CRISPR/Cas9 Gene Therapy Holds Promise to Treat Epidermolysis Bullosa

Results of a study of 3 patients with the monogenic fragile skin disorder recessive dystrophic epidermolysis bullosa (RDEB) show that the removal of exon 31 containing a pathogenic mutation restored the adhesion properties of RDEB fibroblasts and led to accurate type VII collagen (C7) deposition to the basement membrane in a 3D skin model, according to research published in Bioengineering & Translational Medicine.¹

The excision of exon 31 from RDEB keratinocytes and fibroblasts represents the first demonstration of exon skipping in dermal fibroblasts, the authors stated. The other novel COL7A1 exons targeted were 68 and 109.

Hillary Sheppard, PhD | Image Credit: University of Auckland

“The COL7A1 gene, which produces collagen 7 protein, is frequently mutated in individuals with the rare but debilitating fragile skin condition, epidermolysis bullosa. This condition could be treated with a gene-editing approach whereby skin cells are taken from an affected individual, the single broken gene is repaired using CRISPR-based gene editing, and skin equivalents are engineered using gene-edited, patient-specific cells,” study senior author Hilary Sheppard, PhD, The University of Aukland, told The American Journal of Managed Care^® (AJMC^®). “These edited skin sheets can then be used as long-lived skin grafts to cover problematic wounds.”

All 3 donors had compound heterozygous mutations in the COL7A1 gene. Researchers designed dual single-guide RNAtargeted to the introns on either side of each mutation. Nonhomologous end joining repair achieved fusion of the breakpoints and generated deletions that spanned each exon.

In keratinocytes, the total proportion of alleles with a deletion spanning the target exon (but not additional exons) was 89.67% for patient 1, 94.98% for patient 2, 89.03% for patient 3 keratinocytes, and 90.76% for patient 3 fibroblasts.

Results showed that 107 of 118 exons can be removed without disrupting the COL7A1 open-reading frame. The finding suggests that exon skipping has broad applicability to patient-specific mutations, the authors stated.

“The simplicity of RNP [ribonucleoprotein]-based delivery and the lack of requirement for positive selection mechanisms represents a simplified editing workflow, which could enable a faster route into the clinic,” the authors wrote.

They also found that dual Cas9-RNP exon skipping did not lead to detectable offtarget insertion and deletion formation, but did generate large on-target structural variants; deletion of exon 31 in patient 3’s skin cells led to predicted mRNA splicing and increased C7 transcription; the deletion of exons 31 restored C7 protein expression in patient 3’s keratinocytes.

The study’s editing protocol and cell culture system did not negatively impact cell growth. Proliferation analysis showed that patient 3’s keratinocytes had similar growth rates following Cas9-RNP editing compared with untreated and wild type keratinocytes over a 1-month period, the authors wrote.

Future research should perform in vivo experiments to evaluate the long-term efficacy of gene-editing techniques, according to the study.

Patients with RDEB present a significant risk for squamous cell carcinoma, and their life expectancy is typically 30 years.² The condition is caused by biallelic, loss-of-function mutations in the COL7A1 gene encoding the C7 alpha-1 chain, and C7 impairments can cause severe blisters, which can lead to systemic and life-threatening complications, the authors wrote.

“Our research adds to a growing body of evidence demonstrating that this gene lends itself to a highly efficient approach whereby faulty regions can be simply removed, producing a slightly truncated, but still functional, protein,” Sheppard told AJMC. “This approach has therapeutic potential. However, our study also highlights the importance of comprehensively assessing on-target editing events, as our analysis revealed the presence of deleted regions of DNA that would not be captured using standard methods.”

References

1. Du Rand A, Hunt J, Samson C, et al. Highly efficient CRISPR/Cas9-mediated exon skipping for recessive dystrophic epidermolysis bullosa. Bioeng Transl Med. 2024;9(4):e10640. doi:10.1002/btm2.10640

2. Dystrophic EB (DEB). EB Research Partnership. Accessed July 31, 2024. https://www.ebresearch.org/dystrophic.html