Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Goto, Maki; Sawamura, Daisuke; Ito, Kei; Abe, Masataka; Nishie, Wataru; Sakai, Kaori; Shibaki, Akihiko; Akiyama, Masashi; Shimizu, Hiroshi

doi:10.1038/sj.jid.5700117

Cited by 74 publications

(74 citation statements)

References 24 publications

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“…The long size of the COL7A1 cDNA hinders the generation of high-titer vectors (Goto et al, 2006;Siprashvili et al, 2010;Titeux et al, 2010). In our study, the titer obtained with the HA-COL7A1 vector from the 293Vec-Ampho population was 2 × 10 5 ivp/mL.…”

Section: Discussionmentioning

confidence: 79%

“…As described in our study for RDEB cells (Fig. 4), and by others (Georgiadis et al, 2016;Goto et al, 2006;Jackow et al, 2016;Titeux et al, 2010), transduced fibroblasts produced COLVII that relocated at the DEJ. However, the long-term persistence of these cells in our TESs remains to be proven.…”

Section: Discussionmentioning

confidence: 90%

“…Also, SIN retroviral vectors without viral enhancers are safer and less prone to transformation upon integration when compared to vectors with intact LTRs (Cavazza et al, 2013;Hacein-Bey-Abina et al, 2008;Howe et al, 2008;Modlich et al, 2006). Fibronectin and retronectin enhance retroviral gene transfer in keratinocytes (Bajaj et al, 2005;Goto et al, 2006), but the cost of the good manufacturing practices (GMP)-grade fibronectin or retronectin and the numerous manipulation steps limit the clinical development of a gene therapy strategy for RDEB. One alternative could be the use of a molecule easier to manipulate than retronectin or fibronectin that could be added simultaneously with the vector, without further manipulations.…”

Section: Discussionmentioning

confidence: 99%

“…Compared to the LAMB3 cDNA (3.6 kb) (Mavilio et al, 2006), the large size of the COL7A1 cDNA (8.9 kb) has limited the generation of high-titer retroviral vectors from stable retrovirus-producer cells (Chen et al, 2002;Goto et al, 2006;Siprashvili et al, 2010). The use of high-titer retroviral packaging cell lines (293Vec cells), constructed in our laboratory, could circumvent the titer issue.…”

Section: Introductionmentioning

confidence: 99%

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Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

A¹,

Larouche²,

Ghani³

et al. 2018

eCM

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The combination of gene therapy and tissue engineering is one of the most promising strategies for the treatment of recessive dystrophic epidermolysis bullosa (RDEB). RDEB is a rare genetic disease characterised by mutations in the COL7A1 gene, encoding type VII collagen (COLVII), which forms anchoring fibrils at the dermal-epidermal junction of the skin. This disease causes severe blistering and only palliative treatments are offered. In this study, the base of a strategy combining gene therapy and a tissue-engineered skin substitute (TES), which would be suitable for the permanent closure of skin wounds, was set-up. As a high transduction efficiency into fibroblasts and/or keratinocytes seems to be a prerequisite for a robust and sustained correction of RDEB, different envelope pseudotyped retroviral vectors and the transduction enhancer EF-C were tested. When green fluorescent protein (GFP) was used as a reporter gene to evaluate the retroviral-mediated gene transfer, the fibroblast infection efficiency was 30 % higher with the Ampho pseudotyped vector as compared with the other pseudotypes. At least a 3.1-fold and a 1.3-fold increased transduction were obtained in fibroblasts and keratinocytes, respectively, with EF-C as compared with polybrene. A continuous and intense deposit of haemagglutinin (HA)-COLVII was observed at the dermal-epidermal junction of self-assembled TESs made of cells transduced with a HA-tagged COL7A1 vector. Furthermore, HA-tagged basal epidermal cells expressing keratin 19 were observed in TESs, suggesting stem cell transduction. This approach could be a valuable therapeutic option to further develop, in order to improve the long-term life quality of RDEB patients.

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Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

A¹,

Larouche²,

Ghani³

et al. 2018

eCM

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“…Several studies have targeted keratinocytes, because the cells that secrete COL7 are mainly keratinocytes and to a lesser extent fibroblasts. 5,6 However, we and others have recently reported that injection of gene-transferred fibroblasts into the skin can efficiently restore COL7 expression in the dermal-epidermal junction in vitro. 6 -8 Furthermore, intradermal injection of allogeneic fibroblasts into skin of patients with RDEB skin was shown to result in enhanced COL7 expression in selected patients.…”

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confidence: 99%

Keratinocyte-/Fibroblast-Targeted Rescue of Col7a1-Disrupted Mice and Generation of an Exact Dystrophic Epidermolysis Bullosa Model Using a Human COL7A1 Mutation

Ito

Sawamura

Goto

et al. 2009

The American Journal of Pathology

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Recessive dystrophic epidermolysis bullosa (RDEB) is a severe hereditary bullous disease caused by mutations in COL7A1, which encodes type VII collagen (COL7). Col7a1 knockout mice (COL7 m؊/؊ ) exhibit a severe RDEB phenotype and die within a few days after birth. Toward developing novel approaches for treating patients with RDEB, we attempted to rescue COL7 m؊/؊ mice by introducing human COL7A1 cDNA. We first generated transgenic mice that express human COL7A1 cDNA specifically in either epidermal keratinocytes or dermal fibroblasts. We then performed transgenic rescue experiments by crossing these transgenic mice with COL7 m؉/؊ heterozygous mice. Surprisingly, human COL7 expressed by keratinocytes or by fibroblasts was able to rescue all of the abnormal phenotypic manifestations of the COL7 m؊/؊ mice, indicating that fibroblasts as well as keratinocytes are potential targets for RDEB gene therapy. Furthermore, we generated transgenic mice with a premature termination codon expressing truncated COL7 protein and performed the same rescue experiments. Notably, the COL7 m؊/؊ mice rescued with the human COL7A1 allele were able to survive despite demonstrating clinical manifestations very similar to those of human RDEB, indicating that we were able to generate surviving animal models of RDEB with a mutated human COL7A1 gene. This model has great potential for future research into the pathomechanisms of dystrophic epidermolysis bullosa and the development of gene therapies for patients with dystrophic epidermolysis bullosa. Dystrophic epidermolysis bullosa (DEB) is clinically characterized by mucocutaneous blistering in response to minor trauma, followed by scarring and nail dystrophy. The blistering occurs along the epidermal basement membrane zone (BMZ) just beneath the lamina densa at the level of the anchoring fibrils. The inheritance of DEB can be autosomal dominant (DDEB) or autosomal recessive (RDEB), each comprising subtypes of different clinical presentations and severities.1 Both DDEB and RDEB are known to be caused by mutations in the COL7A1 gene encoding type VII collagen (COL7), the major component of anchoring fibrils.2 The most severe RDEB subtype, the Hallopeau-Siemens subtype, shows a complete lack of expression of type VII collagen, whereas a less severe RDEB subtype, the non-Hallopeau-Siemens subtype, shows some collagen expression. The clinical fea-

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Gene Therapy for Inherited Skin Disorders

Lwin

McGrath

2017

Encyclopedia of Life Sciences

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Inherited skin disorders are caused by pathogenic mutations in over 500 genes. These mutations result in a spectrum of cutaneous and systemic abnormalities that generate considerable morbidity, and occasionally mortality. To date, however, there are no effective treatments. Nevertheless, the easy accessibility of skin, together with advances in molecular genetics and biotechnology, has paved the way for gene therapy development and testing. Gene therapy aims to reverse the pathology and phenotype of a particular disease through supplementation of the defective gene with a single normal full‐length copy or correction of the underlying mutant gene via genome editing or RNA (ribonucleic acid)‐based methods. Key Concepts Inherited skin disorders (also known as genodermatoses) encompass a group of genetic skin diseases in which single‐ or multiple‐gene defects account for a wide spectrum of clinical phenotypes with significant physical and psychosocial impact. Epidermolysis bullosa (EB) is a diverse group of autosomal dominant and recessive blistering skin diseases that affects ∼500 000 people worldwide. In EB, relatively minor trauma to the skin causes blistering which can be complicated by delayed healing and scarring. There is a desperate clinical need for effective therapies, including gene therapy. Gene therapy involves manipulation of cellular DNA or RNA to provide therapeutic benefit using various strategies. These include gene modification through gene addition or replacement, and gene correction through RNA‐based technologies or genome editing tools. Single‐gene disorders are therefore good candidates for gene therapy. The choice of gene therapy strategy in genodermatoses depends on the mode of inheritance and the nature of the pathogenic mutations of a particular disorder. Given that most autosomal recessive skin disorders result in loss of function, deficiency or absence of the wild‐type protein, restoring protein function through the addition of a wild‐type copy of the mutant gene, or correction of the mutant gene, is an appropriate therapeutic goal. In contrast, heterozygous mutations in dominant skin disorders typically result in dominant‐negative interference of the mutant/wild‐type proteins. Thus, therapeutic benefits are best sought by knockdown/silencing of the mutant allele while preserving the functional wild‐type allele, through applying gene correction techniques using small interfering RNA (siRNA) or gene editing endonucleases.

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Fibroblasts Show More Potential as Target Cells than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa

Cited by 74 publications

References 24 publications

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

Translating the combination of gene therapy and tissue engineering for treating recessive dystrophic epidermolysis bullosa

Keratinocyte-/Fibroblast-Targeted Rescue of Col7a1-Disrupted Mice and Generation of an Exact Dystrophic Epidermolysis Bullosa Model Using a Human COL7A1 Mutation

Gene Therapy for Inherited Skin Disorders

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