Cell: Engineered particles can efficiently deliver gene-editing proteins into mouse cells

Gene editing technology holds promise for treating a range of diseases, but delivering editors safely and effectively into animal models and human cells has proven challenging
.

Now, researchers led by a team at the Broad Institute of MIT and Harvard have developed a way to efficiently implant gene-editing proteins into animal models cells to show the therapeutic effect

In a new study published in the journal Cell, the team shows how they engineered virus-like particles to deliver base editors (proteins that make programmable single-word changes in DNA) and CRISPR-Cas9 nucleases ( A protein that cleaves DNA at targeted sites in the genome)
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In a collaboration with research teams led by Krzysztof Palczewski of the University of California, Irvine and Kiran Musunuru of the Perelman School of Medicine at the University of Pennsylvania, the team used their particles, called engineered virus-like particles (eVLPs), to make small In mice, genes associated with high cholesterol levels were disabled and partially restored visual function in mutant mice that caused genetic blindness

Scientists have long studied virus-like particles as potential drug delivery vehicles
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Virus-like particles (VLPs) are small structures of viral proteins that carry molecular carriers, but do not contain viral genetic material and do not cause infection

The Broad team identified several properties of VLPs that limit their transport efficiency and overcome these bottlenecks by engineering changes in the particle's structure
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The resulting eVLPs, they say, are the first virus-like particles to deliver therapeutic levels of gene-editing proteins to various tissues in adult animals

In their study, when they used eVLPs to deliver the gene-editing machinery in protein form, they did not detect any off-target editing, but when the editor was delivered as DNA, off-target editing was detected
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Their observations confirm previous research showing the benefits of using gene editors in protein form, and show that eVLPs can deliver them safely

"In vivo delivery has proven to be a recurring challenge, although it is a crucial aspect of any future gene editing that plays an important role," said David Liu, senior author of the study Richard Meggin The professor and director calls the Institute for Transformative Technologies Broad Health Care
.

Liu is also a Howard Hughes Medical Institute investigator and a professor at Harvard University

"VLPs have been one of the most attractive delivery technologies, but delivery of proteins in vivo is inefficient," Liu said
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"By rationally designing molecular solutions to address specific challenges in VLP delivery, we have developed eVLPs that greatly enhance delivery in cultured cells and also enable efficient delivery in animals

Viroid particles have been of interest to researchers for decades because they behave like viruses and can enter cells and deliver things like therapeutic proteins
.

Researchers can influence the final destination of VLPs in the body—for example, the liver or neurons—by using different molecules on the particle surface

To take advantage of these properties and improve delivery, Liu's team systematically designed different parts of the VLP architecture to optimize several key steps - how VLPs are produced, how goods are packaged into VLPs, and how goods are released and distributed within cells
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The final version of their eVLPs packaged loaded proteins 16 times larger than previous designs and enabled 8 to 26 times higher editing efficiency in cells and animals
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As the team hypothesized, they saw little evidence of editing in unwanted locations, and no viral DNA entry into cells treated with eVLPs
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"Because eVLPs provide powerful on-target editing and minimize off-target editing, we hope they will become a safer method for delivering gene editing agents in vivo," said Liu, co-first author of the study.
said Aditya Raguram, a doctoral student in the Ph.
D.
lab
.

eVLPs in action

The team used the optimized eVLP system to correct mutations in a range of mouse and human cells, and in some cases observed 95 percent editing efficiency
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The scientists then used eVLPs to deliver the base editors into the livers of mice, where they effectively edited Pcsk9, a gene that, when mutated, can significantly reduce blood levels of "bad" cholesterol and, in some patients, risk of heart disease
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The researchers found that a single injection of eVLPs programmed to install this mutation resulted in an average 63 percent editing rate of Pcsk9 and a 78 percent drop in Pcsk9 protein levels
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The team said they expect these results to significantly reduce an individual's risk of coronary heart disease
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The researchers also used a single eVLP injection to restore visual function in mice with the blinding mutation
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They corrected mutations in the Rpe65 gene with editing efficiency comparable to other base-editing delivery techniques, but with a lower risk of off-target editing and viral DNA integration
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The team also injected eVLPs directly into the brains of mice and observed about 50 percent editing efficiency in cells exposed to eVLPs
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Future efforts will focus on improving the distribution of eVLPs in the brain, but the results show promise for delivering gene-editing agents to a very difficult-to-target organ
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"eVLPs combine the advantages of viral and non-viral delivery systems," said study co-first author Samagya Banskota, a postdoctoral researcher in Liu's lab
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They can also be programmed and are relatively easy to produce, making them promising tools for protein delivery
.
We look forward to the scientific community adopting our eVLPs and using them to improve therapeutic macromolecular drug delivery in patients
.
"

Liu's team is now expanding the range and cell types of eVLPs in animals
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They will also continue to characterize eVLPs to better predict and mitigate any unwanted immune responses the particles may generate
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"Now that we know some of the key eVLP bottlenecks and how to solve them, even if we have to develop a new kind of unusual eVLP protein cargo, we can do so more efficiently," said Liu, who noted that their eVLP efforts in 2018 Begin at the beginning of the year
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"We badly need a better way to deliver proteins into various tissues in animals and patients, and we hope that these eVLPs can be used not only to deliver base editors, but also other therapeutically relevant proteins
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"