Phase I Study of Lentiviral-mediated COL7A1 Gene-modified Autologous Fibroblasts in Adults With Recessive Dystrophic Epidermolysis Bullosa.
Phase: PHASE1
Conditions: Recessive Dystrophic Epidermolysis Bullosa
Sponsor: King's College London

  
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PROTOCOL FULL TITLE 
 
Phase I study of lentiviral-mediated COL7A1 gene-modified 
autologous fibroblasts in adults with recessive dystrophic 
epidermolysis bullosa (RDEB). 
 
Trial Acronym: LENTICOL-F 
 
CONFIDENTIAL 
 
TRIAL IDENTIFIERS 
EudraCT Number: 2014-004884-19 
MHRA: 14523/0258/001-0002 
GTAC: 15/LO/0665 
ClinicalTrials.gov Identifier: NCT02493816 
 
Protocol Version 4.0, dated 1 March 2016 
Protocol Version (V) 
Date Created 
1.0 
10/FEB/2015 
2.0 
23/MAR/2015 
3.0 
01/JUN/2015 
3.1 
07/SEP/2015 
4.0 
01/MAR/2016 

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Sponsors 
Lead Sponsor: Professor Robert Lechler 
Vice Principal - Health, King’s College London 
1.4 Hodgkin Building, Guy’s Campus 
London. SE1 1UL 
Tel: +44 (0) 207 188 8794 
Fax: +44 (0) 207 848 6982 
Email: robert.lechler@kcl.ac.uk 
 
Co-Sponsor: Dr Kate Blake 
Guy’s and St Thomas’ NHS Foundation Trust 
R&D Department 
16th Floor, Tower Wing 
Guy’s Hospital, Great Maze Pond 
London. SE1 9RT 
Tel: +44 (0) 207 188 7188 
Fax: +44 (0) 207 188 1295 
Email: kate.blake@gstt.nhs.uk  
 
Contact Person on behalf of Sponsors: 
Jackie Pullen 
Acting Director & Quality Manager  
Clinical Trials Office,  
16th Floor Tower Wing, Guy’s Hospital,  
Great Maze Pond, London. SE1 9RT 
Tel : +44 (0) 207 188 5732 
Fax: +44 (0) 207 188 8330 
Email: jackie.pullen@kcl.ac.uk    
Chief Investigator 
Professor John A McGrath 
Professor of Molecular Dermatology and 
Consultant Dermatologist 
Department of Genetics and Molecular Medicine, 
St John’s Institute of Dermatology, King’s 
College London, 9th Floor Tower Wing, Guy’s 
Hospital, Great Maze Pond, London. SE1 9RT 
Tel: +44 (0) 207 188 6409 
Fax: +44 (0) 207 188 8050 
Email: john.mcgrath@kcl.ac.uk 
 
Principal Investigator 
Professor John A McGrath 
Professor of Molecular Dermatology and 
Consultant Dermatologist 
Department of Genetics and Molecular Medicine, 
St John’s Institute of Dermatology, King’s 
College London, 9th Floor Tower Wing, Guy’s 
Hospital, Great Maze Pond, London. SE1 9RT 
Tel: +44 (0) 207 188 6409 
Fax: +44 (0) 207 188 8050 
Email: john.mcgrath@kcl.ac.uk 
Principal Scientific Investigator 
Dr Waseem Qasim 
Reader in Cell & Gene Therapy and Consultant 
in Paediatric Immunology & Bone Marrow 
Transplantation 
Molecular Immunology Unit 
UCL Institute of Child Health and Great Ormond 
 
Co-Investigator  
Dr Jemima Mellerio 
Consultant Dermatologist 
St John’s Institute of Dermatology 
Guy’s and St Thomas’ NHS Foundation Trust 
Westminster Bridge Road, London. SE1 7EH 
Tel: +44 (0) 207 188 6399 
Fax:  +44 (0) 207 188 6379 

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Street Hospital, 30 Guildford Street, 
London. WC1N 1EH 
Tel: +44 (0) 207 905 2270  
Fax: +44 (0) 0207 905 2810 
Email: w.qasim@ucl.ac.uk 
Email: jemima.mellerio@kcl.ac.uk  
 
Co-Investigator  
Dr Su M Lwin 
Clinical Research Fellow & PhD Student 
Department of Genetics and Molecular Medicine, 
St John’s Institute of Dermatology, King’s 
College London, 9th Floor Tower Wing, Guy’s 
Hospital, Great Maze Pond, London. SE1 9RT 
Tel: +44 (0) 207 188 6353 
Fax: +44 (0) 207 188 8050 
Email: su.m.lwin@kcl.ac.uk 
 
Co-Investigator  
Dr Alya Abdul-Wahab 
Clinical Research Fellow and Dermatology 
Registrar, Department of Genetics and 
Molecular Medicine,  
St John’s Institute of Dermatology, King’s 
College London, 9th Floor Tower Wing, Guy’s 
Hospital, Great Maze Pond, London. SE1 9RT 
Tel: +44 (0) 207 188 6353 
Fax: +44 (0) 207 188 8050 
Email: alya.wahab@kcl.ac.uk 
Co-Investigator  
Dr Ellie Rashid 
Clinical Research Fellow 
Department of Genetics and Molecular Medicine, 
St John’s Institute of Dermatology, King’s 
College London, 9th Floor Tower Wing, Guy’s 
Hospital, Great Maze Pond, London. SE1 9RT 
Tel: +44 (0) 207 188 6353 
Fax: +44 (0) 207 188 8050 
Email: ellie.rashid@kcl.ac.uk 
Scientific Co-Investigator  
Dr Wei-Li Di 
Non-Clinical Senior Lecturer in Skin Biology 
Immunobiology Unit 
UCL Institute of Child Health and Great Ormond 
Street Hospital, 30 Guildford Street, 
London. WC1N 1EH 
Tel: +44 (0) 207 905 2369 
Fax: +44 (0) 0207 905 2810 
Email: w.di@ucl.ac.uk 
Scientific Co-Investigator  
Professor Adrian Thrasher 
Professor of Paediatric Immunology and 
Wellcome Trust Principal Clinical Fellow 
Molecular Immunology Unit 
UCL Institute of Child Health and Great Ormond 
Street Hospital, 30 Guildford Street, 
London. WC1N 1EH 
Tel: +44 (0) 207 905 2660 
Fax: +44 (0) 0207 905 2810 
Email: a.thrasher@ucl.ac.uk 
Scientific Co-Investigator  
Dr Farhatullah Syed 
Post-Doctoral Research Associate 
Molecular and Cellular Immunology Unit 
UCL Institute of Child Health and Great Ormond 
Street Hospital, 30 Guildford Street, 
London. WC1N 1EH 
Tel: +44 (0) 207 905 2369 
Fax: +44 (0) 0207 905 2810 
Email: farhatullah.syed@ucl.ac.uk  

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Statisticians 
Professor Janet Peacock 
Professor of Medical Statistics 
 
Fiona Reid 
Lecturer in Medical Statistics 
 
Division of Health and Social Care Research 
King’s College London, 7th floor, Capital House 
42 Weston Street, London. SE1 3QD 
Tel: +44 (0) 207 848 6651 
Email: janet.peacock@kcl.ac.uk 
IMP Manufacturer 
Cellular Therapies 
Dr Wei-Li Di 
Great Ormond Street Hospital (GOSH) for 
Children NHS Foundation Trust 
Great Ormond Street, London. WC1N 3JH 
Tel: +44 (0) 207 905 2369 
Fax: +44 (0) 0207 905 2810 
Email: w.di@ucl.ac.uk  
 
Lentiviral Vector Manufacturer 
Rayne Cell Therapy Suite (RCTS) 
Professor Farzin Farzaneh 
Department of Haematological Medicine 
King’s College London School of Medicine 
123 Coldharbour Lane 
London. SE5 9NU 
Tel: +44 (0) 207 848 5902 
Email: farzin.farzaneh@kcl.ac.uk  
The Robin Eady National Diagnostic 
Epidermolysis Bullosa Laboratory, Viapath  
Head of Laboratory: Dr Lu Liu  
St Thomas’ Hospital 
Guy’s and St Thomas’ NHS Foundation Trust 
Westminister Bridge Road, London. SE1 7EH 
Tel: +44 (0) 207 188 7229 
Email: lu.liu@viapath.co.uk 
Viapath Pathology Services, GSTT 
John Roberts 
Senior Business Development Manager 
Guy’s and St Thomas’ NHS Foundation Trust 
4th Floor Southwark Wing, Guy’s Hospital, 
Great Maze Pond, London. SE1 9RT 
Email: john.roberts@viapath.co.uk  
ELISPOT Laboratory 
Institut Imagine, INSERM UMR1163 
Équipe du Pr. Hovnanian 
2ème étage, pièce 221-B2 ou 220-B1 
142 rue du Cherche-Midi 
75015 PARIS 
Email: matthias.titeux@inserm.fr  
Immunodermatology Laboratory 
St John’s Institute of Dermatology 
Head of Laboratory: Balbir Bhogal  
Guy’s and St Thomas’ NHS Foundation Trust 
St Thomas’ Hospital 
Westminister Bridge Road, London. SE1 7EH 
Tel: +44 (0) 207 188 6364 
Trial Monitoring and Pharmacovigilance 
Clinical Research Associate 
King’s Health Partners Clinical Trials Office 
16th Floor Tower Wing, Guy’s Hospital 
Great Maze Pond, London. SE1 9RT  

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Fax: +44 (0) 207 188 6259 
Email: immunodermatology@gstt.nhs.uk  
Funding Sources 
Sohana Research Fund (SRF) and Dystrophic Epidermolysis Bullosa Research Association 
(DEBRA), UK. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Chief Investigator’s Signature 
 
Phase I study of lentiviral-mediated COL7A1 gene-modified autologous 
fibroblasts in adults with recessive dystrophic epidermolysis bullosa 
(RDEB). 
Trial Acronym: LENTICOL-F 
I, the undersigned, have read and understood the protocol specified above and agree to 
conduct the study as outlined herein. I will provide copies of this protocol and all 
pertinent information to the study personnel under my supervision. I will discuss this 
material with them and ensure they are fully informed regarding the investigational agent 
and the conduct of the study according to GCP as described in the GCP Directive and to 
the relevant ethics committee (IRB/IEC) requirements. 
Investigator Name: Professor John McGrath 
                   Investigator Title: Professor of Molecular Dermatology 
 
Site Address: St. John’s Institute of Dermatology 
9th floor, Tower Wing, Guy’s Hospital  
Great Maze Pond 
London 
SE1 9RT 
 
Site Telephone Number: + 44 (0) 207 188 6409 
Site Fax Number: + 44 (0) 207 188 8050 
 
Investigator Signature: 
 
 
 
Date:  

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1 Study Synopsis .............................................................................................. 10 
2 Glossary of Terms ......................................................................................... 13  
3 Background & Rationale ............................................................................... 15 
3.1 Epidermolysis bullosa (EB) ........................................................................... 15 
3.2 Recessive dystrophic epidermolysis bullosa (RDEB) .................................... 16 
3.3 COL7A1 mutations leading to RDEB ............................................................ 18 
3.4 Management of RDEB .................................................................................. 18 
3.5 Gene therapy for inherited skin disorders ..................................................... 18 
3.6 Rationale for using lentiviral vectors for gene delivery .................................. 19 
3.7 Rationale for targeting fibroblasts for gene therapy in RDEB ........................ 20 
3.8 Previous studies supporting the fibroblast therapy ........................................ 23 
3.9 Experience from clinical gene therapy studies .............................................. 24 
3.10 GMP grade lentiviral vector expressing C7 ................................................. 25 
3.11 Fibroblast harvest, transduction, expansion and cryopreservation ............. 25 
3.12 Restoration of C7 expression in gene-corrected fibroblasts ........................ 26 
3.13 In vitro function tests for gene-corrected fibroblasts .................................... 27 
3.14 In vivo skin graft mouse model to test gene-corrected fibroblasts............... 29 
3.15 Detection of shortened C7 protein in gene-modified keratinocytes ............. 32 
4 Trial Objectives and Design .......................................................................... 36 
4.1. Trial objectives ............................................................................................. 36 
 4.1.1 Primary objectives ................................................................................... 36 
 4.1.2 Secondary objectives .............................................................................. 36 
 4.1.3 Primary endpoint ..................................................................................... 36 
 4.1.4 Secondary endpoints .............................................................................. 36 
4.2 Trial design ................................................................................................... 37 
4.3 Trial timeline.................................................................................................. 38 
5 Trial Medication .............................................................................................. 40 
5.1 Investigational Medicinal Product .................................................................. 41 
5.2 Dosing regimen ............................................................................................. 41 
5.3 Description of IMP ......................................................................................... 42 
5.4 Packaging and labelling ................................................................................ 43 

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5.5 Distribution and shipment ............................................................................. 43 
5.6 Storage, dispensing and return of IMP .......................................................... 43 
5.7 IMP administration ........................................................................................ 44 
5.8 Drug accountability ....................................................................................... 44 
5.9 Subject compliance. ...................................................................................... 45 
5.10 Concomitant medication ............................................................................. 46 
6 Selection and Withdrawal of Subjects ......................................................... 46 
6.1 Inclusion criteria ............................................................................................ 46 
6.2 Exclusion criteria ........................................................................................... 46 
6.3 Selection of participants ................................................................................ 47 
6.4 Randomisation Procedure / Code Break ....................................................... 47 
6.5 Withdrawal of subjects .................................................................................. 47 
6.6 Expected duration of trial. ............................................................................. 48 
6.7 End of trial definition. .................................................................................... 49 
7  Trial Procedures ............................................................................................ 49 
7.1 Trial course ................................................................................................... 49 
7.2 Long-term follow-up ...................................................................................... 49 
7.3 Informed consent .......................................................................................... 49 
7.4 Procedures by visit ........................................................................................ 51 
7.5 Laboratory tests ............................................................................................ 54 
8 Assessment of Efficacy ................................................................................. 56 
8.1 Primary efficacy parameters ......................................................................... 56 
8.2 Secondary efficacy parameters..................................................................... 56 
8.3 Procedures for assessing efficacy parameters ............................................. 56 
9 Assessment of Safety .................................................................................... 57 
9.1 Assessment of benefits and risks. ................................................................. 57 
9.2 Specification, timing and recording of safety parameters .............................. 60 
9.3 Procedures for recording and reporting Adverse Events .............................. 62 
9.4 Reporting responsibilities .............................................................................. 63 
9.5 Adverse Events that do not require reporting ................................................ 64 
9.6 Treatment stopping rules .............................................................................. 67 

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10 Statistics ....................................................................................................... 67 
10.1 Sample Size ................................................................................................ 67 
10.2 Analysis ...................................................................................................... 67 
11 Trial Steering Committee............................................................................. 68 
12 Direct Access to Source Data and Documents ......................................... 68 
13 Ethics & Regulatory Approvals .................................................................. 69 
14 Quality Assurance ....................................................................................... 69 
15 Data Handling ............................................................................................... 70 
16 Data Management ........................................................................................ 71 
17 Publication Policy ........................................................................................ 71 
18 Insurance & Indemnity ................................................................................ 71 
19 Financial Aspects ........................................................................................ 71 
20 Signatures .................................................................................................... 72 
21 References.................................................................................................... 72 
Appendix 1: Development of gene therapy for inherited skin diseases ...... 77 
Appendix 2: Summary of published clinical studies of allogeneic and 
autologous fibroblasts used to treat skin disorders  ................................. 82 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1. Study Synopsis 
Title of clinical trial 
Phase 1 study of lentiviral-mediated COL7A1 gene-modified 
autologous fibroblasts in adults with recessive dystrophic 
epidermolysis bullosa (RDEB).  
Protocol Short 
Title/Acronym 
LENTICOL-F 
Study Phase 1   
Sponsors 
Lead Sponsor: King’s College London (King’s Health Partners) 
Co-Sponsor: Guy’s and St Thomas’ NHS Foundation Trust 
Chief Investigator Professor John A McGrath 
Medical condition or 
disease under 
investigation 
Recessive Dystrophic Epidermolysis Bullosa (RDEB) 
 
Purpose of clinical 
trial 
 
To assess whether intradermal injections of self-inactivating (SIN) lentiviral 
vector (LV)-mediated ex vivo transduced autologous fibroblasts expressing 
codon-optimised COL7A1 (investigational medicinal product – IMP), are 
safe and have potential efficacy in adults with RDEB. 
 
Primary objective 
 
To evaluate the safety of intradermal injections of SIN LV-mediated 
COL7A1 gene-modified autologous fibroblasts in adults with RDEB. 
 
Secondary 
objectives 
 
1. To evaluate the potential efficacy of intradermal injections of SIN LV-
mediated COL7A1 gene-modified autologous fibroblasts in adults with 
RDEB at week (W) 2, month (M) 3 and M12 after the IMP injections. 
2. To evaluate the immune response against the recombinant type VII 
collagen (C7) at W2, W4, M3, M6 and M12 after the IMP injections 
compared to baseline.  
Trial Design  
Phase 1, non-randomised, open-label, single-centre, proof-of-concept 
study. 
 
Endpoints 
Primary Endpoints 
 
Adverse events (AEs), Serious Adverse Events (SAEs), Adverse 
Reactions (ARs) and Serious Adverse Reactions (SARs) at each visit 

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over a 12-month follow-up period. 
 
Secondary Endpoints 
 
Skin biopsy analysis of treated skin at W2, M3 and M12 compared to 
untreated skin: 
I. 
C7 protein expression by immunofluorescence microscopy (IF) 
II. 
Morphology of anchoring fibrils at the dermal-epidermal junction 
(DEJ) by transmission electron microscopy (TEM) 
III. 
Vector copy number by quantitative polymerase chain reaction 
(qPCR) 
 
Serum analysis for: 
IV. 
Detection of anti-C7 antibodies by enzyme-linked immunosorbent 
assay (ELISA) (against NC1 and NC2 domains of C7) and indirect 
immunofluorescence (IIF) at W2, W4, M1, M3, M6 and M12 post-
injections 
V. 
Detection of T-cell responses to the full length C7 by enzyme-linked 
immunosorbent spot (ELISPOT) assay at W4, M6 and M12 post-
injections.  
Sample Size 5–10 patients 
Summary of 
eligibility criteria 
 
Inclusion criteria 
1. Clinical and genetic diagnosis of RDEB with confirmed bi-allelic 
COL7A1 mutations. 
2. A reduced number or morphologically abnormal anchoring fibrils 
confirmed by TEM. 
3. At least 5x3cm of intact skin on the trunk and/or extremities that is 
suitable for cell injections. 
4. Able to undergo local anaesthesia. 
5. Subjects aged ≥ 17years and able to give informed consent prior to the 
first study intervention. 
 
Exclusion criteria 
1. Subjects who received other investigational medicinal products within 6 
months prior to enrolment into this study. 

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2. Past medical history of biopsy proven skin malignancy. 
3. Subjects 
who 
have 
received 
immunotherapy 
including 
oral 
corticosteroids (Prednisolone >1mg/kg) for more than one week 
(intranasal and topical preparations are permitted) or chemotherapy 
within 60 days of enrolment into this study. 
4. Known allergy to any of the constituents of the investigational 
medicinal product (IMP). 
5. Subjects with BOTH: 
 
positive serum antibodies to C7 confirmed by ELISA and 
 
positive IIF with binding to the base of salt split skin.  
6. Subjects with positive results for HIV, Hepatitis B, Hepatitis C, HTLV or 
Syphilis. 
7. Subjects who are pregnant or of child-bearing potential who are neither 
abstinent nor practising an acceptable means of contraception when 
this is in line with the usual and preferred lifestyle of the subject, as 
determined by the Investigator, for 12 months after the cell injections. 
IMP, dosage and 
route of 
administration 
 
IMP: SIN LV-mediated ex vivo transduced autologous fibroblasts 
expressing codon-optimised COL7A1. 
DOSAGE:  0.8–1.2 million cells suspended in 0.25ml of 0.9% saline per 
injection over 1 cm2 of intact skin (3 injections of IMP at a single timepoint). 
ROUTE OF ADMINISTRATION:  Intradermal injections. 
 
Active comparator 
product (Control) 
 
None. 
Maximum duration 
of study 
participation 
32 months (each patient will be followed up for a total of 12 months post-
IMP injections and screening will take place up to 4 months prior to the 
intervention). 
Version and date of 
protocol 
Version 4.0, 01/MAR/2016 
 
 
 

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2. Glossary of Terms 
AE 
 
 
Adverse Event 
AR 
 
 
Adverse Reaction 
ATMP  
 
Advanced Therapy Medicinal Product 
BEBSS 
 
Birmingham Epidermolysis Bullosa Severity Score 
C7 
 
 
Type VII Collagen 
CRF 
 
 
Case Report Form 
DEB 
 
 
Dystrophic Epidermolysis Bullosa 
DEJ 
 
 
Dermal-Epidermal Junction 
DIF 
 
 
Direct Immunofluorescence 
DMC  
 
Data Monitoring Committee 
DMEM  
 
Dulbecco’s Modified Eagle Medium 
DMSO  
 
Dimethyl sulphoxide 
EB 
 
 
Epidermolysis Bullosa 
EBS 
 
 
Epidermolysis Bullosa Simplex 
EDTA  
 
Ethylenediaminetetraacetic acid 
EUD 
 
 
European Union Directives 
FACS  
 
Fluorescence activated cell sorting 
FCS 
 
 
Fetal calf serum 
GCP 
 
 
Good Clinical Practice 
GMP  
 
Good Manufacturing Practice 
GOSH  
 
Great Ormond Street Hospital 
GSTT  
 
Guy’s and St Thomas’ NHS Foundation Trust 
GTAC  
 
Gene Therapy Advisory Committee 
GvHD  
 
Graft versus Host Disease 
HIV 
 
 
Human immunodeficiency virus 
HLA 
 
 
Human Leucocyte Antigen 
HTLV  
 
Human T-lymphotropic virus 
IB 
 
 
Investigator’s Brochure 
ICF 
 
 
Informed Consent Form 
ICH 
 
 
Institute of Child Health 
IEC 
 
 
Independent Ethics Committee 
IMP 
 
 
Investigational Medicinal Product 
IMPD  
 
Investigational Medicinal Product Dossier 

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iPS 
 
 
Induced Pluripotent Stem Cells 
ISA 
 
 
Integration site analysis  
JEB 
 
 
Junctional Epidermolysis Bullosa 
KHP-CTO 
 
King’s Health Partners-Clinical Trials Office 
LV 
 
 
Lentiviral Vector 
MHC  
 
Major Histocompatibility Complex 
MHRA  
 
Medicines and Health Products Regulatory Agency 
QP 
 
 
Qualified Person 
qPCR  
 
Real Time Polymerase Chain Reaction 
RCL 
 
 
Replication-competent Lentivirus 
RDEB  
 
Recessive Dystrophic Epidermolysis Bullosa 
RV 
 
 
Gamma-Retroviral Vector 
SAE 
 
 
Serious Adverse Event 
SAR 
 
 
Serious Adverse Reaction 
SDs 
 
 
Standard Deviations 
SIN 
 
 
Self-inactivating 
SOPs  
 
Standard Operating Procedures 
SUSAR 
 
Suspected Unexpected Serious Adverse Reaction 
TMF 
 
 
Trial Master File 
UCL 
 
 
University College London 
 
 
 
 
 
 
 
 
 

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3 Background & Rationale 
3.1 Epidermolysis bullosa (EB) 
Epidermolysis bullosa (EB) is a heterogeneous group of inherited disorders 
characterised by skin blisters and mucosal fragility. EB affects ~1 in 17,000 live births 
and ~500,000 individuals worldwide (Fine et al. 2014). EB is classified into 4 major 
subtypes based on the new classification system: epidermolysis bullosa simplex (EBS), 
junctional epidermolysis bullosa (JEB), dystrophic epidermolysis bullosa (DEB), and 
Kindler 
syndrome 
although 
the 
full 
classification 
extends 
to 
~20 
different 
clinicopathological variants (Fine et al. 2008, and 2014). The new classification system 
adopts the ‘onion skin approach’ that takes into account the EB type, mode of 
inheritance, phenotype, immunofluorescence antigen mapping findings, and mutation(s) 
present in each patient (Fine et al. 2014). The main forms of EB are distinguished by 
different levels of cleavage underlying the blister formation as well as the adhesion 
proteins that are genetically mutated. In EBS, the level of separation occurs is within the 
epidermis, in JEB cleavage is within the lamina lucida, and in DEB blisters develop 
below the lamina densa within the upper papillary dermis. In Kindler syndrome, the 
plane of cleavage is variable.  
Figure 1: The molecular basis of the major forms of EB. Top left is a light microscopy image 

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of the skin; the boxed area indicates the dermal-epidermal junction: the section is stained with 
haematoxylin & eosin (scale bar=50 μm). Bottom left is a transmission electron micrograph of 
the dermal-epidermal junction; hemidesmosome attachment complexes are boxed (scale 
bar=0.1 μm). Right side is a schematic representation of the protein organisation of dermal-
epidermal attachment complexes, the intrinsic proteins and the genes encoding them, and the 
associated forms of EBS, JEB and DEB. (IC=intracellular; PM=plasma membrane; LL=lamina 
lucida; LD=lamina densa; EC=extracellular).  
 
3.2 Recessive dystrophic epidermolysis bullosa (RDEB) 
One of the most severe forms of EB is recessive DEB (RDEB) that affects ~800 people 
in the UK (source: www.debra.org.uk). RDEB is caused by bi-allelic loss-of-function 
mutations in the type VII collagen gene (COL7A1), leading to reduced or absent 
basement membrane type VII collagen (C7) and poorly formed or absent anchoring 
fibrils at the dermal-epidermal junction (DEJ) (Hilal et al. 1993). Poor anchoring fibril 
function leads to lifelong severe skin blistering and erosions following minor mechanical 
trauma from birth or early infancy. Wound healing is often slow, leading to chronic 
erosions, secondary infection and progressing to widespread, mutilating scars and 
contractures. Pseudosyndactyly (mitten deformities) occurs in nearly every patient with 
the most severe subtype of RDEB (Fine et al. 2005).  
 
Figure 2: "Mitten-like" appearance of the left hand and feet (pseudosyndactyly) in the 
patient with severe generalized RDEB. 

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Figure 3a: Erosions and fragile blisters on the dorsum of the right hand of a child with 
severe RDEB. Figure 3b: Extensive oral ulceration in a child with RDEB–generalised other 
(Figures courtesy of Dr Jemima Mellerio, co-investigator). 
 
Although cutaneous involvement is the most obvious clinical feature, extracutaneous 
features of children with severe RDEB include profound anaemia, failure to thrive, 
growth retardation, severe mucosal involvement, ocular involvement (corneal erosions 
and scarring) and oesophageal strictures (Pillay et al. 2008; Fine et al. 2009).  
 
Figure 4: Erosions on the background of extensive scarring on the back of a young adult 
with severe generalised RDEB (Figure courtesy of Dr Gabriela Petrof). 
 
Currently, there is no effective treatment for RDEB and many individuals develop life-
shortening squamous cell carcinomas by the age of 40 years (Martins et al. 2009).  
a 
b 

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3.3 COL7A1 mutations leading to RDEB 
C7 is synthesised mainly by epidermal keratinocytes and, to a lesser extent by dermal 
fibroblasts (Bruckner-Tuderman et al. 1987), Over 250 mutations in the COL7A1 gene 
have been so far identified. Jarvikallio et al (1997) has described that to some extent the 
severity of blistering and epidermal involvement can be correlated with the mutation 
profile in COL7A1, with more severe forms of the disease having nonsense or frameshift 
mutations on both alleles with less severe forms of RDEB harbouring missense or in-
frame splice site mutations, at least on one COL7A1 allele (Jarvikallio et al. 1997). 
 
3.4 Management of RDEB 
Best practice treatment involves a multi-disciplinary team of healthcare workers, with 
daily dressings often taking 1–4 hours to perform (Grocott et al. 2013). Total healthcare 
costs for individuals with severe RDEB living in the UK are estimated to be in excess of 
£60,000 per year (source www.debra.org.uk), with repeated applications of dressings to 
large wounds accounting for much of the overall expense. Currently, there is no effective 
treatment for RDEB and the management is mainly palliative and supportive. RDEB has 
a significant medical and socio-economic impact for all patients and their families 
(Tabolli et al. 2009). Chronic skin blistering affects the personal, physical, emotional and 
professional aspects of the patient and family (Abercrombie et al. 2008).  
 
3.5 Gene therapy for inherited skin disorders 
Gene therapies aim to provide therapeutic benefit through manipulation of DNA or RNA. 
Single gene disorders can be corrected by gene addition or augmentation, sequence 
correction, gene knockdown or exon skipping. To date, gene therapies using viral 
vectors have largely been conducted ex vivo, on cells harvested, engineered and then 
returned to recipients (Appendix 1). This has allowed issues of toxicity, including 
immune responses against viral antigens and worries about bio-distribution to be 
circumvented. 
 
Monogenic recessive disorders with a clinically severe or life-threatening phenotype 
provide the best candidate diseases for the introduction of a single normal copy of the 

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gene into the target cell. Pre-clinical studies have shown remarkable results in terms of 
gene correction using both in vivo and ex vivo approaches. These include JEB (Ferrari 
et al. 2006; Mavilio et al. 2006), RDEB (Titeux et al. 2010; Baldeschi et al. 2003; 
Woodley et al. 2003; Chen et al. 2002), lamellar ichthyosis (Choate et al. 1996), X-linked 
ichthyosis (Freiberg et al. 1997; Choate et al. 1997), xeroderma pigmentosum (XP) 
(Marchetto et al. 2006; Magnaldo et al. 2004), and Netherton syndrome (Di et al. 2011 
and 2013).  
 
With regard to skin studies relevant to EB, target cells have included both fibroblasts 
and keratinocyte stem cells. Of note, fibroblasts have been transduced and injected into 
recipient immunodeficient mice and shown to secrete C7 (Chen et al. 2002; Ortiz-Urda 
et al. 2003; Goto et al. 2006; Ito et al. 2009; Titeux et al. 2010). Moreover, correction of 
keratinocyte stem cells using integrating viral vectors has been shown in small and large 
animal models (Chen et al. 2002; Ortiz-Urda et al. 2003; Ito et al. 2009; Titeux et al. 
2010; Siprashvili et al. 2010; Gache et al. 2011).  
 
Recently, the Stanford group has successfully begun grafting ex vivo autologous 
epidermal sheets in an adult with RDEB in a phase I clinical trial (ClinicalTrials.gov 
Identifier: NCT01263379). In this study, autologous keratinocytes were transduced with 
GMP grade gamma-retrovirus carrying the full-length COL7A1. Six ~35cm2 autologous 
epidermal sheets were grafted in an adult with RDEB with complete epidermal 
regeneration 30 days post grafting with clinically normal skin appearance and no signs 
of immunological rejection (Siprashvili et al. 2014).  
 
3.6 Rationale for using lentiviral vectors for gene delivery 
RVs are the most widely used vehicles in human gene trials (Khavari et al. 2002; Gache 
et al. 2004; Gache et al. 2011), and have been used to transduce keratinocytes with 
COL17A1 (Seitz et al. 1999), LAMB3 (Vailly et al. 1998), ITGB4 (Dellambra et al. 2001) 
as well as COL7A1 (Titeux et al. 2010; Baldeschi et al. 2003; Woodley et al. 2003; Chen 
et al. 2002; Siprashvili et al. 2014). The use of these vectors in RDEB has been limited 
by the large size (9.2kb) of the COL7A1 cDNA (Gotto et al. 2006). One study comparing 

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adenoviral vectors, adenovirus-associated virus, RVs, and LVs concluded that LVs were 
the most suitable for ex vivo gene therapy due to their ability to transduce clonogenic 
primary keratinocytes (Gagnoux-Palacios et al. 2005). Keratinocytes have been 
successfully transduced in vivo with a C7 LV-containing full-length COL7A1 transgene 
(Chen et al. 2002; Baldeschi et al. 2003; Ortiz-Urda et al. 2002). Limitations of 
oncoretroviral systems include poor translocation across intact nuclear membranes and 
a tendency for transgene ‘silencing’ over time (Fenjves et al. 1996). In addition, 
conventional murine RVs preferentially integrate close to transcriptional start sites and 
have been shown capable of mediating insertional mutagenesis in clinical gene therapy 
studies (Hacein-Bey-Abina et al. 2003). In contrast, LVs can transduce quiescent or 
minimally stimulated cells. LVs are less likely to mediate insertional mutagenesis if 
suitable internal promoter elements are used (Montini et al. 2006). The investigators 
have considerable experience in lentiviral technology and have the ability to generate 
bespoke vectors suitable for fibroblast modification. The vectors are replication-
defective, self-inactivating (SIN) and incorporate a central polypurine tract sequence and 
post-transcriptional regulatory element (Demaison et al. 2002; Qasim et al. 2007).  
 
The investigators are also conducting a Phase I clinical study of ex vivo lentiviral 
modified skin grafts in Netherton Syndrome (ClinicalTrials.gov Identifier: NCT01545323). 
Vector titre and gene expression levels have remained within target ranges during 
scaled up production, providing an important precedent for the work for this study. Our 
preclinical data for expression of COL7A1 from this gene therapy platform is detailed in 
the Investigational Medicinal Product Dossier (IMPD) accompanying this protocol, and 
crucially we have modified cryptic splice donor and acceptor sites within the transgene, 
eliminating splice variants reported by other groups and affording high levels of gene 
expression in keratinocytes and fibroblasts.  
 
3.7 Rationale for targeting fibroblasts for gene therapy in RDEB 
Skin wounds in RDEB are typically very inflamed and often colonised or infected with 
numerous bacteria or yeasts and thus the application of any topical bio-material (e.g. 
keratinocytes or skin bioequivalents) runs a substantial risk of partial or complete loss of 

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the grafted material and failure to generate adequate safety or feasibility data. By 
contrast, intradermal injections of fibroblasts into intact, unwounded RDEB skin, offers a 
“closed” system, one that is not influenced by external organisms or other factors that 
may damage or destroy the fragile graft. The choice of fibroblasts for gene correction is 
also supported by transgenic COL7A1 murine data that found gene-corrected fibroblasts 
alone were able to restore C7 expression to the basement membrane, i.e. without the 
need for gene-correction of keratinocytes (Ito et al. 2009). Fibroblast targeted gene 
therapy also has potential clinical extrapolation: some patients with RDEB have 
localised disease for which localised therapies (rather than systemic measures) may be 
relevant. Moreover, the data obtained from studies of gene therapy in fibroblasts may 
have pertinent implications for gene therapy targeting of MSCs and clinical trials of 
systemic gene therapy for RDEB. 
 
Fibroblast cell therapies have been used to treat a multitude of skin disorders (Appendix 
2).The rationale for the use of fibroblasts in RDEB is based on the earlier work in murine 
models evaluating skin integrity following intradermal fibroblast administration. 
Fibroblasts are able to secrete C7, and therefore the hypothesis was that intradermal 
administration of exogenous allogeneic fibroblasts would lead to increased levels of C7 
at the DEJ, resulting in new anchoring fibrils. Ortiz Urda et al (2002) first showed that 
intradermally administered genetically engineered human fibroblasts can home to 
murine skin and generate new C7 at the DEJ.  This group used a φC31 based integrase 
vector (non-viral vector approach) to transduce RDEB fibroblasts. These were 
administered alongside RDEB(-) fibroblasts and wild type fibroblasts into RDEB human 
skin tissue regenerated on immune-deficient  scid/scid mice. Both RDEB(-) and normal 
fibroblasts failed to restore C7 at the cutaneous BMZ or correct sub-epidermal blistering 
in any of the injected RDEB(-) grafts. Injected RDEB+ cells, however, achieved both 
goals with results for the 4-month duration of the experiment in all patient tissue 
studied. Chen et al (2002) used a LV (SIN-LV expressing COL7A1 cDNA under the 
control of retroviral-MND promoter) to express C7 in RDEB keratinocytes and fibroblasts 
(in which C7 was absent) and demonstrated a reversion of the RDEB cellular 
phenotype in vitro (Chen et al. 2002). These gene-corrected cells were then used to 

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create a composite human skin equivalent transplanted onto scid/scid mice. The 
transplanted human skin exhibited a restoration of type VII collagen and anchoring fibrils 
at the DEJ (Chen et al. 2002). The same group then went onto investigate whether 
gene-corrected fibroblasts alone would be able to generate sufficient C7 to correct the 
phenotype (Woodley et al. 2003). Gene-corrected RDEB fibroblasts, parental RDEB 
fibroblasts, and normal human dermal fibroblasts were used. 5 x 106 fibroblast cells 
were suspended in Phosphate Buffer Solution (PBS) were injected into the dorsal back 
skin of 6 week old athymic nude mice. This study demonstrated that gene-corrected 
fibroblasts alone are able to produce more C7 at the DEJ than wild type in vivo. The 
expression of human type VII collagen by gene-corrected RDEB fibroblasts was 
sustained for at least 4 months in the mouse DEJ after a single intradermal injection of 
gene-corrected RDEB fibroblasts (Figure 5).  
 
 
 
Figure 
5: 
Images 
of 
immunofluorescence 
staining showing deposition of human type VII 
collagen 
at 
the 
mouse 
dermal-epidermal 
junction after a single intradermal injection of 
fibroblasts (figures courtesy of Woodley et al. 
2003) 
 (A) Immunofluorescence staining of mouse skin 
intradermally injected with fibroblasts 2 wk after 
injection was performed with either a monoclonal 
antibody specific for human type VII collagen 
(green, panels A–C) or a rabbit polyclonal antibody 
recognizing both mouse and human type VII 
collagen (red, panels D-F). (A,D,G) Skin injected 
with gene corrected RDEB fibroblasts. (B,E,H) Skin 
injected with normal human fibroblasts. (C,F,I) Skin 
injected with parental RDEB fibroblasts. Merged 
images (panels G-I) demonstrate the colocalization 
of human type VII collagen with mouse type VII 
collagen in the mouse's BMZ. (B) Skin from mice 
injected intradermally with gene-corrected RDEB 
fibroblasts was stained with a monoclonal antibody 
specific for human type VII collagen. (A,D) Biopsies 
taken 2 mo after injection; (B,E) biopsies taken 3 
mo after injection; (C,F) biopsies taken 4 mo after 
injection. e, epidermis; d, dermis. 

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3.8 Previous studies supporting the fibroblast therapy  
The McGrath group has undertaken first-in-man and early phase studies investigating 
the use of intradermal allogeneic fibroblasts in patients with RDEB (Wong et al. 2008; 
Petrof et al. 2013). The first administration of allogeneic intradermal fibroblasts in 
patients with RDEB was performed by Wong et al. in 2008. Five adult RDEB subjects 
(3 with severe generalised RDEB and 2 with a milder phenotype) received six 
injections: 2 of autologous cultured fibroblasts, 2 of cultured fibroblasts from 1 parent, 
and 2 injections of cultured fibroblasts from an unrelated donor (Wong et al. 2008) 
(Figure 6). Biopsies were carried out at 2 weeks and 3 months. All five subjects 
demonstrated an increase in C7 immunolabeling at the DEJ at 2 weeks after allogeneic 
fibroblast injection, compared with baseline. Importantly, the subjects in whom these 
data were obtained tended to have some C7 expression at baseline, in keeping with 
upregulation of mutant but partially functional endogenous C7. 
 
 
Figure 6: Fibroblast injection sites and biopsy time points (Figure courtesy of Wong et 
al. 2008) 
 
Subsequently, a phase II clinical trial demonstrated that a single intradermal injection 
of allogeneic fibroblasts increases the initial rate of erosion healing in subjects with 
RDEB within the first 28 days but not thereafter (Petrof et al. 2013), although that trial 
did not involve further injections of fibroblasts or optimisation of cell dose or dosing 
Grids measuring 1 cm2 were drawn 
onto intact, non-blistered skin of each 
subject. Six injections were given as 
shown and biopsies were then taken 
either at 2 weeks or at 3 months. Four 
of the five baseline biopsies were 
taken 
from 
adjacent 
skin, 
as 
illustrated. 

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regimens.  In this study, allogeneic fibroblasts increased the COL7A1 gene expression 
for up to 3 months and the protein levels for up to 9 months (Petrof et al. 2013). In 
some individuals, injection of allogeneic fibroblasts into intact skin led to increased C7 
at the DEJ within two weeks of a solitary injection, and increased (though not 
normalised) anchoring fibrils were visible on transmission electron microscopy (TEM). 
Despite this beneficial effect, donor fibroblasts were not detected in the patient skin 
biopsies at 2 weeks and 3 months, therefore suggesting that the allogeneic fibroblasts 
were cleared relatively quickly by the hosts’ immune response. It is likely that the 
injection of allogeneic fibroblasts elicit an immune reaction that leads to synthesis of 
heparin binding–EGF-like growth factor (HB-EGF), which upregulates the synthesis 
and assembly of the patient’s own mutated C7 (Nagy et al. 2011). Importantly, this 
immune response however did not result in any adverse events or pathogenic 
consequences.  
 
In all murine studies of gene, cell and protein therapy for RDEB, although circulating 
anti-C7 antibodies have been identified, none of the positive sera have shown binding 
to the dermal-epidermal junction (DEJ) and therefore their pathogenic significance (if 
any) is unknown. Clinically, thus far, there are no reports of any patients with RDEB 
developing EB acquisita (EBA) as a consequence of an interventional therapy or a 
natural event such as revertant mosaicism. In Wong et al. (2008) study,the 
immunostaining for anti-C7 antibodies at three months showed that none of the five 
subjects with RDEB receiving intradermal injections of autologous, related donor 
allogeneic and unrelated donor allogeneic fibroblasts, developed circulating anti-C7 
antibodies at three months. 
 
3.9 Experience from clinical gene therapy studies  
Qasim is leading clinical trials of T cell therapies after haematopoietic stem cell 
transplant (HSCT), and has completed a Phase I study of gene-modified T cells, which 
required production of good manufacturing practice (GMP) grade vector in London 
(Qasim et al. 2007; Qasim et al. 2009). The experience has been applied to the 
production of T cell receptor modified cells for two additional studies which are now 

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open, and forthcoming trials of lentiviral engineered T cells for malignancy. Thrasher 
has undertaken successful gene therapy studies using gamma-retroviral vectors for 
inherited immune deficiency and is leading next generation trials including lentiviral 
gene therapy for Wiskott Aldrich syndrome (Galy et al. 2011), chronic granulomatous 
disease (Grez et al. 2010) and SCID-X1 (Gaspar et al. 2004). There is now an 
established core of expertise in vector production, cell transduction, regulatory issues 
and trial management.  
 
Di and Qasim have taken a novel gene-based therapy for NS from bench to bedside. 
Initial studies using second generation lentiviral vectors showed that it was possible to 
transduce and correct keratinocyte stem cells from patients with NS. Di has produced 
in vitro organotypic cultures showed restoration of defective LEKTI (the protein 
encoded by SPINK5, the NS gene) production and correction of abnormal NS skin 
architecture. In vivo modelling using human:murine skin graft chimeras confirmed 
durability of gene correction (Di et al. 2011). Limitations associated with methylation-
mediated repression of gene expression were successfully addressed by incorporating 
the human involucrin promoter, which mediated compartment specific and high level 
expression of transgene in human keratinocytes (Di et al. 2011). A Phase I study of ex 
vivo lentiviral gene therapy for the NS is currently underway (ClinicalTrials.gov 
Identifier: NCT01545323). This study uses a third generation lentiviral configuration, 
identical to the vector for this study, to generate SPINK5 gene-modified epidermal skin 
sheets which will then be grafted on the individuals with NS. 
 
3.10 GMP grade lentiviral vector expressing C7 protein  
Using similar processes, we have generated a third generation lentiviral vector 
(detailed in the accompanying Investigational Medicinal Product Dossier) expressing a 
modified COL7A1 transgene, devoid of cryptic splice sites and repressive sequences, 
but including a Kozak consensus sequence. Expression is driven by the human PGK 
promoter. The vector has been used to transduce both fibroblasts and keratinocytes 
and mediates restoration of migratory function in vitro.  In a human:murine chimeric 
skin model the vector supports reconstitution of basement membrane C7 expression, 

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and recovery of anchoring fibrils (seen by TEM). Previous reports had indicated that 
retroviral vectors expressing COL7A1 were prone to a high frequency of re-
arrangements in KC. We isolated and expanded clonal populations of keratinotyes that 
had been transduced with pCCL-COL7A1 and found 3/49 clones expressed truncated 
protein bands as a result of deleted sequences within the transgene. It is uncertain 
whether the occurrence of such deletions can be completely eradicated from vectors 
which undergo reverse transcription, but functional studies in these clones found no 
additional aberrant effects.  Subsequently a batch of GMP compliant lentiviral vector 
expressing COL7A1 has been generated with a titre of 1.16x107 TU/ml, sufficient for 
transduction of fibroblasts. Release testing has been completed, including the 
exclusion of adventitious pathogens and replication-competent lentivirus (RCL). 
  
3.11 Fibroblast harvest, transduction, expansion and cryopreservation  
In our GMP validation studies, a 6mm fresh skin biopsy from subjects with RDEB was 
treated with collagenase to extract fibroblasts which were then expanded until sufficient 
to fill a T25 flask. Approximately one million autologous fibroblasts were then 
transduced by exposure to pCCL-PGK-COL7A1 vector, before further expansion over 
3-4 weeks to reach >8x106 cells.  Samples were tested for vector copy number and 
collagen expression by western blot, flow cytometry and in situ staining, and archived 
for RCL and integration site analysis (ISA). Immediate recovery after a freezing-
thawing procedure was evaluated to range from 45% to 75%, rising to 100% viability 
after one week of subsequent culture. We anticipate similar recovery after in vivo 
inoculation. Cells thawed and re-suspended in saline remained viable for over 8 hours 
in a 1ml syringe, and the plan for the clinical trial is to inject within this time window. 
 
3.12 Restoration of C7 expression in gene-modified fibroblasts 
C7 restoration in genetically modified RDEB primary fibroblasts was further confirmed 
by Western blot and in situ immunohistochemistry (Figure 7a & b).   
 
  
 

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Figure 7: Fibroblasts isolated from RDEB patient were transduced with pCCL-PGK-
COL7A1 vector. a. The Collagen VII expression was detected by Western blot with specific 
polyclonal anti-Collagen VII antibody. Full-length type VII collagen at the size of 290Kda was 
detected in cells transduced with therapeutic vector pCCL-PGK-COL7A1(line 1). A similar 
detection pattern was also seen in normal fibroblasts from normal skin (Lane 2). In contrast, no 
Collagen VII was detected in untransduced RDEB fibroblasts (Lane 3). b. Primary fibroblasts 
were stained with COL7A1 antibody (Red) and cell nuclei were stained with DAPI (blue). There 
was C7 protein expression (red) in transduced cells (C7) and normal fibroblasts (FB) compared 
to untransduced cells (Unt). 
 
3.13 In vitro function tests for gene-modified fibroblasts 
Primary fibroblasts from RDEB modified for C7 showed comparable viability/metabolic 
activity assessed using WST-1 kit (Figure 8). Importantly, migration assays show gene 
modified RDEB cells efficiently closed the wound compared to unmodified RDEB cells, 
indicating functional restoration in transduced cells (Figure 9). 
 
DAPI + C7
10X
10X
10X
Unt
C7                                Normal FB
RDEB- FB
Vinculin
C7
a.                              b.

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0
0.5
1
1.5
2
2.5
3
3.5
4
Absorbance [A450nm-A690nm]
72hr
C7+Ve   C7-Ve
RDEB-1
C7+Ve  C7-Ve
RDEB-2
C7+Ve  C7-Ve
RDEB-3
0
0.5
1
1.5
2
2.5
3
3.5
4
96hr
C7+Ve  C7-Ve
RDEB-1
C7+Ve  C7-Ve
RDEB-2
C7+Ve  C7-Ve
RDEB-3
Absorbance [A450nm-A690nm]
 
Figure 8: Metabolic activity and cell viability in cells transduced with pCCL-PGK-
COL7A1 vector. Transduced and untranduced RDEB fribroblasts from three patients were 
plated in 96 well plate (1X104 cells/well) and cultured for 72 and 96 hours. Cell viability and 
metabolic activity using WST-1 assay was performed and results showed there were no 
differences between transduced and untransduced cells. 
  
RDEB FB-C7, 
t=0 hrs
RDEB FB-Unt., 
t=30 hrs
RDEB FB-C7,
t=30 hrs
Normal skin 
FB, t=30 hrs
a.
 

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RDEB FB-Unt
t=0 hrs
RDEB FB-Unt
t=30 hrs
RDEB FB-C7
t=0 hrs
RDEB FB-C7
t=30 hrs
*p<0.01
*p<0.01
No. of cells migrated in 
the migration zone
Normal skin 
FB, t=0 hrs
Normal skin 
FB, t=30 hrs
b.
 
Figure 9: Normalised migration and wound closure in cells transduced with pCCL-PGK-
COL7A1. Panel a. shows migration pattern in 2mm migration zone at 30 hrs which were taken from 
example micrographs of untransduced and transduced RDEB fibroblasts. The migration speed was 
analyzed by in three independent experiments and results showed increase in migration of C7 
modified RDEB primary fibroblasts towards normal compared to unmodified RDEB primary fibroblasts 
at 30 hrs (Panel b).  
 
3.14 In vivo skin graft mouse model to test gene-modified fibroblasts  
A human-murine chimeric mouse model (Larcher et al. 2007; Di et al. 2010; Di et al. 
2011) was used to evaluate the efficacy of the C7 lentiviral vector. NOD/SCID/
knockout mice which are immunodeficient and tolerate human skin organotypic grafts 
were grafted with human transduced or untransduced RDEB fibroblasts and 
keratinocytes to allow C7 expression at dermal-epidermal junction to be examined in 
conditions that resemble skin development in vivo.  
 
The results showed that in the skin grafts generated by gene modified cells, the 
epidermis tightly attached to the dermis, whereas, in the skin grafts generated by 

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unmodified cells, there was detached epidermis in the grafts and there was restoration 
of C7 expression in dermal-epidermal junction in the skin graft generated by gene 
modified RDEB fibroblasts (Figure 10). 
 
 
WT KC
WT FB
RDEB (CK) KC
LV-C7 (CK) FB
RDEB (CK) KC
RDEB (CK) FB
d
c
e
b
a
★
★
★
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
Epidermis
Dermis
Human skin
Mouse skin
 
 
Figure 10: Restoration of C7 expression at DEJ in the grafts generated by gene modified 
RDEB cells. Bio-engineered skin sheets generated from normal, non-gene modified or gene 
modified RDEB keratinocytes and fibroblast cells were grafted onto NSG mice. Grafts were 
harvested 8 weeks post grafting. The morphology showed a splitting phenotype between 
epidermis and dermis in the graft generated by non-modified RDEB keratinocytes/fibroblasts 
(marked as dotted line on middle image, lower panel), whereas, there was no such phenotype 
in grafts generated by normal keratinocytes/ fibroblasts (left lower panel) or unmodified RDEB 
keratinocytes/modified fibroblasts (right lower panel). Immunofluorescent staining using human 
specific anti-C7 antibody showed C7 to be expressed at the DEJ in normal skin (left upper 
panel) and graft generated by normal cells (left lower panel) but not in the mouse skin (right 
upper panel). The staining also confirmed that the expression of C7 in the graft generated by 
modified fibroblasts (right lower panel). In contrast, there was no C7 expression in the graft 
generated by unmodified RDEB cells (left lower panel). These results indicate the restoration 
of C7 in the gene modified skin grafts. 

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To evaluate whether there was the formation of anchoring fibrils which a sign of 
functional correction of C7 protein in the skin graft generated by RDEB transduced 
keratinocytes and transduce fibroblasts, ultrathin tissue sections from the graft were 
analysed by transmission electron microscopy. The results clearly demonstrate an 
abundance of rope-like structures of type VII collagen anchoring fibrils throughout the 
DEJ. Similar to the healthy control graft they exhibit normal cross-banding with the NC-
2 domain extending to 200nm into the dermis and looping around type I and III 
collagen fibres (Figure 11).  
 
 
Figure 11: Restoration of type VII collagen anchoring fibrils in the skin graft generated 
by gene-modified RDEB keratinocytes and fibroblasts. Bioengineered human skin 
equivalents grafted onto NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice and harvested after 8-weeks. A-
C: low magnification (1200x) EM micrographs and D) WT human keratinocyte and fibroblast 
combination showing well formed DEJ with numerous large hemidesmosomes. Thick, cross-
banded type VII anchoring fibrils (arrows) extend ~200nm into the dermis looping round type I 
and III collagen. High proportion of C7 protein depositing plasmalemmal vesicles; E) 
Untransduced RDEB patient (CK) keratinocyte and fibroblast combination showing extensive 
tissue cleavage at the DEJ with lamina densa reduplication. Complete absence of type VII 
collagen fibrils resulting in separation of epidermis from underlying dermis; F) Untransduced 

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RDEB patient (CK) keratinocyte and LV-COL7A1 transduced fibroblast combination showing 
restoration of dermal-epidermal adhesion after LV-COL7A1 mediated correction of RDEB 
fibroblasts. Several thick, looping, arching and cross-banded type VII collagen anchoring fibrils 
shown providing attachment at the DEJ. Anchoring fibril (AF), collagen type I and III (C), 
dermal–epidermal junction (DEJ), hemidesmosome (HD), keratin filament (KF), lamina densa 
(LD), lamina lucida (LL), melanin (MN), plasmalemmal vesicle (PV). Images taken at x13,000 
and x30,000 magnification. Scale bar =300nm. 
 
3.15 Detection of shortened C7 protein in gene-modified keratinocytes 
Abnormal, shorted C7 protein bands have previously been reported in keratinocytes 
following viral vector transduction at a frequency of 25-33% (Titeux et al. 2010; Chen et 
al. 2000). There is a known risk of variant splicing or rearrangements of repetitive 
sequences during reverse transcription or other aberrations.  
 
We used a codon optimisation COL7A1 gene and had anticipated that coCOL7A1 
would minimize aberrant splicing and may reduce the frequency of shortened C7 
protein in gene modified cells. We assessed the frequency of fragmented C7 protein in 
RDEB keratinocytes rather than fibroblasts, as this allowed clonal populations to be 
efficiently derived and studied. Cells were transduced with pCCL-PGK-COL7A1 and a 
small proportion found to express a shortened form of C7 protein assessed by western 
blot (Figure 12a). Following single cell cloning we found the majority of transduced 
clones expressed full length C7 but about ~6% (3/49; Clone-34, Clone-E8 and Clone-
33) of clones had a shortened form of the protein (Figure 12b). Direct Sanger 
sequencing on these clones showed that there were deletions in these clones, for 
example, 5280-6446bp (1166bp) deletion in clone-34; 5516-6681 (1165bp) and 
7329-7827bp (498bp) deletion in clone-E8; 5532-5654bp (122bp) and a further 
deletion between 6139-6628bp (498bp) in clone-C33 (Figure 13). Interestingly, all 
three deletions occurred within between 5200bp to 7900bp. Further studies will be 
carried out to understand the mechanism underlying these deletions but we suspect 
they arise during reverse transcription due to repeat sequences within the C7 gene.  
 

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255 kD
150 kD
102 kD
76 kD
Full
length C7
Vinculin
Abnormal 
C7 protein
~230 kD
EBKC clones expressing full 
length C7, ~290kD
EBKC clones 
expressing
truncated C7
Clone-34, Clone-E8 and 
Clone-C33
1       2        3       4
Full
length C7
Vinculin
Abnormal 
C7 protein
255 kD
150 kD
102 kD
76 kD
a.
b.
 
Figure 12: Normal and abnormal expression of C7 in RDEB keratinocytes transduced 
with LV-COL7A1. a. Lane 1: Heterogeneous populations of transduced EBKC keratinocytes 
showing both full length and shortened C7 expression; Lane 2: EBKC Clone C34 expressing 
shortened C7; Lane 3: human normal skin keratinocytes showing expression of full length C7; 
Lane 4: untransduced EBKC keratinocytes with no expressed C7. b. EBKC clones, expressing 
full length or shortened C7 protein (Clone C34, E8 and C33).  
 
1
8882bp
hPGK promoter
~500bp
1000
2000
3000
4000
5000
6000
7000
8000
5280                              6446
1166bp
1
8882bp
hPGK promoter
~500bp
1000
2000
3000
4000
5000
6000
7000
8000
5516                            6681
1165bp
1
8882bp
hPGK promoter
~500bp
1000
2000
3000
4000
5000
6000
7000
8000
5532     5654
6139       6628
7329           7827
498bp
498bp
122bp
1
8882bp
hPGK promoter
~500bp
1000
2000
3000
4000
5000
6000
7000
8000
Normal full length 
C7 EBKC clone
Clone- C34
Clone-E8
Clone-C33
abnormal C7 
clones
 
Figure 13: Direct sequencing of normal and abnormal clones detected on western 
blotting. Direct Sanger sequencing of entire C7 showed deletions in three clones expressing 
shortened C7 protein, whereas the majority had the expected full length COL7A1 sequences 
(uppermost readout). 
 
In vitro functional assessment for these shortened C7 protein clones included viability 
and metabolic activity using a WST-1 assay and xCELLigence Real-Time Cell 

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Attachment (RTCA) assay. Results showed increased viability/metabolic activity in C7 
transduced RDEB Keratinocytes clones with normal protein compared to unmodified 
RDEB keratinocyte clones or clones transduced and expressing shortened C7 protein. 
Better cell attachment, spreading and proliferation were also observed in appropriately 
modified RDEB keratinocyte clones compared to untrasduced RDEB keratinocytes or 
transduced clones expressing abnormal C7 (Figure 14 and 15). 
     
Absorbance [A450nm-A690nm]
24hr
Full length C7-EBKC 
Clones 
48hr
Absorbance [A450nm-A690nm]
Absorbance [A450nm-A690nm]
72hr
96hr
Absorbance [A450nm-A690nm]
Full length C7-EBKC 
Clones 
Full length C7-EBKC 
Clones 
Full length C7-EBKC 
Clones 
 
Figure 14: Viability/metabolic activity of normal and abnormal C7 expressing RDEB 
keratinocytes. Cells expressing shortenedC7 protein showed a decreased viability/metabolic 
activity compared to cells expressing full length C7 protein. 
 

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6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
-0.5
0             8.0            16.0          24.0          32.0          40.0          48.0         56.0
Cell Index ((CI)
Time  (in Hours)
Nor_KS (N-Tert)
Full length C7-EBKC 
Clones 
Abnormal C7 EBKCClones
COL7A1 Untransduced EBKC Cells
 
Figure 15: Assessment of cell attachment, spreading and proliferation on Real-Time Cell 
Analyzer. Clones expressing shortened C7 protein and untransduced RDEB keratinocytes 
showed decreased cell attachment, spreading and proliferation compared to normal cells 
assessed by Real-Time Cell Analyzer (RTCA). 
Although there were about 6% shortened C7 protein expressed in transduced RDEB 
keratinocyte, shortened C7 protein has not been detected in transduced RDEB 
fibroblasts in our experiments to date. Studies by others also reported the frequency of 
shortened C7 proteins in transduced fibroblasts is much lower compared to around 
25% keratinocytes (personal communication with Prof A Hovnanian).   
 
In summary, the codon optimised COL7A1 vector is susceptible to residual aberrant 
protein expression effects, but at a notably reduced frequency compared to previously 
described, non-optimised configurations. This is likely to be related to regions with 
clusters of repeat sequences, but we found no evidence that the consequential 
truncated proteins caused any further deterioration in function.  
 
 
 
 
 
 

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4. Trial Objectives and Design  
4.1 Trial objectives 
4.1.1 Primary objective 
To evaluate the safety of intradermal injections of SIN LV-mediated COL7A1 gene-
modified autologous fibroblasts in adults with RDEB.  
 
4.1.2 Secondary objectives 
I. 
To evaluate the potential efficacy of intradermal injections of SIN LV-mediated 
COL7A1 gene-modified autologous fibroblasts in adults with RDEB at week 
(W) 2, month (M) 3 and M12 after the IMP injections. 
II. 
To screen for immune response against the recombinant C7 at W2, W4, M3, 
M6 and M12 after the IMP injections compared to baseline.  
 
4.1.3 Primary endpoint 
 Adverse events (AEs), Serious Adverse Events (SAEs), Adverse Reactions (ARs) 
and Serious Adverse Reactions (SARs) at each visit after screening over a 12-
month follow-up period. 
 
4.1.4 Secondary endpoints 
 Skin biopsy analysis of treated skin at W2, M3 and M12 compared to untreated 
skin: 
I. C7 protein expression by immunofluorescence microscopy (IF) 
II. Morphology of anchoring fibrils at the dermal-epidermal junction (DEJ) by 
transmission electron microscopy (TEM) 
III. Vector copy number by quantitative polymerase chain reaction (qPCR) 
 Serum analysis for: 
IV. Detection of anti-C7 antibodies by enzyme-linked immunosorbent assay 
(ELISA) 
(against 
NC1 
and 
NC2 
domains 
of 
C7) 
and 
indirect 
immunofluorescence (IIF) at W2, W4, M3, M6 and M12 post-injections 
V. Detection of T-cell responses to the full length C7 by enzyme-linked 

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immunosorbent spot (ELISPOT) assay at W4, M6 and M12 post-injections.  
 
4.2 Trial design  
This is a prospective phase 1, non-randomised, open label, single-centre, proof-of-
concept study. Each study participant will receive three intradermal injections of ex 
vivo transduced autologous fibroblasts expressing codon-optimised COL7A1 as the 
IMP on Day 0 only. Each injection of the IMP containing 0.8–1.2x106 cells suspended 
in 0.25ml of 0.9% saline, will be administered intradermally into 1cm2 area of intact 
skin (x3). Participants will be followed up with study interventions for a 12-month 
period at various time points as outlined in the trial timeline (Figure 16). All follow-
ups, where possible, will be co-ordinated to try to coincide with the individuals’ routine 
clinic reviews.  
Each subject will undergo an initial screening including a physical examination and 
assessment of disease severity. Blood analyses and skin biopsies will be performed 
at various time points as per the monitoring schedule (Table 2 and Figure 16). The 
second participant will be treated only if there is no safety concern 4 weeks after the 
first participant’s IMP injections. All patients with RDEB are followed up on a lifelong 
basis, and we will therefore be able to capture any long-term possible adverse effects 
related to the IMP. 

  
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4.3 Trial timeline 

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Figure 16: LENTICOL-F trial timeline. *Each injection consists of 0.8–1.2 million cells suspended in 0.25ml of 0.9% saline. Each of 
three syringes containing the IMP will be administered over 1 cm2 area of intact skin. . Virology tests done at V1A & V1B = HIV, 
HepBsAg, HepBcAb, HepCIgG, HTLV 1&2 and Treponema pallidum serology; Skin procurement and biopsy at V1B = 2 to 3 biopsies 

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(one 6mm procurement for tissue culture for IMP production; and one to two 4mm for C7IF, TEM, qPCR and histology); Skin biopsy 
@ W2, M3 and M12 = Two 6-8mm biopsies for C7 IF, TEM, qPCR and histology (one from injected site and one from non-injected 
site). All skin biopsies will be archived for integration site analyses in the rare event of clinically indicated serious adverse events. 
Pregnancy tests at V1A, V1B and V2: if serum is not obtainable, urine pregnancy test may be performed instead. Historical samples/ 
results may be used for C7 ELISA & IIF, ELISPOT, C7 IF microscopy & TEM and virology tests. 
NB: If W2 and M3 skin biopsy results are negative then M12 biopsy may not be performed.Abbreviations: BEBS score=Birmingham 
Epidermolysis Bullosa Severity Score; IMP=Investigational medicinal product.  
 
 
 
 
 
 
 
 
 

  
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5 Trial Medication 
5.1 Investigational Medicinal Product 
The patient specific gene-modified autologous fibroblasts are classified as an 
Investigational Medicinal Product (IMP) that comes under the European Union 
Directives (EUDs) on medicinal products and are subject to advanced therapy medicinal 
product (ATMP) guidelines and will be manufactured and expanded according to GMP 
regulations. Each participant will be screened for infectious diseases in accordance with 
the EU directive 2006/17 (Commission Directive 2006/17/EC of 8 February 2006) prior 
to skin biopsy for cell culture to produce autologous fibroblasts under GMP conditions. 
The pCCL-PGK-COL7 lentiviral vector gene modified autologous fibroblasts which are 
suspended in saline will be produced under MIA(IMP) 17328 at Cellular Therapies, 
Great Ormond Street Hospital for Children NHS Foundation Trust (GOSH), Great 
Ormond Street, London, WC1N 3JH. The IMP manufacturing site is non-commercial and 
part of an academic institution with a certified manufacturer/ importer’s licence as well as 
GMP and QP certification. The unit also has a HTA licence and JACIE accreditation for 
transplantation. 
 
Release of the IMP will require qualified person (QP) examination of the manufacturing 
record, liquid nitrogen temperature monitor recorder and other tests undertaken on cells 
to confirm transduction (copy number). Assays for information include evidence of 
reconstitution of C7 expression by flow cytometry, immunostaing and immunoblot (Table 
1). 
 
If the genetically modified autologous RDEB primary fibroblasts do not meet 
specification, the transduced cells will not be released. Efficacy and safety assessments 
will be carried out and data will be recorded in the Case Report Forms (CRFs). 

  
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Test 
Day/week during 
manufacturing 
Specification 
BACTalert 
Weekly 
No organisms detected 
 
Cell number 
Before release 
2.4-3.6X106 viable cells for gene 
modified fibroblasts 
Stable lentiviral 
transduction 
 
specific qPCR  
Copy number = 0.01-1.0 copy/cell 
C7 protein 
expression* 
Flow cytometry, immunostaining  
and immunoblot 
restoration of C7 expression 
compared to baseline C7 
Cell storage 
temperature 
Daily record 
Less or equal to -700C 
 
*depending on the mutation, some subjects may have residual protein expression 
Table 1: Specification for releasing transduced RDEB autologous fibroblasts.  
 
5.2 Dosing regimen 
Each trial participant will receive three intradermal injections of gene-modified 
autologous fibroblasts on Day 0. Each 1cm2 of intact skin will be injected with 0.8–
1.2x106 cells/ 0.25ml of normal saline. Once all 3 syringes are received, they will be 
injected as described in Section 5.7. The person handling and injecting the IMP will 
have appropriate training. Trial subjects will be monitored closely for any immediate 
allergic reactions and their vitals monitored every 15mins for half an hour. Vital signs 
will include blood pressure, respiratory rate, heart rate, pulse oximetry and 
temperature. 
 
 
 

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5.3 Description of IMP 
 
The patient-specific gene modified autologous fibroblasts (IMP) are generated from 
primary RDEB fibroblasts using pCCL-PGK-COL7 in its final formulation which is 
intended for injection. 
 The pCCL-PGK-COL7A1 vector is a third generation lentiviral vector encoding the 
gene codon optimised (co)COL7A1 (Figure 17). It is replication defective, does not 
encode pathogenic genes and is only packaged in the presence of minimal viral 
proteins provided in trans by transient transfection. It is self-inactivating (SIN), which 
restricts transcriptional activity to the internal expression cassette incorporating a 
human promoter PGK. The whole production phase for pCCL-PGK-COL7 involved 
one production run from which ~20 litres of virus supernatant were produced and 
then concentrated.  
Plasmid-1
Plasmid-2
Plasmid-3
Plasmid-4
 
Figure 17: Structure of the pCCL-PGK-COL7A1 lentiviral vector delivery system. 
 
The appearance of the final medicinal product is cell/saline suspension of 0.25ml in 
1ml luer-screw capped syringes. The product is labelled with a completed label 
corresponding to that in the batch manufacturing records (BMR) and further kept in a 
clean container sealed with clinical tape Tagaderm (from 3M) and then placed in a 
transport container for release. GMP compliant manufacturing and quality control 
procedures used to produce the IMP at the Cellular Therapies at GOSH are detailed in 
the accompanying IMPD. 

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5.4 Packaging and labelling 
Gene-modified fibroblasts are thawed, washed, counted and then packed into syringes 
capped with luer-screw. A total of 3 labelled syringes containing gene-modified 
autologous fibroblasts are then put into a petri dish, and sealed with clinical tape. Both 
syringes and petri dishes are clearly labelled with the study ID and IMP information.  
The product is finally placed into a medical cooler bag to maintain the product at 
ambient temperature. 
 
5.5 Distribution and shipment 
The IMP will be transported from the Cellular Therapies at GOSH to the trial site – 
clinical research facility at 15th Floor, Tower Wing, Guy’s Hospital by a specialised 
courier at the ambient temperature with a temperature monitor within 8 hrs of the IMP 
release. 
 
5.6 Storage, dispensing and return of the IMP 
Cultured transduced autologous fibroblasts are cryopreserved and store in a liquid 
nitrogen vessel with vapour phase. On the day of therapy, frozen cells will be removed 
from liquid nitrogen and quickly thawed in the Cellular Therapies at ICH/GOSH.  
Thawed cells are recovered and counted. After counting, cells are re-pelleted, and 
resuspended in 0.9% saline with an approximate concentration of 2-4x106 viable 
cells/ml. 0.25ml cell suspensions containing approximately 0.8-1.2x106 viable cells are 
packed into syringes capped with luer-screw. There will be three syringes of 
transduced cells provided. All syringes are clearly labelled with patient specific 
identifiers and IMP information and put into a transfer container for transport.  
 
 A Proof of Receipt form will be completed to confirm receipt of the product and faxed 
back to the sponsor and manufacturer. A copy of the form will be filed in the 
Investigator Site File. If it is found that the packaging has been tampered with or is 

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damaged, the sponsor will be notified immediately. The product(s) affected will not be 
used. Each dispensing will be recorded on the IMP Accountability Record. Any unused 
IMP may be returned to GOSH and stored for research use only.  
 
5.7 IMP administration 
The selected area of intact skin of each trial participant will be marked with an ink 
tattoo marker that is normally used for to mark the sites of radiotherapy on cancer 
patients. No radiotherapy or irradiation will be involved here. The schema of the tattoo 
sites may vary depending on the individuals but the investigator will aim to provide the 
minimal number of tattoos required in order to aid the accurate identification of the 
injection sites at future visits. Prior to injections, the intact skin will be cleaned with 
Chlorhexidine solution and the IMP administered by the trained investigator. Each 
injection of the IMP containing 0.8–1.2x106 cells suspended in 0.25ml of 0.9% saline, 
will be administered intradermally into 1cm2 area of intact skin (x3). Trial subjects will 
be monitored closely for any immediate allergic reactions and their vitals monitored 
every 15mins for half an hour.  
 
5.8 Drug accountability 
Each IMP dispensing will be documented on the IMP accountability form, along with 
unused returned study treatments. The study monitor will check all dispensing against 
the study schedule and record all administrations to the trial participants. A full review 
of the actual dispensed, administered and returned study treatments will be performed. 
 
   

  
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Figure 18: Map showing the sites of IMP injection and the timeline of the biopsies to be 
taken.  
 
5.9 Subject compliance 
The IMP will be administered to the trial participants attending St John’s Institute of 
Dermatology at GSTT in the designated Clinical Research Facilities, 15th floor, Guy’s 
Tower. Non compliance issues are not expected as the treatment is administered in 
hospital setting by a member of the research team. Skin biopsies and blood tests will 
also be performed by a member or members of the clinical team. However, because 
the study population involves adults with a severe blistering skin disease, if a blood 
test is unobtainable at a particular time, the investigators may use a blood sample 
obtained at a more convenient time (but chronologically related). Participants will be 
assessed for any adverse events and concomitant medications at each visit from 
V1Bbut otherwise, no assessments are scheduled to be performed by study subjects 

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at home so no subject compliance issues are expected. Support and guidance will be 
provided by members of the clinical research team as required. 
 
5.10 Concomitant medication 
For management of concomitant therapies, please refer to the Investigators Brochure 
(IB). The study subjects can continue to receive their regular medication(s). A 
complete listing of all concomitant medication received during the treatment phase will 
be recorded in the relevant CRF. 
 
6 Selection and withdrawal of subjects  
6.1 Inclusion criteria  
I. 
Clinical and genetic diagnosis of RDEB with confirmed bi-allelic COL7A1 mutations. 
II. 
A reduced number or morphologically abnormal anchoring fibrils confirmed by TEM. 
III. 
At least 5x3cm of intact skin on the trunk and/or extremities that is suitable for cell 
injections. 
IV. 
Able to undergo local anaesthesia. 
V. 
Subjects aged ≥ 17 years and able to give informed consent prior to the first study 
intervention. 
 
6.2 Exclusion criteria  
I. 
Subjects who received other investigational medicinal products within 6 months prior to 
enrolment into this study. 
II. 
Past medical history of biopsy proven skin malignancy. 
III. 
Subjects who have received immunotherapy including oral corticosteroids (Prednisolone 
>1mg/kg) for more than one week (intranasal and topical preparations are permitted) or 
chemotherapy within 60 days of enrolment into this study. 
IV. 
Known allergy to any of the constituents of the investigational medicinal product (IMP). 
V. 
Subjects with BOTH: 
 
positive serum antibodies to C7 confirmed by ELISA and 
 
positive IIF with binding to the base of salt split skin.  
VI. 
Subjects with positive results for HIV, Hepatitis B, Hepatitis C, HTLV or Syphilis. 

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VII. 
Subjects who are pregnant or of child-bearing potential who are neither abstinent nor 
practising an acceptable means of contraception when this is in line with the usual and 
preferred lifestyle of the subject, as determined by the Investigator, for 12 months after the 
cell injections. 
 
6.3 Selection of participants  
The patient population is under the care of the specialist EB team at St John’s Institute 
of Dermatology, GSTT. As a tertiary care centre, patients may have been referred 
from other regional district hospitals in the first instance. Potential study subjects will 
be identified from the National EB database that includes patients from both GSTT and 
nationally and an initial approach made by members of the clinical team. Potential 
participants may also be recruited from Patient Identification Centres. Study 
information sheets will be provided and individuals interested will be invited for further 
consultation and screening, assessed for eligibility, and consented as necessary. All 
individuals invited to participate in the study will have a minimum of 24 hours to make 
their decision. 
 
 
6.4 Randomisation procedure / code break 
This is a phase 1, non-randomised, open-label, single-centre, proof-of-concept study, 
therefore randomisation procedure/code break is not applicable. 
 
6.5 Withdrawal of subjects  
Participants have the right to withdraw from the study at any time for any reason.  The 
investigator also has the right to withdraw patients from the study drug in the event of 
inter-current illness, AEs, SAEs, SUSARs, protocol violations, cure, administrative 
reasons or other reasons.  It is understood by all concerned that an excessive rate of 
withdrawals can render the study un-interpretable, therefore unnecessary withdrawal 
of patients should be avoided.  Should a patient decide to withdraw from the study, all 
efforts will be made to report the reason for withdrawal as thoroughly as possible.  

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Should a participant withdraw from the study after the injection of the IMP, efforts will 
be made to continue to obtain follow-up data, with the permission of the participant. If 
a participant withdraw before the IMP injection, the participant will be replaced. 
 
6.6 Expected duration of trial 
The planned duration of the study will be 32 months. Each trial participant will be 
followed up for 12 months after the last intradermal injections of the IMP: gene-
modified autologous fibroblasts. We may recruit and produce the IMP for more than 
one participant at a time, though only administer the IMP with minimal intervals 
between individuals. 
 
Each participant will have a screening visit up to 4 months prior to Day (D) 0 when the 
IMP will be administered. There will be 7 scheduled follow-up visits over 12 months 
from Day 0.  Safety and efficacy will be assessed initially using the first 12 month data. 
Participants will then be reviewed every 4 months for the second year and then every 
6 months for the third year by the consultant dermatologist responsible for their clinical 
care (Professor John McGrath or Dr Jemima Mellerio who are the tenured senior 
clinical investigators for this trial) or an experienced EB specialist nurse. If a participant 
does not wish to attend for a 4-month or 6-month hospital visit, we will offer them the 
option of a telephone call to enquire about adverse events which will be recorded in 
the eCRF. All trial participants also have direct access to the EB specialist nurses 
should they develop any untoward symptoms requiring urgent clinical review, or wish 
to discuss any additional aspects of the clinical trial, in the interim between these time 
points. All adult subjects (aged ≥ 17 years) within the EB service are regularly seen at 
St John’s Institute of Dermatology at GSTT. Dr Jemima Mellerio and Professor John 
McGrath provide consultant care dedicated to this service. 
 
 
 
 

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6.7 End of trial definition 
The point will be defined as the final study participant’s last scheduled follow-up visit 
according to the protocol, which will be the 12 month follow-up visit after the 
intradermal injections of the IMP of the last subject entering the trial. 
 
7 Trial Procedures  
7.1 Trial course  
This clinical trial will be conducted within the clinical research facility (CRF), 15th floor, 
Tower Wing, Guy’s Hospital, London whilst some of the screening and follow up visits 
may be carried out in the Dermatology outpatient at GSTT. Subjects will be required to 
visit the clinic 10 times in 12 months. The screening assessment – visit (V1A) will be 
conducted up to 6 months prior to D0 – the day of the IMP injections. The timings of 
the subsequent visits will be relative to D0 (V2) (Table 3). Timing of screening, 
treatment and follow-up visits are summarised in the trial flowchart and monitoring 
schedule. 
 
7.2 Long-term follow-up 
Patients with RDEB are regularly reviewed, often for life, by the consultant 
Dermatologists in the multi-disciplinary setting at the regular EB clinics at GSTT, 
providing opportunities for detection of late adverse events. Once these studies are 
officially closed we will continue to report serious events related to the IMP (SARs and 
SUSARs) to the MHRA and London – West London & Gene Therapy Advisory 
Committee (GTAC) in the same way as during the study. 
 
7.3 Informed consent  
Once the Investigator has determined the subject’s potential eligibility for the study, the 
background of the proposed study, the benefits and risks of the procedures and the 
study, will be explained to the potential study participant both during the consultation 
and through the participant information sheet. Potential participants will be given a 
minimum of 24 hours to review the information received and may take the information 

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away to consider his/her decision to participate in the study. Written informed consent 
will be obtained from the subject by a physician listed on the delegation of duties log 
before any study related procedures, including screening tests, are performed. Prior to 
screening, the subject must first sign the institution's Independent Ethics Committee 
(IEC) approved informed consent form (ICF). Failure to provide informed consent 
renders the subject ineligible for the study. Study participants will be instructed that 
further information can be obtained at any time from the Investigator, and that they are 
free to withdraw their consent and to discontinue participation in the study at any time 
without any impact on the standard of their routine clinical care. 
 

  
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7.4. Procedures by visit 
Visit number 
V1A 
V1B 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
Timeline of each visit relative to the day of gene-modified 
fibroblast injections (D0) 
Up to 6M 
prior to D0 
At least 
6W prior 
to D0 
D0 
W1 
(±3D) 
W2 
(±3D) 
W4  
(±14D) 
M3 
(±1M) 
M6 
(±1M) 
M9 
(±1M) 
M12 
(±1M) 
Approximate duration of each visit (hours) 
2 
2 
1.5 
1.5 
2 
1.5 
2 
1.5 
1.5 
2 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
Inclusion / exclusion 
X 
X 
 
 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
Vital signs 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Serum pregnancy test for female participants with child-bearing 
potential (if serum is not obtainable, urine pregnancy test may 
be used) 
X 
X 
X 
 
 
 
 
 
 
 
aBEBS Score 
X 
 
 
 
X 
 
X 
 
 
X 

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Visit number 
V1A 
V1B 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
V9 
Timeline of each visit relative to the day of gene-modified 
fibroblast injections (D0) 
Up to 6M 
prior to D0 
At least 
6W prior 
to D0 
D0 
W1 
(±3D) 
W2 
(±3D) 
W4  
(±14D) 
M3 
(±1M) 
M6 
(±1M) 
M9 
(±1M) 
M12 
(±1M) 
bClinical photography 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood samples 
cBasic bloods 
X 
 
X 
 
X 
X 
X 
X 
 
X 
dVirology 
X 
X 
 
 
 
 
 
 
 
 
C7 ELISA & IIF 
X 
 
 
 
X 
X 
X 
X 
 
X 
ELISPOT 
 
 
X 
 
 
X 
 
X 
 
X 
eRCL 
X 
 
 
 
X 
X 
X 
 
 
X 
 
 
Skin biopsies 
 
 
C7 IF microscopy & TEM 
 
X 
 
 
X 
 
X 
 
 
X 
fqPCR 
 
X 
 
 
X 
 
X 
 
 
X 
gIntegration site analysis 
 
X 
 
 
X 
 
X 
 
 
X 
Skin procurement 
(1x6mm) 
Tissue culture for IMP production 
 
X 
 
 
 
 
 
 
 
 
hIntradermal injections of IMP  
 
 
X 
 
 
 
 
 
 
 
 
Adverse event(s) assessment 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant medications assessment 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 

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Table 2: LENTICOL-F trial flowchart with details of interventions at each visit. Abbreviations: V=Visit; D=Day; W=Week; 
M=Month. Consent will be obtained at each visit. On Day 0, your vital signs will be monitored every 15mins for 30mins after the gene-
modified fibroblast injections. There will be one-off readings of the vital signs on the rest of the other visits during the study. Historical 
samples/ results may be used for C7 ELISA & IIF, ELISPOT, C7 IF microscopy & TEM. Historical virology results performed within 
the last 3 months may be used. NB: If W2 and M3 skin biopsy results are negative then M12 biopsy may not be performed. 
aBirmingham Epidermolysis Bullosa Severity Score;  
bClinical photography of potential injection sites over an area of the intact skin will be taken at screening on visit 1, post-IMP 
injections on visit 2 and at each visit thereafter;  
cIncludes FBC, U&Es, LFTs, ESR and CRP; dIncludes HIV, HepBcAb, HepBsAg, HepCIgG, HTLV 1&2 and Treponema pallidum 
serology; eBlood samples will be archived for replication-competent lentivirus (RCL) - may be archived for at least 15 years and 
analysed in the event of relevant serious adverse reactions (SAEs) as clinically indicated;  
fVector copy number and COL7A1 gene expression will be measured using quantitative polymerase chain reaction (qPCR) and 
some skin samples retained for integration site analysis in the event of clinically indicated SAEs;  
gDNA will be extracted from part of the skin biopsy and archived for integration site analysis in the event of relevant adverse 
reactions as clinically indicated; 
hEach of 3 intradermal injections of IMP (COL7A1 gene-modified autologous fibroblasts) will be administered to 1cm2 of intact skin  

  
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7.5 Laboratory tests 
Blood samples (approximately 15-20ml) will be taken by venepuncture.  At screening, 
trial subjects will be tested for HIV, hepatitis B surface antigen (HepBsAg), hepatitis B 
core antibody (HepBcAb) and hepatitis C IgG (HepCIgG), HTLV1&2 and Treponema 
pallidum serology. The subjects will also have a blood DNA sample sent for mutation 
analysis for the COL7A1 gene if this result is not already available. All of the blood 
tests will be analysed at the Viapath laboratories apart from the DNA mutation 
analysis, and ELISA and IIF which will be performed at The National Diagnostic EB 
laboratory and Viapath Immunofluorescence Laboratory, both at St Thomas’ Hospital, 
respectively. Blood samples for RCL analysis will be archived. Blood samples for 
ELISPOT will be couriered to Institut Imagine in Paris as shown below. qPCR for the 
COL7A1 expression on the skin biopsy will be performed at Cellular Therapies at 
GOSH (Table 3 and 4). The following blood analyses will be performed at screening 
and at each subsequent review as outlined in the trial flowchart in section 4.3. 
 
Type of Blood 
Analysis 
Tests 
Laboratory 
Haematology 
FBC 
ESR 
Viapath 
Biochemistry 
U&Es 
LFTs 
CRP 
Beta-HCG 
Viapath 
Infection screen 
(at baseline 
HIV 
HepBsAg 
Viapath 

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only) 
HepBcAb 
HepCIgG 
HTLV 1&2 
Treponema pallidum 
 
Serum 
 
ELISA and IIF for C7 antibodies 
Immunodermatology Laboratory 
(Viapath) 
ELISPOT for detection of T-cell 
responses to the full length C7 
Institut Imagine, INSERM UMR1163 
Équipe du Pr. Hovnanian 
2ème étage, pièce 221-B2 ou 220-B1 
142 rue du Cherche-Midi 
75015 PARIS 
Replication competent lentivirus 
Archived at UCL ICH/Guy’s 
Blood for DNA 
analysis 
(screening only 
if not available) 
Mutational analysis 
The Robin Eady National Diagnostic 
EB laboratory 
Table 3: Types of blood analysis and where tests are performed for the trial.  
 
Skin biopsies will be taken at three time points – from injected site and approximately 
3cm away from the injected site (non-injected site); at W2, M3 and M12. NB: If W2 and 
M3 skin biopsy results are negative then M12 biopsy may not be performed. The 
samples will be analysed for the following: 
Type of Analysis Tests 
Laboratory 
Skin biopsies 
1)  C7 IF microscopy (stored in Michel’s 
medium) 
2) TEM of DEJ for anchoring fibrils 
The Robin Eady 
National Diagnostic 
EB laboratory 
3) qPCR for vector copy number and 
COL7A1 gene expression 
Cellular Therapies at 
GOSH/Guy’s/ The 
Robin Eady National 
Diagnostic EB 

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laboratory 
5) Integration site analysis 
Archived at UCL 
ICH/Guy’s 
 
 
Table 4: Table showing where skin biopsy analyses are performed for the trial. 
 
8 Assessment of Efficacy  
8.1 Primary efficacy parameters 
The primary efficacy of this phase 1 trial will be based on the safety of the administration of IMP 
in adults with RDEB, as analysed according to the incidence of Adverse events (AEs), Serious 
Adverse Events (SAEs), Adverse Reactions (ARs) and Serious Adverse Reactions (SARs) at 
each visit after screening over a 12-month follow-up period. 
 
8.2 Secondary efficacy parameters 
 
Skin biopsy analysis of treated skin at W2, M3 and M12 compared to untreated skin: 
I. 
C7 protein expression by IF microscopy 
II. 
Morphology of anchoring fibrils at the DEJ by TEM 
III. 
Vector copy number by qPCR 
 
 
Serum analysis for: 
IV. 
Detection of anti-C7 antibodies by ELISA (against NC1 and NC2 domains of C7) and IIF 
at W2, W4, M3, M6 and M12 post-injections 
V. 
Detection of T-cell responses to the full length C7 by ELISPOT assay at W4, M6 and 
M12 post-injections. 
 
8.3 Procedures for assessing efficacy parameters 
Clinical assessment: Review of adverse events and concomitant medications will be 
performed at each visit from V1B. Physical examination, vital signs and clinical 

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photographyof the injection sites will be performed at each visit. BEBS score will be 
documented at screening, W2, M3 and M12. Efforts will be made for the clinical 
assessment to be performed by the same assessor for each participant throughout the 
trial to minimise inter-observer variations. 
 
Biochemical analysis: Routine blood analysis including FBC, U&Es, LFTs, ESR and 
CRP will be measured at screening, D0, W2, W4, M3, M6 and M12 to monitor any 
potential systemic adverse effects. At the screening visits, virology tests for HIV, 
HepBcAb, HepBsAg, HepCIgG, HTLV1&2 and Treponema pallidum serology will be 
performed to screen for blood borne viral infections and treated as per GSTT 
guidelines. Analysis of antibodies for C7 and IIF will be performed both prior to and 
after IMP administration (screening, W2, W4, M3, M6 and M12) in order to detect any 
potential development of EB acquisita in participants with RDEB as a potential 
adverse effect of introducing new C7 expression from the gene-modified autologous 
fibroblasts. ELISPOT assay for detection of T-cell responses to the full length C7 will 
be performed at screening, W4, M6 and M12. 
Skin Ultrastructural Analysis: Skin biopsies will be taken at four time points 
(screening, W2, M3 and M12) for C7 IF microscopy to assess a change in the level of 
C7 expression. This will be correlated with the presence and morphology of anchoring 
fibrils at the DEJ as assessed by TEM.  

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Vector copy number (VCN) and COL7A1 gene expression: Real-time polymerase 
chain reaction (qPCR) will be used to analyse vector copy in the skin biopsy of the cell 
injection sites at W2, M3 and M12. 
Histology: Skin histology will be used to analyse the difference in the morphology of 
DEJ between treated and untreated skin at W2, M3 and M12. 
 
9 Assessment of Safety  
Adverse events and concomitant medications will be reviewed at every visit after V1A. 
Clinical photographs will be taken at each visit in order to monitor changes in the 
injection sites especially with regards to any suspicious lesions or adverse reactions 
locally. If suspicious lesions are detected then, the participant will be immediately 
assessed and managed by a consultant dermatologist and if found malignant then, the 
lesion will be excised as per GSTT guidelines. Physical examination and vital signs will 
be performed at every visit in order to assess any potential systemic adverse effects 
and treated as appropriate as per GSTT guidelines. 
 
9.1 Assessment of benefits and risks 
Potential benefits 
The injection of fibroblasts expressing COL7A1 may allow localised recovery of 
anchoring fibril production and assembly, and restoration of basement membrane 
integrity. This study is designed to test the safety of injecting autologous gene-
modified fibroblasts, and the three injection sites will allow serial histological 
assessment of skin architecture in direct comparison to a paired site injected with non-
engineered autologous fibroblasts. Whilst no direct benefit is expected for the subjects 
at this stage, establishing safety would allow more extensive therapies to be offered. 
 

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Potential risks 
Insertional mutagenesis 
Gamma-retroviral vector mediated gene therapy for another skin disease, JEB, has 
shown impressive therapeutic efficacy (Mavilio et al. 2006) and long-term safety (>6 
years after skin grafting) (Dr F.Mavilio, personal communication).  Of note in RDEB, 
previous modelling suggests that deficiency off COL7A1 is associated with a 
carcinogenic phenotype. Fibroblasts collected form RDEB patients with down-
regulated COL7A1 or null C7 expression exhibit promotion of substrate adhesion and 
invasion of tumour keratinocytes (Ng et al. 2012).  Correction of this deficiency is 
expected to reduce transformational risk. 
 
Concern regarding the insertional mutagenesis using gamma-RVs has prompted the 
development of safer delivery mechanisms using lentiviral vectors. Although there is 
no published date indicating the safety and efficacy of LVs configured with internal 
promoter elements in fibroblast transduction, LVs encoding the human internal 
promoters driving expression of various transgenes have been used to treat Wiskott-
Aldrich Syndrome  (Galy et al. 2011; Scaramuzza et al. 2012), ADA-SCID (Gasper et 
al. 2004) and chronic granulomatous disease (Grez et al. 2010). Older configurations 
using viral internal LTRs have been reported safe in children treated for 
adrenoleukodystrophy (Cartier et al. 2009; Biffi et al. 2011).  
 
The pCCL-PGK-COL7A1 vector is a GMP grade recombinant LV for ex vivo gene 
therapy. This third generation SIN lentiviral vector lacks viral coding sequences that 
could give rise to the formation of replication competent lentivirus or immunogenic 
peptides. It is also devoid of viral enhancer-promoter sequences that are known to be 
involved in insertional mutagenesis by retroviruses and derived vectors. The internal 
promoter is the 516bp sequence of human PGK gene promoter (Ginn et al. 2010; 
Huston et al. 2011). A mutated woodchuck post-transcriptional regulatory element 
(WPRE) sequence is incorporated in the vector to enhance transgene expression. 
 

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Replication-competent lentivirus (RCL)  
Emergence or transfer of RCL is highly unlikely in this SIN configured vector with a 
four plasmid packaging system. The vector has been found to be free of RCL and has 
not been detected in any preclinical study with similar vectors. Culture supernatant, 
transduced cells and patient samples will however be archived for RCL testing should 
the need to analyse these arise. Blood samples will be taken from the participating 
subjects at the screening visit, week 2, month 3 and month 12. These samples will be 
archived at the research laboratories, 9th floor Tower Wing at Guy’s Hospital for at 
least 15 years and will be analysed for RCL in the event of relevant adverse reactions 
as clinically indicated. 
 
Germ-line transmission of vector sequences 
This is an ex vivo modification procedure with extended culture periods, therefore the 
risk of germ line transmission is negligible. 
 
Allergic/immunological responses  
Whilst the risk of immune responses against excipients is extremely low, immediate or 
delayed host-mediated immune responses against recombinant C7 are possible. All 
injections and monitoring will be given in a clinical area supported by resuscitation 
facilities (Clinical Research Facility).  Participants will be monitored regularly as part of 
the study protocol and asked to report any systemic symptoms to the research team. 
Blood analysis for ELISA and IIF to C7 will be undertaken at baseline, W2, M1, M3, 
M6 and M12 to monitor for a delayed immunological response to the IMP, as well as 
ELISPOT assay for detection of T-cell responses to the full length C7. 
 
Expected Adverse Reactions (ARs) following IMP administration 
The following ARs, such as any untoward and unintended response in a subject to an 
IMP which is related to any dose administered to that subject, are considered 
expected reactions that could occur following administration of the IMP: 

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i) 
Localised mild pain during administration 
ii) 
Mild bruising at the site of the administration, localized erythema, pruritus 
iii) 
Scarring and dyspigmentation at the site of injection 
 
Recognition and treatment of possible overdose 
The intradermal injections will be performed by the Chief Investigator or delegated 
clinician who will have been appropriately trained in this procedure. Only the aliquoted 
amount of fibroblasts in normal saline solution (0.25ml) will be given at each site. The 
IMP volume will be labelled and crosschecked by the clinical research nurse/research 
team member at the time of administration and documented in the participants’ drug 
charts, as per standard SOPs for drug administration used at the clinical site.  
 
Other adverse clinical consequences  
As with any new form of therapy, there may be risks which are unknown or not 
anticipated.  
 
9.2 Specification, timing and recording of safety parameters 
Adverse events (AEs): All researchers involved in this trial must be familiar with the 
assessment of safety for the trial. Researchers are defined as any clinical researcher, 
investigator, research nurse or other personnel member conducting trial related clinical 
research activity. It is imperative that all investigators have a thorough understanding 
of anticipated AEs and the reporting process of these events. All AEs will be recorded 
in accordance with ICH GCP, EU directives and King’s Health Partners Clinical Trials 
Office (KHP-CTO) SOPs. 
   
Medical history: A full medical history will be taken from the patient by a member of 
the clinical team during the screening visit. This ensures that the study participants 
satisfy the eligibility criteria and do not have any medical conditions that would make it 

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inappropriate for them to participate in the study. All known medical history since birth, 
including ongoing conditions, will be recorded in the CRF, together with any other 
clinically important findings that the Investigator considers relevant to the study.  
 
Physical examination: A physical examination of each subject will be conducted by 
the Investigator at all trial visits. All abnormal findings (including gastrostomies and 
surgical scars) will be documented at screening. Any changes that occur during the 
study will be documented.  
 
Vital signs: Vital signs (heart rate, blood pressure, respiratory rate and temperature) 
will be measured at screening, day 0 (treatment day), and at every follow-up visit. 
 
Serum analyses: Routine blood analysis including FBC, U&Es, LFTs, ESR and CRP 
will be measured at screening, D0, W2, W4, M3, M6 and M12 to monitor any potential 
systemic adverse effects. Analysis of antibodies for C7 and IIF will be performed both 
prior to and after IMP administration (screening, W2, W4, M3, M6 and M12) in order to 
detect any potential development of EB acquisita in participants with RDEB as a 
potential adverse effect of introducing new C7 expression from the gene-modified 
autologous fibroblasts. ELISPOT assay for detection of T-cell responses to the full 
length C7 will be performed at screening, W4, M6 and M12.  
9.3 Procedures for recording and reporting Adverse Events 
All researchers involved in this trial must be familiar with the assessment of safety for 
the trial. Researchers are defined as any clinical researcher, investigator, research 
nurse or other personnel member conducting trial-related clinical research activity. It is 
imperative that all investigators have a thorough understanding of anticipated adverse 
events (AEs) and the reporting process of these events. All AEs will be recorded in 
accordance with GCP, EU directives and KHP-CTO SOPs.  
 
The Medicines for Human Use (Clinical Trials) Regulations 2004 and Amended 
Regulations 2006 gives the following definitions: 

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Adverse Event (AE): Any untoward medical occurrence in a subject to whom a 
medicinal product has been administered including occurrences which are not 
necessarily caused by or related to that product. 
Adverse Reaction (AR): Any untoward and unintended response in a subject to an 
investigational medicinal product which is related to any dose administered to that 
subject. 
Unexpected Adverse Reaction (UAR): An adverse reaction the nature and severity of 
which is not consistent with the information about the medicinal product in question set 
out in the Investigator's Brochure (IB) relating to the trial in question (for any other 
investigational product). 
Serious Adverse Event (SAE), Serious Adverse Reaction (SAR) or Unexpected 
Serious Adverse Reaction (USAR): Any adverse event, adverse  reaction or 
unexpected adverse reaction, respectively, that 
 
Results in death; 
 
Is life-threatening; 
 
Required hospitalisation or prolongation of  existing   hospitalisation; 
 
Results in persistent or significant disability or incapacity; 
 
Consists of a congenital anomaly or birth defect. 
Suspected Unexpected Serious Adverse Reaction (SUSAR): Is defined as a serious 
adverse drug reaction, the nature and severity of which is not consistent with the 
information about the medicinal product in question set out in the IB relating to the trial 
in question, and which results in any of the outcomes set above. 
Important Medical Events (IME) & Pregnancy: Although not a serious adverse event, 
any unplanned pregnancy will also be reported via the SAE reporting system as stated 
below.  
Important medical events that may not be immediately life-threatening or result in death 
or hospitalisation but may jeopardise the patient or may require intervention to prevent 
one of the other outcomes listed in the definition above should also be considered 
serious.  

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9.4 Reporting Responsibilities 
All AEs, SAEs, SARs and SUSARs will be documented in the patients’ notes. Events 
defined as serious will be reported in as much detail as possible to the sponsor as soon 
as possible (or immediately) within 24 hours after the Investigator has been notified. 
King’s College London (KCL) has delegated the delivery of the Sponsor’s responsibility 
for Pharmacovigilance (as defined in Regulation 5 of the Medicines for Human Use 
(Clinical Trials) Regulations 2004 to the King’s Health Partners Clinical Trials Office 
(KHP-CTO). The latest version of the SAE reporting form is available on the KHP-CTO 
website. 
 
The Chief Investigator (CI) has overall responsibility for the conduct of a study/trial. 
The CI has coordinating responsibility for reporting SAEs to the study/trial Sponsor. 
 
The Principal Investigator (PI) has overall responsibility for the study/trial being 
conducted in the site. The PI is responsible for informing the CI, or the organising 
research team, of all SAEs that occur. 
 
The Sponsor of the study/trial is responsible for ensuring that all relevant information 
about a SUSAR is reported to the Competent Authority (the Medicines and Healthcare 
products Regulatory Agency). 
 
All SAEs, SARs and SUSARs (except those specified in this protocol as not requiring 
reporting) will be reported immediately (and certainly no later than 24hrs) by the 
Investigator to the KHP-CTO and CI for review in accordance with the current 
Pharmacovigilance Policy. All medical emergencies including severe allergic reaction, 
anaphylaxis, pulmonary embolism, pneumonia and myocardial infarction will be 
managed according to GSTT SOPs.  
 
Death as a result of disease progression and other events that are primary or secondary 

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outcome measures are not considered to be SAEs and should be reported in the normal 
way, on the appropriate CRF. 
 
The KHP-CTO will report SUSARs to the regulatory authorities (MHRA, competent 
authorities of other EEA (European Economic Area) states in which the trial is taking 
place. The CI will report to the relevant ethics committee. Reporting timelines are as 
follows: 
 SUSARs which are fatal or life-threatening must be reported not later than 7 days 
after the sponsor is first aware of the reaction. Any additional relevant information 
must be reported within a further 8 days. 
 SUSARs that are not fatal or life-threatening must be reported within 15 days of the 
sponsor first becoming aware of the reaction.   
 The CI and KHP-CTO (on behalf of the co-sponsors), will submit a Development 
Safety Update Report (DSUR) relating to this trial IMP, to the MHRA and REC 
annually.  
 
9.5 Adverse Events (AEs) and Serious Adverse Events (SAEs) that do not 
require reporting 
The following will not require reporting although will be documented on the eCRF. 
Adverse event as a result of venesection and cannulation include: 
i) Mild bruising at site of needle puncture 
Adverse event as a result of the skin biopsy include: 
i) Mild bruising at the site of the skin biopsy 
ii) Cutaneous skin infection requiring oral course of antibiotics 
A small scar will result after each skin biopsy, resembling an old chickenpox 
scar. 
All hospitalisations which are expected to take place as a result of disease progression 
including any planned elective surgeries will not be reported at the interim and the 

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end-of-trial safety data analysis but will be documented in the patient notes. This may 
include but not limited to: 
 Skin 
o Skin infection 
o Review of a wound 
o Skin blisters/ erosions as part of EB wound 
 Teeth 
o Dental extractions/ abscess  
 Hand 
o Hand surgery 
o De-gloving injury  
o Occupational Therapy review and splints 
 Transfusions 
 Overnight hospital stay for reviews 
 Blood monitoring, routine blood tests 
 Corneal abrasions 
 Eye Infections 
 Chronic eye problems due to EB 
 Gastrointestinal problems  
o Dysphagia, oesohageal stricture and dilation 
o Gastrostomy insertion, leakage or blockage/  jejunal tube insertion 
 leakage/ blockage 
o Regurgitation or vomiting as a result of oesophageal blisters/ stricture 
o NG tube insertion 
o Constipation 
 Vertebral or other fractures 
 IV bisphosphonates 

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 IV iron infusion 
 Contractures requiring physiotherapy 
 Hydrotherapy 
 ENT 
o Tonsillitis 
o Otitis externa 
o Otitis media 
 Pain 
o pain assessment for acute or chronic pain 
o pain from ongoing EB wounds 
 Accidental injuries causing minor skin wounds 
Thus, any hospitalisation that is not associated with the IMP will not be reported, 
unless the IMP results in a prolongation of existing hospitalisation. Unscheduled 
and/or emergency hospitalisations that are not expected due to the natural course of 
the disease will be reported via the sponsor’s normal serious adverse event (SAE) 
reporting practice. AEs will be reviewed at each visit both from the patient’s report as 
well as documentation from medical notes. 
 
9.6 Treatment stopping rules 
The trial may be prematurely discontinued by the Sponsor, Chief Investigator or 
Regulatory Authority on the basis of new safety information or for other reasons given 
by the Data Monitoring & Ethics Committee/ Trial Steering Committee regulatory 
authority or ethics committee concerned. 
 
The trial may also be prematurely discontinued due to lack of recruitment or upon 
advice from a Trial Steering Committee, who will advise on whether to continue or 
discontinue the study and make a recommendation to the sponsor. If the trial is 
prematurely discontinued, active participants will be informed and no further participant 

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data will be collected. The Competent Authority and Research Ethics Committee will 
be informed and no further participant data will be collected. 
 
Treatment stopping rules for individual patients will include voluntary discontinuation 
by the patient, a change in inclusion or exclusion criteria of the clinical trial or the 
development of a SUSAR/SAE/SAR. 
 
10 
Statistics 
10.1 Sample size 
The aim of this phase 1 trial will be to recruit approximately 5–10 patients from GSTT 
EB clinical database. As described previously this is a rare disease with an incidence 
of 1 in 17,000 live births. As per our statistician’s advice, even if no subjects 
experienced adverse reactions (i.e. 0% AR), a sample size of 10 would give a 95% 
confidence interval of 0% to 31% (exact CI from Stata®).  A sample of 10 could still 
potentially provide reassurance that no more than 3 in 10 subjects should experience 
a given side-effect. 

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10.2 Analysis 
All study participants will be included in all analyses unless they have withdrawn 
consent. For subjects who drop-out rather than withdrawn consent, a request will be 
made to use data collected prior to drop-out. Descriptive statistics such as means, 
standard deviations (SDs), minimum and maximum frequencies and proportions, as 
appropriate will be presented. No hypothesis tests will be performed. When all 
participants complete Visit 7 (Month 6), an interim analysis of the collected data will be 
performed and the results will be published in a peer-reviewed journal and there will be 
another analysis at the end of the trial (12 months after the last participant’s cell 
injections).  
 
11 Trial Steering Committee  
The Trial Steering Committee (TSC) consists of a panel of scientists specialised in the 
field of EB who will provide constructive criticism and suggestions to improve both the 
clinical trial implementation and data evaluation. 
The Steering Committee shall be responsible for: 
 Providing feedback and routine assessment of progress of LENTICOL-F as a 
whole. 
 Ensuring all the work meets the functional requirements and is in accordance 
with the project deliverables and where necessary GCP and/or GMP compliant. 
 Maintaining a complete overview of the scientific and process development 
progress of the project in relation to external progress in the various fields 
related to the project. 
 Collecting and reviewing aggregate safety data, with the authority to 
recommend premature termination of the trial if felt necessary.  
 
12 Direct Access to Source Data and Documents 
The Investigator(s) will permit trial-related monitoring, audits, IREC review, and 

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regulatory inspections by providing the Sponsors, Regulators and REC direct access 
to source data and other documents (e.g. patients’ case sheets, blood test reports, X-
ray reports, histology reports etc.). The Investigators will also permit authorised 
inspectors to inspect all facilities and records relating to the study and aid the 
inspector(s) to perform the audit in a timely manner. 
 
13 Ethics & Regulatory Approvals 
The trial will be conducted in compliance with the principles of the Declaration of 
Helsinki (1996), the principles of GCP and in accordance with all applicable 
regulatory requirements including but not limited to the Research Governance 
Framework and the Medicines for Human Use (Clinical Trial) Regulations 2004, as 
amended in 2006 and any subsequent amendments. 
 
Prior to the initiation of the trial, the Investigator will submit the protocol, subject 
information sheet, subject consent form, letter to the GP and any other related 
documents to the appropriate Independent Research Ethics Committee (IREC), 
London – West London & GTAC, and to the MHRA for Clinical Trial Authorisation. 
The Chief Investigator will submit a final report at conclusion of the trial to the KHP-
CTO (on behalf of the Sponsor), the REC and the MHRA within the timelines defined 
in the Regulations. 
 
14 Quality Assurance 
Monitoring of this trial will be to ensure compliance with Good Clinical Practice and 
scientific integrity will be managed and oversight retained by the KHP-CTO Quality 
Team. The relevant regulatory authorities also have the right to conduct an inspection 
of the study, that the study was, in fact, performed at stated sites and that the data 
reported to the authorities reflects the data in the records of the Investigator. The 
authorities also inspect such studies to verify that the studies were conducted in 
accordance with government regulations relating to the IEC and informed consent. It is 

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the joint responsibility of the sponsor, co-sponsor and the Investigators to ensure that 
the study has been conducted in line with all relevant regulations and KCL standard 
operating procedures. 
 
15 Data Handling  
The Chief Investigator will act as custodian for the trial data. The following guidelines 
will be strictly adhered to: 
 Patient data will be anonymised and coded. 
 This clinical trial will use the Biomedical Research Centre’s (BRCs) industry-
standard secure database called MedSciNet. All access to the MedSciNet data 
system is controlled using a Username/Password login. Passwords are encrypted 
before 
storing 
to 
database, 
using 
SHA-1 
hash 
(MS 
.NET 
SHA1CryptoServiceProvider). These are created and controlled by Administrative 
users of the system as identified by the Chief Investigator. The data is stored in the 
BRC eCRF servers which are located in Holland and meet or exceed all MHRA 
requirements for CTIMP data storage. Only the server administrator has access to 
this server and the core database, via remote connection. The back up process is 
twofold: every 24hrs the database is backed-up to the server and every 7 days the 
entire server is backed up to an archival tape system.  
 The final database will be stored in a read-only format for 5 years before being 
archived onto the server for a further 2 years. At this point the archived version will 
be compressed and will remain in the server and a copy will be sent to the CI. The 
documents that relate to ATIMP traceability will be retained for a minimum of 30 
years after the expiry date of the IMP. In order to meet regulatory requirement, at 
GSTT, hard copies of Care Report Forms will be kept in a locked filing cabinet in a 
locked office with the Trial Site File as per King’s Health Partners-Clinical Trial 
Office (KHP-CTO) Standard Operating Procedure (SOP) – The Creation and 
Maintenance of Trial Master Files and Essential Documentation (available on the 
KHP-CTO website). The Trial Master File will be archived as per current KHP-CTO 
SOPs. All trial data will be stored in line with the Medicines for Human Use (Clinical 

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Trials) Amended Regulations 2006 and the Data Protection Act and archived in line 
with the Medicines for Human Use (Clinical Trials) Amended Regulations 2006 as 
defined in the KHP-CTO Archiving SOP. 
 
16 Data Management 
Electronic Case Report Forms (eCRFs) will used to capture the data of each subject 
enrolled in the study. These will be standardised and protocol specific and create a 
time stamped electronic record of any amendments or additions to the document. 
eCRF pages will be monitored on an on-going basis by the study monitors.  
 
17 Publication Policy  
It is intended that the results of the study will be reported and disseminated through 
posters and oral presentations at international conferences, scientific meetings and in 
high impact peer-reviewed scientific journals. Publicity will be performed through press 
releases at the local, national and international levels. Press releases will be co-
ordinated through King’s College London, Sohana Research Fund (SRF) and DEBRA 
UK. SRF and DEBRA UK will also inform patients and potential participants through 
their website and newsletters. 
 
18 Insurance / Indemnity  
The trial is co-sponsored by King’s College London and Guy’s and St. Thomas’ NHS 
Foundation Trust. The co-sponsors will at all times maintain adequate insurance in 
relation to the study independently. King’s College London, through its own 
professional indemnity (Clinical Trials) and no fault compensation and the Trust having 
duty of care to patients via NHS indemnity cover, in respect of any claims arising as a 
result of clinical negligence by its employees, brought by or on behalf of a study 
patient. 
 

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19 Financial Aspects  
Funding to conduct this trial is provided by Sohana Research Fund (SRF) and 
registered charity DEBRA UK. 
 
20 Signatures 
Chief Investigator: Professor John McGrath 
 
 
Signature:  
 
 
 
 
 
 
Date: 
 
 
21 References 
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Pillay E: Epidermolysis bullosa. Part 1: causes, presentation and complications. Br J Nurs 2008; 17: 
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Titeux M, Pendaries V, Zanta-Boussif MA et al. SIN retroviral vectors expressing COL7A1 under human 
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Dermatol 2003; 121: 1021–28. 
 
 

  
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Appendix 1: Development of gene therapy for inherited skin diseases 
Method 
Target 
gene 
Condition 
Target cell 
Comments 
Reference 
Self-inactivating 
lentiviral vector 
COL7A1 
Recessive 
dystrophic EB 
In vivo 
In vivo inoculation of skin equivalents grafted onto mice, C7 
expression up to 3 months 
1 
Self-inactivating 
lentiviral vector 
COL7A1 
Recessive 
dystrophic EB 
Fibroblast 
Gene corrected fibroblasts injected into murine skin able to 
produce C7 at basement membrane zone 
2 
Gamma 
retroviral vector 
COL7A1 
Recessive 
dystrophic EB 
Keratinocyte 
Construction of skin equivalents using classical retroviral gene 
transfer; transduction efficiency 40% 
3 
Self-inactivating 
gamma 
retroviral vector 
COL7A1 
Recessive 
dystrophic EB 
Keratinocyte + 
Fibroblast 
Generation of human skin equivalents showing in vivo 
functional correction of phenotype; C7 expression of 
recombinant C7 ranging between 30-80% keratinocytes and 
40-70% fibroblasts 
4 
Retroviral vector 
COL7A1 
Recessive 
dystrophic EB 
Keratinocyte 
Genetic correction of both dog RDEB and human primary 
RDEB cells 
5 
φC31 integrase 
based 
plasmid 
COL7A1 
Recessive 
dystrophic EB 
Keratinocyte 
Nonviral approach into primary RDEB cells; regenerated skin 
(40% efficiency); stable correction required short-term drug 
selection and enrichment 
6 
3’ pre-
transplicing 
molecule 
COL7A1 
Recessive 
dystrophic EB 
Keratinocyte 
Trans-splicing used to reduce size of COL7A1 transcript and 
therefore risk of genetic rearrangement 
 
 
 
7 
φC31 integrase 
based 
LAMB3 
Junctional EB 
Keratinocyte 
Nonviral approach showing corrected hemidesmosome 
formation in JEB primary cells 
8 

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plasmid 
Transposase 
(sleeping 
beauty) 
LAMB3 
Junctional EB 
Keratinocyte 
Sleeping beauty transposable element used to integrate 
LAMB3 cDNA into epidermal cells in 6 JEB patient primary 
cells; transfected cells enriched using drug selection. 
9 
PAC (P1-based 
artificial 
chromosome 
clone) 
COL7A1 
Recessive 
dystrophic EB 
Keratinocyte 
Gene transfer using micro-injection of entire human locus 
COL7A1 in a P1 derived artificial chromosome  
10 
TALEN gene 
editing 
COL7A1 
Recessive 
dystrophic EB 
Fibroblasts 
Transciption activator-like endonucleases (TALEN) gene 
editing of RDEB fibroblasts subsequently reprogrammed into 
iPS cells (induced pluripotent stem cells) 
11 
Gamma 
retroviral vector 
COL17A1 
Junctional EB 
Keratinocyte 
Restoration of full-length BP180 protein expression in primary 
keratinocytes then used to regenerate human skin on 
immunodeficient mice 
12 
Gamma 
retroviral vector 
 
LAMB3 
Junctional EB 
Keratinocyte 
Only case of successful gene transfer in an inherited skin 
disorder in an adult patient with non-herlitz junctional EB; 
restored phenotype and laminin 332 expression 7 years post 
graft 
13 
Self-inactivating 
lentiviral 
vector 
 
LAMB3 
Junctional EB 
Keratinocyte 
Pre-clinical study using potentially safer LV 
14 
Gamma 
retroviral vector 
XPC 
Xeroderma 
pigmentosum 
Keratinocyte + 
Fibroblast 
Functional in vitro correction of XPC keratinocytes 
15 
Adenoviral 
vector 
XPA 
Xeroderma 
pigmentosum 
In vivo 
In vivo inoculation of recombinant AV vector in UV irradiated 
murine skin 
16 
Self-inactivating 
lentiviral vector 
XPA/ 
XPC/ 
XPD 
Xeroderma 
pigmentosum 
Fibroblast 
Efficient SIN LV vector able to correct cellular phenotype in XP 
17 
Gamma 
retroviral vector 
XPC 
Xeroderma 
pigmentosum 
Keratinocyte 
MLV derived vector able to transduce primary cells and 
maintain XPC expression in up to 130 population doublings 
18 

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ABBREVIATIONS: AV, adenoviral; C7, type VII collagen; EB, epidermolysis bullosa; LI, lamellar ichthyosis; LV, lentiviral; PC, 
pachyonychia congenita; RDEB,recessive dystrophic epidermolysis bullosa; SIN, self-inactivating vectors. 
 
Appendix 1 References 
1. Woodley D T, Keene DR, Atha T, Huang Y, Ram R, Kasahara N and Chen M (2004). Intradermal injection of lentiviral vectors 
corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo. Mol Ther 2004; 10(2): 318-326 
2. Woodley DT, Krueger GG, Jorgensen CM, et al. Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone 
can produce type VII collagen at the basement membrane zone. J Invest Dermatol. 2003;121(5):1021-1028. 
3. Gache Y, Baldeschi C, Del Rio M, et al. Construction of skin equivalents for gene therapy of recessive dystrophic epidermolysis 
bullosa. Human Gene Ther. 2004;15(10):921-933. 
4. Titeux M, Pendaries V, Zanta-Boussif MA, et al. SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo 
gene therapy of recessive dystrophic epidermolysis bullosa. Mol Ther. 2010;18(8):1509-1518. 
5. Baldeschi C, Gache Y, Rattenholl A, et al. Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral 
vectors. Hum Mol Genet. 2003;12(15):1897-1905. 
TALEN gene 
editing 
XPC 
Xeroderma 
pigmentosum 
Fibroblast cell 
line 
Successful targeted gene correction of XPC cell line 
19 
Self-inactivating 
lentiviral 
vector 
SPINK5 
Netherton 
syndrome 
Keratinocyte 
Pre-clinical work demonstrating full length LEKTI expression in 
organotypic cultures grafted onto mice. Currently recruiting for 
phase I clinical trial 
20 
Retroviral vector 
STS 
X-linked 
ichthyosis 
Keratinocyte 
Restoration of enzymatic activity in cultured primary 
keratinocytes deficient in steroid sulfatase 
21 
siRNA 
KRT5 
EB Simplex 
Keratinocyte 
Pre-clinical study, specific knockdown of mutant keratin 5 
22 
siRNA 
KRT6A 
Pachyonychia 
congenital 
Keratinocyte 
Phase 1b trial injecting TD101 siRNA locally for plantar 
keratoderma in PC 
23 
Direct injection 
of plasmid 
TGM1 
Lamellar 
Ichthyosis 
Keratinocyte 
Non-uniform restoration of gene expression on regenerated LI 
skin 
24 

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6. Ortiz-Urda S, Thyagarajan B, Keene DR, et al. Stable nonviral genetic correction of inherited human skin disease. Nat Med, 
2002;8(10):1166-1170. 
7. Murauer EM, Gache Y, Gratz IA et al. Functional Correction of Type VII Collagen Expression in Dystrophic Epidermolysis 
Bullosa. J Invest Dermatol. 2010; 131(1):74-83 
8. Ortiz-Urda S, Thyagarajan B, Keene DR et al. PhiC31 integrase-mediated nonviral genetic correction of junctional epidermolysis 
bullosa.” Hum Gen Therapy 200314(9): 923-928. 
9. 11. Ortiz-Urda S, Thyagarajan B, Keene DR et al. 2002). Stable nonviral genetic correction of inherited human skin disease. Nat 
Med. 2002 8(10): 1166-1170. 
10. Mecklenbeck S, Compton SH, Mejia JE et al, microinjected COL7A1-PAC vector restores synthesis of intact procollagen VII in a 
dystrophic epidermolysis bullosa keratinocyte cell line. Hum Gen Therapy 2002; 13(13): 1655-1662. 
11. Osborn MJ, Starker CG, McElroy AN et al. TALEN-based gene correction for epidermolysis bullosa. Mol Ther 2013 21(6): 1151-
1159. 
12. Seitz CS, Giudice GJ, Balding SD et al. BP180 gene delivery in junctional epidermolysis bullosa. Gene Ther. 1999 6(1): 42-47. 
13. Mavilio F, Pellegrini G, Ferrari S, et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified 
epidermal stem cells. Nat Med. 2006;12(12):1397-1402. 
14. Di Nunzio F, Maruggi G, Ferrari S, et al. Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally 
targeted lentiviral vectors. Mol Ther. 2008;16(12):1977-1985. 
15. Arnaudeau-Begard C, Brellier F, Chevallier-Lagente O, et al. Genetic correction of DNA repair-deficient/cancer-prone xeroderma 
pigmentosum group C keratinocytes. Hum Gene Ther. 2003;14(10):983-996. 
16. Marchetto MC, Muotri AR, Burns DK, Friedberg EC, Menck CF. Gene transduction in skin cells: preventing cancer in xeroderma 
pigmentosum mice. Proc Natl Acad Sci USA. 2004;101(51):17759-17764. 
17. Marchetto MC, Correa RG, Menck CF, Muotri AR. Functional lentiviral vectors for xeroderma pigmentosum gene therapy. J 
Biotechnol. 2006;126(4):424-430. 
18. Warrick E, Garcia M, Chagnoleau C, et al. Preclinical corrective gene transfer in xeroderma pigmentosum human skin stem cells. 
Mol Ther. 2012;20(4):798-807. 

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19. Dupuy A, Valton J, Leduc S, et al. Targeted gene therapy of xeroderma pigmentosum cells using meganuclease and TALEN. 
PLoS One. 2013;8(11):e78678. 
20. Di WL, Larcher F, Semenova E, et al. Ex-vivo gene therapy restores LEKTI activity and corrects the architecture of Netherton 
syndrome-derived skin grafts. Mol Ther. 2011;19(2):408-416. 
21. Freiberg RA, Choate KA, Deng H, Alperin ES, Shapiro LJ, Khavari PA. A model of corrective gene transfer in X-linked ichthyosis. 
Hum Mol Genet. 1997;6(6):927-933. 
22. Atkinson SD, McGilligan VE, Liao H, et al. Development of allele-specific therapeutic siRNA for keratin 5 mutations in 
epidermolysis bullosa simplex. J Invest Dermatol. 2011;131(10):2079-2086. 
23. Leachman SA, Hickerson RP, Schwartz ME, et al. First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin 
disorder. Mol Ther. 2010;18(2):442-446. 
24. Choate KA, Medalie DA, Morgan JR and P. A. Khavari. Corrective gene transfer in the human skin disorder lamellar ichthyosis. 
Nat Med. 1996; 2(11): 1263-1267. 
 
 
 
 
 
 
 
 
 
 
 

  
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Appendix 2: Summary of published clinical studies of allogeneic and autologous fibroblasts used to treat skin 
disorders 
 
Cell 
Study type 
Clinical 
indication 
No. Of 
patients 
Cell type 
Outcome 
Reference 
Fibroblast 
Prospective pilot 
study 
Prominent facial 
scar 
10 
Intradermal cultured 
autologous fibroblasts 
Size reduction between 10%-
85%; histologically ñ dermal 
thickness 
1 
 
Fibroblast 
Prospective 
placebo 
controlled Phase 
III trial 
Facial Contour 
deformities 
215 
Intradermal cultured 
autologous fibroblasts 
Most significant difference evident 
in acne scars; continued response 
rates up to 12 months post 
treatment 
2 
 
 
Fibroblast  
 
 
 
 
 
Interventional 
non-blinded 
prospective study 
Adult RDEB 
5 
Intradermal injection 
allogeneic cultured 
fibroblasts;  intact skin 
↑COL7A1 and C7 for 3 months; 
can remain raised for up to 9 
months 
3, 4 
Fibroblast 
Phase II placebo 
controlled double 
blind RCT 
Adult RDEB 
5 
Intradermal cultured 
allogeneic fibroblasts 
No significant difference between 
placebo; improvement in QOL 
5 
Fibroblast 
Interventional 
Ageing skin 
5 
Intradermal cultured 
Benefits limited to slight reduction 
6 

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non-blinded study 
autologous fibroblasts 
in skin fragility 
Fibroblast 
Phase II open 
label dose 
escalation pilot 
study 
Ageing skin 
10 
Intradermal cultured 
allogeneic fibroblasts 
Slight reduction in nasolabial 
crease 
7 
Fibroblast 
Single centre 
interventional 
study 
Ageing skin and 
scars 
20 
Intradermal cultured 
autologous fibroblasts  
Variable improvement at 6 months 
8 
Keratinocyt
Phase II placebo 
controlled double 
blind RCT 
Chronic Venous 
Ulcers 
228 
Spray allogeneic 
neonatal keratinocyte 
and fibroblast cell 
applied therapy  
Greater mean reduction of wound 
size compared with placebo 
9 
Fibroblast 
Prospective 
interventional 
study 
Burns (third 
degree) 
14 
Allogeniec fibroblasts 
on meshed split 
thickness skin grafts 
Improved healing time and 
hypertrophic scar formation 
compared with conventional 
method 
10 
Fibroblast 
Multicentre 
double blind 
placebo 
controlled Phase 
II RCT 
Ageing skin 
372 
Intradermal cultured 
autologous fibroblasts 
Moderate improvement in 
nasolabial fold wrinkles compared 
to placebo, only 1 point subjective 
difference 
11 
Cultured dermal substitute 
CDS 
Prospective 
interventional 
study 
RDEB 
3 
Allogeneic cultured 
dermal substitute 
using cryopreserved 
normal human 
++Granulation found on wound 
surface within 1 week; 
epithelialisation at margins of 
ulcer at 4 weeks 
12 

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fibroblasts on matrix of 
hyaluronic acid and 
Atelo-collagen 
CDS 
Case report 
Digital gangrene 
associated with 
hypereosinophili
c syndrome 
1 
Allogeneic cultured 
dermal substitute 
using cryopreserved 
normal human 
fibroblasts on matrix of 
hyaluronic acid and 
Atelo-collagen; 
replaced every 3 days 
for 5 weeks 
Pain reduction at site; healthy 
granulation tissue during 
treatment and so replaced with 
split skin graft 
13 
CDS 
Prospective 
interventional 
study 
Full thickness 
skin defects 
following tumour 
resection 
12 
Allogeneic cultured 
dermal substitute 
using cryopreserved 
normal human 
fibroblasts on matrix of 
hyaluronic acid and 
Atelo-collagen 
9/10 patients developed healthy 
granulation tissue allowing split 
skin graft; Others showed rapid 
re- epithelialisation 
14 
CDS 
Case report 
Chronic venous 
ulcer 
1 
Allogeneic cultured 
dermal substitute 
using cryopreserved 
normal human 
fibroblasts on matrix of 
hyaluronic acid and 
Atelo-collagen 
Granulation tissue within 2 weeks; 
completely healed by 9 weeks 
15 

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CDS 
Multi-centre 
prospective 
interventional 
study 
Chronic venous 
and arterial 
ulcer 
13 
Allogeneic cultured 
dermal substitute 
using cryopreserved 
normal human 
fibroblasts on matrix of 
hyaluronic acid and 
Atelo-collagen 
9/13 cases (arterial + venous) 
healed well, one complicated by 
local infection; larger ulcers still 
required grafting 
16 
CDS 
Case report 
Ulcer associated 
with Anti-
Phospholipid 
syndrome + 
stasis dermatitis 
1 
Allogeneic cultured 
dermal substitute 
using cryopreserved 
normal human 
fibroblasts on matrix of 
hyaluronic acid and 
Atelo-collagen; 
replaced every week 
All ulcers healed within 4 weeks 
17 
CDS 
Prospective 
interventional 
study 
Intractable 
ulcers lower leg 
5 
Allogeneic cultured 
dermal substitute 
using cryopreserved 
normal human 
fibroblasts on matrix of 
hyaluronic acid and 
Atelo-collagen 
Wound size decreased in all 
cases (33% reduction in 7 ulcers, 
complete resurfacing in 2 cases); 
no control comparator 
18 
CDS 
Prospective 
interventional 
study 
Wound donor 
site following 
skin graft 
14 
Comparitive between 
fresh and 
cryopreserved human 
fiboblasts in allogeneic 
cultured dermal 
No difference noted in re-
epithelialisation between the 
preparation methods 
19 

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Appendix 2 References 
1 
Watson D, Keller GS, Lacombe V et al. Autologous fibroblasts for treatment of facial rhytids and dermal depressions. A pilot 
study. Arch Facial Plast Surg 1999; 1: 165-170. 
2 
Weiss RA, Weiss MA, Beasley KL et al. Autologous cultured fibroblast injection for facial contour deformities: a prospective, 
placebo-controlled, Phase III clinical trial. Dermatol Surg 2007; 33: 263-268. 
3 
Wong T, Gammon L, Liu L et al. Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa. J Invest 
Dermatol 2008; 128: 2179-2189. 
4 
Nagy N, Almaani N, Tanaka A et al. HB-EGF induces COL7A1 expression in keratinocytes and fibroblasts: possible 
mechanism underlying allogeneic fibroblast therapy in recessive dystrophic epidermolysis Bullosa. J Invest Dermatol 2011; 
131: 1771-1774. 
5 
Venugopal SS, Yan W, Frew JW et al. A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for 
recessive dystrophic epidermolysis bullosa. J Am Acad Dermatol 2013; 69: 898-908 e897. 
6 
Eca LP, Pinto DG, de Pinho AM et al. Autologous fibroblast culture in the repair of aging skin. Dermatol Surg 2012; 38: 180-
184. 
7 
Lowe NJ, Lowe PL, St Clair Roberts J. A phase IIa open-label dose-escalation pilot study using allogeneic human dermal 
fibroblasts for nasolabial folds. Dermatol Surg 2010; 36: 1578-1585. 
8 
Nilforoushzadeh MA, Siadat AH, Arianrad M et al. Soft tissue augmentation by autologous cultured fibroblasts transplantation 
for treatment of wrinkles and scars: a case series of 20 patients. J Res Med Sci 2010; 15: 167-171. 
9 
Kirsner RS, Marston WA, Snyder RJ et al. Spray-applied cell therapy with human allogeneic fibroblasts and keratinocytes for 
the treatment of chronic venous leg ulcers: a phase 2, multicentre, double-blind, randomised, placebo-controlled trial. Lancet 
2012; 380: 977-985. 
10 
Moravvej H, Hormozi AK, Hosseini SN et al. Comparison of the Application of Allogeneic Fibroblast and Autologous Mesh 
Grafting With the Conventional Method in the Treatment of Third-Degree Burns. J Burn Care Res 2012. 
substitute 
CDS 
Prospective, 
open label proof 
of concept clinical 
trial 
Diabetic foot 
ulcer 
5 
Autologous fibroblast 
seeded artificial dermis 
using animal product 
free serum 
3/5 complete wound healing 
within 12 weeks; no deleterious 
effects using animal free serum 
20 

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11 
Smith SR, Munavalli G, Weiss R et al. A multicenter, double-blind, placebo-controlled trial of autologous fibroblast therapy for 
the treatment of nasolabial fold wrinkles. Dermatol Surg 2012; 38: 1234-1243. 
12 
Hasegawa T, Suga Y, Mizoguchi M et al. Clinical trial of allogeneic cultured dermal substitute for the treatment of intractable 
skin ulcers in 3 patients with recessive dystrophic epidermolysis bullosa. J Am Acad Dermatol 2004; 50: 803-804. 
13 
Ohtani T, Okamoto K, Kaminaka C et al. Digital gangrene associated with idiopathic hypereosinophilia: treatment with 
allogeneic cultured dermal substitute (CDS). Eur J Dermatol 2004; 14: 168-171 
14  
Moroi Y, Fujita S, Fukagawa S et al. Clinical evaluation of allogeneic cultured dermal substitutes for intractable skin ulcers 
after tumor resection. Eur J Dermatol 2004; 14: 172-176. 
15 
Hasegawa T, Suga Y, Mizoguchi M et al. Intractable venous leg ulcer treated successfully with allogeneic cultured dermal 
substitute. Scand J Plast Reconstr Surg Hand Surg 2007; 41: 326-328. 
16 
Yonezawa M, Tanizaki H, Inoguchi N et al. Clinical study with allogeneic cultured dermal substitutes for chronic leg ulcers. Int 
J Dermatol 2007; 46: 36-42. 
17 
Nishimoto J, Amoh Y, Tanabe K et al. Intractable leg ulcers associated with antiphospholipid syndrome with stasis dermatitis: 
treatment with allogeneic cultured dermal substitute. Eur J Dermatol 2007; 17: 350-351. 
18 
Yamada M, Kubo H, Kobayashi S et al. Bone Marrow-Derived Progenitor Cells Are Important for Lung Repair after 
Lipopolysaccharide-Induced Lung Injury. The Journal of Immunology 2004; 172: 1266-1272. 
19 
Yamada N, Uchinuma E, Matsumoto Y et al. Comparative evaluation of re-epithelialization promoted by fresh or 
cryopreserved cultured dermal substitute. J Artif Organs 2008; 11: 221-224. 
20 
Morimoto N, Ito T, Takemoto S et al. An exploratory clinical study on the safety and efficacy of an autologous fibroblast-
seeded artificial skin cultured with animal product-free medium in patients with diabetic foot ulcers. Int Wound J 201