Spot On Treatment Team Stockholm iGEM 2010 Andreas Constantinou - - PowerPoint PPT Presentation

Spot On Treatment

Team Stockholm iGEM 2010

Andreas Constantinou Hassan Foroughi Asl Emmelie Lidh Johan Nordholm Nina Schiller

Vitiligo

• Complex skin disorder
• Loss of pigment cells
• 0.5-2 %
• No cure or effective treatment
• Wikipedia. The Free Encyclopedia
• Wikipedia. The Free Encyclopedia

IgG accumulation

Autoimmune hypothesis

Oxidative stress

O2

Biochemichal hypothesis

Different hypotheses

• ­‑

IgG accumulation

Autoimmune hypothesis

Oxidative stress

O2

Biochemichal hypothesis

Different hypotheses

Protein A (Z-domain) IgG protease

• ­‑

Proteins masking the autoimmune response

IgG accumulation

Autoimmune hypothesis

Oxidative stress

O2

Biochemichal hypothesis

Different hypotheses

Protein A (Z-domain) IgG protease +

• ­‑

Proteins masking the autoimmune response Genes involved in regulation of melanogenesis

SOD bFGF

IgG accumulation

Autoimmune hypothesis

Oxidative stress

O2

Biochemichal hypothesis

Different hypotheses

Tyrosinase Protein A (Z-domain) IgG protease MITF yCCS +

• ­‑

Proteins masking the autoimmune response Genes involved in regulation of melanogenesis

SOD bFGF

IgG accumulation

Autoimmune hypothesis

Oxidative stress

O2

Biochemichal hypothesis

Different hypotheses

Tyrosinase Protein A (Z-domain) IgG protease MITF ( ) ( ) yCCS +

• ­‑

Proteins masking the autoimmune response Genes involved in regulation of melanogenesis

Vision

Vision

Bacteria applied

1

Vision

Bacteria applied

1

Proteins secreted

2 2

Vision

Bacteria applied

1

Proteins secreted

2 2

Internalization

3 3

Vision

Bacteria applied

1

Proteins secreted

2

Re-pigmentation

4 2

Internalization

3 3

Skin penetration

• Effective barrier
• Needle-free approach
• Cell-penetrating peptides (10-20 a.a.)
• 3 types tested
• Able to carry cargo into cells/skin

Huang, et al. (2010)

CPP Cargo

Wet-lab approach

Cloning Expression Purification Skin & cell penetration Activity assays

1 2 3 4 5

Cloning

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

Cloning

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

Cloning

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

Cloning

3 ×

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

5

Cloning

3 ×

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

3 × 5 + 5

Cloning

3 × +

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

3 × 5 + 5 ( ) 2

Cloning

3 × +

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

3 × 5 + 5 ( ) 2 7× +

Cloning

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

Protein expression

• Low/no pEX expression
• Experimental bottleneck

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

Commercially available Ptac expression vector

BioBrick pEX Ptac expression vector bFGF + GST bFGF + 6x His

0 h 1 h 2 h 3 h λ 0 h 1 h 2 h 3 h λ

Protein purification

• Nickel resin columns
• 6x His tag

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

Protein activity

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

IgG digestion O2 conversion

SOD activity IgG protease activity

• SOD determination kit

(Sigma-Aldrich)

• Digestion of IgG

secondary antibodies

+ substr.

• söndag den 7 november 2010

Skin & cell penetration

• Immunohistochemistry
• α-His-tag antibodies
• Cultured cells
• Keratinocytes
• Melanocytes
• Skin
• Cultured artificial (human)
• Extracted live (mouse)

Cloning Expression Purification Skin & cell penetration Activity

1 2 3 4 5

Fixation + Cross-section

Modeling

• Model for safe induction of protein expression
• Toy model based on lac operon
• Well studied
• LacI regulated expression vector

Aim

Modeling

lac operon

mRNA mRNA Repressor β-gal Perm. Allolactose Internal lactose External lactose

Expression vector

Modeling

lac operon

mRNA mRNA Repressor β-gal Perm. Allolactose Internal lactose External lactose

Expression vector

Ahmazadeh, et al. (2005)

Future prospects

• Transfer to Lactobacillus
• Allows for secretion
• ”Skin-friendly”
• Other applications

Future prospects

• Transfer to Lactobacillus
• Allows for secretion
• ”Skin-friendly”
• Other applications

Expr. vector

Lactobacillus Escherichia coli

Our special thanks to

Our sponsors

Faculty ¡of ¡Science, Stockholm ¡University Swedish ¡Vi:ligo ¡Associa:on Thermo ¡Fisher ¡Scien:fic Sigma-­‑Aldrich

Elisabeth Haggård Gunnar von Heijne Robert Daniels

Our supervisors

Sridhar Mandali Richard Odegrip Sergey Surkov Jaroslav Belotserkovsky

...and everyone else who made this project possible

...and

Inspira:onal ¡SoBware ¡for ¡Biologists