Abstract
In Neurofibromatosis 1 (NF1) germ line loss of function mutations result in reduction of cellular neurofibromin content (NF1+/−, NF1 haploinsufficiency). The Ras-GAP neurofibromin is a very large cytoplasmic protein (2818 AA, 319 kDa) involved in the RAS-MAPK pathway. Aside from regulation of proliferation, it is involved in mechanosensoric of cells. We investigated neurofibromin replacement in cultured human fibroblasts showing reduced amount of neurofibromin. Full length neurofibromin was produced recombinantly in insect cells and purified. Protein transduction into cultured fibroblasts was performed employing cell penetrating peptides along with photochemical internalization. This combination of transduction strategies ensures the intracellular uptake and the translocation to the cytoplasm of neurofibromin. The transduced neurofibromin is functional, indicated by functional rescue of reduced mechanosensoric blindness and reduced RasGAP activity in cultured fibroblasts of NF1 patients or normal fibroblasts treated by NF1 siRNA. Our study shows that recombinant neurofibromin is able to revert cellular effects of NF1 haploinsuffiency in vitro, indicating a use of protein transduction into cells as a potential treatment strategy for the monogenic disease NF1.
Highlights
In monogenic dominantly inherited diseases such as Neurofibromatosis type 1 (NF1), a loss of function mutation in one Neurofibromatosis 1 (NF1) allele leads to a reduced amount of the functional corresponding protein in all body cells expressing this gene
We showed that the problems can be overcome in vitro by using endo-lysosome disrupting techniques as specialized transduction reagents along with light triggered photochemical internalisation (PCI)
We tested the impact of transduced neurofibromin on the mitogen activated protein kinase (MAPK) pathway and the capability of the cells to orientate themselves to nano-micro structured surfaces
Summary
In monogenic dominantly inherited diseases such as Neurofibromatosis type 1 (NF1), a loss of function mutation in one NF1 allele leads to a reduced amount of the functional corresponding protein in all body cells expressing this gene. Restoring functional neurofibromin in protein transduction approaches in all patient body cells as an alternative might be a clue to avoid the risks of genetic gene replacement therapies[13]. In autosomal recessive transmitted diseases protein replacement therapies have been tested in patients with Morbus Gaucher[14], Fabry’s disease[15,16], Mucopolysaccharosidosises, Morbus Pompe or Hemophilia A17–19. These diseases are caused by an entire lack of a protein with extracellular effects or a function in lysosomes in cells. Cotreating cells with a photosensitizer and light induced disruption of endosomes and lysosomes called photochemical internalization (PCI) leads to a cytosolic distribution of the internalized proteins in vitro
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