transglutaminase 1 deficient lamellar ichthyosis - ucsf school of

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


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


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”


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


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


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


CloningCloning of of TGaseTGase 1 1 constructsconstructsPlasma

membrane

catalyticregion

His-tag

93R 94G

FL hTGK His

67/33 hTGK His His-tag


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


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+


PurificationPurification::NiNi--NTANTA--affinityaffinity--chromatographychromatography

Imidazol

Histidin

Interaction between neighbouring residues in the 6x His-tag and Ni-NTA matrix.


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


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


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


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


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


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)


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

transglutaminase 1 deficient lamellar ichthyosis - ucsf school of

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