A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa

doi:10.1016/j.jaad.2013.08.014

Journal of the American Academy of Dermatology

Volume 69, Issue 6, December 2013, Pages 898-908.e7

This project was presented and peer reviewed at several international conferences: European Society for Dermatologic Research (ESDR), Budapest, Hungary, 2009 (concurrent session, preliminary data); DebRA international invitation-only research conference (Vienna, Austria, September 2009); American Academy of Dermatology, San Francisco, 2010 (residents and fellows symposium); ESDR Helsinki 2010 (plenary presentation); World Congress of Dermatology, Seoul, Korea, 2011; European Academy of Dermatovenereology, Lisbon, Portugal, 2011; Australasian Society for Dermatology Research, Perth, WA 2012; International Investigative Dermatology Meeting, Edinburgh, 2013.

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Background

Chronic wounds are a major source of morbidity and mortality in generalized severe recessive dystrophic epidermolysis bullosa (RDEB-GS).

Objective

This was a phase II double-blinded randomized controlled trial of intralesional allogeneic cultured fibroblasts in suspension solution versus suspension solution alone for wound healing in RDEB-GS.

Methods

Adult patients with RDEB-GS were screened for chronic ulcers and reduced collagen VII expression. Up to 6 pairs of symmetric wounds were measured and biopsied at baseline, then randomized to cultured allogeneic fibroblasts in a crystalloid suspension solution with 2% albumin or suspension solution alone. Ulcer size, collagen VII protein and messenger RNA expression, anchoring fibril numbers, morphology, and inflammatory markers were measured at 2 weeks and at 3, 6, and 12 months.

Results

All wounds healed significantly more rapidly with fibroblasts and vehicle injections, with an area decrease of 50% by 12 weeks, compared with noninjected wounds. Collagen VII expression increased to a similar degree in both study arms in wounds from 3 of 5 patients.

Limitations

The number of patients with RDEB-GS who met inclusion criteria was a limitation, as was 1 trial center rather than multicenter.

Conclusions

The injection of both allogeneic fibroblasts and suspension solution alone improved wound healing in chronic nonhealing RDEB-GS wounds independently of collagen VII regeneration. This may provide feasible therapy for wound healing in patients with RDEB-GS.

Section snippets

Methods

Ethical approval was granted in 2007 (Southeastern Sydney Area Health Service Human Research Ethics Committee 07/28). It was listed with the Australian and New Zealand Clinical Trials Registry (http://www.anzctr.org.au/ACTRN12610000760077).

Patients

Of 12 patients with RDEB in our EB registry⁶ aged 18 years or older, 5 were excluded because of squamous cell carcinoma and 2 because of negative collagen VII expression. All 5 remaining patients with RDEB consented to the trial (Fig 1 and Table I). All had mitten hand deformities, had a history of surgery to the fingers, had chronic anemia, were wheelchair dependent, and patient 3 had significant renal impairment. There was no relation between the HLA types of the donor and the recipients,

Discussion

This phase II double-blind randomized controlled trial of allogeneic cultured fibroblasts in RDEB-GS has demonstrated that the injection of either allogeneic fibroblasts or suspension solution alone encouraged wound healing. This study demonstrates that intradermal wound injections of suspension solution has a similar effect to injections of cultured allogeneic fibroblasts in encouraging wound healing and increased collagen VII expression in the basement membrane zone. The trial was presented

Conclusion

Injection of allogeneic fibroblasts and suspension solution are potential future therapeutic options for chronic wounds in patients with RDEB. It is not known if suspension solution alone has an equally beneficial effect because of possible systemic fibroblast effects, but would have significant cost benefit over fibroblasts even if used for half the injections, as in this study. The injections could be given during general anesthesia when patients with RDEB-GS underwent other routine

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Cytosine Deaminase Base Editing to Restore COL7A1 in Dystrophic Epidermolysis Bullosa Human: Murine Skin Model
2023, JID Innovations
Recessive dystrophic epidermolysis bullosa is a debilitating blistering skin disorder caused by loss-of-function mutations in COL7A1, which encodes type VII collagen, the main component of anchoring fibrils at the dermal−epidermal junction. Although conventional gene therapy approaches through viral vectors have been tested in preclinical and clinical trials, they are limited by transgene size constraints and only support unregulated gene expression. Genome editing could potentially overcome some of these limitations, and CRISPR/Cas9 has already been applied in research studies to restore COL7A1 expression. The delivery of suitable repair templates for the repair of DNA cleaved by Cas9 is still a major challenge, and alternative base editing strategies may offer corrective solutions for certain mutations. We show highly targeted and efficient cytidine deamination and molecular correction of a defined recessive dystrophic epidermolysis bullosa mutation (c.425A>G), leading to restoration of full-length type VII collagen protein expression in primary human fibroblasts and induced pluripotent stem cells. Type VII collagen basement membrane expression and skin architecture were restored with de novo anchoring fibrils identified by electron microscopy in base-edited human recessive dystrophic epidermolysis bullosa grafts recovered from immunodeficient mice. The results show the potential and promise of emerging base editing technologies in tackling inherited disorders with well-defined single nucleotide mutations.
Current topics in Epidermolysis bullosa: Pathophysiology and therapeutic challenges
2021, Journal of Dermatological Science
Epidermolysis bullosa (EB) is a group of inherited skin and mucosal fragility disorders resulting from mutations in genes encoding basement membrane zone (BMZ) components or proteins that maintain the integrity of BMZ and adjacent keratinocytes. More than 30 years have passed since the first causative gene for EB was identified, and over 40 genes are now known to be responsible for the protean collection of mechanobullous diseases included under the umbrella term of EB. Through the elucidation of disease mechanisms using human skin samples, animal models, and cultured cells, we have now reached the stage of developing more effective therapeutics for EB. This review will initially focus on what is known about blister wound healing in EB, since recent and emerging basic science data are very relevant to clinical translation and therapeutic strategies for patients. We then place these studies in the context of the latest information on gene therapy, read-through therapy, and cell therapy that provide optimism for improved clinical management of people living with EB.
Highly branched poly(β-amino ester)s for gene delivery in hereditary skin diseases
2021, Advanced Drug Delivery Reviews
Non-viral gene therapy for hereditary skin diseases is an attractive prospect. However, research efforts dedicated to this area are rare. Taking advantage of the branched structural possibilities of polymeric vectors, we have developed a gene delivery platform for the treatment of an incurable monogenic skin disease – recessive dystrophic epidermolysis bullosa (RDEB) – based on highly branched poly(β-amino ester)s (HPAEs). The screening of HPAEs and optimization of therapeutic gene constructs, together with evaluation of the combined system for gene transfection, were comprehensively reviewed. The successful restoration of type VII collagen (C7) expression both in vitro and in vivo highlights HPAEs as a promising generation of polymeric vectors for RDEB gene therapy into the clinic. Considering that the treatment of patients with genetic cutaneous disorders, such as other subtypes of epidermolysis bullosa, pachyonychia congenita, ichthyosis and Netherton syndrome, remains challenging, the success of HPAEs in RDEB treatment indicates that the development of viable polymeric gene delivery vectors could potentially expedite the translation of gene therapy for these diseases from bench to bedside.
Comparison of similar cells: Mesenchymal stromal cells and fibroblasts
2020, Acta Histochemica
Almost from all organs, both mesenchymal stromal cells and fibroblasts can be isolated. Mesenchymal stromal cells (MSCs) are the most preferred cellular therapeutic agents with the regenerative potential, and fibroblasts are one of the most abundant cell types with the ability to maintain homeostasis. Because of the promising properties of MSCs, they have been well studied and their differentiation potentials, immunomodulatory potentials, gene expression profiles are identified. It has been observed that fibroblasts and mesenchymal stromal cells have similar morphology, gene expression patterns, surface markers, proliferation, differentiation, and immunomodulatory capacities. Thus, it is hard to distinguish these two cell types. Epigenetic signatures, i.e., methylation patterns of cells, are the only usable promising difference between them. Such significant similarities show that these two cells may be related to each other.
Phase I/II open-label trial of intravenous allogeneic mesenchymal stromal cell therapy in adults with recessive dystrophic epidermolysis bullosa
2020, Journal of the American Academy of Dermatology
Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder due to a lack of type VII collagen. At present, treatment is mainly supportive.
To determine whether intravenous allogeneic bone marrow–derived mesenchymal stromal/stem cells (BM-MSCs) are safe in RDEB adults and if the cells improve wound healing and quality of life.
We conducted a prospective, phase I/II, open-label study recruiting 10 RDEB adults to receive 2 intravenous infusions of BM-MSCs (on day 0 and day 14; each dose 2-4 × 10⁶ cells/kg).
BM-MSCs were well tolerated with no serious adverse events to 12 months. Regarding efficacy, there was a transient reduction in disease activity scores (8/10 subjects) and a significant reduction in itch. One individual showed a transient increase in type VII collagen.
Open-label trial with no placebo.
MSC infusion is safe in RDEB adults and can have clinical benefits for at least 2 months.
Footprint-free gene mutation correction in induced pluripotent stem cell (iPSC) derived from recessive dystrophic epidermolysis bullosa (RDEB) using the CRISPR/Cas9 and piggyBac transposon system
2020, Journal of Dermatological Science
Recessive dystrophic epidermolysis bullosa (RDEB) is a monogenic skin blistering disorder caused by mutations in the type VII collagen gene. A combination of biological technologies, including induced pluripotent stem cells (iPSCs) and several gene-editing tools, allows us to develop gene and cell therapies for such inherited diseases. However, the methodologies for gene and cell therapies must be continuously innovated for safe clinical use.
In this study, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to correct the pathogenic mutation in RDEB-specific iPSCs, and the piggyBac transposon system so that no residual gene fragments remained in the genome of iPSCs after correcting the mutation.
For homologous recombination (HR)-based gene editing using CRISPR/Cas9, we designed guide RNA and template DNA including homologous sequences with drug-mediated selection cassette flanked by inverted repeat sequences of the transposon. HR reaction using CRISPR/Cas9 was induced in RDEB-specific iPSCs, and mutation-corrected iPSCs (MC-iPSCs) was obtained. Consequently, the selection cassette in the genome of MC-iPSCs was removed by transposase expression.
After CRISPR/Cas9-induced gene editing, we confirmed that the pathogenic mutation in RDEB-specific iPSCs was properly corrected. In addition, MC-iPSCs had no genetic footprint after removing the selection cassette by transposon system, and maintained their “stemness”. When differentiating MC-iPSCs into keratinocytes, the expression of type VII collagen was restored.
Our study demonstrated one of the safer approaches to establish gene and cell therapies for skin hereditary disorders for future clinical use.

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: Drs Venugopal and Yan contributed equally to this article.

: Funding for this study was provided by Australian Blistering Diseases Foundation; Research Infrastructure Support Services, Australia; Caroline Quinn Trust Fund; Australian Postgraduate Awards for PhD studies (Drs Venugopal and Yan); DebRA Australia, Ireland, and New Zealand; Premier Specialists, Sydney, Australia; Office of Research Administration, Palo Alto Department of Veterans Affairs Medical Center; the Epidermolysis Bullosa Medical Research Fund; and American Australian Association.

: Conflicts of interest: None declared.

View full text

Original articleA phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa

Background

Objective

Methods

Results

Limitations

Conclusions

Section snippets

Methods

Patients

Discussion

Conclusion

The classification of inherited epidermolysis bullosa (EB): report of the third international consensus meeting on diagnosis and classification of EB

J Am Acad Dermatol

Mutation analysis and characterization of COL7A1 mutations in dystrophic epidermolysis bullosa

Exp Dermatol

Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa

J Invest Dermatol

Bone marrow transplantation for recessive dystrophic epidermolysis bullosa

N Engl J Med

HB-EGF induces COL7A1 expression in keratinocytes and fibroblasts: possible mechanism underlying allogeneic fibroblast therapy in recessive dystrophic epidermolysis bullosa

J Invest Dermatol

Epidemiology of EB in the antipodes: the Australasian EB registry with a focus on Herlitz junctional

Arch Dermatol

Quality of life measurements in epidermolysis bullosa: tools for clinical research and patient care

Dermatol Clin

Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue

J Clin Invest

Original article
A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa