophage therapy called crPhage is a new way of killing bacteria, a new paradigm for antibacterials, can You tell me how it works?

Yes, you're familiar with CRISPR-Cas9 used for genome editing. CRISPR-Cas3 is similar to that in the way it finds a particular spot in the genome. But Cas3 and Cas9 differ in what they do when they get there. Cas9 creates a double-stranded break, but Cas3 is an enzyme that degrades the DNA. Once it gets its shredding teeth into the bacterial genome Cas3 moves along one strand of the DNA and shreds it.

- So you have to direct Cas3 to the bacterial genome, and it begins destroying the DNA?

Right, and just like the other gene-editing technologies, you need a way to get the components into the cells. We quickly settled on bacteriophages - viruses that naturally infect and replicate in bacteria - as the preferred delivery vector. As it happens, nature invented the perfect technology for inserting DNA into bacterial cells - bacteriophages - and we took advantage of that and put the two mechanisms together. We combined the Cas3 DNA-shredding mechanism and the bacteriophage lytic capability into one dual mechanism of action that is very effective at killing bacterial cells.

- Great, and how do you make it specific to the 'bad' bacteria that you want to target and not the 'good' bacteria that you want to spare?

That's the key differentiator of our approach. With traditional antibiotics - even the relatively selective ones - you kill many different species of bacteria, and we are killing just one species. The selectivity comes from the two separate pieces. One is the bacteriophage that tends to be quite specific to the bacterial species, and the other is the targeting of the CRISPR-Cas3 construct. Currently, we target it to genes that are conserved in that bacterial species, but we also have the option to target genes that are specific to a strain or set of strains if desired.

How to make room for Cas3

- Excellent, and can You tell me about some of the challenges on the way?

The biggest scientific challenge was figuring out how to get the CRISPR-Cas3 system into the bacteriophage genome. So initially, we had a lot of success inserting just the RNA guide into the bacteriophage. That allowed the phage to activate endogenous Cas3 that was already present in the bacteria.

But a lot of bacterial species don't have that CRISPR system in them, and to target those species we need to bring the complete system into the cell.

- So the phage needs to carry the Cas3 as well?

Yes, Cas3, along with a protein complex called CASCADE that helps to target Cas3 to the appropriate spot in the bacterial genome. The challenge is that bacteriophages have minimal genomes and generally don’t carry a lot of 'extra' genes, so we needed to find those ’extra’ genes that could be deleted without disrupting the lifecycle of the phage. There wasn't a lot of literature on that, so we had to learn a lot more about the phage biology and phage genomics in order to deliver the entire CASCADE-Cas3 complex along with the guide RNA.

When we managed that during 2019 it was a significant milestone scientifically. Now we have a platform that, at the least, in theory, can target pretty much any bacterial species that we would want to remove from the body.

How to make the perfect bacterial killer.

Compared to natural bacteriophage Locus' CRISPR enhanced phage, crPhage, is typically 1-3 orders of magnitude more efficient in bacteria-killing.

This is how they are made.

Bacteriophages are the most common organisms on the planet, and the first step is to collect them from the environment. Locus Bioscience uses automated robotics and a high-throughput screening system acquired from EpiBiome in 2018 to find bacteriophages that infect and kill the target bacteria, for example, E. coli.

Then they sequence the genomes to determine which genes can be deleted to make room for the CRISPR-Cas3 construct. The genes are removed and if the phages are still able to replicate the researchers put in CRISPR-Cas3 system and a new enhanced bacterial killer is born.

Pharma back the new antibacterials

- Were there other challenges?

Yes. The biggest challenge for most technology companies is financing, and our collaboration with Johnson and Johnson was a watershed moment for us and the field as well.

A lot of pharmaceutical companies have moved away from developing antibiotics, and this agreement showed a large pharmaceutical company bought into our technology approach, as well as the commercial opportunities for CRISPR phage products downstream.

We signed an agreement that could be worth $818 million in milestones to develop two crPhage products targeting respiratory pathogens.

A phage cocktail for urinary tract infections

- This current trial is for urinary tract infection, what are You targetting?

For this trial, we are targetting E. coli, because the vast majority of urinary tract infections are caused by E. coli colonization into the bladder. Our product is a cocktail of three CRISPR engineered bacteriophages that collectively kill about 95% of all E. coli bacterial strains that are relevant for urinary tract infections.

- And how will patients take the phage-cocktail, will it be a pill or an injection?

For this initial trial, we are delivering the product directly into the bladder because we want to make sure we know the precise dose of crPhage being delivered relative to the level of bacterial load.

But obviously, we don't consider that a commercially viable route of administration. The next trial will have either intravenous delivery or intramuscular injection, and ultimately, our long term goal is to make it a pill that can be taken orally.

Trial to market in 2027-28

Urinary Tract Infections (UTIs) are infections of the urinary tract, including the urethra, bladder, ureters, or kidneys. Worldwide, 150 million people are affected by UTIs each year, with 70-90% of these caused by E. coli.

The current treatment using a cocktail of three CRISPR enhanced bacteriophage is in a 30 patient phase 1b placebo-controlled clinical trial. The first 10 patients are through the dosing portion of the trial. Next step will be a phase II trial enrolling 250 patients with recurrent urinary tract infections and finally a phase III trial to compare efficacy with best currently available therapy.

If all progresses according to plan, the treatment should get to market in 2027-2028.

CRISPR phage therapy for Alzheimer's

- To round off maybe, you can tell me a little bit about future perspectives?

We look at the world in two different ways.

The first is traditional bacterial infections like urinary tract infections, skin infections and respiratory infections, where there are a very clearly defined pathogenic bacterial species that need to be removed to fight that infection.

That has been our focus up until now.

But research around the world on the microbiome - the collection of bacteria, viruses and fungi in the body - show it is important for health.

- Ah, so you also aim to manipulate the microbiome as a treatment?

Our technology specifically targets bacteria, but there are a lot of bacterial species that are now being associated with diseases, such as Crohn's disease, colorectal cancer, and even things in the central nervous system like Alzheimer's disease and autism.

As scientists more clearly establish a causative relationship between those bacteria and the diseases, we feel we're very well-positioned to develop products that kill those bacteria and therefore treat those diseases.

- Sounds great, thank you very much.

Explainer of the CRISPR Cas3-enhanced bacteriophage (crPhage) technology. Courtesy of Locus Bioscience.

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