Gene Addition (Supplementation) Therapy
The most common form of gene addition therapy for RDEB utilizes viral vectors and is used in both in vivo and ex vivo gene therapy (Figure 2). RVs, LVs, HSV type-1 (HSV-1; NCT03536143) and AAV vectors have been developed for RDEB gene therapy.[25,51] RVs and LVs are RNA viruses that can integrate their genetic material into the target cell genome following reverse transcription to double-stranded DNA. Conversely, AAV is a small, nonenveloped virus lacking pathogenicity that possesses a linear single-stranded DNA genome capable of being engineered to deliver therapeutic transcription units to target cells. When adapted as a gene therapy agent, AAV vectors do not integrate their genetic material into the target cell genome (i.e. episomal).
Ex Vivo Gene Therapy
In 2000, one of the first in vitro studies of viral-mediated gene therapy utilized an engineered miniature version of COL7A1 cDNA (minigene). This in vitro study demonstrated that RV harbouring a COL7A1 minigene resulted in expression of a truncated (shorter) but at least partially functional C7 protein. With improvement in vector technology over the ensuing years, various preclinical studies began to develop full-length COL7A1 cDNA counterparts using similar RV approaches targeting either keratinocytes (or epidermal stem cells) alone or in combination with fibroblasts (Table 1).[10,11,54,55] Findings from these studies were promising as C7 expression was maintained for at least a year in vivo and was the basis for the first human gene therapy trial targeting keratinocytes in RDEB.
In 2006, the first human trial of EB gene therapy was reported in JEB. In this early-phase trial, RV-mediated full-length LAMB3 cDNA-supplemented patient keratinocytes were successfully grafted as epithelial sheets onto a 36-year-old man affected with intermediate JEB. Transgenic epidermal grafts were transplanted onto the patient's upper thighs, and stable disease correction in treated areas was shown for at least 6 years.[39,40] Eleven years later, the same group reported a landmark study in the field of gene therapy where autologous transgenic epithelial sheets regenerated an entire, fully functional epidermis on a 7-year-old child suffering from JEB with a homozygous mutation in LAMB3.
This successful clinical application of gene-augmented keratinocyte grafts laid the foundation for further clinical trials for RDEB using the same ex vivo approach. Indeed, subsequent to promising preclinical data, Siprashvili et al. reported the first clinical trial of ex vivo gene therapy for RDEB in 2016. In this trial, classical RV-harbouring full-length COL7A1 cDNA was successfully used to transduce patient keratinocytes and generated gene-modified epidermal sheets, which were grafted as autologous therapy onto six wound sites in each of four adults with RDEB. The grafts were tolerated for 12 months, and C7 expression was restored at variable levels among the patients without toxicity. However, clinical benefits, such as wound healing, generally declined over the first year after treatment.
A further report of the same trial was published in 2019 (NCT01263379) with three additional patients grafted with COL7A1-supplemented keratinocyte sheets using the same vector technology (EB-101, now licensed by Abeona Therapeutics Inc., Dallas, TX, USA), with 2–5 years' follow-up.[37,38] None of the participants experienced any serious adverse events.[37,38] Specifically, there was no immune reaction against the therapeutic transgene or the newly synthesized C7 (i.e. no EB acquisita) and no cancer at treated sites.[37,38] The latter is of particular concern as the use of classical RV (i.e. old-generation viral vector) is associated with a phenomenon called insertional mutagenesis, leading to malignant transformation. This is due to the activating vector randomly integrating near to proto-oncogenes. In terms of efficacy, there was variable wound healing between participants but encouragingly, treated wounds with 50% or greater healing resulted in improvement in patient-reported pain, itch and wound durability, accompanied by C7 expression persisting for up to 2 years after treatment in 2/7 participants. An open-label multi-centre phase III clinical trial of gene-modified keratinocyte sheets for RDEB is in progress (NCT04227106).
Using a safer form of SIN RV encoding full-length COL7A1 cDNA with a transduction efficiency (percentage of cells containing the transgene) of ~40–60%, gene-corrected skin equivalents were grafted onto immunodeficient mice, restoring C7 production and functional AFs. This study laid the foundation for the EBGraft trial[13,36] (NCT04186650), an open-label phase I/II clinical trial in which the first patient was grafted with gene-corrected skin equivalents in September 2020 (Alain Hovnanian, personal communications). Indeed, recent in vivo data suggest that both keratinocytes and fibroblasts need to be targeted in COL7A1 gene therapy to achieve therapeutic durability and functional restoration of the skin.
Following the observation that genetically corrected fibroblasts alone were sufficient to produce functional C7 in mice and subsequently in humans,[11,57–59] SIN LV and SIN RV were used to produce gene-modified fibroblasts that could be injected intradermally. The intradermal delivery circumvented the risks posed by skin grafts such as engraftment failure and infections, and risks associated with general anaesthesia, while the SIN viral vector technology further reduces the risk of oncogenic events.[62,63]
However, the risk of autoimmune reactions against the newly synthesized C7 remains. Further to promising preclinical data, albeit with a transduction efficiency of ~10%, a phase I clinical trial of intradermally injected gene-modified fibroblasts in adults with RDEB (NCT02493816) demonstrated a good safety profile and early efficacy, with a significant increase in C7 expression in six of 12 injected sites compared with noninjected skin in three of four patients with the presence of a transgene in one patient a year after treatment.
In parallel, Fibrocell Technologies (now owned by Castle Creek Biosciences, Exton, PA, USA) is currently evaluating gene-corrected fibroblasts (FCX-007) in an ongoing phase III trial (NCT04213261) after favourable outcomes from an earlier phase I/II trial (NCT02810951).
In Vivo Gene Therapy
Another viral vector used for COL7A1 gene therapy is nonintegrating (episomal) HSV-1, developed by Krystal Biotech, Inc. (Pittsburgh, PA, USA). In a phase I/II clinical trial named GEM-1 (NCT03536143), a topical gel containing HSV-1 harbouring two copies of COL7A1 cDNA was applied to RDEB wounds, which healed completely after an average of 17·4 days. Treatment was well tolerated with repeated dosing without causing either inflammation or infection, but longer-term safety data are awaited (NCT03536143).
Nonviral strategies targeting keratinocytes or fibroblasts are also used to correct RDEB cells, including highly branched poly(β-amino ester)s (HPAEs) to deliver COL7A1 cDNA.[64–68] Amryt Pharma Plc (London, UK) is currently developing a nonintegrating in vivo gene therapy (AP103) involving HPAE-harbouring full-length COL7A1 cDNA as repeated topical applications.
The British Journal of Dermatology. 2022;186(4):609-619. © 2022 Blackwell Publishing