Feng Zhang

Nature’s Needle: Feng Zhang’s Team Re-engineers Bacterial “Syringes” for Programmable Protein Delivery

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Contractile injection systems from symbiotic bacteria that deliver protein payloads into insect hosts have been adapted by the Zhang lab at the Broad Institute to target human cells for biomedical applications

Joseph “Joe” Kreitz wants to find ways to make sure that molecular tools go to the right tissues and cell types. And now, the MIT graduate student and his colleagues in Feng Zhang’s lab at the Broad Institute have unveiled a new tool that could be a game changer for the therapeutic delivery of biomolecules: a bacterial “syringe.”

Across the biosphere, there is incredible diversity in extracellular contractile injection systems (eCIS) used to mediate symbiosis between bacteria and their eukaryotic hosts. This seems to be a general strategy among symbionts—bacteria that live within larger organisms.

Kreitz and colleagues focused on an eCIS called a Photorhabdus virulence cassette (PVC), which is produced by a bacteria of insects to target their host, deliver a toxin to kill that insect, and use the carcass of that insect to drive its own reproduction. “We thought it might be possible to actually modify this PVC to target human cells and then deliver a therapeutic payload,” Kreitz told GEN. “It’s a very beautiful strategy for delivering payloads into cells.”

In a new report published in Nature, Kreitz and colleagues from Zhang’s lab reveal how they took a naturally occurring eCIS and turned it into a highly efficient and specific biomolecular delivery tool. “We really need new approaches in the delivery space,” Zhang told GEN. “So much emphasis has been put on viral vectors or LNPs, and new approaches are really important.” Just last month, Zhang launched a new company called Aera Therapeutics that focuses on moving the cargo of genetic medicines—RNAi, antisense RNA, mRNA, or a genetic editing payload—based on protein nanoparticles (PNPs).

“Mechanistically, they are different, as they use proteinaceous effectors to inject through the membrane, as opposed to lipids (e.g., for lipid nanoparticles, or LNPs) that merge with and deliver into the cell,” said Rodolphe Barrangou, distinguished professor and CRISPR lab lead at North Carolina State University, and chief editor of The CRISPR Journal. “The sheath is very much reminiscent of phage tails and is more akin to a prokaryotic viral vector, though rather than package nucleic acid, it delivers proteins.”

AlphaFold adaptations

In the new report, Zhang’s team started by cloning the PVC from nature and expressing it in Escherichia coli, then taking this purified protein and showing that it is active in insect cells. Kreitz and colleagues then demonstrated that PVCs could be modified to target human cells.

“These PVCs naturally target insect cells, so they don’t target human cells at all,” Zhang said. “This is beneficial for us because that’s how Joe is able to make it very specific—you can remove the part that binds to an insect cell and replace it with something that binds to a specific thing on a human cell surface and get it to go into a human cell.”

While it’s not really known what the PVC targets on the insect cells, Zhang’s team used the AI-based protein structure prediction system AlphaFold to identify the region of the tail fiber mediating that interaction that binds to something on insect cells.

“[AlphaFold] gave us the information we needed to make a new delivery strategy that can be changed to target different cells,” said Kreitz. “All it takes is the addition of a very simple modification to its tail fiber protein that extends from the base to the part near the spike that actually gets driven through the membrane. We can add a novel binding domain to this tail fiber that would trick the syringe into binding a human cell instead of an insect.”

Kreitz and his colleagues were able to modify the binding domain so that it targets a specific human receptor, showing different strategies to reprogram these tail fibers. As examples, they retargeted these tail fibers against the human epidermal growth factor receptor (EGFR) and then showed that the whole system produced activity in EGFR-positive cell lines. They could also attach binding domains from human viruses to repurpose these PVCs to bind to human cells.

This system is really effective at killing cancer cells in a very specific way. The PVCs can target cancer epitopes and have the system inject toxins to have the cell die in a very programmable manner that doesn’t appear to show a lot of off-target effects. “It doesn’t seem to kill cells that don’t contain the receptor that you’re targeting,” said Kreitz. “So, you could imagine an application where you’re giving patients a PVC that targets a cancer epitope and then having the system go and deactivate cells that express that cancer epitope.”

Primed for protein delivery

Additionally, the Broad team showed that this re-engineered eCIS could deliver payloads beyond toxins. “One of the things we discovered pretty quickly is that, in terms of protein delivery, the system is actually quite powerful,” said Kreitz. “It can load and deliver a really versatile set of protein payloads. The system naturally delivers toxins because, of course, it’s trying to kill the insect. Those are around 300 amino acids [in size].”

In terms of protein delivery, the system is highly versatile in the types of payloads that it can load and deliver. Zhang’s team reprogrammed the system to deliver a diverse array of proteins—from some very small proteins up to Cas9, which is many times larger than the typical PVC toxin

As to how this works, Kreitz thinks that there may be some protein unfolding at play. “This system likely unfolds these proteins to some extent because it needs to fit the protein into this tube,” said Kreitz. “It is quite remarkable that the system can load such large payloads and then actually deliver them and retain the function of those payloads in target cells.”

A major sticking point, however, is that the eCIS, at least in its current form, is strictly a protein delivery system. That is useful in some circumstances, but if you wanted to deliver a different biomolecule, it would probably require additional engineering.

While Zhang’s group tested to see if they could rewire the PVC to load nucleic acids, the system initially did not cooperate. “Right now, it’s a protein delivery system only, so it’ll be great to get it to deliver RNA or DNA,” Zhang told GEN. “There are a lot of different phage or secretion system mechanisms that deliver things like RNA or DNA, so we’ll be trying to engineer that.”

But Barrangou told GEN that the protein delivery aspect is the major appeal. “Being able to deliver Cas9 and expand this to other genome effectors is exciting and useful at a time when delivery is the challenge for genome editing’s next step into the clinic,” said Barrangou. “Being able to program these molecular machines for cell- and tissue-specific delivery is very intriguing and potentially very useful, so editing strategies can be targeted to specific cell types and tissues.”

The University of Pennsylvania assistant professor Michael J. Mitchell, PhD, who played an integral part in developing the LNP platform during a postdoc in Bob Langer’s lab, agrees that this is an exciting advancement for the field of protein delivery. “LNPs are great for administering RNAs, but protein delivery using LNPs for applications such as gene editing remains challenging,” Mitchell told GEN. “It is difficult to encapsulate proteins into LNPs and deliver them efficiently into cells; this PVC platform can potentially overcome those challenges.”

Barrangou says he’d like to see future research looking into expanding the protein payloads (to other genome editing effectors beyond Cas9) and determining payload capacity (how large can payloads be and how can this be multiplexed to deliver more than one protein, either within the same payload or by delivering cocktails).

Delivery beyond the dish

The rest of the Nature report is devoted to characterizing the efficiency and specificity of this delivery system at targeting cells without creating off-target effects. Using intracranial injections, the team introduced the re-engineered eCIS into the brain of a live mouse; it is not yet known where the PVC goes, whether it has good tissue penetration, or if it just gets cleared.

“Because this is quite a large complex, it’d be interesting to study exactly whether it’s able to move through tissues and reach cells that are farther away from where you’re actually injecting,” said Kreitz. “In our paper, we’re injecting it right into a very defined, small region of the brain that we’re interested in. But for some diseases, you might want it to go in a more systemic way throughout the body. The PVC’s behavior in vivo is a place that will require more work in the future.”

Both Barrangou and Mitchell would like to see the system tested more broadly. Mitchell is keen to see is if the PVCs can be administered systemically (IV) and deliver proteins to therapeutically relevant target cells and tissues. “They show an initial in vivo proof of concept as a direct injection into the brain, but demonstrating IV delivery could open up many therapeutic opportunities in organs such as the liver and lungs,” said Mitchell. Barrangou also wants to see the system tested in many cell types and organisms, including plants, given the challenges of deploying genome editing in plants.

According to Zhang, there’s a lot of diversity in eCIS systems. “You can also find them in the human gut, so it would be very interesting to see what they may do inherently to target human cells,” said Zhang. “There may also be invading properties that they may have because they’ve been living in the human gut. Those are just some examples, but there’s a lot that we can do.”

Zhang thinks that there are probably many things in nature that cells have coopted to be able to transfer molecules back and forth.

“There are also many other versions of this in other bacteria, so you can either engineer this one or explore all the others, which will probably have different properties, to achieve a set of capabilities, and I think we’ll have to do both,” said Zhang.

“We’re just looking at the tip of the iceberg,” he continued. “It’s pretty trivial to re-engineer and is similar to producing any recombinant protein, but in terms of developing this into a biomedical tool, there’s still a lot of work to be done across the board, from discovery to manufacturing. You can grow a lot of bacteria using fermentation processes, and we’ll have to make sure that they’re pure because we need to get really endotoxins and other bacterial contaminants, which could be immunogenic,” said Zhang. “So, that could be part of the challenge, but that’s more about the manufacturing. It’s still early days for this as a technology.”

CRISPR pioneer muses about long journey from China to pinnacle of American science

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Scientist Feng Zhang has done revolutionary research on the gene-editing technique CRISPR from his lab at the Broad Institute in Massachusetts.
 Feng Zhang occupies a corner office on the 10th floor of the gleaming, modern biotechnology palace called the Broad Institute. He is one of the most acclaimed young scientists in the United States, regularly mentioned, even at 35, as a possible Nobel laureate.
That’s because of CRISPR, the gene-editing technique that lets scientists manipulate the genetic code of organisms almost like revising a sentence with a word processor. Zhang was one of its pioneers, and on Wednesday he emerged victorious after a bitter patent dispute.The ruling, by judges with the U.S. Patent Office, declared that Zhang’s work on living plant and animal cells was sufficiently original to deserve its own protection. It was a decisive outcome that will surely prove lucrative for Zhang and the Broad Institute, but he did not do anything special to celebrate. He made no immediate public comment. He did not even read the news coverage, he said.

“The patent stuff is not so interesting, and it can be distracting,” the soft-spoken scientist offered a day later, finally addressing the case as he sat down with a Washington Post reporter for a previously scheduled interview. “Now we can get back to work.”

The patent dispute was closely followed in the triangle of geography marked by the institute, Harvard University and the Massachusetts Institute of Technology. Here, in what has become the Silicon Valley of the life sciences, Zhang and his colleagues have spun off ventures that can commercialize their inventions.

CRISPR is an all-purpose tool that promises great advances in the prevention of diseases caused by genetic mutations. In China, Zhang’s birth country, it is already being used in human clinical trials.

Yet the technique has also raised unsettling possibilities for cosmetic human enhancements and “designer babies.” Earlier this week, the National Academy of Sciences and National Academy of Medicine produced a long report on the ethics of gene editing, arguing for extreme caution when dealing with heritable human traits but leaving open the possibility of use to remove disease-causing genes.

Some critics worry about a slippery slope, but Zhang thinks the bioethics committee got it just right.

“I think these are important issues, but I don’t think right at this second we should be overly concerned about it. It’s too far off,” he said.

The politics of science

Even with the patent case behind him, however, there is another significant distraction these days. It arises not through the courts but from the White House.

Science is inherently an international enterprise, built around a universal language of discovery and methodology. Zhang’s lab, like similar facilities across the country, has a large percentage of foreign-born scientists drawn to research opportunities in the United States.

President Trump’s executive order banning entry from seven Muslim-majority countries has alarmed this global community. The Broad, as it is commonly called, put out a statement of opposition, saying the order “turns its back on one of America’s greatest sources of strength: the flow of visitors, immigrants and refugees who have enriched our nation with their ideas, dreams, drive, energy, and entrepreneurship.”

 Zhang talks of his own life story when asked about Trump’s action.

“From my own experience, America has been an amazing place,” he said. “And it sort of gives opportunities for immigrants to realize what they want to do, to reach for their potential, and also, by doing that, make the world a better place. I’m very fortunate to have had the opportunity to move here.”

He was 11 when he first came to the United States in 1993. He spoke almost no English, arriving with his father to at last rejoin his mother. The teeming city of Shijiazhuang, in the north of China, was replaced by the alien landscape of Des Moines.

His mother had not intended to stay following her studies here, but Iowans embraced her. She got a good job with a company called the Paper Corp. She decided to start a new life and bring her son and husband to the United States. They each received a series of visas and green cards. She eventually became a citizen, as did her son. Her husband remains a Chinese citizen.

“I never felt I was discriminated against. I never felt we weren’t welcome there,” Zhang said of his youth in the heartland. And there were other immigrants, too, many of them Vietnamese refugees from war zones. He spent half the day learning English and then playing word bingo to hone his vocabulary.

He hung out with other kids interested in science. “We were all nerds,” he said. As a teenager, he got a position working after school at the Human Gene Therapy Research Institute. He could call himself a bench scientist, often working late into the evening while his mother waited for him in the parking lot.

Elite institutions soon recognized his brilliance. His résumé includes a degree from Harvard, then a doctorate from Stanford. He learned about the natural bacterial immune system, CRISPR, an acronym for clustered regularly interspaced short palindromic repeats.

Bacteria evolved a defense mechanism against viral invaders that would insert genetic material into bacterial DNA. The system functions like molecular scissors, snipping away the invasive material.

Two other researchers, who would become rivals in the patent case, published the first paper describing the gene-editing technique and applied for patents. Jennifer Doudna and Emmanuelle Charpentier showed how to turn the natural bacterial system into a laboratory tool, but initially they did not apply it to plant and animal cells. That was Zhang’s breakthrough, published in 2013 at the same time as a similar paper by Harvard geneticist George Church.

“Feng was very early in recognizing the importance of reducing it to practice in mammalian cells,” Church said this week.

Doudna and Charpentier can still receive patents on their original discovery. In an email Friday to The Post, Doudna wrote, “Obviously the Broad Institute is happy that their patent didn’t get thrown out, but we are pleased that our patent can now proceed to be issued.”

But she raised another concern. The judges’ decision was based in part on public comments she made, expressing uncertainty about whether CRISPR would work in cells with nuclei. Because of that, she fears the ruling could have a chilling effect on scientific communication.

“Must every scientist now factor in a potential patenting strategy and alter how transparent they are about their work?” Doudna wrote.

Doudna and Charpentier have already received the $3 million Breakthrough Prize funded by Silicon Valley tech tycoons. Then earlier this year they won the Japan Prize, each receiving the equivalent of about $420,000.

And lurking out there somewhere is the Nobel.

‘Why do we age?’

On Thursday, the morning after the ruling, Zhang drove his 2004 BMW to work as always, arriving at 7:30 to meet with a student and help him prepare for a class presentation. Then he had a call with an oil executive in the United Arab Emirates who is funding research on a genetic disease that affects the executive’s daughter.

He still has a spot in his lab for experiments, though he does those during the summer since right now he’s busy teaching two classes. The lab work is in the hands of about 20 researchers, some already with doctorates and medical degrees.

CRISPR gets all the publicity these days, but it is not the only game in town. Life is a complex chemical system that over billions of years has developed all sorts of tricks and mechanisms. Most of the microbes in the human gut have never been cultured or characterized. Basic questions remain unanswered.

“Why do we age?” Zhang asked.

The CRISPR system is itself a work in progress. It’s an inexact editor still.

“It cuts very well,” he said. “To insert something, it doesn’t work very well at all.”

But he’s working on that. Everyone stand by.

Source:www.washingtonpost.com