Forget UPS and FedEx: Tiny golden delivery trucks created at Fred Hutchinson Cancer Research Center can ship CRISPR into human blood stem cells, offering a potential way to treat diseases like HIV and sickle cell anemia.
And the researchers behind those trucks have even bigger distribution dreams.
Gene therapy — the editing of our DNA to treat disease — is a clinical reality today, but only in a handful of rich countries. Fred Hutch scientists think their new CRISPR courier could help deliver gene therapy to patients around the world.
A new paper published in Nature Materials describes how the scientists loaded CRISPR onto spherical gold nanoparticles. These tiny shuttles then deposited the gene-editing tool into blood stem cells donated by healthy individuals and isolated in test tubes, where CRISPR altered genes related to HIV and certain blood disorders.
It is the first time that nanoparticles have successfully ferried CRISPR into blood stem cells to edit DNA, the researchers said. And it’s a promising step toward addressing CRISPR’s critical delivery problems.
The first of these problems has vexed the field since the gene-editing technique was discovered. Scientists need to deliver CRISPR into the right spot in a cell. That is proving tricky enough. DNA represents the body’s crown jewels, and CRISPR must sneak past all sorts of security systems to gain access.
And then CRISPR must go global. Gene editing could benefit millions of people worldwide. But as the treatment process stands right now, the vast majority won’t. That process depends almost entirely on highly engineered viruses made in high-tech, multimillion-dollar facilities.
The researchers think their golden nanoparticles can solve both problems. As efficient couriers, they could reduce the need for engineered viruses and specialized research centers. And that could help make these emerging, high-tech treatments accessible and affordable, said senior scientist Dr. Jennifer Adair of Fred Hutch.
“Gene therapy has a lot of potential across many diseases, but the process we have right now is just not feasible in every place in the world,” Adair said. “We want to end up delivering gene therapy in a syringe. This gold nanoparticle represents the first possibility we have to do that for blood stem cells.”
The Fred Hutch team’s CRISPR system consists of several components. First is a genetic GPS called guide RNA. It’s a molecule that beelines for a specific section of DNA. Next up is a DNA-cutting protein called CRISPR-Cpf1. It acts like a pair of molecular scissors, snipping away at the precise spot where the guide RNA takes it. Finally, there’s a DNA template that makes the necessary edits in the DNA code at the snipped site.
When it comes to delivering CRISPR into cells, scientists’ mileage has varied, said Dr. Reza Shahbazi, a postdoctoral researcher at Fred Hutch and the study lead. “It’s been a hard problem for the field.”
Indeed. Scientists typically use two main strategies to get CRISPR inside a cell. They can use an electric shock to briefly open a door in a cell’s membrane. But cells unsurprisingly don’t like to get zapped, and the process can harm them.
Researchers can also make couriers out of viruses, longtime pros at cellular breaking and entering. But that process is expensive and slow. The rough cost of engineering enough viral couriers to treat a dozen patients? Half a million dollars and 12 months of hard work.
Viruses are also unruly. Scientists can load them up and send them on their way, but they have little control over where that payload gets delivered. That’s risky. Several studies have shown that viral vectors can linger in a cell and turn on cancer-causing genes or silence cancer-suppressing ones.
So Shahbazi turned to gold. Specifically, gold ions suspended in water. (Other research groups have used gold nanoparticles to deliver CRISPR, Adair and Shahbazi note, but none have targeted blood stem cells.)
“Gold has lots of special characteristics that we can take advantage of,” Shahbazi said. Scientists can easily play around with the size of the particles, for example. That’s important because different sizes are used to target different parts of the body, he explained.
Gold’s surface chemistry provides another bonus. It enables several complex chemical tricks that make the nanoparticle stable, Shahbazi said. He had to tinker with positive and negative charges of the different components, making small molecular modifications to prevent the different parts from repelling one another.
The overall charge of the gold nanoparticle helps the delivery truck drive up to the cell membrane. There, the nanoparticle gets folded into the cell and wrapped up in a pocket called an endosome. That’s part of a natural process a cell uses when it wants to look at what’s inside foreign molecules.
Gold’s chemistry now comes into play once more, at the very end of the delivery process. The cell essentially floods the endosome pocket with acid to break down what’s inside. But the charge on Shahbazi’s delivery vehicle interacts with that acid in a way that it bursts free from the pocket. It’s now parked inside the nucleus, the home for DNA, ready to unload CRISPR.
After CRISPR makes its cut, a cell has a few standard plays in its playbook to repair the damaged DNA. The first: Panic. Cellular maintenance crews are called in and frantically try to fix the mess. The results are random insertions or deletions of DNA “letters,” also called indels.
Indels essentially break a gene, and that can help treat certain diseases. But many therapies aim to precisely rewrite the DNA code, which requires template DNA to travel along with the CRISPR system. The challenge: getting the cell to correct that new DNA exactly.
Adair and Shahbazi’s research demonstrated that the molecular scissors they used — Cpf1 — cut DNA in a specific way that leads to more precise editing than the more familiar Cas9 enzyme. This kind of cut tells the cell to stitch the new DNA code into the space where the cut was made.
“We get very few indels with our system,” Adair said. “That’s a big deal because now we can be very precise in how we edit the genome.”
Moving forward, Adair and Shahbazi will work to boost the editing performance of their technique. Their CRISPR system edited between 10% and 20% of the cells they targeted. While that’s not yet comparable to delivery via electric shock, all of the cells survived and performed better than untreated blood stem cells, which is a significant improvement, Adair said.
Better understanding of the biology of blood stem cells will help them fine-tune their approach. Blood stem cells hold the promise for a one-and-done gene therapy treatment, Adair explained. These special cells produce every white and red blood cell in the body — billions every day. In theory, edited genes within blood stem cells would be passed along to their progeny for the lifetime of a patient.
In this study, the Fred Hutch team targeted two genes in the DNA of these stem cells. Both targets are tied to diseases that afflict millions worldwide — most of whom live nowhere near the fancy research facilities that currently crank out viral couriers for CRISPR treatments.
The first experiments aimed to disrupt the CCR5 gene, which has been proposed as a strategy to render cells resistant to HIV infection. The second aimed to create a naturally occurring mutation in the on/off switch for gamma hemoglobin. While this switch is typically turned off, turning it on can protect against inherited blood disorders like sickle cell anemia.
The edited human cells were transplanted into mice and were still around four months after they were infused. Surprisingly, the gold-treated cells performed better than untreated cells. Those early results suggest that the researchers’ all-in-one gene-editing package has the potential to treat patients in the clinic — and without the need for those expensive, heavily engineered viruses.
Adair has been working to simplify the CRISPR process for years. In 2016, she published research on “gene-therapy-in-a-box.” That mobile system provided a proof of concept that gene therapy could be done anywhere, even the poorest of countries. Her work into synthetic nanoparticles is another step toward realizing that dream.
This research was supported by a grant from The Hartwell Foundation as well as with funding from Fred Hutch donors through the Evergreen Fund. A Cooperative Centers of Excellence in Hematology grant from the National Institute of Diabetes and Digestive and Kidney Diseases supported the collection of human blood stem cells used in this study. A Cancer Consortium Support Grant from the National Cancer Institute supports shared resources used in the conduct of these studies.
Jake Siegel is a former staff writer at Fred Hutchinson Cancer Center. Previously, he covered health topics at UW Medicine and technology at Microsoft. He has an M.A. from the Missouri School of Journalism.