CRISPR/Cas9 RNP System Shown to Be Effective for Gene Editing in Epidermolysis Bullosa Mice

Researchers have successfully applied the CRISPR/Cas9 system in a mouse model of recessive dystrophic epidermolysis bullosa (RDEB) to restore collagen VII protein function, a new study shows. RDEB is caused by mutations in the Col7a1 gene, which leads to either a lack of collagen VII or dysfunctional collagen VII protein.

The study reporting this experiment, “Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model,” was published in the journal Proceedings of the National Academy of Sciences.

In healthy individuals, the epidermal keratinocytes (cells of the epidermis) and dermal fibroblasts (cells of the dermis) secrete collagen VII proteins, which play a role in anchoring these two layers together. The absence or dysfunction of this protein in RDEB patients causes abnormal epidermal-dermal adhesion.

Current treatments for RDEB include protein therapy or combined gene and cell therapy. However, despite advances in RDEB research, there is still no cure for the disease.

CRISPR/Cas9 genome editing has emerged as a powerful method for editing genes, and it has the potential to treat patients with genetic diseases. Unfortunately, the delivery method of CRISPR/Cas9 has remained challenging as most researchers use a viral-based delivery system which comes with a host of limitations.

Now, researchers at the National Institute of Biological Sciences in Beijing, China, set out to demonstrate the potential to use CRISPR/Cas9/sgRNA ribonucleoproteins (RNPs) in treating RDEB.

First, the team created an RDEB mouse model based on a specific point mutation present in an RDEB patient. They showed that this mouse model exhibited the typical pathologic features of patients with RDEB.

Then, researchers determined the feasibility and safety of using a method called exon skipping as a gene-correction method.

The gene Col7a1 has 119 exons (coding regions) that come together to ultimately be used to generate collagen VII proteins. The mutation present in this specific mouse model resides on exon 80 and causes the protein to become dysfunctional. If that specific exon is deleted using the CRISPR/Cas9 system (think of the system as “molecular scissors” that cut off segments of genetic material), then theoretically a slightly smaller but functional version of the collagen VII protein would be made.

Researchers found that mouse mutants with the exon 80 deletion were no different from normal mice, having an intact epidermis-dermis anchoring. Therefore, exon skipping, the scientists believe, would be a valid and appropriate method to restore the function of collagen VII protein.

The team set out to define an optimal method for delivering the CRISPR/Cas9/sgRNA into the skin of the animals. RNPs, which refer to a complex of both protein and RNA, is a new delivery method for CRISPR/Cas9/sgRNA into cells that have shown to be effective in previous studies. So, researchers targeted exon 80 using this CRISPR/Cas9/sgRNA RNP system in a population of skin stem cells.

They found that the system was successful in gene editing stem cells from the skin, and that it was able to correct skin defects. In fact, researchers demonstrated that the average adhesion area of mice with the Col7a1 mutation increased from ~30% to ~60% after just one treatment with the CRISPR/Cas9/sgRNA RNPs.

“Using this method, we show that Cas9/sgRNA ribonucleoproteins efficiently excise exon80, which covers the point mutation in our RDEB mouse model, and thus restores the correct localization of the collagen VII protein in vivo. The skin blistering phenotype is also significantly ameliorated after treatment,” the team wrote.

“Our study provides proof-of-principle evidence that Cas9/sgRNA ribonucleoprotein-based gene therapies can be applied to restore collagen VII protein function in postnatal RDEB mice, suggesting that the Cas9/sgRNA ribonucleoprotein-based gene editing system may offer curative treatment for RDEB and other genetic disorders,” the researchers concluded.