Evaluation of miglustat as maintenance therapy after enzyme therapy in adults with stable type 1
Towards Enzyme Substitution Therapy of …obstacles.medschool.ucsf.edu/pdf/Traupe.pdfEnzyme...
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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
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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
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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”
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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
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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
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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
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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
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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
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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+
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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.
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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
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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
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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
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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
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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
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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)
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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