New methodology makes use of virus-like protein to bundle, ship RNA | Spectrum
Cre load: Similar to viral vectors (center), protein capsules deliver mRNA that codes for the Cre protein to cells, which causes them to glow green (left), unless the mRNA lacks flanking sequences that the protein recognizes (right) .
A new gene delivery system attacks a protein found in humans in order to encapsulate messenger RNA (mRNA), the mediator between genes and proteins, and to transport it into cells. The technology can help researchers deliver gene therapies or the CRISPR gene editor to tissues throughout the body.
“We’ve been working on gene therapy and gene editing for a long time, and the tools are getting pretty good,” said Feng Zhang, professor of neuroscience at the Massachusetts Institute of Technology who led the work. Researchers have only been able to get CRISPR into cells in some parts of the body, but this limits the diseases it can fight.
Empty virus envelopes often supply genes that are used in research and therapy. But these viral vectors can trigger immune responses, and they only carry genes up to a certain size, making it difficult to transport sequences that encode large enzymes – like Cas9, the main DNA-cutting protein that CRISPR uses.
Another delivery approach involves encasing genes in lipid nanoparticles, but targeting specific tissues in this way remains a challenge. Nanoparticles often accumulate in the liver.
The new method called Selective Endogenous eNcapsidation for cellular Delivery (SEND) uses a human protein that normally carries mRNA in a protective capsule, similar to a virus. Since the protein occurs naturally in humans, it can trigger a lower immune response than viral vectors. It can also carry larger payloads and, unlike nanoparticles, can be tailored to specific cells. The researchers described the method in Science in August.
Some proteins derived from retrotransposons – stretches of DNA that copy and paste throughout the genome – form virus-like protein envelopes, previous studies show. A signaling protein called ARC involved in autism, for example, encloses its own mRNA and transports it between cells.
To find other virus-like proteins, Zhang and colleagues searched the genome of mice and humans for capsule-forming sequences similar to those found in some viruses and retrotransposons. The team then expressed the most promising candidate genes – with emphasis on those found in both mice and humans – in bacteria and cultured human cells.
Seven of the proteins the team analyzed formed virus-like structures, the researchers reported. The bacteria and cultured cells secreted a candidate called PEG10 in higher concentrations than the others. Further analysis showed that PEG10 binds to its own mRNA and often attaches to regions at either end of the sequence that are not translated into protein.
The team used these binding sequences to reprogram PEG10 to carry a different mRNA load. They included a sequence encoding a protein called Cre between the two untranslated regions in PEG10 and introduced the flanked Cre sequence into a batch of human cells, along with mRNA encoding PEG10 and a fusogen, a protein on the Surface of a virus-like capsule that enables the capsule to enter a cell. The researchers then harvested the virus-like proteins that the cells secreted two days later.
When the team added the secreted capsules to a second batch of cells – this time mouse stem cells engineered to express a green fluorescent protein in the presence of Cre – the cells glowed green, suggesting that the system was successfully using Cre mRNA had given up.
Some fusogens target specific cell types and could be used to deliver genetic cargo to specific cells or tissues, the researchers say. For example, in one experiment, they delivered Cre mRNA to mouse skin cells using virus-like particles equipped with a fusogen that targets these cells.
In another test, the team used the technique to introduce CRISPR into cultured cells from humans. They enveloped mRNA that codes for Cas9 and a sequence that directs the protein to cut a specific DNA target. DNA sequencing revealed that the system modified the target gene in about 40 percent of the cells.
SEND could help scientists develop therapies for autism and provide a way to safely and effectively conduct gene therapy or gene editing treatments, the researchers say. It could also be used as a research tool to study the effects of certain genetic changes.
“We really believe we can turn this into a very powerful toolbox,” says Zhang. The researchers plan to develop a repertoire of fusogens that target different cell types and test the system’s ability to transport a range of genetic loads.
Quote this article: https://doi.org/10.53053/BZTX8458