What’s new

in EB research

Giovanna Zambruno

Istituto Dermopatico dell'Immacolata Rome, Italy

Fourth International Consensus Meeting on Diagnosis and Classification of EB, June 2013, London

1. Intraepidermal: EB simplex (EBS)

The fourth EB consensus classification: novel genes and EB subtypes

3. Sub-lamina densa: dystrophic EB (DEB)

4. Mixed: Kindler syndrome (KS)

2. Intra-lamina lucida: junctional EB (JEB)

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DSuprabasal EBS Basal EBS

TGM5

JUPDST,

EXPH5

ITGA3

Towards molecular therapies for EB

Heterogeneous disease group

Gene expression and function(well-characterized, animal models available) PROGNOSIS

DIAGNOSIS

Causative genes(18 genes identified to date)

Therapeutic approaches

GENE THERAPY

PROTEIN THERAPY

CELL THERAPY

Mouse models of dystrophic EB show therole of collagen VII in wound healing

Nyström A et al. J Clin Invest 2013;123:3498-509

• Mice defective for collagen VII show delayed wound closure

• Loss of collagen VII perturbs laminin-332organization during wound healing

• Collagen VII is needed for normal fibroblastmigration and cytokine production during wound healing

• Findings validated in human dystrophic EB wounds

Knowledge of the role of collagen VII in wound healing is instrumental to facilitate development of novel therapeutic strategies for dystrophic EB

Limits and challenges:

• Retroviral vectors safety (and regulatory) issues

• Costs (GMP legislation)

• Immunogenicity of the transgene product

Nat Med 2006; 12: 1397-1402

Pilot clinical trial in a single patient, affected with

generalized non-Herlitz JEB due to lamini-332

deficiency, showing that ex vivo gene therapy with

retroviral vectors is feasible and curative

Biopsy

Grafting of transducedepidermal

sheets

In vitro

epidermal stem cell

transduction with wt cDNA

Biopsy

Grafting of transducedepidermal

sheets

In vitro

epidermal stem cell

transduction with wt cDNA

Biopsy

Grafting of transducedepidermal

sheets

In vitro

epidermal stem cell

transduction with wt cDNA

Biopsy

Grafting of transducedepidermal

sheets

In vitro

epidermal stem cell

transduction with wt cDNA

• Ex vivo gene therapy with retroviral vectors of a

laminin-332 defective non-Herlitz generalized

JEB patient (H Hintner & JW Bauer, Salzburg,

Austria) fall 2013

• Phase 1 trial of gene transfer for recessive

dystrophic EB (A Lane, Stanford, CA, USA)

(http://clinicaltrial.gov/, n. NCT01263379)

2014 ?

• Phase I/II ex vivo gene therapy clinical trial for

recessive dystrophic EB (GENEGRAFT, A

Hovnanian, Paris, France) 2015 ?

Gene therapy trials for EB

“Local” therapy approach aimed to correct the gene defect in specific skin areas, no effect on internal disease manifestations

Protein therapy for dystrophic EB: preclinical studies

• Different dystrophic EB mouse models have shown that both local (intradermal injection) and systemic delivery (intravenous injection) of human recombinant collagen VII restore its expression in the skin and reverse the disease phenotype

• However, development of anti-human collagen VII circulating antibodies

Limits and challenges:

• Transient therapeutic effect

• Possible pathogenic effect of autoantibodies

• Amount of protein needed

Woodley DT et al. Nat Med. 2004;10:693-5Remington J et al. Mol Ther; 2009; 17: 26-33Woodley DT et al. J Invest Dermatol 2013;133:1910-3

Towards systemic protein therapy for dystrophic EB

• LOTUS Tissue Repair was founded in 2010 to develop recombinant collagen VII as protein replacement therapy for dystrophic EB

Presentation by Dr. Márcia Séllos-Moura, Shire HGT – Sunday, 22

• 8 January 2013, Shire plc announces the acquisition of Lotus Tissue repair. Press release: “….Shire’s Human Genetic Therapies business will undertake the further development of … recombinant form of human collagen Type VII (rC7), an intravenous protein replacement therapy for the treatment of DEB. The product is in late pre-clinical development and has the potential to be a first-in-class systemic therapy for the treatment of DEB….”

Cell therapies for dystrophic EB

• Fibroblasts

• Mesenchymal stem cells

• Hematopoietic stem cells

Cell types in clinical studies:

Fibroblast cell therapy for dystrophic EB: preclinical studies

Limits and challenges:

• Local treatment

• Transient therapeutic effect

Fritsch A et al. J Clin Invest 2008; 118: 1669-79

Kern JS et al. Mol Ther 2009; 17: 1605-15

Intradermal injection of wild-type fibroblasts in a mouse model of DEB:

• Locally increased collagen content correctly located in the skin, improved skin integrity and resistance to mechanical stress lasting more than3 months

• Mild transient skin inflammation

Col7a1flNeo/flNeo

Allogeneic fibroblasts in recessive dystrophic EB: clinical studies

• Increased collagen VII expression and improved anchoring fibril morphology lasting up to 3-12 months in some subjects

• Disappearance of allogeneic fibroblasts within two weeks

• Mild inflammatory response, no anti-collagen VII antibody production

A single intradermal injection of allogeneic cultured fibroblasts in 5 RDEB patients:

Wong et al. J Invest Dermatol

2008; 128: 2179-89

Nagy N et al. J Invest Dermatol

2011; 131: 1771-74

Phase II clinical study of injectable allogeneic human dermal fibroblasts in dystrophic EB

• Aimed to assess the effect on wound closure of a singleintradermal injection of human dermal fibroblasts (VAVELTA®, Intercytex Ltd., Manchester, UK), as compared to placebo (vehicle alone), in recessive dystrophic EB patients. The fibroblasts were injected at the margin of chronic erosions

• Out of 104 adult patients with RDEB invited to participate, 11 were treated difficulty in recruiting a sufficient number of patients meeting the inclusion criteria

• An injection of allogeneic fibroblasts increases the initial rate of erosion healing within the first 28 days (statistically significant difference only at day 7). No difference at 6 months

• Further studies needed to better define the effectiveness of allogeneic fibroblasts and the potential benefits of re-treatment

Petrof G et al. Br J Dermatol e-pub 2013 Aug 21

Mesenchymal stem cells in dystrophic EB: clinical studies

• Replenishment of type VII collagen and re-epithelialization of

chronically ulcerated skin after intradermal administration of

allogeneic mesenchymal stromal cells in two patients with

recessive dystrophic epidermolysis bullosa.

Conget P et al. Cytotherapy 2010;12:429-31

• EBSTEM – A phase I/II non randomised open-label single-centre proof of concept trial to assess safety and reduced disease severity following intravenous infusion of allogeneic

mesenchymal stem cells in children affected with recessive dystrophic EB (J.A. McGrath, London, UK). 6-10 patients to be included. Start: 2013.

Hematopoietic stem cells forEB treatment: preclinical studies

• Production of collagen VII, reduced blister formation and improved survival

• Presence of collagen VIIproducing donor cells in the skin and mucosae

Injection of hematopoietic stem cell-enriched bone marrow cellsfrom GFP+ transgenic mice intomice lacking collagen VII:

Tolar J et al. Blood 2009; 113: 1167-1174

GFP+ Col7a1+ cell in tongue

• Trial of immunomyeloablative therapy and transplantation of allogeneic bone marrow stem cells (ClinicalTrials.gov no. NCT00478244) in generalized RDEB

• 7 children (15 mo-14.5 yrs) entered the trial; 6 underwent bone marrow transplantation; 2 patients died

• Improved wound healing and decreased blister formation in 6 patients

• Systemic treatment acting also on oral and esophageal mucosa

Limits and challenges:

• Life-threatening complications due to combined risks of HCT and severe EB

• Disease improvement, not cure

• Duration of therapeutic effect?

Hematopoietic stem cell transplantation forsevere EB treatment: novel strategies

To decrease the risk linked to the procedure and

improve the clinical outcome:

• Assessment of non-myeloablative conditioning

regimen for transplant

• Assessment of co-infusion of mesenchymal stem

cells and hematopoietic stem cells

-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1

Trial 1: MA (N=6)

TBI

BU BU BU BU

Flu FluFlu

HCTGVHD

Prophylaxis:

Cyclosporine +

MMFCY CY CY CY

-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1

Trial 2: MA, MSC (N=7)

BU BU BU BU

Flu FluFlu

HCT +

MSCGVHD

Prophylaxis:

Cyclosporine +

MMFCY CY CY CY

-9 -8 -7 -6 -5 -4 -3 -2 -1 0 1

Trial 3: NMA, MSC (N=7)

Flu FluFluGVHD

Prophylaxis:

Cyclosporine +

MMFCY

FluFlu

HCT +

MSC

Improving HCT in Epidermolysis Bullosa

Kindly provided by Dr. J. Tolar

Epigenetic reprogramming: a novel strategy for obtainment of pluripotent stem cells and

treatment of EB

Somatic cells

Hochedlinger K & Plath K Development 136: 509, 2009

• Induced pluripotent stem cells (iPSC) can be generated from adult somatic cells (e.g. fibroblasts and keratinocytes) by transduction of four transcription factors

• Genetically-corrected iPS cells as a possible tool for future treatmentof EB

Oct4 Sox2 cMycKlf4 Intermediate cells

(transient population)

Partially programmed cells(stable cells line)

iPS cells

Knockdown of lineage genes,Inhibition of DNA methylation

• Revertant mosaicism results in areas of unaffected skin,

i.e. skin which does not blister even after minor trauma

• Described in several subtypes of EB (EB simplex,

junctional EB, dystrophic EB and Kindler syndrome)

• Is it possible to use revertant cells for therapy?

First experiments of culturing keratinocytes from a

revertant skin area and then grafting the cultured

epithelium on affected skin were unsuccessful.

Alternative strategy: use patient keratinocytes from

revertant skin patches to obtain iPS to be re-

differentiated and employed for grafting

Pasmooij et al. Discov Med 2012;14:167-79

Junctional epidermolysis bullosa

Somatic revertant mosaicism in EB: an example of natural gene therapy

EB-CLINET is a clinical network of EB centres and

experts, which aims at establishing EB centres

worldwide to improve medical care for people with

EB through exchange of knowledge and

experience about EB and, by providing a basis for

clinical trials, to accelerate the way to a cure for

this disease

http://www.eb-clinet.org

1st Conference of EB-CLINET, Salzburg, October 5-7 2012

Presentation by Dr. Gabriela Pohla-Gubo, project leader – Sunday, 22

Crucial instruments towards EB cure

• Development of recommendations and guidelines for patient care (wound care, oral health care available at http://www.debra-international.org)

• Studies on natural disease course

• International EB registry

• Definition of therapy outcome measures

Thank you !