1 Institute of Biophysics, Faculty of medicine, University of Ljubljana, Ljubljana, Slovenia
2 Department of Dermatovenereology, University Medical Centre Ljubljana, Ljubljana, Slovenia
3 Medical Center for Molecular Biology, Faculty of medicine, University of Ljubljana, Ljubljana, Slovenia
Correspondence/
Korespondenca:
Mirjana Liovic, e: mirjana.
liovic@mf.uni-lj.si Key words:
epidermolysis bullosa;
mutation; therapy Ključne besede:
bulozna epidermoliza;
mutacija; zdravljenje Received: 12. 3. 2019 Accepted: 5. 8. 2019
en article-lang
10.6016/ZdravVestn.2937 doi
12.3.2019 date-received
5.8.2019 date-accepted
Metabolic and hormonal disorders Metabolne in hormonske motnje discipline
Professional article Strokovni članek article-type
Epidermolysis bullosa, wound management
and new therapeutic approaches Dedne bulozne epidermolize, oskrba ran in nove možnosti zdravljenja
article-title
Epidermolysis bullosa Dedne bulozne epidermolize alt-title
epidermolysis bullosa, mutation, therapy bulozna epidermoliza, mutacija, zdravljenje kwd-group The authors declare that there are no conflicts
of interest present. Avtorji so izjavili, da ne obstajajo nobeni
konkurenčni interesi. conflict
year volume first month last month first page last page
2020 89 1 2 107 117
name surname aff email
Mirjana Liovic 3 mirjana.liovic@mf.uni-lj.si
name surname aff
Špela Zemljič-Jokhadar 1
Vlasta Dragoš 2
eng slo aff-id
Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia
Inštitut za biofiziko, Medicinska fakulteta, Univerza v ljubljani, Ljubljana, Slovenija
1
Department of
Dermatovenereology, University Medical Centre Ljubljana, Ljubljana, Slovenia
Dermatovenerološka klinika, Univerzitetni klinični center ljubljana, Ljubljana, Slovenija
2
Medical Center for Molecular Biology, Faculty of medicine, University of Ljubljana, Ljubljana, Slovenia
Medicinski center za molekularno biologijo, Medicinska fakulteta, Univerza v Ljubljani, Ljubljana, Slovenija
3
Epidermolysis bullosa, wound management and new therapeutic approaches
Dedne bulozne epidermolize, oskrba ran in nove možnosti zdravljenja
Špela Zemljič-Jokhadar,1 Vlasta Dragoš,2 Mirjana Liovic3
Abstract
Epidermolysis bullosa is a hereditary skin fragility disorder, which is linked to mutations in 18 genes that are expressed in the skin. Today we distinguish 4 main EB types, which include about 30 different disease subtypes with a variety of clinical symptoms. The main types are classified according to the skin layer that is affected: EB simplex linked to the epidermis; junctional EB linked to the basal membrane, and dystrophic EB linked to the dermis. Kindler syndrome is the fourth and very rare type of EB. In all cases, the patient’s skin is very fragile, so the basic clin- ical symptoms are skin blisters and wounds that heal with difficulty. EB affects patients both physically and psychologically, and the chronic inflammation accompanying the disease often leads to aggressive forms of squamous cell carcinoma. This is a group of rare genetic diseases with an incidence of 1:40000. In Slovenia there are 60 patients with EB. In this article we present the current standard of care of patients with this still incurable disorder, as well as the newest experimental methodologies aiming at the development of possible genetic, regenerative and pharmacological treatments of EB.
Izvleček
Bulozna epidermoliza (EB) je dedna bolezen krhkosti kože, povezana z mutacijami 18 različnih genov, ki se izražajo v koži. Zaradi raznovrstnosti simptomov se razvršča v 4 osnovne tipe s 30 različnimi podtipi. Osnovni tipi EB so razdeljeni glede na plast kože, ki je z mutacijo prizadeta:
EB simpleks, ki je vezana na epidermis, junkcijska EB, ki je vezana na bazalno membrano, ter distrofična EB, ki je vezana na dermis. Poznamo še Kindlerjev sindrom, ki je izredno redka ob- lika te bolezni. Pri vseh primerih je koža bolnikov zelo krhka, zato so osnovni simptomi mehurji in odprte rane, ki se težko celijo. Bolezen prizadene bolnike telesno in duševno, kronični vnet- ni procesi pa pogosto povzročijo nastanek agresivnih oblik ploščatoceličnega karcinoma kože.
Gre za skupino redkih bolezni z incidenco okoli 1 : 40.000. V Sloveniji imamo 60 bolnikov z EB. V članku predstavljamo trenutno uveljavljen način oskrbe kroničnih ran bolnikov z EB in pregled najnovejših in najsodobnejših genetskih, regenerativnih in farmakoloških pristopov za razvoj načina zdravljenja te še vedno neozdravljive bolezni.
Cite as/Citirajte kot: Zemljič-Jokhadar Š, Dragoš V, Liovic M. Epidermolysis bullosa, wound management and new therapeutic approaches. Zdrav Vestn. 2020;89(1–2):107–17.
DOI: https://doi.org/10.6016/ZdravVestn.2937
Copyright (c) 2020 Slovenian Medical Journal. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Slovenian Medical
Journal
1 Introduction
Epidermolysis bullosa (epidermolysis bullosa – EB) is a hereditary skin disor- der with characteristic blisters, erosions and wounds arising from a mild mechan- ic skin stimulation (Figure 1). EB patients frequently have numerous comorbidities, depending on the type of epidermolysis bullosa, such as dehydration, secondary lesion infections, onset of granulated tis- sues, atrophic scarring, post-inflammato- ry pigmentation, as well as poikiloderma, hypertrophic scars, milia, palmoplantar keratodermas, cutis aplasia. Late conse- quences of the basic diseases are skin car- cinoma, both basal cell carcinoma (BCC), as well as squamous cell carcinoma (SCC) (1). Accessory structures of the skin can manifest dystrophy or a complete loss of nails, scarring alopecia or alopecia univer- salis or hypotrichosis. In the mucosa of the mouth, patients may develop microsto- mia, obliteration of the vestibulum, enam- el hypoplasia, numerous cases of caries, missing teeth, or periodontitis. There may be an onset of corneal erosions, eyelid in- fections, corneal scarring, symblepharon,
ectropion, corneal infection and vision impairment with blindness, and conjunc- tival granulomata. In the area of the inner ear, there is a tightening of the outer ear canal, conductive hearing loss and, in the area of the nose, a tightening of the nos- trils resulting from granulations.
Other possible complications on oth- er organ systems with some types of epi- dermolysis bullosa occur in the digestive tract: pyloric atresia, oesophageal stric- ture, chronic constipation, gastroesoph- ageal reflux, anal fissures, protein losing enteropathy, colitis. In the genitourinary system, there can be urethral strictures, malformation of the female genital tract, vesicoureteral obstructions and stenoses, scarring in the vulva, kidney failure. In the upper respiratory tract, there are la- ryngotracheal stenoses. In the musculo- skeletal system, the following may occur:
osteopenia, osteoporosis, flexural limb contractures, finger contractures and fu- sion (pseudosyndactyly), webbed fingers and toes, muscle atrophy. Patients may also suffer from anaemia from different conditions, most often from lack of iron.
There are also cases of dilated cardiomy-
Figure 1: Some clinical symptoms of EB. a) aplasia cutis congenita; b) blood-fluid-filled blisters; c) erosions.
the functional and structural integrity of the epidermis, basal membrane or dermis.
EB phenotypes are expressed in a broad spectrum of symptoms (4). We differenti- ate between 4 basic EB types: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and the Kindler syndrome (Table 1). This classification is based on the lay- er in which the skin tissue cleavages. In the scope of basic types, there are at least 30 clinically different EB subtypes, differ- entiated by scope and severity of changes to the skin and mucous membranes, lo- cation and impact on the internal organs and tissues (2). The disease can be inher- ited either dominantly or recessively. EBS is characteristically inherited dominantly, however there are 18 known families that have the recessive type of the disease in the world (5-13). It is mostly tied to the muta- tions in the genes for keratin 5 or 14. On the other hand, JEB is exclusively recessive in nature and is linked to the mutations in the genes for α6β4 integrin, collagen 17 or laminin 5. Unlike those, DEB is inherited both along the dominant and the recessive path, and is tied to mutations in the genes which encode collagen 7. Kinder syn- drome is an exceptionally rare type of the disease, which is linked to the mutations in the KIND1 (or FERMT1) gene, which encodes the kindlin protein. Kindlin has a special role in connecting actin filaments with the extracellular matrix. The disease is inherited recessively.
The estimated extent of EB in USA (the National Epidermolysis Bullosa Register – NEBR, which has analysed the data for 16 years) is estimated at 11 cases per mil- lion, with a frequency of approximately 20 cases per million children born (15). In Slovenia, the incidence is approximately 30 cases per million. It is estimated that currently there are approximately 500,000 patients suffering from EB globally.
In 2014, a new classification system for diagnosing EB was developed, which in- cludes the EB type, method of inheritance, phenotype characteristic (scope of wounds and various extracutaneous symptoms), Table 1: Main types and subtypes of hereditary epidermolysis bullosa (14).
Level of skin
cleavage Major EB
type Major EB
subtype Targeted protein(s)
Intraepidermal EBS Suprabasal EBS transglutaminase 5; plakophilin 1;
desmoplakin; plakoglobin Basal EBS transglutaminase 5; plakophilin 1;
desmoplakin; plakoglobin Intralamina
lucida JEB JEB,
generalised laminin-332, collagen XVII; α6β4 integrin; α3 integrin subunit
JEB, localised collagen XVII; laminin-332; α6β4 integrin Sublamina
densa DEB DDEB collagen VII
RDEB collagen VII
Mixed Kindler
syndrome — Fermitin family homolog 1 (kindlin-1)
DDEB – Dominant dystrophic epidermolysis bullosa; DEB – dystrophic epidermolysis bullosa; EB – epidermolysis bullosa; EBS – simplex epidermolysis bullosa, JEB – junction epidermolysis bullosa;
RDEB – recessive dystrophic epidermolysis bullosa
ectropion, corneal infection and vision impairment with blindness, and conjunc- tival granulomata. In the area of the inner ear, there is a tightening of the outer ear canal, conductive hearing loss and, in the area of the nose, a tightening of the nos- trils resulting from granulations.
Other possible complications on oth- er organ systems with some types of epi- dermolysis bullosa occur in the digestive tract: pyloric atresia, oesophageal stric- ture, chronic constipation, gastroesoph- ageal reflux, anal fissures, protein losing enteropathy, colitis. In the genitourinary system, there can be urethral strictures, malformation of the female genital tract, vesicoureteral obstructions and stenoses, scarring in the vulva, kidney failure. In the upper respiratory tract, there are la- ryngotracheal stenoses. In the musculo- skeletal system, the following may occur:
osteopenia, osteoporosis, flexural limb contractures, finger contractures and fu- sion (pseudosyndactyly), webbed fingers and toes, muscle atrophy. Patients may also suffer from anaemia from different conditions, most often from lack of iron.
There are also cases of dilated cardiomy-
Figure 1: Some clinical symptoms of EB. a) aplasia cutis congenita; b) blood-fluid-filled blisters; c) erosions.
opathy. Among endocrine disorders, there may be a delay in the onset of puberty and amenorrhea. Other system complications include sepsis, delayed growth and de- velopment, and resulting from any of the numerous above comorbidities also a dis- ability (2).
The reason for the onset of fragility of the skin and epithelial tissue is one of 18 different genes which encode 18 structural and adhesive proteins of the cells and ex- tracellular matrix. They are manifested in a part of skin and have a width of just a few hundred micrometres (Figure 2).
The impacted layer includes the basal part of the epidermis, the basal membrane and partially also dermis. Mutations in tar- get genes most often cause the change in one amino acid in a protein (missense) or premature termination of gene expression, which results in a protein folding problem and consequently incorrect expression or lack of protein (3). Mutations can lead to irregularities in these proteins that impact
the functional and structural integrity of the epidermis, basal membrane or dermis.
EB phenotypes are expressed in a broad spectrum of symptoms (4). We differenti- ate between 4 basic EB types: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB) and the Kindler syndrome (Table 1). This classification is based on the lay- er in which the skin tissue cleavages. In the scope of basic types, there are at least 30 clinically different EB subtypes, differ- entiated by scope and severity of changes to the skin and mucous membranes, lo- cation and impact on the internal organs and tissues (2). The disease can be inher- ited either dominantly or recessively. EBS is characteristically inherited dominantly, however there are 18 known families that have the recessive type of the disease in the world (5-13). It is mostly tied to the muta- tions in the genes for keratin 5 or 14. On the other hand, JEB is exclusively recessive in nature and is linked to the mutations in the genes for α6β4 integrin, collagen 17 or laminin 5. Unlike those, DEB is inherited both along the dominant and the recessive path, and is tied to mutations in the genes which encode collagen 7. Kinder syn- drome is an exceptionally rare type of the disease, which is linked to the mutations in the KIND1 (or FERMT1) gene, which encodes the kindlin protein. Kindlin has a special role in connecting actin filaments with the extracellular matrix. The disease is inherited recessively.
The estimated extent of EB in USA (the National Epidermolysis Bullosa Register – NEBR, which has analysed the data for 16 years) is estimated at 11 cases per mil- lion, with a frequency of approximately 20 cases per million children born (15). In Slovenia, the incidence is approximately 30 cases per million. It is estimated that currently there are approximately 500,000 patients suffering from EB globally.
In 2014, a new classification system for diagnosing EB was developed, which in- cludes the EB type, method of inheritance, phenotype characteristic (scope of wounds and various extracutaneous symptoms), Table 1: Main types and subtypes of hereditary epidermolysis bullosa (14).
Level of skin
cleavage Major EB
type Major EB
subtype Targeted protein(s)
Intraepidermal EBS Suprabasal EBS transglutaminase 5; plakophilin 1;
desmoplakin; plakoglobin Basal EBS transglutaminase 5; plakophilin 1;
desmoplakin; plakoglobin Intralamina
lucida JEB JEB,
generalised laminin-332, collagen XVII; α6β4 integrin; α3 integrin subunit
JEB, localised collagen XVII; laminin-332; α6β4 integrin Sublamina
densa DEB DDEB collagen VII
RDEB collagen VII
Mixed Kindler
syndrome — Fermitin family homolog 1 (kindlin-1)
DDEB – Dominant dystrophic epidermolysis bullosa; DEB – dystrophic epidermolysis bullosa; EB – epidermolysis bullosa; EBS – simplex epidermolysis bullosa, JEB – junction epidermolysis bullosa;
RDEB – recessive dystrophic epidermolysis bullosa
discoveries in mapping immunofluores- cent tagged antigens and mutations pres- ents in an individual patient (14).
2 Diagnosing EB
The first step in diagnosing EB type in a patient is to determine the level of the location of the occurrence of blisters in the skin, usually by immunofluorescent anti- gen mapping and/or transmission elec- tronic microscopy (1). Using monoclonal antibodies focused on the components of the epidermal basement membrane and an epidermal antigen, we can additionally improve subclassification.
At the level of the skin cleavage, we can classify them into one of the four EB groups. Further diagnostics require a ge- netic analysis of the mutation, as it makes it possible to classify the disease in more detail and to provide further genetic con-
Figure 2: A schematic of a part of the skin (frame) with EB. This is a thin layer encompassing the lower part of the epidermis, basal membrane and the surface part of the dermis in which 18 proteins are express – potential generators of hereditary EB. Some of the most frequent ones are marked in the schematic.
sultation (14). If genetic analysis is not available, EB is classified by phenotype characteristics, such as dispersion (local- ised or general), severity and the presence of extracutaneous complications. The pa- tient’s family history can provide an in- sight into how it was inherited (1).
3 Wound care
Pope et al. (16) developed a procedure for treating wounds, consisting of four steps:
1. EB type, comorbidities, age, nutritional status, systemic comorbidities of other organ systems.
2. Care is focused on the patient and man- agement of their pain, itching, day-to- day activities and consulting regarding therapy.
3. Local treatment of the wound: deter- mining the location and the charac-
teristics of the wounds, regular gentle cleaning, removing necrotic tissue so that the lesion heals sooner, treating colonisation/inflammation/infection, selecting the appropriate local therapy and dressing, assessment of healing.
4. With wounds that heal poorly or not at all, we conduct a biopsy and a histolog- ical verification when suspecting skin carcinoma.
It is important to have the support of various medical providers, such as special- ised nurses or cooperation with various clinics when treating patients.
Local treatment of wounds depends on the type of lesion we are dealing with.
There are numerous dressings available, and we divide them into three groups:
non-occlusive, partially occlusive and ac- tive or biological dressings (16,17). Select- ing the appropriate dressing depends on the level the wound is healing. A necrot- ic wound must be treated with a dressing that uses hydrogel and in combination with polyurethane film or alginate. When a wound is inflamed, an alginate dressing will provide appropriate conditions for the wound to heal. In the proliferative phase and in the maturation phase, a dressing is needed to regulate moisture by soaking in the excess exudation (polyurethane or silicon foam and other). With an infect- ed wound, topical antimicrobial drugs and antimicrobial dressings (with silver, honey, charcoal, polyaminopropyl bigua- nide, dialkylcarbomoyl chloride) must be used (16,17). To prevent the onset of new blisters, silicon dressings are often used (16).
4 Patient care in Slovenia
The rare occurrence of EB and the im- pact on numerous other organs and sys- tems (beyond the skin) are a special chal- lenge for appropriate treatment of these patients (2). In the past few decades in Slo- venia, an interdisciplinary patient treat- ment has been developed, where groups
include dermatologists as well as other medical specialists. These include neo- natologists, paediatricians and internists, pathologists, medical geneticist, otorhino- laryngologists, ophthalmologists, plastic surgeons, orthopaedic surgeons, paedi- atric gastroenterologists, haematologists, dentists, anaesthesiologists, endocrinolo- gists, neurologists, radiologists, cardiolo- gists, nephrologists, specialists of physical therapy. The treatment also includes dis- trict nurses and a dietician from early on.
The main concern of the group is to reg- ularly monitor the skin and the wounds, diagnosing skin carcinoma early, regularly monitoring the complications on other organ systems, itching relief management and pain prevention.
5 Development of new therapy approaches
The main reason for the onset of EB is genetic changes to key structural genes which encode different structural proteins that connect the three layers of skin. For a complete recovery, the mutation would have to be fixed. Skin in our biggest organ by size. A therapeutic approach for treat- ing this disease would have to fix all the cells that for the damaged proteins. Unlike other organs, such as lungs, heart, liver, etc., skin is readily accessible. Therefore, therapy of hereditary skin diseases could potentially utilize an approach that would mitigate the clinical signs. In the past ten years, we have seen a major breakthrough in this area, even though none of numer- ous promising and tested approaches has proven to provide routine EB therapy.
Besides the long-term procedure of intro- ducing new medications and clinical stud- ies, one of the reasons is probably the high price of genetic therapy: up to 6 million euros per patient.
Past new therapeutic approaches are divided into: cellular therapy, gene ther- apy, protein therapy and system therapy (Figure 3). Below, we present a few of the most interesting approaches.
5.1 Cellular therapies
Cellular therapies include past trials with allogeneic fibroblasts, mesenchymal stem cells, stem cells from the umbili- cal cord and bone marrow transplant. In 2009, Kern et al. (18) did a study on more than 50 mice with recessive dystrophic EB; in separate experiments, they subder- mally injected healthy mouse and human fibroblasts. They established that the pro- cedure was safe (there was a mild tempo- rary inflammation). The level of collagen 7 (which is tied to the dystrophic EB type) increased at the dermo-epidermal con- nection by 3.5- to 4.7-fold. It is interesting that the injected cells produced healthy collagen 7 for a month, while the protein was stable for longer. Consequently, the skin of the mice maintained the improved phenotype for more than 100 days after the procedure. Injected cells remained at the injection point, did not divide, form
tumours or fibrotic tissue. In 2010, a study was published (10) in which two patients with RDEB were subcutaneously injected allogenic mesenchymal stem cells, isolated from bone marrow. After a while, the cells disappeared; however, their effect was visi- ble already after one week (the dermo-epi- dermal barrier was re-established), and lasted up to 4 months. In 2013, another study was conducted (20), in which a sim- ilar approach to the one performed on the mice was tested on patients with RDEB.
Allogenic fibroblasts were injected into the skin around the wounds. Cells survived a few weeks in the tissue, then disappeared, while their positive effect lasted up to 28 days after the procedure. In 2015, Petrof et al. (21) published a new study based on ten RDEB patients. They delivered allo- genic mesenchymal stem cells from bone marrow by infusion. The effect was visible in two months after several repeated pro- cedures. The surface of the affected skin
grew back faster, and the skin inflamma- tion was significantly reduced. Patients said that the positive effect was present for 4 – 6 months. El-Darouti et al. (22) published a study in 2016, in which they conducted a cellular infusion of non-he- matopoietic bone marrow cells, which are proven to be able to differentiate into fi- broblasts. They had two groups of RDEB patients: the first received an infusion of stem cells with added ciclosporin, while the second group received stem cells with- out ciclosporin. In both groups, the effect was very similar (there were no statistical- ly significant differences between them):
the patients’ health condition apparently improved, their wounds grew back faster, and one year after the test was completed, there were no major negative consequenc- es. It has been established that in proce- dures which capture mesenchymal stem cells, they do not divide or differentiate into healthy cells after the transplant, but operate indirectly by secreting numerous growth factors and cytokines which affect the surrounding tissue and thereby accel- erate wound healing and inflammation re- duction. Unfortunately, their effect, albeit very positive, was again just transitory and not long-lasting. We also should mention an exceptional experiment in which a se- lect group of children with RDEB received bone marrow transplants (23). Among the seven patients, one died before the transplant because of exceptionally ag- gressive therapy (immuno-myeloablative therapy). After bone marrow transplant, another patient died as a result of the re- jection of the implant and an infection.
With the remaining 5 patients, the phy- sicians established that within half a year after the procedure, there was a significant improvement to their condition, resulting from the build-up of healthy collagen 7 in the basal membrane area. Two years after the procedure, there was still a presence of foreign cells in the skin (chimerism) and an improved health condition.
Figure 3: Basic principles of some new therapeutic approaches for EB.
Protein therapy Cellular therapy
Gene therapy
Growing donor cells in culture
Tissue biopsy
Healthy donor
Isolation and multiplication of donor stem cells
Delivery of healthy donor
stem cells to the patient Transfection
Delivery of the healthy protein to the patient Bioreactor,
expression of the recombined
protein Cleaning
the protein Recombined construct
Slicing the mutated gene and replacing it with a healthy copy of the gene
Gene editing technology
Retrovirus with a healthy copy of the gene
Growing and multiplication of repaired cells
Delivery of the patient’s repaired cells
Patient’s cells grown in culture
Tissue biopsy Retrovirus with a
healthy copy of the gene Healthy copy of the gene Target cells for in vitro
expression of the healthy gene
Vector
Patient
grew back faster, and the skin inflamma- tion was significantly reduced. Patients said that the positive effect was present for 4 – 6 months. El-Darouti et al. (22) published a study in 2016, in which they conducted a cellular infusion of non-he- matopoietic bone marrow cells, which are proven to be able to differentiate into fi- broblasts. They had two groups of RDEB patients: the first received an infusion of stem cells with added ciclosporin, while the second group received stem cells with- out ciclosporin. In both groups, the effect was very similar (there were no statistical- ly significant differences between them):
the patients’ health condition apparently improved, their wounds grew back faster, and one year after the test was completed, there were no major negative consequenc- es. It has been established that in proce- dures which capture mesenchymal stem cells, they do not divide or differentiate into healthy cells after the transplant, but operate indirectly by secreting numerous growth factors and cytokines which affect the surrounding tissue and thereby accel- erate wound healing and inflammation re- duction. Unfortunately, their effect, albeit very positive, was again just transitory and not long-lasting. We also should mention an exceptional experiment in which a se- lect group of children with RDEB received bone marrow transplants (23). Among the seven patients, one died before the transplant because of exceptionally ag- gressive therapy (immuno-myeloablative therapy). After bone marrow transplant, another patient died as a result of the re- jection of the implant and an infection.
With the remaining 5 patients, the phy- sicians established that within half a year after the procedure, there was a significant improvement to their condition, resulting from the build-up of healthy collagen 7 in the basal membrane area. Two years after the procedure, there was still a presence of foreign cells in the skin (chimerism) and an improved health condition.
Figure 3: Basic principles of some new therapeutic approaches for EB.
Protein therapy Cellular therapy
Gene therapy
Growing donor cells in culture
Tissue biopsy
Healthy donor
Isolation and multiplication of donor stem cells
Delivery of healthy donor
stem cells to the patient Transfection
Delivery of the healthy protein to the patient Bioreactor,
expression of the recombined
protein Cleaning
the protein Recombined construct
Slicing the mutated gene and replacing it with a healthy copy of the gene
Gene editing technology
Retrovirus with a healthy copy of the gene
Growing and multiplication of repaired cells
Delivery of the patient’s repaired cells
Patient’s cells grown in culture
Tissue biopsy Retrovirus with a
healthy copy of the gene Healthy copy of the gene Target cells for in vitro
expression of the healthy gene
Vector
Patient
5.2 Gene therapies
Experiments with gene therapy in- clude: delivery of a healthy copy of the gene through a virus under ex vivo con- ditions, retroviral and lentiviral vectors (24-28), and non-viral experiments. The latter include: RNA interference or in- hibition of the damaged gene expression (29), using antisense oligoribonucleotides (AON) for suppressing mutated exons of a gene when copying RNA into the pro- tein, aminoglycosides for suppressing the premature termination codons (30), as well as using various proteins for gene ed- iting, such as zing finger nucleases (ZNF), Talens, Crispr/Cas9 (31).
With ex vivo gene therapy, the patient’s cells are removed in a skin biopsy, then grown under in vitro conditions. Repairing the mutated gene in the cells is also done in vitro in a culture. Then the repaired cells are inserted back into the patient. All these approaches are detailed in review articles (32-34); however, we will provide more information on the recently published first successful gene therapy procedure, which encompassed nearly all of skin sur- face (80%) of a patient with JEB, related to the mutations in the gene for laminin 332 (LAMB3) (35). After skin biopsy, isolating patient’s keratinocytes, growing cells and in vitro repair of the broken gene by deliv- ering a copy of a healthy gene through the lentiviral vector, the authors also complet- ed clonal tracing of autologous repaired cells. It has been proven that human epi- dermis regenerates thanks to progenitor cells, called holoclones. These are long-liv- ing stem cells of the epidermis of the skin, which can be successfully multiplied in vivo and in vitro. They differentiate into other groups of progenitor cells, which in the end differentiate into epidermis kera- tinocytes. After the selection, they were used as basis for growing epidermal sheets of healthy, autologous cells of the patient, which are then planted into the most dam-
aged parts of the skin. Using this proce- dure, an eight-year-old boy had 80% of his skin replaced (transplanted), and now has no more problems, except in the areas that were not included in the transplanta- tion. This is an exceptional accomplish- ment, as the boy was in very poor medical condition, and has had a normal life for a few years now since the procedure. Even though the study was exceptional and highly successful, the chances for using a similar procedure for other EB patients are currently low. Besides the very high costs of such treatment, there is still the concern related to possible side effects related to the viral delivery of the healthy gene, as it could result in malignant changes even later in life, which opens a set of different ethical questions related to using such EB therapy.
5.3 Protein therapy
Protein therapy includes intradermal or intravenous delivery ex vivo and un- der in vitro conditions of recombinantly obtained healthy protein (36,37). Healthy collagen 7 was generally delivered topi- cally, which yielded good results (38). All such studies have been conducted at the level of pre-clinical trials and on mouse models; however, in spite of positive ef- fects, they are still only experimental in character.
5.4 Systemic therapies
Systemic therapy encompasses using existing medications for treating oth- er health issues with the objective that some of its effects to improve the health of EB patients. This is especially related to reducing the inflammation response, reducing wound scarring, faster wound healing, etc. The drugs tested so far in- clude the effects of minocycline (39,40), which has anticollagenase action; howev- er, it unfortunately also causes issues with skin hyperpigmentation. Ciclosporin (41) has anti-inflammatory action; but it can
also cause skin cancer. Etanercept (42), which inhibits TNFaplha (anti-inflam- matory action) and Losartan (43), which inhibits TGFbeta (reduced scarring), can affect all the processes, related to these factors in the body. Researchers also tried bone marrow stimulation with the HMG protein (44) in order to increase the num- ber of stem cells in the skin and improve wound growth, as they believe that with EB patients the number of skin stem cells decreases during their lifetime because of constant activation of wound growth.
6 Research in Slovenia
At the Medical Centre for Molecular Biology (Institute of Biochemistry of the Faculty of Medicine, University of Ljublja- na), we have been conducting EB studies at the genetic, protein and cellular levels for nearly 20 years. We collaborate with the Dermatology & Venereology Clinic of the University Medical Centre Ljubljana and numerous international research and medical centres (Guy’s Hospital, King’s College London, United Kingdom; Miller School of Medicine, University of Miami, USA; Institute of Medical Biology, AStar, Singapore; Department of Medical Scienc- es, Uppsala University, Sweden, etc.). In the past few years, our research has been focused on studying the mechanism of the development of the disease and new deliv- ery systems that can be useful especially for delivering therapeutic molecules into the skin (45-48). An especially important achievement was the breakthrough in re- generative medicine and the preparation of the first induced EBS pluripotent stem cell line (iPSC) with the assistance of re- programming (dedifferentiation) of the keratinocytes of an EBS patient (49). In future studies, this cell line will be used as a cellular model and the basis for in vitro preparation of a tissue equivalent of EBS skin. This will then be used for transplant tests of numerous substances that could contribute to faster wound healing or to reducing certain clinical symptoms of EB
(e.g. inflammation).
7 Conclusion
In the past decade, we have witnessed an exceptional breakthrough in EB re- search mechanisms, understanding of the disease itself and the development of nu- merous strategies for experimental ther- apy approaches that mainly had positive effects. The clinical treatment of these patients has also changed, which contrib- utes to a better quality of living despite the severity of the disease. Support groups play in important contribution, especially the umbrella organization DEBRA Inter- national (dystrophic epidermolysis bullo- sa research association, http://www.deb-
ra-international.org/about-debra.html), which is present in more than 50 coun- tries through local DEBRA organizations, including in Slovenia (Debra Slovenia, http://www.debra-slovenia.si/).
8 Acknowledgment
We would like to thank the Slovenian Research Agency (programme P1–0390), the Ministry of Education, Science and Sports of the Republic of Slovenia, and the EraCoSysMed tender (project 4D-HEAL- ING), the Development Fund of the Uni- versity of Ljubljana, and the institution CELSA Alliance (project Molecular basis of keratopathies) for supporting and fi- nancing our research.
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