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Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Towards Enzyme Substitution Therapy Towards Enzyme Substitution Therapy of Transglutaminase 1 deficient of Transglutaminase 1 deficient
Lamellar IchthyosisLamellar IchthyosisObstacles to translation
Heiko Traupe and Karin AufenvenneMünster, Germany
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Transglutaminase 1 Transglutaminase 1 deficientdeficient Lamellar IchthyosisLamellar Ichthyosis
Oji V. and Traupe H., Ichthyoses: Differential diagnosis and molecular genetics, European Journal of Dermatology, in press, 2006
• Congenital recessive skin disorder characterized by generalized scaling
• Apparent at birth and persisting throughout life
• Prevalence 1:200,000 2,000 patients in Europe 1,300 in USA, 700 in Japan (estimates)
• Deplorable therapeutic situation
• Emollients, urea, lactic acid and retinoids
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Lamellar Ichthyosis Lamellar Ichthyosis –– animal modelanimal model
TGM1-knock-out mouse(Matzuki et al., PNAS, 95: 1044-1049, 1998; Steinert et al. US 2003/0072795)
• 1000 times reduced barrier function• trans epidermal water loss 100 times higher than wild-type • mice die 4 - 5 h after birth
- Difficulties to provide definite proof of concept in a “lethal mouse model”
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
TransglutaminasesTransglutaminases in the Epidermisin the Epidermis
Transglutaminases have key functions in the corneocyte formation process by cross linking the proteins of the cornified envelope.
Transglutaminase 1 (TG1) has a key function in the catalysis of the first steps of cornified envelope formation and in the covalent fixation of the lipid envelope to the cornified envelope
image source: Beiersdorf
Horny layer
Transition layer
Granular layer
Prickle-cell layer(with Odland-Bodies)
Basal layer
Basal membrane
TG5TG1
TG2
TG3
TG2
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Transglutaminase 1Transglutaminase 1
• Synthesized as a 817 residue membrane-anchored precursor
• Ca2+ dependent • Intracellular• Expressed in the upper differentiated
layers of the epidermis• Attachment of long chain omega
hydroxyceramides to involucrin e.g.• In situ enzyme test established -
shows pericellular distribution and most marked activity in stratum granulosum
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Transglutaminase 1 in the skinTransglutaminase 1 in the skin
3367
10 10
33
67
10
33
67
10
3367
ProliferatingKeratinocytes Fully differentiated Keratinocytes
acylation Proteolyticprocessing
33
67
10
According to Steinert et al., 1996
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
CloningCloning of of TGaseTGase 1 1 constructsconstructsPlasma
membrane
catalyticregion
His-tag
93R 94G
FL hTGK His
67/33 hTGK His His-tag
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
The Gateway TechnologyThe Gateway Technology
• Universal cloning method • provides rapid and highly
efficient way to move DNA sequences into multiple vector systems
• based on site-specific recombination of bacteriophage lambda
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
BaculovirusBaculovirus--ExpressionsystemExpressionsystem
attB1 attB2TGase 1 cDNA
• Cloning of TGase 1 cDNA into the entry-vector pENTR 3C• LR-reaction with linear BaculoDirect DNA
High titervirus stock
Virus amplification
Infektion of HighFiveinsect cells in liquid culture
TGase 1 Expression
Direct transformation of Sf9-cells with recombinant BaculoDirect DNA and selection with ganciclovir
Primary VirusInfection of
Sf9 insect cells
Insertion of TGase 1 cDNA
pENTR 3C
attL1 ccdB attL2
PPH HSV1 tkattR1 attR2 PPH+
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
PurificationPurification::NiNi--NTANTA--affinityaffinity--chromatographychromatography
Imidazol
Histidin
Interaction between neighbouring residues in the 6x His-tag and Ni-NTA matrix.
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
0
0,05
0,1
0,15
0,2
0,25
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
FL hTGK HisO
D 2
80nm
Fraction (2 ml)
0
0,05
0,1
0,15
0,2
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
67/33 hTGK His
Fraction (2 ml)
OD
280
nm
FL hTGK His(92 kDa)
67/33 hTGK His(80 kDa)
50 mM imidazol 100 mM imidazol
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Activity of recombinant Activity of recombinant TGaseTGase 11
165
1042
0
200
400
600
800
1000
1200
FL hTGkHis
67/33 hTGkHis
Specific Activity [U/mg]
0
20
40
60
80
0 1 2 3 4 5 6
Time [min]
Inte
nsity
[int
]
Fluorescence Spectroscopy
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Transglutaminase 1 activity in normal Transglutaminase 1 activity in normal skin and lamellar ichthyosisskin and lamellar ichthyosis
NH Lamellar ichthyosis NH
Lamellar ichthyosis+ 67/33 hTGK His+ EDTA
Lamellar ichthyosis+ FL hTGK His
Lamellar ichthyosis+ 67/33 hTGK His
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
ObstaclesObstacles to to translationtranslation (1)(1)• No definite proof of concept yet (TG1 deficient mice die
shortly after birth....)– Keratinocyte-cultures of TGase 1-deficient patients / treatment
with recombinant TGase 1 constructs– inducible mouse model– skin equivalents
• Stabilization and formulation– liposome or micelle formulation needed and specialists for this
• Upscale production under GMP conditions needed– requires investor /classical problem of orphan drugs
• Preclinical toxicity studies (ten dogs, ten mice...)• Clinical studies to prove benefit over emollients and
retinoids
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
ObstaclesObstacles to to translationtranslation (2)(2)Lamellar Ichthyosis Enzyme Lamellar Ichthyosis Enzyme replacementreplacement TherapyTherapy
ConsortiumConsortium LIERTHECLIERTHEC
A novel approach to treat Lamellar Ichthyosis
Heiko Traupe N-Zyme BioTec GmbHPoliklinik für Hautkrankheiten Riedstrasse 7Universitätsklinikum Münster 64265 Darmstadt
Hans C. Korting Pharma CompanyKlinik und Poliklinik N. N.für Dermatologie und AllergologieLudwig-Maximilians-Universität München
• It is intended to make use of a recombinant transglutaminaseconstruct of N-Zyme BioTec, a leading company in transglutaminase technology
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
Economical ConsiderationEconomical ConsiderationCost estimation: 6.0 Mio Euro
• Production of GMP-grade enzyme (CMO) • Formulation and Toxicology • Clinical trials / approval (orphan drug - status) • Project coordination and consulting
Market estimation• Prevalence 1:200,000; about 2,000 patients in Europe,
1,300 in the US and 700 in Japan 4,000 patients• Treatment costs per year and patient 50,000 EURO
• Market penetration of 25 % (peak sales) 50 Mio EUROreference e.g. Cerezyme (Genzyme)
Enzyme substitution of lamellar ichthyosis - Heiko Traupe
AcknowledgementsAcknowledgements
Münster• Karin Aufenvenne• Tatjana Walker• Markus Höwel• Vinzenz Oji• Brigitte Willis
Cardiff• Daniel Aeschlimann
Bundesministeriumfür Bildung
und Forschung
Network for Ichthyosisand related
keratinisation disorders