Gene Therapy for Epidermolysis Bullosa: Sticky Business

Despite efforts to promote development of therapies for rare diseases, the number of available treatments remains low and progress is too slow to have a significant impact on patients. This does not necessarily reflect a lack of interest in rare disease research, and significant efforts have been made toward the development and clinical testing of molecular therapies for these diseases. The intrinsic challenges of understanding the mechanisms underlying rare diseases render the design of efficacious therapies laborious. Importantly, a better understanding of disease mechanism is often only revealed by clinical trials. The efforts behind developing therapies for dystrophic epidermolysis bullosa (DEB), a rare genetic skin blistering disorder, serve as a good illustration. Recently, early data from the first gene therapy trial for recessive DEB were reported by Siprashvili et al. in the Journal of the American Medical Association.¹ The results—the culmination of an effort of more than a decade to develop a topical keratinocyte-based gene therapy to treat recessive DEB—provide support for cautious optimism.

DEB is caused by mutations in COL7A1 encoding type VII collagen (C7), which links the epidermal basement membrane to the dermal extracellular matrix.² Intracellularly, three C7 α1 chains fold into a trimeric molecule.² After secretion, two C7 molecules align in an antiparallel fashion, undergo proteolytic maturation, and become stabilized by intramolecular disulfide bonds. Subsequently, they aggregate laterally to superstructures called anchoring fibrils, which can simultaneously interact with multiple laminin-332 and type IV collagen molecules in the epidermal basement membrane and entrap dermal collagen fibrils.²

In DEB, the anchoring fibrils are either missing or reduced in number and functionally abnormal, resulting in chronic skin fragility, painful wounds, and progressive soft tissue fibrosis. A feared complication of DEB is development of aggressive squamous cell carcinoma at repeatedly wounded, scarred, and inflamed sites.² Because C7 is also present in extracutaneous organs, DEB patients would profit most from systemic therapy.² However, given that chronic wounds and squamous cell carcinoma at chronically scarred inflamed sites are major concerns,² topical causal therapies may also provide significant benefit.

The skin is an attractive organ for gene therapy because it is easily accessible and its major cells can be propagated and manipulated in vitro. Furthermore, it is estimated that more than 500 genetic diseases affect the skin, so once a robust therapeutic protocol has been established, it may be modified and applied to many genodermatoses.³ The treatment protocol employed by Siprashvili et al. involved retroviral transduction of COL7A1 cDNA into cultured DEB keratinocytes to restore C7 synthesis, followed by generation of autologous epidermal grafts and transplantation onto wounds. The approach appeared to be safe and well tolerated, but long-term efficacy was variable. No neutralizing immune response was observed to de novo synthesized C7. The recombinant retrovirus was not detected in the circulation, and malignant transformation of transplanted gene-corrected keratinocytes was not observed over a 12-month follow-up period. However, owing to the limited follow-up and the low number of patients (N = 4), the results must be regarded as preliminary and interpretation should be cautious.

This study presents the most convincing data to date on restoration of C7 deposition in patients. The authors ensured that normal full-length C7 was expressed by the gene-corrected keratinocytes. To this end, they used an antibody directed to the C terminus of C7 and not the N terminus, as was used in most previous studies (e.g. refs. 4 5 6). In addition, restoration of anchoring fibrils was studied by immunoelectron microscopy in order to exclude potential false positives, e.g. electron-dense fibers of microfibrillar networks. Even so, the correlation between the presence of anchoring fibrils and functional skin stability was imperfect,⁷ and future studies should assess resistance to frictional forces as a functional criterion.

It is intriguing that the life span of the transplanted gene-corrected epidermal grafts seemed to be limited. Three of the four patients showed a decline in C7 and a tendency toward increased blistering at transplant sites after six months. The fourth patient exhibited a decrease in C7 and anchoring fibrils at 12 months postgrafting, suggesting that the benefit was tapering off. These data suggest patient-dependent responses to the treatment and underscore the need to better understand the underlying intrinsic factors determining successful long-term efficacy of gene-corrected skin grafts in DEB. Secondary mechanisms, such as altered transforming growth factor-β bioavailability and inflammation, contribute to disease severity and disease progression in DEB.^8,9,10 A key question is whether younger patients with less advanced disease might have responded better to the treatment.

The first gene therapy for a genodermatosis, junctional EB (JEB), involved a single patient harboring LAMB3 mutations.¹¹ In a subsequent study another patient was treated with the same approach and with similar efficacy.¹² The grafted gene-corrected epidermal sheets promoted skin integrity and maintained stable laminin-332 secretion beyond six years.^11,13 Siprashvili et al. speculate that the difference in graft life span may be linked to the number of epidermal stem cells in the skin biopsy. The strong regenerative pressure in DEB skin has been suggested to lead to depletion of skin stem cells, but this has not been conclusively studied. A better understanding of the role of epidermal stem cells in DEB will impact not only approaches to keratinocyte gene therapy but other efforts as well, such as attempts to use bone marrow transplantation for treatment of DEB.^4,10

The distinct functions of the defective proteins in DEB and JEB may also influence the long-term therapeutic benefit of gene-corrected epidermal sheets. Laminin-332 can directly regulate cell behavior and survival and does not disperse far from the cells that produce it.¹⁰ This may create a local growth advantage for keratinocytes expressing laminin-332, as has been suggested by studies of naturally corrected revertant mosaic skin patches in JEB.¹⁴ For C7, which is not directly associated with growth factor signaling, no such advantage may exist. In addition, C7 is produced by both keratinocytes and fibroblasts,¹⁰ raising the question of whether skin grafts containing both corrected cell types might prove to be better therapeutics for DEB than purely epidermal grafts, as were used by Siprashvili et al.

Another issue is the nature of a heterotrimeric vs. homotrimeric gene product. Siprashvili et al. expressed COL7A1 from a retroviral promoter. Although this also applied to LAMB3 in the aforementioned trials,¹¹ this was a milder concern there because secretion of laminin-332 requires all three chains (α3, β3, and γ2), so control over synthesis and secretion was maintained by the other chains.¹¹ By contrast, C7, a homotrimeric single-gene product, has no such control of secretion. Posttranslational modifications are crucial for the function of C7, and supraphysiological expression may lead to secretion of undermodified C7 with impaired functionality. Most efforts in the gene therapy field have focused on achieving targeted gene expression without detailed assessment of the function of the transgene product, and future studies must address this before embarking on translation into the clinic on a large scale.¹⁵

In conclusion, the study by Siprashvili et al. underscores the challenges of designing effective molecular therapies for genetic diseases. It shows how something as seemingly straightforward as applying epidermal grafts after successful gene correction may in fact be much more complex than anticipated. These strategies can be perfected only with a better understanding of cellular and molecular disease mechanisms.