Engineered particles efficiently deliver gene-editing proteins into cells in mice

Credit: Ricardo Job Reese, Extensive Communications

Gene-editing approaches promise to treat a range of diseases, but delivering editing factors to cells in animal models and humans safely and efficiently has proven to be challenging. Now, researchers led by a team at the Broad Institute at MIT and Harvard University have developed a way to get gene-editing proteins into cells in animal models efficiently enough to show therapeutic benefit.

In a new work published in cellIn the study, the team shows how they engineered virus-like molecules to deliver key editors — proteins that make single-letter programmable changes in DNA — and CRISPR-Cas9 nuclease, a protein that cuts DNA at target sites in the genome. In collaboration with research teams led by Krzysztof Palczewski at the University of California, Irvine, and Kieran Mosonoro at the Perelman School of Medicine at the University of Pennsylvania, the team used their molecules, called virus-like particles (eVLPs), to disrupt a gene in mice that can be associated with high levels of cholesterol, restoring visual function. Partially to mice harboring a mutation that causes genetic blindness.

Scientists have long studied virus-like particles as potential drug delivery vehicles. Virus-like particles (VLPs) are small structures of viral proteins that carry a molecular charge, but do not contain viral genetic material and do not cause infection. Because VLPs lack viral genetic material, they may be safer than other delivery methods that use actual viruses, which can introduce their genetic material into a cell’s genome and may cause cancer.

Broad’s team identified several features of VLPs that limit their delivery efficiency, and engineered changes to the particle structure to overcome these bottlenecks. They say the resulting eVLPs are the first virus-like particles to deliver therapeutic levels of gene-editing proteins to a variety of tissues in adult animals.

In their study, the team detected no off-target modification when they used eVLPs to deliver the gene-editing machinery as a protein, but they did so when the editors were delivered as DNA. Their observations confirm previous research showing the benefits of using protein forms for gene editors and showing that eVLPs can deliver them safely. The scientists add that eVLPs could potentially be used not only for gene editing, but also to deliver other therapeutic proteins.

David Liu, senior author of the study and Richard Merkin Professor and Director of the Merkin Institute for Transformative Technologies in Healthcare at Broad. Liu is also a researcher at the Howard Hughes Medical Institute and a professor at Harvard University.

“VLP has always been one of the most attractive delivery technologies but it has suffered from inefficient protein delivery in vivo,” said Liu. “By rationally engineering molecular solutions to address specific challenges in the VLP delivery process, we developed eVLPs that significantly increased the potency of delivery in cultured cells, and also enabled efficient delivery in animals.”

Delivery Obstacles

For decades, virus-like particles have been of interest to researchers because they behave like viruses in terms of their ability to enter cells and transport cargo like therapeutic proteins. Researchers can influence the final destination of VLPs in the body – the liver or nerve cells, for example – by using different molecules on the particle’s surface.

To take advantage of these features and improve delivery, Liu’s team systematically designed different parts of the VLP architecture to optimize several critical steps – how VLPs are produced, how goods are packaged into VLPs, and how goods are released and distributed within cells.

The latest version of eVLP contains 16 times more cargo proteins than previous designs, and allows for an eight to 26-fold increase in editing efficiency in cells and animals. As the team hypothesized, they saw little evidence of modification at undesirable sites, and they saw no incorporation of viral DNA in cells treated with eVLPs.

“As eVLPs offer robust on-target editing and reduced off-target editing, we hope that they will serve as a safer way to deliver gene-editing factors in vivo,” said Aditya Raguram, co-first author of the study and a Ph. Dr.. A student in Liu’s lab.

eVLPs in action

The team used the improved eVLP system to correct mutations in a group of mice and human cells, noting an editing efficiency of 95 percent in some cases.

The scientists then used eVLPs to deliver key editors to the liver in mice, where they efficiently edited a gene that, when mutated, can significantly lower levels of “bad” cholesterol in the blood, reducing the risk of heart disease in some patients. The researchers showed that a single injection of eVLPs programmed to stabilize such a mutation resulted in an average of 63% modification of Pcsk9 and a 78% reduction in levels of Pcsk9 proteins. The team says they expect these findings will significantly reduce an individual’s risk of coronary heart disease.

The researchers also used a single injection of eVLP to restore visual function in mice carrying the blinding mutation. They corrected a mutation in the Rpe65 gene with editing efficiency similar to other basic editing delivery techniques but with less off-target editing and the risk of viral DNA integration.

The team also injected eVLPs directly into the brain of mice and observed a release efficiency of about 50 percent in cells exposed to eVLPs. Future efforts will focus on improving the distribution of eVLP throughout the brain, but the results show promise for delivering gene-editing factors to an organ known to be difficult to target.

“eVLPs combine the advantages of viral and nonviral delivery systems,” said Samagya Banskota, co-first author of the study and a postdoctoral fellow in the Liu lab. “They are also programmable and relatively easy to produce, making them promising tools for protein delivery. We look forward to the scientific community adopting and using eVLPs to improve delivery of therapeutic molecules to patients.”

Liu’s group is now working to expand the range of organs and cell types that eVLP can target in animals. They will also continue to characterize eVLPs to better predict and mitigate any unwanted immune responses that the particles may produce.

“Now that we know some of the major bottlenecks to eVLP and how we can address them, even if we have to develop a new eVLP for an unusual type of protein charge, we can probably do it more efficiently,” Liu said, noting that eVLP’s efforts have begun to early 2018.

“There is an urgent need for a better way to deliver proteins to different tissues in animals and patients,” Liu said. “We hope that eVLPs will be useful in delivering not only essential editors, but also other relevant therapeutic proteins.”

Mastering a mule viral package

more information:
Samagya Banskota et al, Virus-like molecules engineered for efficient delivery of therapeutic proteins in vivo, cell (2022). DOI: 10.1016 / j.cell.2021.12.021

Journal information:

Submitted by the Broad Institute of MIT and Harvard University

the quote: Engineered Particles Efficiently Deliver Gene-Editing Proteins to Cells in Mice (2022, Jan 12) Retrieved Jan 12, 2022 from

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