Topic Article:
Potential of Fibroblast Cell Therapy for Recessive Dystrophic Epidermolysis Bullosa
Tracy Wong, Luke Gammon, Lu Liu, Jemima E Mellerio, Patricia J C Dopping-Hepenstal, John Pacy, George Elia, Rosemary Jeffery, Irene M Leigh, Harshad Navsaria and John A McGrath
Journal of Investigative Dermatology (2008) 128, 2179-2189; doi:10.1038/jid.2008.78

Allogeneic Cell Therapy for Epidermolysis Bullosa

Varee Poocheron ¹, Shasa Hu ¹ and Robert S. Kirsner ¹

Journal of Investigative Dermatology (2008) 128, 2134. doi:10.1038/jid.2008.188

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe mechanobullous disease characterized at the molecular level by mutations in the COL7A1 gene, leading to reduced type VII collagen and defective anchoring fibrils at the dermal–epidermal junction (DEJ) (Fine et al., 2000). Patients who inherit this condition suffer from recurrent blistering, cutaneous ulcers, severe scarring, and deformities, and they are at high risk for developing life-threatening squamous cell carcinoma (Mallipeddi, 2002). Currently, there is no treatment for RDEB.

One approach to treatment would be to replace or reinforce the absent, reduced, or defective anchoring fibrils made up of type VII collagen, which would result in reduced blistering and ulceration. Among the current approaches under investigation are the use of gene therapy (Mavilio et al., 2006) and tissue-engineered skin (Falabella et al., 1999). Although type VII collagen is largely secreted by keratinocytes, fibroblasts also participate in its production (Varki et al., 2007; Goto et al., 2006). Recent studies have demonstrated that intradermal injections of normal human fibroblasts into affected mice resulted in the presence of persistent type VII human collagen (Ortiz-Urda et al., 2003; Woodley et al., 2007).

Stemming from these observations, Wong and colleagues (2008) report the first clinical experience in using allogeneic fibroblasts to treat RDEB. Each individual in a series of five patients with differing severities of RDEB received separate injections of cultured fibroblasts that were derived from their own skin (autologous), from a parent (allogeneic), and from an unrelated donor (allogeneic). Injection sites were assessed at 2 weeks and 3 months after injection. Described overall as safe and without adverse effects, the injection of allogeneic fibroblasts led to increased amounts of anchoring fibrils at both 2 weeks and 3 months in patients with less severe forms of RDEB. Interestingly, the increase in type VII collagen appeared to be derived from the patient’s own cells as opposed to from donor fibroblasts.

Through the following questions, we examine this paper in greater detail.

QUESTIONS
1. What is the rationale for and what are the potential pitfalls of using allogeneic fibroblasts to treat RDEB?
2. Why were three different formulations of fibroblasts injected into subjects’ skin, and why was a sample taken at 2 weeks and 3 months?
3. What data were presented that suggested utility of the modality, and was clinical improvement measured and correlated with histologic findings?
4. Why might baseline immunostaining have predicted response at 3 months, and why did the injection of fibroblasts from unrelated donors produce better results than the injection of fibroblasts from related donors?
5. What are the potential mechanisms of action of allogeneic fibroblast injections?
6. What may be the clinical implications of this article?

1. DEB is a family of inherited mechanobullous disorders due to mutations in the COL7A1 gene that result in reduced, truncated, or absent type VII collagen and anchoring fibrils at the DEJ, predisposing patients to increased risk for chronic wounds, infection, disfigurement, and malignancy. Although various topical wound-healing agents have been examined, including autologous cultured keratinocytes and skin bioequivalents, none has shown unequivocal benefits in the treatment of dystrophic forms of EB. Alternative approaches are needed.

Despite the belief that keratinocytes account for the majority of type VII collagen production at the DEJ in human skin, fibroblasts are also capable of synthesizing and secreting type VII collagen, thus contributing to the formation of anchoring fibrils. In their examination of COL7A1 gene–corrected fibroblasts and keratinocytes using retroviral gene therapy, Goto et al. (2006) found that fibroblasts assembled more type VII collagen at the basement membrane zone (BMZ) of skin grafts on immunodeficient rats than did keratinocytes, concluding that fibroblasts may be better target cells for DEB gene therapy. Additionally fibroblasts are easier to deal with as they are more robust and easier to propagate (Chen and Woodley, 2006). Woodley et al. (2004) also demonstrated that recombinant human type VII collagen directly injected into mouse skin or DEB human skin equivalents localizes to the BMZ, organizes into anchoring fibrils, and reverses the features of DEB disease in skin equivalents. The rate of turnover of type VII collagen delivered by this method, however, is not known. Based on the supposition that the administration of actual fibroblasts would result in more sustained type VII collagen deposition, several murine studies have demonstrated that injections of both gene-corrected RDEB fibroblasts and normal human fibroblasts into immunodeficient mouse skin or transplanted human skin equivalents result in generation of new human type VII collagen and formation of anchoring fibrils at the DEJ (Woodley et al., 2003; Ortiz-Urda et al., 2003). This appears to be dependent on the number of cells injected, i.e., 5 × 10^6^ cells versus 1 × 10^6^ cells (Woodley et al., 2003; Ortiz-Urda et al., 2003). The investigators in the present study focused on the effects of a single injection of 5 × 106 normal human fibroblasts directly into intact skin of moderately and severely involved RDEB patients, demonstrating increased type VII collagen expression and numbers of anchoring fibrils, especially among the patients with milder RDEB disease activity at baseline.

2. A total of six injections into six discrete nonblistered sites were administered. The sites of intact skin of patients with RDEB were subdivided for three different treatments—two of the six injection sites received autologous cultured fibroblasts, two received injections of cultured fibroblasts from one parent, and two received injections of what was considered the active treatment group of cultured fibroblasts from an unrelated (normal) donor. Autologous cultured fibroblasts served as a control, decreasing the possibility that type VII collagen expression or anchoring fibril formation was triggered by physical injury to the skin induced by actual injections or generic cytokine stimulus released from cultured fibroblasts. Parental fibroblasts, carrying one loss-of-function mutation in the COL7A1 gene, were used to determine whether a dose or threshold effect of type VII collagen expression was present and would positively impact the production of de novo type VII collagen in test subjects. Samples at 2 weeks were taken to assess early changes in the expression of type VII collagen and anchoring fibril formation among the test subjects. These variables were again evaluated at 3 months to determine whether initial responses would be sustained, attenuated, or augmented with time.

The investigators treated patients with a spectrum of severity of RDEB and included both moderately and severely involved RDEB patients. They demonstrated increased type VII collagen expression and numbers of anchoring fibrils, especially among the patients with milder RDEB disease activity at baseline. By selecting patients on different ends of the RDEB disease spectrum, the investigators avoided a major potential pitfall. Baseline biopsies of subjects with severe RDEB demonstrated markedly decreased or absent type VII collagen and anchoring fibrils compared with patients with milder phenotypes, who later had greater or more sustained responses to intradermally administered fibroblasts. Intentionally or unintentionally, the investigators showed a graded response to treatment via the inclusion of patients with both mild and severe disease. Whether the number of injected fibroblasts, although based on earlier studies, was enough to produce a lasting or meaningful outcome is also unknown, especially in patients with low levels of type VII collagen at baseline. Furthermore, it is still unclear how long newly generated type VII collagen will persist, even in the skin of milder RDEB patients, and whether increased amounts of mutated type VII collagen will in fact have measurable therapeutic value, as suggested by the investigators. Lastly, although the investigators did not note significant clinical, histopathologic, or serologic evidence of inflammation, it is yet unknown whether larger or subsequent doses of allogeneic fibroblasts would lead to a more exuberant host response.

3. Wong et al. (2008) show that, compared with baseline immunofluorescence, there was a 1.5- to 2-fold increase in expression of type VII collagen following injection of allogeneic, but not autologous, fibroblasts. At 3 months, this increased type VII collagen (later found to be of the patient’s own mutant form) was still present in the three subjects who had greater baseline type VII collagen expression, and further increased in sites injected with unrelated, normal donor cells. Transmission electron microscopy also indicated an increase in anchoring fibrils (albeit abnormal-appearing) at sites treated with allogeneic fibroblasts, but it was more marked in subjects 4 and 5, who had milder baseline phenotypes. These findings imply that allogeneic fibroblasts stimulate increased expression and deposition of mutant type VII collagen, which may itself play a role in maintaining adhesion at the DEJ.

Although promising information on surrogate endpoints was collected, no data were shown specifically measuring clinical outcome. The investigators did note subjective differences in the degree of epidermal–dermal adhesion during punch biopsies. Biopsies from sites injected with allogeneic fibroblasts appeared grossly to have less detachment than those from autologous cell injection sites. This may correlate with the histologic findings of increased type VII collagen expression and abnormal anchoring fibrils at the DEJ, which the investigators suspect still aids in maintaining adhesion but is a clinical outcome that has not yet been proven.

4. Immunostaining for type VII collagen showed that subjects 4 and 5, with milder (non-Hallopeau–Siemens) phenotypes, had more baseline type VII collagen at the DEJ than their more severely involved counterparts. Greater numbers of anchoring fibrils in patients 4 and 5 were also found at baseline than in subjects 1, 2, and 3. Accordingly, both patients had better sustained responses at 3 months, perhaps due to already increased baseline type VII collagen production capacity that allogeneic fibroblasts would further stimulate or possibly to a better ultrastructural scaffolding on which new collagen, although mutated, could be deposited. Baseline studies in future patients may predict which patients would receive the most benefit from this therapy. Furthermore, administration of fibroblasts from unrelated donors may have resulted in more significant type VII collagen production than that of parental donors owing to greater expression of COL7A1 genes in normal versus heterozygous donors.

5. Previous murine studies have indicated that human type VII collagen produced from injected fibroblasts, as well as recombinant type VII collagen itself, possibly homes toward and directly incorporates at the DEJ, restoring normal DEJ structure and function (Woodley et al. 2004). However, through examination of reverse transcriptase–PCR for COL7A1, the investigators found the recipient’s own mutant gene expression was increased following injection of allogeneic fibroblasts. Furthermore, fluorescent Y-probe in situ hybridization (of tissue from patient 5, a female) indicated no detectable allogeneic fibroblasts 2 weeks after administration. These findings allow Wong et al. (2008) to surmise that allogeneic fibroblasts exert a paracrine effect on the recipients’ own cells (fibroblasts or keratinocytes, or both), although the investigators were unable to detect any significant upregulation of specific cytokine gene expression that may induce type VII collagen expression.

Of interest was the finding of limited inflammatory response, especially in light of the fact that allogeneic fibroblasts were not detected at an early time point (2 weeks after intradermal injection). There was an increase in lymphocytes and dermal dendritic cells, as well as a minor increase in dermal macrophages, which normalized by 3 months.

6. Wong et al. (2008) have reported promising early results in human subjects injected with allogeneic fibroblasts, although much work is now required to elucidate specific mechanisms by which these fibroblasts stimulate further type VII collagen production and why larger quantities of abnormal type VII collagen and anchoring fibrils would augment adhesion at the DEJ, as the investigators conclude. Further study, probably in animal models, is required to clarify the suggested paracrine mechanism by which allogeneic fibroblasts stimulate subjects’ own cells, as well as the fate of the injected fibroblasts themselves. Optimization of this process may have significant clinical benefit, and would possibly be safer than administration of cells that have been gene-corrected via retroviruses or lentiviruses, although inflammatory markers and host responses (especially to unrelated donor cells) in this model have not yet been fully examined.

Lastly, although application of allogeneic cells to wounds is not a new concept, incorporation of greater numbers of or more targeted allogeneic cells in existing biosynthetic topical products or skin equivalents may greatly aid the management of RDEB patients.

REFERENCES
Chen M, Woodley DT (2006) Fibroblasts as target cells for DEB gene therapy. J Invest Dermatol 126:708–10

Falabella AF, Schachner LA, Valencia IC, Eaglstein WH (1999) The use of tissue-engineered skin (Apligraf) to treat a newborn with epidermolysis bullosa. Arch Dermatol 135:1219–22

Fine JD, Eady RA, Bauer EA, Briggamen RA, Bruckner-Tuderman L, Christiano A et al. (2000) Revised classification system for inherited epidermolysis bullosa: report of the Second International Consensus Meeting on Diagnosis and Classification of Epidermolysis Bullosa. J Am Acad Dermatol 42:1051–66

Goto M, Sawamura D, Ito K, Abe M, Nishie W, Sakai K et al. (2006) Fibroblasts show more potential as target cells than keratinocytes in COL7A1 gene therapy of dystrophic epidermolysis bullosa. J Invest Dermatol 126:766–72

Mallipeddi R (2002) Epidermolysis bullosa and cancer. Clin Exp Dermatol 27:616–23

Mavilio FG, Pellegrini G, Ferrari S, Di Nunzio F, Di Iorio E, Recchia A et al. (2006) Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 12:1397–402

Ortiz-Urda S, Lin Q, Green CL, Keene DR, Marinkovich MP, Khavari PA (2003) Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue. J Clin Invest 111:251–5

Varki R, Sadowski S, Uitto J, Pfendner E (2007) Epidermolysis bullosa. II. Type VII collagen mutations and phenotype–genotype correlations in the dystrophic subtypes. J Med Genet 44:181–92

Wong T, Gammon L, Liu L, Mellerio JE, Dopping-Hepenstal PJC, Pacy J et al. (2008) Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa. J Invest Dermatol 128:2179–89

Woodley DR, Krueger GG, Jorgensen CM, Fairley JA, Atha T, Huang I et al. (2003) Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone. J Invest Dermatol 121:1021–8

Woodley DT, Keene DR, Atha T, Huang Y, Lipman K, Li W et al. (2004) Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa. Nat Med 10:693–5

Woodley DT, Remington J, Huang Y, Hou Y, Li W, Keene DR et al. (2007) Intravenously injected human fibroblasts home to skin wounds, deliver type VII collagen, and promote wound healing. Mol Ther 15:628–35

¹ Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, USA