L7 - Gene therapy for skin disorders

Question 1

Background

Answer 1

7 year-old with epidermolysis bullosa - genetic disorder with severe skin blistering.
BM doesn’t adhere to dermis due to mutations in laminin (interacts with dermis)

Question 2

The patient

Answer 2

Mutation in intron 14 of LAMB3 gene. First treated with antibiotics and skin graft from father - didn’t work.

Question 3

The procedure

Answer 3

Autologous cell therapy won’t work alone - need to correct mutation

Question 4

Gene therapy

Answer 4

Retroviral vector expressing full length LAMB3 cDNA. Done ex vivo - cells were corrected and then skin substitute was made. Extensive gene characterisation to ensure that the virus hadn’t integrated somewhere bad. Corrected cells were expanded and made into epithelial sheets for graft.

Question 5

Follow up

Answer 5

Next gen seq, in situ hybridisation, clonal analysis. Epidermis consisted of GM cells. Restoration of epidermal-dermal junction. IHC to look at laminin 332 expression. Normal skin functionality and elasticity

Question 6

Explain study - Fabrication of 3D skin substitutes and appendages could contribute to regenerative therapies

Answer 6

Hypothesis - PSCs can be used to mimic developmental patterning.
iPSC cultured in suspension and then aggregated and allowed to differentiate in SCID mouse. Hair shaft from EB was TP into nude mouse - outgrowth of hair cells from the donor mouse.
A step toward complete regeneration of skin.