Lamellar Ichthyosis(LI) is a severe type of ichthyosis that is often caused by a deficiency of the enzyme transglutaminase-1. Transglutaminase (TGase)-1 is important for the formation of the stratum corneum (horny layer) which is thickened very much in LI. One of the tasks of TGase-1 is to attach lipids to the so called "cornified envelope," i.e. a protein envelope which surrounds the cells in the stratum corneum. Current thinking is that when the lipids cannot be attached to the cornified envelope the lipid mantle of the stratum corneum becomes disturbed and this will result in considerable problems such as increased water loss and in the end ichthyosis. It is important to know that within the keratinocytes the TGase-1 is located at the inner side of the cell membrane.
Long-term aim In the long-term we want to develop an enzyme replacement therapy for TGase-1 deficient LI by making use of a cream that contains the enzyme. An essential intermediate step for this aim is a successful packing of TGase-1 in a suitable liposome vehicle.
Short term aims of the current liposome formulation project The first aims of the liposome formulation project which is funded by F.I.R.S.T. are:
- a) to produce large quantities of the enzyme TGase-1
- b) to develop suitable lipid vesicles (liposomes) into which the enzyme can be packed
- c) to actually encapsulate TGase-1 within liposomes
- d) to cultivate skin cells (keratinocytes) from patients with established TGase-1 deficiency
- e) to show that the liposomes are taken up by the keratinocytes and can pass the cell membrane and release the enzyme at the inner side of the cell membrane.
So far we successfully expressed large amounts (more than 10 mg) of TGase-1 in our lab making use of insect cells and the so called baculovirus system. For the second goal we developed together with our partner Dr. Dathe from Berlin a liposome prototype which contains a protein named apolipoprotein E peptide. This peptide was added to the liposome in order to facilitate the uptake of the liposome into the keratinocytes. Moreover this liposome prototype also has a fluorescent marker in order to 1) visualize the liposome within the cell, 2) demonstrate that it can be taken up by keratinocytes and, 3) show that this vehicle releases the enzyme to the inner side of the cell membrane where TGase-1 should go. To sum up, we have so far achieved in our current project steps a) to c) and are currently working to show that the liposomes are indeed taken up by (normal) keratinocytes and release the enzyme to the inner side of the cell membrane. In initial cell culture studies we isolated primary keratinocytes from three patients with transglutaminase-1 deficient lamellar ichthyosis. We also observed that keratinocytes from these patients grow very slowly when compared to normal keratinocytes and are difficult to cultivate.
Perspectives and work packages for the second work period In the second work period we want to
- a) continue with expression and purification of TGase-1 protein
- b) further investigate the liposome protein complexes and further characterize them with respect to stability and size distribution.
- c) further investigate the uptake of the liposome-TGase-1 complex to normal keratinocytes
- d) attempt to develop a three dimensional ichthyosis model in cell culture called skin equivalent model and to treat this model with a liposome/TGase-1 complex.
This last aim (d) will start to address a major potential hurtle: getting the liposome/TGase-1 complex through the thick stratum corneum and into the proper skin cells.
