The Potential of Gene Therapy for Recessive Dystrophic Epidermolysis Bullosa

K.S. Subramaniam; M.N. Antoniou; J.A. McGrath; S.M. Lwin


The British Journal of Dermatology. 2022;186(4):609-619. 

In This Article

RNA-based Therapies

Exon-skipping Antisense Oligonucleotides

Targeting the RNA instead of the cDNA, antisense oligonucleotides (AONs) are used to remove the exon containing the pathogenic mutation within COL7A1. This strategy has a large therapeutic potential, as nearly 84 of 118 COL7A1 exons are in frame (thus the number of deleted or inserted base pairs is a multiple of three, resulting in a change in only a few amino acids), such that their removal would result in a truncated but still functional C7.[69] AONs are small molecules comprising single-stranded nucleic acids that can bind to their target RNA sequence. In RDEB, AON-mediated exon-skipping is used to bypass COL7A1 mutations in 'hotspot' exons where mutations are more prevalent. ProQR Therapeutics (Leiden, the Netherlands) has launched a phase I/II multicentre trial in patients with RDEB to evaluate proof-of-mechanism, efficacy, systemic exposure and safety following topical application of an AON specifically designed to induce skipping of exon 73 during pre-mRNA splicing, as many mutations (~11%) are clustered in this exon (NCT03605069).

Spliceosome-mediated RNA Trans-splicing (SMaRT)

Another RNA-based technology for RDEB gene therapy is spliceosome-mediated RNA trans-splicing (SMaRT), which combines the original mutated COL7A1 pre-mRNA and a synthetic 3' trans-splicing molecule, resulting in a full-length COL7A1 mRNA, thereby improving C7 production.[70–75] This approach has the potential to circumvent a large number of COL7A1 mutations, which is important given that most mutations are specific to individuals or families.[76] Despite the promising preclinical data in SMaRT technology demonstrating ~40–50% efficiency of transduction and restoration of C7 in xenograft mouse models,[70,71] clinical translation has not been achieved thus far, possibly due to safety and logistical challenges. Specifically, the risk of nonspecific trans-splicing resulting in off-target events, and hence the possibility of mutagenesis, is still largely unknown.

Premature Termination Codon Readthrough Drugs

Although not strictly gene therapy, pharmaceutical intervention to overcome the effects of PTC mutations in COL7A1 are also under investigation. PTCs usually arise from single-nucleotide mutations that produce a stop codon, resulting in loss of full-length C7 protein production. Considering that approximately 20% of the >1000 known COL7A1 mutations related to DEB are nonsense mutations, repurposed PTC-readthrough drugs such as gentamicin hold promise as a widely applicable and affordable treatment for individuals harbouring this type of genetic defect.[17]

Aminoglycosides, such as gentamicin, promote re-establishment of full-length C7 production in RDEB-associated nonsense mutations.[77] Gentamicin administered topically or intradermally, in a pilot clinical trial with patients harbouring nonsense mutations (NCT02698735), led to improved wound closure and decreased new blister formation, underscored by functional restoration of C7 and AFs.[17] An early-phase trial of intravenous gentamicin is ongoing (NCT03392909). Potential adverse effects include toxicity and contact sensitization.

In vitro, Amlexanox, a new anti-inflammatory and antiallergic drug that inhibits the release of histamine and leukotrienes, is demonstrated to induce PTC-readthrough to skip nonsense mutations in COL7A1 mRNA during translation, with subsequent C7 production in patient cells.[78] Due to its safety profile, approved for the treatment of mouth ulcers and currently in clinical trials for type 2 diabetes mellitus, Amlexanox is another attractive PTC-readthrough option for patients with RDEB harbouring nonsense mutations.[78]

Genome Editing Therapies

Genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) have enabled the correction of COL7A1 mutations at the DNA level.[79–81,83–88] These nucleases induce targeted single- or double-strand breaks in the DNA, followed by repair with correction of deleterious mutations through activation of the endogenous DNA repair machinery. The mutation in DNA can then be corrected via one of two processes: nonhomologous end joining (NHEJ), or homology-directed repair (HDR). HDR offers more precision than NHEJ in gene correction, as it utilizes a donor template to restore the wildtype sequence. By contrast, NHEJ comprises rejoining of the double-strand breaks, thereby introducing small insertions or deletions.

The ability to generate iPSCs, which have unlimited potential for self-renewal, from somatic cells such as fibroblasts, using a combination of transcription factors, has propelled the progress of genome editing techniques.[49] Gene-corrected iPSCs can be further differentiated into clinically relevant cell types, such as keratinocytes and/or fibroblasts, to generate epithelial sheets and skin equivalents for grafting. Sebastiano and colleagues established iPSC lines which on correction were differentiated into corrected keratinocytes and used to create autologous epithelial grafts with normal levels of C7.[80]

TALEN-mediated genome editing has also been used in keratinocytes[81] and epidermal stem cells[82] from patients carrying COL7A1 mutations. Skin grafts on immunodeficient mice using cells that had undergone genome editing mutation correction showed phenotypically normal human skin and C7 at the BMZ as well as formation of AFs.[82] More recently, CRISPR/Cas9-edited RDEB fibroblasts, keratinocytes and iPSCs[83–88] were successfully grafted in an RDEB mouse model with functional correction of COL7A1 mutations.[84] In 2020, CRISPR/Cas9-mediated excision of mutation-carrying COL7A1 exon 80 (prevalent in Spanish and Latin American populations) was granted orphan drug designation by the European Medicines Agency as a potential new therapy for RDEB.[89,90]

An adaptation of CRISPR/Cas9 genome editing is to deliver DNA base editors to a desired target site.[91,92] Adenine base editors (ABEs) were first introduced in 2017 by Gaudelli et al. to convert an A–T base pair to G–C to correct mutations in human cells.[93] In 2019, this method was applied to correct RDEB mutations using patient fibroblasts and subsequently to generate iPSCs.[94] Skin equivalents derived from these cells showed deposition of C7 at the BMZ in a xenograft mouse model, demonstrating that base editing has efficient and precise genome editing potential for RDEB.[94] The observation that gene editing can induce both off-target and on-target unintended mutations makes for limited clinical translation. However, the latest development of prime and base editing (which mediate targeted modifications to DNA without the need for double-strand breaks or donor DNA templates)[91] reduces such risks.