The complexity of EB requires a variety of different therapeutic approaches
EB is a systemic disease, and for some severe subtypes systemic treatments or cures are essential to ensure survival and quality of life. Step-change improvements to tissue integrity for EB patients will be made by addressing the underlying defects in the structural proteins. To remedy these defects, diverse technologies currently being developed include topical and systemic treatments to provide a functional protein through correction of the primary genetic defect at the DNA or mRNA level, or by the direct provision of protein.
Therapeutic strategies explored in clinical studies range from different types of stem cells either donor-derived, or patient-derived and gene-corrected, to corrected fibroblast or keratinocyte treatments, injected intradermally or engineered into skin grafts. Other approaches include the application of a healthy protein, gene or gene transcript, antisense oligonucleotides, and premature termination (PTC) readthrough drugs using various routes.
Symptom-relieving or disease-modifying therapies using small molecular drugs address secondary disease sequelae such as itch, pain, inflammation, fibrosis, or oncogenesis to improve quality of life in affected individuals.
Gene therapies and combined gene/cell therapies
Gene therapy aims to treat EB by replacing, inactivating, or repairing the affected gene in the skin cells. Gene therapy strategies can thus be used to reduce levels of the disease-causing protein or increase production of the healthy protein. The genetic material is usually transferred through a carrier or viral vector into the skin cells. This can be done either by treating the cells directly in the body (in vivo), for example by injecting the genetic material, or outside of the body (ex vivo). In the latter case, the gene therapy protocol is considered a combined gene/cell therapy, since the cells are removed from the patient’s body, genetically modified in tissue culture, expanded and then returned to the patient.
Gene replacement therapy is currently the most promising gene therapy for individuals with EB. A functional gene or gene transcript (cDNA) is delivered into the skin cells in order to supplement the mutant gene. Ex vivo gene replacement strategies involve isolation of the patient’s skin stem cells, genetically correcting them by the introduction of a wild-type cDNA copy of the affected gene, expanding the corrected cells into epidermal sheets and grafting these back onto wounds. The headline-making skin graft treatment of 80% of a JEB child’s body surface in 2017 indicated the feasibility of ex vivo gene replacement therapy, building on earlier proof-of-concept transplantations in 2005 and 2016, also in two patients with laminin-deficient JEB. Nevertheless, extensive grafting is surgically traumatic for patients. While current ex vivo gene therapy technology is useful for treating non-healing wounds of limited size, it is still an invasive and expensive option.
The development of ex vivo grafting technologies continues to be refined, and multiple clinical trials are in progress.
Epidermal stem cells for gene therapies
To develop a durable ex vivo grafting therapy, a persistent epidermal stem cell population will need to be provided. The human epidermis consists of a stratified squamous epithelium that is replenished constantly, with a complete turnover every 3–4 weeks by resident stem cells in the epidermis. However, in EB, recurrent blistering depletes the stem cell pool, which can be a major obstacle for a successful outcome.
Induced pluripotent stem cells (iPSC) for gene therapies
To overcome the limitation of stem cell-based gene therapy, induced pluripotent stem cells (iPSC) can be developed from small biopsies of a patient’s skin. IPSCs have been produced from both fibroblasts and keratinocytes. Following correction of the EB mutation, the iPSCs can then be redifferentiated into either keratinocytes or fibroblasts. iPSC-derived therapies are currently being developed for all major forms of EB.
Other ex vivo approaches use intradermal injection of genetically engineered fibroblasts overexpressing the gene of interest. Although the use of fibroblasts has advantages over keratinocytes such as robustness and engraftment upon delivery into intact skin, the injection itself is a painful procedure. Alternative approaches to delivering gene corrected cells to treat skin and mucosal surfaces are being developed, notably through the use of ‘spray-on’ technologies. All such treatments need to address the problem of the severe microbial colonisation of infected chronic wounds, usually involving some form of debridement to allow cell therapies to take.
Current in vivo gene therapy approaches for EB use topical administration of non-integrating vectors expressing a wild-type copy of the affected EB gene. In these approaches, the introduced transgene is not integrated into the cell’s genome, increasing the safety but making repeated application necessary.
Gene editing uses programmable nucleases like CRISPR/Cas9, to permanently correct the genetic defect at the DNA level by inserting, deleting, modifying or replacing DNA in the genome at site specific locations. Gene correction can occur either via non-homologous end joining (NHEJ) or homology direct repair (HDR) and is applicable to both autosomal recessive and dominant diseases. The major concern with the use of gene editing technologies is their unpredictable off-target effect. These could lead to unintended mutations, which might have harmful side effects. In EB, these technologies are evaluated at the preclinical level, where great progress has been made for precise ex vivo gene correction in primary skin cells and in induced pluripotent stem cells. Major bottlenecks still exist for the development of efficient, safe and targetable in vivo delivery systems for gene editing molecules.
Cell- and extracellular vesicle-based therapies
Cell Therapy is the transfer of intact, living cells into a patient with the goal of improving the disease. The cells may originate from the patient (autologous cells) or from a HLA-matched donor (allogeneic cells). The cells used in cell therapy are classified by their potential to transform into different cell types. Pluripotent cells can transform into any cell type in the body and multipotent cells can transform into other cell types of a limited repertoire. In EB, hematopoietic stem cells (HSC), Mesenchymal stromal cells (MSC) derived from various sources such as bone marrow, adipose tissue, and umbilical cord blood, as well as dermal fibroblasts and keratinocytes are used for therapeutic approaches.
Extracellular vesicles (EV) including exosomes, have emerged as a novel cell-free strategy for the treatment of many diseases. EV are produced by cells and contain bioactive components such as nucleic acids, proteins, and lipids. They can be found in various biological fluids, including blood, breast milk, and urine. In general, EVs have several potential advantages over cells with regard to clinical application, including increased safety, since EV cannot divide or differentiate after administration, and improved reproducibility.
Autologous and allogeneic cell therapies
Mesenchymal stromal cells (MSC) intravenously or intradermally injected or topically applied onto lesions are expected to send signals that reduce inflammation and thus contribute to skin regeneration and accelerated wound healing. Most studies focus on treatment of chronic DEB wounds. Allogeneic fibroblasts and keratinocytes are mainly used in wound dressings, as a topical spray or intradermal injection in DEB patients, aiming to restore type VII collagen expression. Local injection of allogeneic fibroblasts into RDEB wounds did not provide evidence for improved wound healing.
Bone marrow transplantation
Early bone marrow transplant treatments with hematopoietic stem cells and mesenchymal stroma cells for both RDEB and JEB showed proof of principle of systemic stem-cell treatment. However, the rigour of pre-transplant chemotherapy to ablate the patient’s bone marrow was fatal for many compromised patients. Subsequent protocol refinements to reduce the severity of chemotherapy enhanced survival in RDEB patients greatly, and the chimerism of bone marrow following treatment did not impact on beneficial outcome. Surviving patients showed very variable results however: some sustained improved wound healing and reduced skin blistering over a period of years, while others showed minimal benefit – these benefits correlated with persistence or loss of transplants and consequent levels of wild-type collagen and correctly assembled anchoring fibrils in the skin.
Revertant mosaicism cell therapy
Revertant mosaicism often referred to as a natural gene therapy, is due to spontaneous correction of the disease-causing mutation in some skin cells manifesting in healthy skin patches. These can be leveraged as a source of naturally corrected autologous cells to treat skin lesions. The first approach in treating chronic ulcers by grafting revertant epidermis was performed in a JEB patient carrying a LAMB3 mutation. Grafted sites exhibited protein expression similar to healthy skin, however only small areas can be treated by this method. Attempts to expand the revertant cells in vitro to generate skin sheets for grafting onto lager wound areas in RDEB patients did not result in long-term closure of the wounds. Main reason might be the limited number of revertant stem cells in the skin graft. In order to ensure long-term clinical benefit, solid protocols must be developed to amplify revertant stem cells in cell culture.
Using patient cells from revertant skin patches for the generation of iPSC followed by differentiation into keratinocytes for cell therapy approaches constitutes a promising tool, as the risk of immune rejection is low.
EV (extracellular vesicles) play a key role in cell-cell communication by transferring their content to target cells and tissues, and can thus be exploited as delivery vehicles for a variety of therapeutic agents. Studies for DEB test MSC-derived extracellular vesicles for topical delivery for COL7A1 mRNA or type VII collagen protein delivery onto wounds.
In the context of cancer, tumor-derived EVs that circulate in the blood represent a source of biomarkers that can be used for early cancer detection and diagnosis, which is explored for EB-associated Squamous cell carcinoma.
Although chronic wounds are a fundamental issue for patients with EB, wound management is still challenging. Despite the large number of wound care products available, dressings suitable for EB wounds are very limited due to the fragile skin and co-morbidities of the patients. The selection of wound dressing is thus very individual and dictated by the type of EB, presence of infection, availability of products and personal preference. Most adhesive dressings may result in skin stripping. Thus, one focus in clinical research for EB is the development of nonadherent and biological wound dressings, containing cells, collagens or amonitic membranes. Several clinical studies assess the efficacy and clinical effects of such tissue engineered skin substitutes for different types of EB. Improved, more rapid, and less painful healing of wounds was reported for Helicoll Collagen I and Apligraf wound dressings compared to conventional dressings.
RNA-based strategies offer a series of therapeutic applications, including modulation of the mRNA transcript, targeting genetic defects through mRNA repair, and the silencing of mutation-bearing transcripts. Several technologies are exploited for modulation of mRNA in EB, all of them aiming to restore functional protein expression. Targeting the genetic defect at the RNA level induces only a transient effect, and thus requires repeated application of the treatment. This limitation might on the other hand enhance the safety of RNA-based approaches by reducing the risk of mutagenesis. Most of these strategies are sequence-dependent and thus represent a highly individualized treatment option.
Antisense oligonucleotide (AON)-mediated exon skipping
A promising approach of mRNA modulation is the antisense oligonucleotide (AON)-mediated skipping of exons harboring the disease-causing mutation.
AONs are short pieces of DNA or RNA, designed to bind the mutated exon and thus inhibit its inclusion into the mature mRNA during the splicing process. AON-mediated exon skipping has been successfully applied to remove various mutation-bearing COL7A1 exons in vitro and in vivo, leading to the expression of a truncated, yet functional type VII collagen protein. Topical application of an ASO targeting COL7A1 exon 73 is currently being evaluated in a DEB clinical trial.
Small interfering RNA (siRNA)
Small interfering RNA (siRNA) offers a potential therapy route for dominant EB forms by specifically inhibiting expression of the mutant mRNA without silencing the functional allele. This approach relies on the design of highly specific siRNA, which are short RNA molecules, that bind the mutant mRNA leading to its degradation after transcription. Preclinical studies have shown to increase the ratio of functional proteins in DDEB and EBS. Main remaining issue in developing an effective siRNA therapy approach is finding the best way to deliver the drug to where it is needed.
Spliceosome-mediated RNA trans-splicing (SMaRT)
Spliceosome mediated RNA trans-splicing (SMaRT) replaces a mutated sequence of agene transcript with a functional copy delivered by an engineered RNA repair molecule. In contrast to AON exon skipping and siRNA, SMaRT can replace larger regions of the gene transcript and is thus more broadly applicable to more EB patients with a defect in the respective gene. Efficient and specific correction of EB mutations have been demonstrated for the PLEC1, KRT14, COL7A1, and COL17A1 genes in preclinical studies in vitro and in vivo. Future investigations to enhance a clinical application must address the risk of off-target effects and the development of a feasible in situ delivery method.
Depending on the EB type and protein, as little as 20-30 % of wild-type levels of the missing protein can be effective in strengthening tissues. As early as 2006, intravenous recombinant human collagen VII was shown in an RDEB mouse model to enhance wound healing and strengthen skin. Systemic infusion of recombinant human collagen VII has previously been tested in a phase 1/2 and a phase 2 clinical trial for safety and tolerability and wound healing efficacy in RDEB patients. The large size of the protein and a tendency to form aggregates, however, impairs skin homing, thereby compromising the clinical feasibility of this therapeutic strategy. Intradermal injections of recombinant human collagen VII are also progressing to clinical trial. At present, for laminin-deficient JEB, attempts to develop protein therapy are hindered by the tendency of the protein to form misfolded aggregates during manufacture, and is not currently being pursued.
The most severe EB forms are caused by nonsense mutations which result in premature termination codons (PTC), which results in near complete loss of functional protein. A promising approach for these patients involves the use of drugs to force transcriptional read-through of PTCs and thus increase production of full-length protein. The aminoglycoside antibiotic gentamicin has been described as an active PTC read-through drug. Several clinical studies of topical and intravenous gentamicin application involving RDEB and JEB patients with PTC mutations in COL7A1, COL17A1 and LAMB3, resulted in increased full-length protein expression and clinical improvement. Studies to assess the efficacy and safety of systemic gentamicin administration, especially with regards to potential severe side effects such as renal and ototoxicity are ongoing. The anti-inflammatory drug Amlexanox, also shown to be effective at driving PTCs readthrough is entering clinical trials for RDEB.
Apart from the primary structural-functional defect causing skin blistering, molecular mechanisms cause secondary symptomes such as pain and itch. Further, it has become evident that chronic tissue damage leads to dysregulation of inflammatory pathways and fibrotic processes which predispose to aggressive and fatal squamous cell carcinoma (SCC) in certain subtypes. Deeper insight into pathogenic mechanisms have enabled the characterisation of potential therapeutic targets, that can be addressed by small molecule drugs. Such symptom-relieving and disease-modifying drugs use either innovative agents from the bench, specifically developped for EB, or compounds that were already approved for other diseases and have been repurposed for the treatment of EB. Drug repurposing for EB has been examined since many decades. It represents a much more cost-effective and rapid route to clinical application over a new compound, and allows safer clinical validation protocols, since the safety studies have already been conducted. Various small molecule drugs, focusing on immune-regulatory traits involved in pain, itch, chronic wounding as well as inflammaion, scarring and SCC, have reached the stage of clinical investigation in EB.