Emerging Gene Therapeutics for Epidermolysis Bullosa under Development

Abstract

The monogenetic disease epidermolysis bullosa (EB) is characterised by the formation of extended blisters and lesions on the patient's skin upon minimal mechanical stress. Causal for this severe condition are genetic mutations in genes, leading to the functional impairment, reduction, or absence of the encoded protein within the skin's basement membrane zone connecting the epidermis to the underlying dermis. The major burden of affected families justifies the development of long-lasting and curative therapies operating at the genomic level. The landscape of causal therapies for EB is steadily expanding due to recent breakthroughs in the gene therapy field, providing promising outcomes for patients suffering from this severe disease. Currently, two gene therapeutic approaches show promise for EB. The clinically more advanced gene replacement strategy was successfully applied in severe EB forms, leading to a ground-breaking in vivo gene therapy product named beremagene geperpavec (B-VEC) recently approved from the US Food and Drug Administration (FDA). In addition, the continuous innovations in both designer nucleases and gene editing technologies enable the efficient and potentially safe repair of mutations in EB in a potentially permanent manner, inspiring researchers in the field to define and reach new milestones in the therapy of EB.

Keywords: base editing; epidermolysis bullosa; gene editing; gene replacement; gene therapy; prime editing.

Figure 1

Epidermolysis bullosa subtypes. The three major epidermolysis bullosa (EB) subtypes are defined by the level of tissue separation (***) within the skin. EB simplex (EBS) is characterised by intra-epidermal blistering. In junctional EB (JEB), skin cleavage occurs within the lamina lucida of the basement membrane zone (BMZ). Dystrophic EB (DEB) is caused by the loss of anchoring fibrils leading to skin cleavage directly underneath the lamina densa of the BMZ. The extremely rare Kindler EB (KEB), in which intra-epidermal, junctional, or dermal blister formation can occur. Created with BioRender.com (accessed on 20 December 2023).

Figure 2

Gene Therapies for EB. Gene replacement therapies rely on viral-vector-based gene delivery, in contrast to gene editing therapies, where a nuclease is delivered to specifically target a mutated gene locus. CRISPR/Cas9 represents the most advanced gene editing tool today. Gene therapies can be applied ex vivo or in vivo. In ex vivo gene therapies the patient’s skin cells are isolated from skin biopsies and subsequently treated in vitro in order to induce re-expression of the corrected protein. The gene-corrected cells are then used for the generation of autologous skin grafts, which are subsequently transplanted back onto the patient. In contrast, in vivo gene therapy is based on the direct local treatment of the patient’s skin. Created with BioRender.com (accessed on 20 December 2023).

Figure 3

CRISPR/Cas9-based gene editing tools. Classical CRISPR/Cas9-based editing is based on DSBs (DNA cleavage site(s) indicated via red scissors) created by spCas9. The cell’s own repair machinery creates indels, allowing for gene knock-out or -reframing. By providing a repair template, the outcome can by directed towards the HDR pathway, ultimately leading to traceless correction. Repair templates can also be combined with Cas9n, avoiding possibly adverse off-target effects arising from the creation of DSBs. Double nicking is another possibility for using Cas9n. In this case, in order to create a (staggered) DSB, it is necessary to use two sgRNAs binding and cleaving in close proximity on opposite strands. Similar to the default CRISPR/Cas9-approach, DN can be used for gene knockout and gene reframing as well as HDR. Base editing is possible via the fusion of Cas9n (initially dCas9) to adenosine/cytosine deaminases. While adenosine deaminases allow for the conversion of adenosine to guanosine, cytosine deaminases are able to convert cytidine to thymidine. The nick created by the Cas9n (D10A) mutant recruits the cell’s repair machinery, ultimately increasing the chances of installing the desired edit on the second DNA strand. In contrast to base editing, prime editing utilizes the Cas9n (H840A) mutant fused to a reverse transcriptase. PE uses a pegRNA, combining a default sgRNA with 3’ extensions consisting of PBS and RTT. While the PBS binds to the DNA strand initially untargeted by the spacer/sgRNA 3’ of the nick, the RTT provides a template for the RT and includes the desired edit. This edit can vary in length and composition, making PE a hugely versatile gene editing tool. If the newly RT-synthesised strand is incorporated, the edit is successfully installed on one DNA strand. In order to install the edit on the second strand, a so-called nicking sgRNA can be used. Similar to base editing, the nicking of the unedited strand recruits the cellular repair machinery. This machinery then uses the edited strand as a template for the repair. Prime editing is also the basis for PASTE. However, PASTE uses the PE reaction only to install an Bxb1 integrase attB site at a desired locus. The Cas9n (H840A) mutant is fused not only to a RT but also to a Bxb1 integrase. Once the attB site is in place, a gene or sequence of interest with an attP site can be incorporated at this site. This theoretically allows for the integration of sequences with up to several kilobases. UGI = uracil glycosylase inhibitor. Created with BioRender.com (accessed on 20 December 2023).