Scientists rewrite hookworm DNA to produce therapeutic antibodies

Rewriting a hookworm’s DNA had for a long time been beyond scientists’ capabilities. Its resilient outer layer, intricate life cycle and insufficiently understood genome placed it outside the scope of gene-editing techniques that function consistently in bacteria, mice and human cell lines.

A group headed by Makedonka Mitreva, Gordon R. Miller Professor in the Division of Infectious Diseases within the John T. Milliken Department of Medicine at Washington University School of Medicine (WashU Medicine), has now surmounted those barriers.

After introducing new genetic instructions into the hookworm genome, the researchers transformed the parasite into a producer of a therapeutic antibody that releases it into its host’s bloodstream.

Rethinking parasites

Hookworms infect several hundred million people across tropical areas, generally producing mild digestive symptoms in otherwise healthy adults. In contrast with most parasites, they are unable to reproduce within the human body.

A managed infection therefore remains managed, and, should treatment become necessary, one oral dose of antiparasitic medicine eliminates the worms in roughly 24 hours.

For many decades, hookworms were primarily investigated as a cause of disease. Researchers later found that infected people experienced less inflammation linked to certain intestinal illnesses.

Secretions from the worms seemed to dampen the immune response that drives disorders such as ulcerative colitis. This finding changed the focus: rather than merely treating hookworm infection, scientists started to explore whether the parasites could have a therapeutic role.

The new study takes this concept a step further. Instead of depending on substances naturally made by a hookworm, the team modified it to manufacture and secrete a selected protein.

Choosing the target

The researchers selected Ancylostoma ceylanicum, a species of hookworm that infects people.

As a proof of concept, they engineered it to make a small antibody capable of neutralising tetrodotoxin, a paralysing neurotoxin found in pufferfish for which no antidote currently exists.

The work was funded by the Defense Advanced Research Projects Agency (DARPA), which was interested in biological countermeasures for soldiers deployed in remote areas.

Rewriting hookworm DNA

Creating a genetically modified hookworm required the team to address challenges that had not previously been tackled. Gene-editing methods effective in other organisms had not been adapted to the biology of hookworms.

Over several months, the researchers identified a safe harbour in the hookworm genome – a location where new DNA could be inserted without interfering with nearby genes or harming the worm’s survival.

They employed CRISPR-Cas9, a targeted gene-editing technique, to cut hookworm DNA and add the gene encoding the anti-tetrodotoxin antibody.

They also included a routing signal to send the resulting protein outside the worm’s body, rather than into its own tissues. The genetic material was delivered into hookworm eggs by electroporation – an electrical pulse that temporarily creates pores in cell membranes.

Engineered hookworms complete the cycle

The altered eggs matured into larvae, which were introduced into hamsters to establish whether the worms could finish their life cycle while producing the protein. They could.

The added gene was transmitted to the following generation of worms, demonstrating stable inheritance.

Importantly, its insertion did not turn off neighbouring genes, and the worms’ biology was not disrupted.

Antibodies reach the blood

Blood taken from hamsters 22 days after they had been infected with the engineered worms contained the human antibody made by parasites residing in their intestines.

In laboratory tests, that blood neutralised approximately 16% of the tetrodotoxin.

Although this figure may appear limited, Mitreva stressed that it is an initial proof of concept.

The researchers are continuing work to boost protein production and secretion, while the present findings probably understate the potential of the platform.

As the worms live in the gut and release most of their products there, levels of therapeutic protein in the intestine could be considerably greater than those that entered the bloodstream in this study.

This could make the platform particularly appropriate for gut-targeted disorders, including inflammatory bowel disease and food allergies.

Potential medical uses

The potential reaches further than tetrodotoxin. In controlled numbers, hookworms can already live unobtrusively in a host for years without causing severe illness. They offer ongoing, consistent secretions and can deliver substances directly to the gut lining or the bloodstream.

For long-term conditions that otherwise require repeated injections or infusions, this combination could provide something unavailable from conventional drug platforms.

Crohn’s disease and ulcerative colitis are obvious potential targets. Other candidates include conditions in which low, continuous doses of a therapeutic protein might prevent symptoms or help manage them.

In remote places or settings with limited resources, people could take a single oral dose of modified larvae and have a living pharmacy operating within them for years.

Safety and next steps

Before any transition towards use in humans, safety and containment remain essential issues.

One option being considered is modifying the worms so that they cannot lay eggs, stopping their spread or reproduction in the environment. Injectable medicines do not offer such an inherent off switch.

“What we demonstrated is that the concept works end to end,” said Mitreva. “You can insert a gene, the worm produces the protein, the protein gets out of the worm, and it is functionally active in the host.”

The platform can now be refined for particular diseases and protein concentrations according to the intended use.

A biological system that evolved to colonise humans is being redirected for human benefit. Achieving this depended on the first successful rewriting of a hookworm’s genome.