Exon Skipping Therapies May Be Effective for Some Mutations

Using exon skipping therapies may be an easy, affordable, and effective approach for people with epidermolysis bullosa (EB) with amenable mutations, an early study suggests.

Such therapies would promote the production of a slightly shorter, but working version of the skin-related protein that is missing in people with EB, researchers said.

This conclusion was based on the fact that this type of therapy effectively skipped the mutated portion, or exon, of the disease-causing gene and allowed the production of a working protein in skin cells collected from a child with junctional EB or JEB.

The study, “Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa,” was published in the International Journal of Molecular Sciences.

EB is primarily caused by mutations in genes that provide the instructions to produce key proteins for the development of the skin layers. A deficiency in either of these proteins makes the skin extremely fragile, easily injured, and prone to fluid-filled blisters.

Exons are the sections of genes that contain the genetic information needed to make proteins. Much like pieces of a puzzle, exons are linked together in the messenger RNA (mRNA), an intermediate molecule derived from DNA that is used to guide protein production.

Mutations in a given exon may break the ability of the remaining exons to link together properly in the mRNA, affecting the generation of a working protein.

Increasing research efforts have been focusing on the development of gene therapies for EB, as they deliver a healthy version of the mutated gene to cells, potentially restoring the production of the missing skin protein.

However, “first marketing approvals are still being awaited and are not expected for all EB types in the near future,” the researchers wrote.

Thus, exon skipping therapies have the potential to be an important alternative for this rare skin condition. They work by forcing the cell’s machinery to skip a particular exon, at or next to the mutation site, so the remaining exons do fit together in the gene’s mRNA. That allows the production of a smaller, but functional version of the relevant protein.

This is achieved with antisense oligonucleotides or ASOs, lab-made molecules designed to be complementary to a specific region in the premature mRNA, altering the mature mRNA and the resulting protein.

Notably, exon-skipping approaches are being developed for JEB and dystrophic EB given that several exons of COL17A1 and COL7A1 — two genes often mutated in these subtypes — are dispensable and not required for normal or sufficient protein function.

Now, researchers in Austria and Belgium have provided evidence of the feasibility of this therapeutic approach. They used lab-grown skin cells collected from an 8-year-old child with JEB due to a mutation in exon 7 of the COL17A1 gene.

COL17A1 provides the instructions to produce type XVII collagen (C17), a key protein for the proper bonding of two skin layers, the epidermis and the dermis. The patient’s mutation resulted in an mRNA change that promoted a premature stop in protein production, leading to a shorter, nonfunctional C17.

The team hypothesized that treating patient-derived skin cells with ASOs targeting the mutated exon 7 of COL17A1 would restore exon alignment in the gene’s mRNA and result in a working, although slightly shortened, C17 protein.

Three such ASOs were designed and then delivered to cells grown in the lab using liposomes, which are tiny fatty vesicles made of the same material as that found in cell membranes. This process allowed the ASOs to easily enter and deliver their cargo to the cells.

These vesicles also were chosen based on their “low toxicity, as well as properties well-suited for skin transfection,” the researchers wrote.

The results showed that all three ASOs resulted in the successful skip of exon 7 during COL17A1 mRNA maturation. That, in turn, increased the levels of the nearly full-length mRNA and promoted the production of a slightly shorter C17 protein.

This smaller protein was found to be appropriately integrated in the cell surface of skin cells. It also was found to be located in the appropriate skin layer in cells grown in three dimensions, more closely resembling the skin’s natural structure.

To the researchers’ surprise, mRNA lacking both exons 6 or 7 also was detected in untreated and treated patient-derived cells, as well as molecules missing either exon 6 or 7 in healthy skin cells.

Notably, further analysis showed that the protein resulting from the loss of both exons 6 and 7 also was functional.

“The observation that exons 6 and 7 are also naturally skipped in healthy [skin cells] supported our hypothesis that these exons may be dispensable,” the team wrote.

These findings suggest that “both the application of our [ASO-filled liposomes] onto wounds or onto intact skin might be feasible treatment strategies,” they added.

“The presented approach may thus pose a versatile, easy to design and relatively cheap way to provide this therapy to patients, even if they have an ultra-rare [genetic profile] that requires a personalized approach,” the researchers wrote.

As such, exon skipping therapies “could bridge the gap until a permanent cure can be provided for EB patients,” they concluded.