A synthetic protein carrier delivered a gene editor into the muscle of one pig
A synthetic protein carrier delivered a gene editor into the muscle of one pig
On September 2, Nature published a paper on STV-C8, a protein particle that transports RNA. In an experiment involving one healthy pig, STV-C8 delivered a CRISPR–Cas9 system into muscle to remove part of a gene. The team had previously tested the carrier in cells and mice.
For RNA to function in a target cell, its carrier must leave the producer cell, enter the target cell, and release the RNA inside it. STV-C8 was assembled from protein components with distinct roles. One protein region binds RNA, another binds the cell membrane, and a third helps the particle leave the cell inside a membrane vesicle. The scaffold is a protein assembly consisting of eight identically arranged subunits, designed using the RFdiffusion protein design model. The VSV-G protein helps the membrane vesicle fuse with the target cell.
The researchers selected the scaffold shape through screening. The team produced more than one hundred variants and separately measured how many particles left the producer cells, how many entered the target cells, and whether the delivered RNA functioned. Based on these three tests, the team selected STV-C8. In a laboratory human cell line, STV-C8 produced more of the test protein than the other protein carriers included in the comparison. Compared with the lipid nanoparticles used by the authors, which are shells made from lipid molecules that deliver RNA, the proportion of cells in which the delivered RNA functioned was more than a thousand times higher. Producing the same amount of test protein required an RNA dose that was more than ten thousand times smaller.
The team also tested STV-C8 with a gene editor. The CRISPR–Cas9 system uses a guide RNA to direct the Cas9 protein to a selected region of DNA. In pig connective tissue cells, the CRISPR–Cas9 system delivered by STV-C8 removed exon 51, a region of the dystrophin gene, in approximately 40% of cases. After mice received the test RNA intravenously, its signal was concentrated mainly in the lungs. After a local injection of STV-C8 into the muscle of one healthy pig, sequencing of the muscle tissue confirmed the removal of exon 51.
The choice of exon 51 was based on a pig model of muscular dystrophy. When exon 52 is absent, the cell begins reading the DNA sequence in groups of three from the wrong position and produces an incorrect dystrophin protein. Removing the adjacent exon 51 restores the correct grouping into triplets and allows the cell to produce a shortened dystrophin protein. A 2023 study tested this pair of changes in pigs, which is why Cas9 was directed specifically to exon 51.
Coauthor Christoph Gruber described the team’s aim as follows:
“We wanted to design new structures for a specific purpose, namely the efficient delivery of RNA, rather than recreate structures found in nature.”