Michele De Luca | Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena, Italy

Over three decades of research on both adult stem cells and somatic derivatives of pluripotent stem cells are culminating in remarkable clinical results. The combined disciplines of cell therapy, gene therapy and tissue engineering, broadly known as regenerative medicine, have the potential to revolutionize the treatment of previously incurable injuries and rare, orphan diseases.¹

In 1975, the first human epidermal keratinocyte culture was established by Prof. Howard Green. Less than 10 years later, the lives of two children suffering from full-thickness burns on over 95% of their body surface were saved using autologous keratinocyte cultures.² Since then, such cultures have been used worldwide to treat thousands of patients with massive third-degree burns.³

This stunning achievement heralded the age of modern regenerative medicine and paved the way toward the development of ex vivo cell therapies of other epithelial injuries, as, for instance, severe ocular burns.⁴ Corneal transparency is critical for visual acuity and relies on stromal avascularity, proper organization of collagen fibers and epithelial integrity. Repair and renewal of the corneal epithelium are sustained by stem cells located in the basal layer of the limbus, the narrow zone between the cornea and the bulbar conjunctiva.⁵ Ocular burns affecting the central part of the cornea can be treated by a keratoplasty, but extensive chemical burns affecting also the limbus lead to limbal stem cell deficiency (LSCD), making keratoplasty ineffective.⁵ LSCD is characterized by neovascularization, chronic inflammation, stromal scarring, and invasion of the cornea by bulbar conjunctival cells, with consequent corneal opacification and loss of vision. In unilateral LSCD, an option to prevent the conjunctival over-growth is to restore the limbus by grafting limbal fragments taken from the uninjured eye. The finding that limbal cultures include corneal stem cells fostered the therapeutic application of autologous limbal cultures, which led to regeneration of a fully functional corneal epithelium and restoration of visual acuity not only in unilateral LSCD but also in severe bilateral corneal damage.^4,5 Indeed, 1-2 mm² of healthy limbus in one eye suffices to generate limbal cultures able to restore the corneal epithelium of both eyes^4,6 (Figure 1A).

The knowledge acquired during the implementation of such advanced therapies fostered the development of combined cell and gene therapy for genetic skin diseases, such as Epidermolysis Bullosa (EB).

Epidermolysis Bullosa

EB is a heterogeneous group of rare, dominantly or recessively inherited, genetic disorders characterized by recurrent blistering of the integument. Blisters arise, spontaneously or upon minimal mechanical stress or trauma, because of the extreme skin fragility caused by mutations in genes encoding various structural proteins of the epidermal-dermal junction.⁷ Common features of many EB forms include damage of ocular surface, upper airways, oral mucosa, and gastrointestinal and renal systems, as well as hair, nail and enamel defects. More than 1,000 mutations on at least 16 structural genes cause distinct clinical manifestations, ranging from mild to severe, with local or generalized involvement and significant morbidity and mortality. This variety depends on several molecular (targeted protein, type of mutation and degree of function loss, mode of inheritance and genetic background) and phenotypic (distribution and severity of the lesions, involvement of mucosae) factors. Severe EB forms can be early lethal and generalized EB frequently leads to aggressive squamous cell carcinoma (SCC).⁷

EB encompasses 4 major forms, primarily based on the level of skin cleavage, that is intraepidermal in EB simplex (EBS), within the lamina lucida in JEB, beneath the lamina densa in DEB and at multiple levels within and/or beneath the basement membrane in Kindler syndrome (KEB).⁷

EBS is the most common form of EB and can arise from mutations in 7 different genes. Over 75% of EBS is due to dominantly inherited genetic changes affecting KRT5 and KRT14, the genes encoding keratin 5 (K5) and keratin 14 (K14), which form the intermediate filament network of basal keratinocytes. Mutations in highly conserved amino acids within the helix initiation or termination motifs lead to severe EBS, which is characterized by blisters, often leading to chronic erosions, covering the entire skin surface and affecting several mucous membranes. Aminoacidic substitutions in other K5/K14 regions lead to localized EBS, marked by milder clinical manifestations usually restricted to the extremities. Very rare, severe (in some cases lethal) forms of EBS are caused by recessively inherited nonsense or missense KRT5 and KRT14 pathogenic variants. EBS can also be caused by mutations in plectin (encoded by PLEC) and dystonin (encoded by DST), which are hemidesmosome proteins that anchor keratin filaments to the plasma membrane.⁷

JEB is one of the most devastating forms of EB. It is due to recessively inherited mutations in genes encoding the heterotrimeric protein laminin 332 (LAMA3, LAMB3, LAMC2), collagen XVII (COL17A1), integrins α6β4 (ITGA6, ITGB4) and integrin α3 (ITGA3). The most severe forms of JEB are caused by mutations affecting laminin 332 and integrins α6β4, whilst mutations in COL17A1 and ITGA3 usually have a milder phenotype. Patients carrying biallelic premature termination codons leading to absence of laminin 332 or α6β4 (severe JEB) usually die within 2 years after birth. Approximately 40% of patients with intermediate JEB die before adolescence, whilst adults have a high risk of developing SCC. Missense or splicing mutations that allow residual expression of the protein, even if truncated and only partially functional, can significantly reduce the severity of the phenotype, suggesting that low expression of one component can still sustain its interactions with the binding partners.⁷

Dystrophic EB (DEB) can be dominantly or recessively inherited and is due to over 200 mutations in COL7A1, the gene encoding collagen VII (C7), the main component of anchoring fibrils. Dominant DEB has a mild phenotype with blisters primarily involving the extremities. In contrast, Recessive DEB (RDEB) can be ravaging, being characterized by massive blistering and scarring, disabling joint contractures and pseudosyndactyly, all of which highly reduce the patients’ quality of life. Severe RDEB usually results from biallelic COL7A1 premature termination codons, but variants include nonsense or splice site mutations, deletions or insertions, ‘silent’ glycine substitutions or non-glycine missense mutations within triple helix or non-collagenous NC-2 domains. The nature and the positions of these mutations correlate with the severity of the phenotype. Patients with generalized RDEB almost invariably develop aggressive, highly metastatic SCC.⁷

KEB is caused by mutations in FERMT1, the gene encoding fermitin family homolog 1 (kindlin-1), an intracellular protein of focal adhesions. Blisters can occur within basal keratinocytes, along the lamina lucida and below the lamina densa of the basement membrane. Features include skin fragility and mild photosensitivity, poikiloderma, palmoplantar hyperkeratosis and high risk of developing SCC in adulthood. As with other EB forms, several mucous membranes can be involved.⁷

Combined Cell and Gene Therapy for Junctional Epidermolysis Bullosa

LAMB3-dependent generalized intermediate JEB was the first genetic skin disease successfully tackled by ex vivo combined cell and gene therapy.⁸ Autologous epidermal cultures transduced with a gamma-retroviral vector (γRV) carrying a LAMB3 cDNA were grafted on patients’ selected skin areas, upon surgical removal of blistering epidermis and proper preparation of the wound bed.

Transgenic grafts restored large non-healing epidermal lesions in two adult JEB patients^8,9 (Figure 1B). More recently, transgenic epidermal cultures proved to be life-saving, as they restored virtually the entire epidermis of a seven-year-old boy suffering from a devastating form of JEB with very poor prognosis.¹⁰ Through the entire 6-year follow-up, his newly formed epidermis remained robust and resistant to mechanical stress, freed from blisters or erosions. It expressed normal levels of laminin-332, had normal thickness and continuity of the basement membrane and, notably, unveiled normal would healing upon injuries. The regenerated epidermis was entirely transgenic, as LAMB3 mRNA and laminin 332 were both uniformly and seamlessly detected in all the analyzed skin sections. No immune response or inflammation were observed.¹¹

In summary, all three patients presented a stable, fully functional, blister-free epidermis with normal expression of laminin 332 at the epidermal-dermal junction and a normal number of mature hemidesmosomes.^8-10 Despite the very high number (between 1x10⁷ to 4x10⁸) of transgenic clonogenic keratinocytes transplanted per patient, no adverse events have been observed (up to 16 years of follow-up).¹² In particular, neither cellular transformation nor aberrant clonal expansion have been so far detected in the regenerated transgenic skin.^11,13 An oncoming multicenter European Phase II/III clinical trial (referred to as Hologene 5) aims to confirm safety and efficacy of transgenic epidermal cultures on a larger number of LAMB3-JEB patients (NCT05111600).¹⁴

Combined Cell and Gene Therapy for Dystrophic Epidermolysis Bullosa

Autologous cultured keratinocytes transduced with a γRV carrying a COL7A1 cDNA (earlier referred to as LEAES and today as EB-101), have been used to restore the expression of C7 on 42 skin wounds on 7 RDEB patients (NCT01263379)¹⁵ (Figure 1B). At 2-year follow-up, more than 70% of the treated wounds healed and expressed C7 assembled in functional anchoring fibrils on at least 50% of their surface, which significantly improved the clinical picture. No adverse events related to the use of γRV-corrected cells were reported.¹⁶ An ongoing Phase III clinical trial aims to confirm safety and efficacy on a larger number of RDEB patients (NCT04227106).

In collaboration with Johann Bauer and colleagues, we obtained similar results in a similar Phase I/II trial (NCT02984085), using autologous epidermal cultures transduced with the same type of yRV used for gene therapy of JEB. However, while the LAMB3-transgenic epidermis exhibits a fully functional, seamless basement membrane and a normal number of mature hemidesmosomes, COL7A1-transduced keratinocytes were able to partially restore the expression of C7, hence they regenerated a sort of ‘mosaic’ patterned epidermis (our unpublished data). This difference could be, at least in part, ascribed to a lower transduction efficiency of γRV-COL7A1, as compared to γRV-LAMB3, and to competition between untransduced and transgenic RDEB keratinocytes, which is unlikely to occur in the JEB scenario. In fact, signals emanating from the interaction of laminin 332 with integrins α6β4 induce nuclear localization YAP, a transcriptional co-activator sustaining human epidermal stem cells.¹⁷ LAMB3-JEB triggers YAP inactivation and leads to epidermal stem cell depletion, supporting the notion that JEB is an adhesion and a stem cell disease. It follows that genetic correction of LAMB3-JEB rescues not only cell adhesion but also epidermal stemness, thus conferring to transgenic JEB keratinocytes a selective advantage over the untransduced counterpart, both in vitro and in vivo.¹⁷ Such a selective advantage does not hold true for RDEB clonogenic keratinocytes. This hurdle might be exceeded by a substantial improvement of the efficiency of RDEB keratinocyte transduction.

Tackling dominantly inherited EB

Gene addition strategies can successfully tackle a significant number of severe, recessively inherited genetic diseases, but are unsuitable to correct dominant mutations. The discovery of the CRISPR/Cas9 gene editing system now allows to precisely target genomic loci, hence discriminate wild-type and mutant alleles. The CRISPR/Cas9 technology is under investigation in keratinocytes, fibroblasts or induced pluripotent stem cells (iPSCs) for gene editing of many forms of EB.¹⁸ Base editing is emerging as potentially suitable for correcting EB point mutations. The refinement of base editing in the form of ‘prime editing’ represents a further progress, potentially able to edit the vast majority of all pathogenic EB mutations.^19,20 However, gene editing approaches are largely at the preclinical stage. Once fully developed, they could also be applied to recessively inherited EB, provided that their efficiency in targeting epidermal stem cells (see below) would be comparable to that of gene addition strategies.

Long-term epidermal regeneration relies on transgenic epidermal stem cells, detected as holoclone-forming keratinocytes

Squamous epithelia are constantly renewed. Being the first protective barrier against the external environment, these epithelia receive daily assaults, such as wounds, that need timely repair. Long-lived keratinocyte stem cells, residing both in the epidermal basal layer and in the bulge of the hair follicle, are responsible for such regeneration and repair processes.²¹ They have the unique capacity to self-renew and to generate committed progenitors – often referred to as transient amplifying (TA) cells – that generate terminally differentiated keratinocytes after a limited number of cell divisions.¹⁰

Even though they were not called stem cells at the time, keratinocytes cultured in 1975 in Green’s laboratory matched the definition of stem cells as we know them today. In fact, human clonogenic keratinocytes are endowed with an impressive proliferative potential and consist of stem cells and TA progenitors. A crucial step towards their identification and isolation was taken in 1987, when Barrandon and Green succeeded in cultivating human keratinocytes at a clonal level, hence identified three types of clonogenic keratinocytes giving rise to clones referred to as holoclones, meroclones and paraclones.²² They can be isolated both from a tissue biopsy and a keratinocyte primary culture.¹²

Initially described in the skin, they were found also in other stratified epithelia, such as cornea, urethra and oral mucosa.²³ All clonal types are endowed with proliferative capacity.²³ But while paraclones can undergo only few population doublings, holoclones and meroclones can produce dozens of cell doublings. The onset of replicative senescence is determined by clonal conversion, namely progressive decline in the proportion of holoclones and meroclones and progressive increase of paraclones, the latter generating only aborted colonies.

Cultured epithelial grafts contain all clonal types. Thorough analysis of data accumulated during over 30 years of clinical application of such cultures have provided compelling, yet indirect, evidence that holoclones and meroclones/paraclones are generated by stem cells and TA progenitors, respectively. For instance, permanent restoration of a transparent, renewing corneal epithelium, as well as a renewing epidermis, strictly requires a defined number of holoclone-forming cells in the culture.⁴ But formal evidence of holoclone-forming cells being authentic, long-lived, self-renewing stem cells was gained only through the in-depth analysis of the transgenic epidermis that restored the skin of JEB patients. Using proviruses as clonal genetic marks, clonal tracing of the newly formed transgenic epidermis has unambiguously shown that holoclone-forming cells are long-lived, self-renewing stem cells, necessary and sufficient to sustain the human epidermis. They continuously generate meroclones and paraclones that, as expected from TA progenitors, are short-lived and, although instrumental for proper tissue regeneration and wound healing, are progressively lost during epidermal renewal. In a nutshell, clonal tracing has shown that the main feature distinguishing the holoclone-forming cell from the other keratinocyte clonal types, is its self-renewal and long-term regenerative capacity.¹⁰

It follows that cultured epidermal grafts must contain an adequate number of holoclone-forming cells to permanently sustain the regenerated epidermis.²⁴ While paraclones could be identified based on their morphology (small irregular colonies containing large and flattened cells), holoclones and meroclones cannot be distinguished based on their growth rate and behavior and/or their shape and size. Thus, by no means a colony forming efficiency assay, which measures the number (and shape) of colonies, would suffice to establish the number of holoclone-forming cells harboring a cultured epidermal graft. Such number can be attained by a formal clonal analysis.¹⁰

Fundamental insights into stem cells of interfollicular epidermis and hair follicle have been gathered from murine studies, but not always murine findings apply to humans. For instance, the murine epidermis does not contain the same types of clonogenic keratinocytes found in the human skin. Nevertheless, an important step toward molecular definition of human holoclones came from the discovery of p63 as a key transcription factor sustaining murine squamous epithelia.^25,26 In fact, p63-null mice lack all stratified epithelia and have major defects in their limb and craniofacial development.^25,26 This phenotype could be explained by either inability of the p63-null ectoderm to develop into epithelial lineages and/or lack of stem cell character necessary to sustain epithelial morphogenesis and renewal. Subsequently, it has been shown that ΔNp63α, a specific p63 isoform, underpins the proliferative, regenerative capacity of mammalian epithelial stem cells.²⁷ In humans, ΔNp63α is highly expressed by epidermal and limbal holoclones and it progressively declines during keratinocyte clonal conversion.²⁸ Quantification of ΔNp63α^bright cells has been used as a pre-transplantation assay to evaluate the number of holoclones contained in a limbal/corneal culture.⁴

Strikingly, permanent restoration of a functional corneal epithelium in patients receiving limbal cultures for the treatment of severe chemical burns requires a defined number of ΔNp63α^bright holoclone-forming cells in the culture.⁴ This assay, however, has not yet been validated for epidermal cultures.

YAP is a transcriptional co-activator driving cell proliferation in many types of stem and progenitor cells and a key regulator of mechanotransduction. Unphosphorylated YAP translocates to the nucleus, where it induces target genes through interaction with TEAD transcription factors.

Phosphorylation of YAP in defined serine residues results in its sequestration, hence functional inactivation, into the cytoplasm by 14-3-3 proteins.²⁹ YAP interacts with ΔNp63α in sustaining self-renewal and proliferative/regenerative capacity of holoclone-forming cells.¹⁷ The transcriptomic profile of single human keratinocytes unveiled that FOXM1, a transcription factor member of the forkhead box family, acts downstream of YAP.³⁰ Nuclear YAP and FOXM1 are highly expressed in epidermal holoclones but virtually undetectable in meroclones and paraclones. In contrast, phosphorylated YAP and 14-3-3σ are barely detectable in holoclones and progressively increase during clonal conversion. Accordingly, the ablation of either YAP or FOXM1 induces the selective disappearance of holoclones, whilst enforced YAP or FOXM1 (or ablation of 14-3-3σ) halt clonal conversion and sustain holoclone-forming cells indefinitely.³⁰

Both microarray and single cell RNA-seq data have also shown that holoclone-forming cells display other common stem cell features, such as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity, and are enriched in genes regulating microtubules and actin polymerization.³⁰

Although a human holoclone molecular signature is thus emerging, further development of single cell genetic and epigenetic analyses is required and should give more insights that could allow to prospectively distinguish epidermal holoclones from the other clonal types.

Conclusion

There is no cure for EB. Available therapies are palliative, only partially alleviating the devastating clinical manifestations, hence they are not sufficient to provide decisive relief from pain, symptoms and mental stress achieve satisfactory living standards for these patients. Long-lasting, curative therapies are urgently needed, and several attempts have been made in this respect. As of today, gene correction in combination with cell-based approaches focus on individually designed treatments, holding promises for more effective results. The increasing number of clinical trials assessing such innovative, advanced molecular therapies resurges new hopes to definitively tackle this devastating disease. But none of these advanced approaches have yet made it to a routine therapy. The genetic and phenotypic EB heterogeneity (and the ambitiousness of a regenerative medicine approach) would require the convergence of multiple expertise and disciplines, including stem cell biology, developmental and molecular biology, genetics, tissue engineering and, not to say, a deep knowledge of all the clinical and surgical features of the different forms of the disease. Hence, a multidisciplinary collaborative effort is critical.

Acknowledgements

This work was supported by the European Research Council (ERC) Advanced Grant HOLO-GT (No. 101019289) to MDL. We thank Michele Palamenghi for Figure 1.

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