Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.

1. First-in-man gene therapy for Netherton syndrome
From laboratory to patient
Wei-Li Di
UCL Great Ormond Street Institute of Child Health
UCL GOS Institute of Child Health
Rare Skin Disease Symposium, Madrid 2016

2. UCL GOS Institute of Child Health
Outline of talk
• Netherton syndrome and the pathogenesis of the disease
• The strategy of ex-vivo gene therapy for Netherton syndrome
• Pre-clinical study in vitro and in vivo
• Phase I clinical trial for Netherton syndrome

3. Netherton syndrome
• Cause
Compound null mutations in SPINK5 gene,
resulting in loss of function of coding protein LEKTI
• Clinical features
Ichthyosiform erythroderma, trichorrhexis
invaginata, atopic manifestations and allergy
• Prognosis
High mortality in the 1st year of life due to
hypernatraemic dehydration secondary to severe
water loss
• Treatment
Treatments are limited to management of symptoms
UCL GOS Institute of Child Health
ibmi.mf.uni-lj.si/.../acta-
apa-00-2/Dragos.html

4. LEKTI
Kallikrein 5 and 7
desmoglein IPro-elastase 2
Pro-filaggrin
+ degradation
Integrity and permeability in the skin barrier
UCL GOS Institute of Child Health
Pathogenesis of Netherton syndrome
-
desquamation

5. Histopathological features of Netherton syndrome
UCL GOS Institute of Child Health
• Absent LEKTI expression
• Over-expression /
over- activity of KLK5
• exfoliated corneocytes
hyperkeratosis/psoriasiform
parakeratosis/hypergranulosis

6. Gene therapy strategy for Netherton syndrome
LEKTI
Controls kallikrein activity
Restores skin barrier function
Adding a wild type SPINK5 gene in
keratinocytes to restore LEKTI
expression and function
UCL GOS Institute of Child Health

7. Delivery platform
1st vertion of lentiviral vector carrying coSPINK5
Vector features:
• HIV-1 derived, replication defective and self inactivating (SIN)
• Central polypurine tract sequence and post transcriptional regulatory element
• Spleen Focus Forming Virus (SFFV) as Internal promoter
• codon optimized SPINK5 cDNA linked to eGFP
• eGFP alone used as control vector
UCL Institute of Child Health

8. Transduction efficiency in primary keratinocytes
UCL GOS Institute of Child Health
Lane 1: untransduced cell
Lane 2: transduced with control vector
Lane 3: transduced with SPINK5 vector
• 40-60% of eGFP positive cells assessed
by FACS
• 0.59-0.68/cell of proviral copy numbers
Measured by qPCR 20 days post transduction

9. Stability of transgene SPINK5
eGFP intensity in cells transduced with LV-SPINK5/eGFP
0
50
100
150
200
250
Day 0 Day 6 Day 13 Day 20 Day 27 Day 34 Day 41 Day 49
Meanintensity
UCL GOS Institute of Child Health
eGFP expression in a longer term culture LEKTI expression in a longer term culture

10. DNA methylation in the transgene
UCL GOS Institute of Child Health
Cells transduced with
SFFV-GFP
Cells transduced
with SFFV-SPINK5
LEKTI expression
Azacidine

11. High DNA Methylation level across the SFFV promoter
and codon optimised SPINK5 boundary
UCL GOS Institute of Child Health
* p<0.05; ** p<0.01; NS p>0.05
• DNA methylation cause transgene silencing,
• SFFV promoter was susceptible to methylation when it is used in
combination with GC enriched codon optimised SPINK5

12. Revised lentiviral vector – human Involucrin promoter (INVO)
UCL GOS Institute of Child Health
• Less CpG sites in INVO compared to SFFV
(9 CpGs on INVO v 25 CpGs on SFFV)
• Well characterised and compartment
expression
• Human origins and more suitable for clinical
applications

13. Lentiviral vector carrying codon optimised SPINK5 cDNA
Linked to eGFP under the control of human involucrin promoter
UCL GOS Institute of Child Health
2nd version of lentiviral vector
carrying coSPINK5
• Test:
(Low methylation level and stable LEKTI expression)

14. UCL Institute of Child Health
Pre-clinical studies in vitro and in vivo
Skin biopsies 4-6 mm
Isolation and expansion of keratinocytes
Transduced with the SPINK5-vector or GFP-vector
In vivo skin grafting
expanding cells in vitro
In vitro 3D culture
Analysis
8 weeks 4 weeks

15. Restoration of LEKTI expression 3D culture in vitro
UCL Institute of Child Health
Red=LEKTI; blue=nuclei

16. Correction of epidermal architectures
in gene modified grafts
UCL GOS Institute of Child Health
H & E
LEKTI staining
eGFP vector SPINK vectorNetherton skin
Grafted mice

17. UCL GOS Institute of Child Health
• Ex vivo gene therapy for Netherton syndrome using skin
sheet generated from genetically corrected autologous
keratinocytes.
• Longitudinal evaluation of safety and efficacy
Phase I clinical trial:
Lentiviral gene therapy for Netherton syndrome

18. Pre-clinical study
Production of Investigational
Medicinal Product
under GMP conditions
GMP facilities,
equipment,
Procedures,
reagents
From laboratory to patient
UCL GOS Institute of Child Health
SPINK5
lentiviral vector
Gene corrected
epithelial sheets

19. 3rd generation pCCL-SIN-cppt/CTS vector offers maximum biosafety
• Packaging constructs are deleted
for all HIV accessory proteins (vpu,
vpr, nef, vif and Tat)
• Segregates gag/pol and rev genes
in two separate plasmids
• The vesicular stomatitis virus (VSV-
g) envelope provides a broad
tropism, mediates effective
keratinocyte stem cell transduction
UCL GOS Institute of Child Health

20. Manufacturing process for gene modified
epithelial sheet
+
6mm biopsy

21. Pre-clinical study
Approvals by authorities Production of Investigational
Medicinal Product
under GMP conditions
GMP facilities,
equipment,
Procedures,
reagents
From laboratory to patient
Gene
Therapy
Advisory
Committee
(GTAC)
Medicines and
Healthcare
Products
Regulatory Agency
(MHRA)
UCL GOS Institute of Child Health
SPINK5
lentiviral vector
Gene corrected
epithelial sheets
The trial was approved in 2013

22. Data from a Netherton patient grafted with
gene modified epithelial sheet
UCL GOS Institute of Child Health

23. Generation of the gene modified epithelial sheet (43 days)
A 6mm skin biopsy
Isolation, expansion
and transduction
cells
Sheet culture
Dispatch for
grafting
+
UCL Institute of Child Health

24. time of release definition
Clarity 1/0 1= media transparent
0= media cloudy
Size 1/0 1= sheet size > target size of 20cm2 for
adults /10cm2 for children
0= sheet size ≤ 20cm2/10cm2
Integrity 1/0 1= visibly intact with no disintegration,
0= visible disintegration
Colour 1/0 1= pale white, otherwise scored as 0
Lift ability 1/0 1= The sheet attached to Tricotex and can
be lifted from the container to graft area.
0= unliftable
The quality of the epithelial sheet for release
UCL GOS Institute of Child Health

25. LEKTI expression in the culture sheet
confirmed by in situ immunofluorescence staining and Western blot
(before graft)
Red=LEKTI, Blue =
nuclei
UCL GOS Institute of Child Health
Line 1&4: normal keratinocytes
Line 2&5: untransduced JM keratinocytes
Line 3&6: transduced JM keratinocytes

26. Item Specification Result
The score of the
epithelial sheet
5 5
Stable lentiviral
transduction
average copy
number/cells=0.1-3
2.2
LEKTI protein
expression
positive LEKTI expression
in cells and sheet
positive
Gram stain Negative Negative
Sterility (weekly
BACTalert test)*
No organisms detected No organism
detected
Release criteria of gene modified epithelial sheet
UCL Institute of Child Health

27. Plan of follow up
Skin biopsy Sheet graft
-2
ma
1d
0
wb
2
1
W
3
2
W
4
1
M
5
2
M
6
3
M
7
6
M
8
12
M
9
15
M
10
18
M
11
21
M
12
24
M
13
30
M
14
36
M
15
Visual
inspection               
Blood
pressure               
IgE     
Punch
biopsy       
ELISPOT      
RCL
sample       
TEWLc
    
Photograph               
a=month; b=week; c= trans-epidermal water loss; d=visit number
UCL GOS Institute of Child Health

28. Follow up study --- post graft
Observation of graft area
Post graft
Week 1 Month 1 Month 2 Month 3
2 months Prior to graft:
UCL GOS Institute of Child Health

29. UCL GOS Institute of Child Health
Virus copy number in skin biopsies
detected by qPCR
Samples Viral copy number per cell
(month 3 post graft)
Base line skin Under detection
Graft skin 0.0015
Outside graft skin Under detection
.

30. UCL Institute of Child Health
Restoration of LEKTI expression in grafted skin
Up to 3 months follow-up
Normal skin Base line skin
Month 1 Month 3

31. UCL Institute of Child Health
Integrated vector DNA sites
detected by LAM-PCR and deep sequencing
Samples:
• Untransduced and transduced cells
• Skin biopsy from the inside graft (month 3 follow up)
Results:
• A polyclonal vector integration profile for transduced cells prior to graft with no preferred
integration in/nearby genes previously involved in serious adverse events
• Skin biopsy from graft site did not find any integration sites

32. Summary:
1. Establishment of a lentiviral delivery platform (internal human
involucrin promoter and codon optimised SPINK5 cDNA) for
delivery of wild type SPINK5 gene to primary keratinocytes
2. Laboratory to patient --- translational development of gene
therapy for Netherton syndrome
3. Successful generation and grafting GMP comliance gene
modified autologous epithelial sheet on the patient
4. Early results (up to 3 months follow-up) showed restoration of
LEKTI expression in the grafted skin, which provides a proof of
principle of gene therapy for other genodermatoses
UCL GOS Institute of Child Health

33. Acknowledgements
UCL Institute of Child Health
Dermatology, ICH:
Dr Anastasia Petrova,
Dr Farhatullah Syed,
Dr Ekaterina Semenova
Dr Gill Talbot
Dr David Almarza
Ms Sumera Ghani
Prof John Harper
GMP Facilities, ICH
Ms Havinder Hara
Dr Sue Swift
Wolfson Centre for Gene therapy, ICH:
Prof Waseem Qasim, Prof Adrian Thrasher
Epithelial Biomedicine
Division,
CIEMAT, Madrid, Spain
Dr. Fernado Larcher
Rayne Institute, KCL
Dr Lucas Chan
Prof Farzin Farzaneh
St John's Institute of
Dermatology, KCL
Prof Jemima Mellerio
Dr Su Lwin
Dr A Abdul-Wahab
Prof John McGrath
Plastic Surgery Unit, St
Thomas’ Hospital, London
Miss Catina Bernadis
The Moulton Charitable Trust
The support from patients
and their families