Gene Correction With Cas9 Nickase Holds Promise for Rare Skin Disorder Junctional Epidermolysis Bullosa

Type XVII collagen is crucial for maintaining stability between different skin layers, particularly between the dermis and epidermis. Mutations in COL17A1, the gene encoding collagen XVII, have been implicated in a debilitating hereditary form of epidermolysis bullosa (EB, see Fact Box).

Although gene correction using CRISPR-Cas9 has emerged as a promising strategy to treat genetic diseases, there are no methods to treat EB by restoring COL17A1 function.

In a recent study published in Molecular Therapy, a team of researchers led by Ulrich Koller at the Department of Dermatology and Allergology, Paracelsus Medical University Salzburg (Austria), developed a patient-specific proximal paired nicking method to reframe a homozygous 2-bp deletion in exon 52 of COL17A1, a mutation commonly found in patients with junctional epidermolysis bullosa (JEB).

Paired nicking-based COL17A1 editing led to COL17A1 reframing in primary JEB keratinocytes and three-dimensional (3D) skin models, restoring collagen XVII localisation and function.

»Our findings show for the first time that COL17A1 gene reframing via paired Cas9 nickases is a very efficient and safe option for the treatment of JEB and potentially other genetic defects caused by pathogenic frameshift mutations,« said Dr. Johannes Bischof, the first author of the study. »Our findings also demonstrate the superiority of Cas9-nickase-based targeting over wild-type Cas9-based strategies for gene reframing, which can be adapted for other diseases beyond EB,« he added.

Paired nicking induces COL17A1 reframing in human keratinocytes

Electroporation of primary JEB keratinocytes with gRNAs and Cas9 nickase led to a high frequency (> 90%) of indels. In contrast, no indels were observed in wild-type keratinocytes. NGS analysis of genomic DNA showed that proximal paired nicking restored the COL17A1 reading frame in 28.3% of JEB keratinocytes.

To gain more insight into the types of indels induced by Cas9 nickase, the team performed NGS analysis of the transcriptomes of treated JEB keratinocytes. COL17A1 transcripts with 25- and 37-nt deletions were the most frequent, accounting for > 42% of edited transcripts.

The frequency of off-target indels with proximal paired nicking was low, ranging from 0% to 0.10%. In contrast, the frequency of off-target indels introduced by the wild-type Cas9 was up to 32.0%.

Paired nicking-mediated COL17A1 reframing restores collagen XVII expression and function in human keratinocytes

Flow cytometry analysis showed that paired nicking-based COL17A1 reframing restored the expression of full-length collagen XVII in 45.9% of the treated JEB keratinocytes.

Commenting on whether this percentage of JEB cells with functional collagen XVII would suffice for a clinically significant improvement in the skin condition, Bischof said: »There is evidence that physiological re-expression of functional COL17A1 may enhance the regeneration potential and longevity of the corrected cells by giving them an advantage over uncorrected cells. This would mean that we might not need to target every cell in a patient if, in the long run, the corrected cells outcompete the mutation-bearing cells.«

Immunofluorescence analysis confirmed that the expression of full-length collagen XVII was restored in JEB keratinocytes treated with gRNA/Cas9 nickase and showed that collagen XVII localised predominantly to the cell surface, where it was also localised in wild-type keratinocytes (Figure 2).

Inefficient keratinocyte adhesion to the basement membrane is central to EB pathogenesis. Cell adhesion analysis revealed that although ~75% of JEB primary keratinocytes adhered to laminin-332-coated dishes, 85–90% of corrected JEB keratinocytes and wild-type keratinocytes could adhere to the plates (p<0.05).

Consistent with their findings in primary keratinocytes, immunofluorescence staining revealed deposition of collagen XVII within the basal membrane zone between the epidermis and dermis in 3D skin equivalents generated using human JEB keratinocytes with paired nicking-based COL17A1 reframing.

»We demonstrated the correct membrane localisation of the restored protein in cells and correct deposition at the dermo-epidermal junction in 3D engineered skins, indicating correct protein function. Confirmation of these findings in a preclinical in vivo setting is still pending,« Bischof noted.

Challenges and future steps

Although this was the first study to show the feasibility of using proximal paired nicking to treat COL17A1-associated JEB by restoring collagen type XVII expression, Bischof said that they must confirm the superiority of this approach over recent gene replacement therapies.

Reflecting on the challenges they might face in advancing their method, Bischof said: »A key challenge is the lack of suitable models that would allow us to adequately assess long-term functional outcomes, such as longevity of the repair, long-term skin stability, level of clonality of the regenerated skin over time, wound healing, and proper repopulation with immune cells.«

The team plans to develop an appropriate in vivo model that would allow them to investigate the skin stability, long-term effects, and additional safety aspects of their proximal paired nicking approach. »Aside from this, we focus on how we can make the repair as perfect or ‘traceless’ as possible and improve the feasibility of an in vivo, rather than an ex vivo, therapy,« Bischof added.

»What’s clear is that there is still much to learn about type XVII collagen biology and how it influences keratinocyte stem cell dynamics and skin regeneration. Therefore, we favour a strategy that keeps regulation of gene expression under the control of its normal promoter rather than using gene replacement approaches,« he concluded.

Link to the original article in Molecular Therapy:

Paired nicking-mediated COL17A1 reframing for junctional epidermolysis bullosa.

Christos Evangelou, Ph.D., is a freelance medical writer and science communications consultant.