title: Recombinant filaggrin is internalized and processed to correct filaggrin deficiency.
abstract: This study was designed to engineer a functional filaggrin (FLG) monomer linked to a cell-penetrating peptide (RMR) and to test the ability of this peptide to penetrate epidermal tissue as a therapeutic strategy for genetically determined atopic dermatitis (AD). A single repeat of the murine filaggrin gene (Flg) was covalently linked to a RMR motif and cloned into a bacterial expression system for protein production. Purified FLG+RMR (mFLG+RMR) was applied in vitro to HEK-293T cells and a reconstructed human epidermis (RHE) tissue model. Immunochemistry demonstrated RMR-dependent cellular uptake of FLG+RMR in a dose- and time-dependent manner in HEK cells. Immunohistochemical staining of the RHE model identified penetration of FLG+RMR to the stratum granulosum, the epidermal layer at which FLG deficiency is thought to be pathologically relevant. In vivo application of FLG+RMR to FLG-deficient flaky tail (ft/ft) mice skin demonstrated internalization and processing of recombinant FLG+RMR to restore the normal phenotype. These results suggest that topically applied RMR-linked FLG monomers are able to penetrate epidermal tissue, be internalized into the appropriate cell type, and be processed to a size similar to wild-type functional barrier peptides to restore necessary barrier function, and prove to be therapeutic for patients with AD.
authors.affiliation: Casey Eye Institute, Oregon Health and Sciences University, Portland, Oregon, USA. Electronic address: stoutt@ohsu.edu.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Stout
authors.affiliation: Casey Eye Institute, Oregon Health and Sciences University, Portland, Oregon, USA.
authors.forename: Trevor
authors.initials: T
authors.lastname: McFarland
authors.affiliation: Department of Restorative Dentistry, School of Dentistry, Oregon Health and Science University, Portland, Oregon, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: Mitchell
authors.affiliation: Casey Eye Institute, Oregon Health and Sciences University, Portland, Oregon, USA.
authors.forename: Binoy
authors.initials: B
authors.lastname: Appukuttan
authors.affiliation: Casey Eye Institute, Oregon Health and Sciences University, Portland, Oregon, USA.
authors.forename: J
authors.initials: J
authors.lastname: Timothy Stout
mesh_terms: D000818:Animals; D003876:Dermatitis, Atopic / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D020405:Dermis / Q000166:cytology / Q000378:metabolism; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism; D000091344:Filaggrin Proteins; D015316:Genetic Therapy / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D007381:Intermediate Filament Proteins / Q000172:deficiency / Q000235:genetics* / Q000493:pharmacokinetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D010641:Phenotype; D011994:Recombinant Proteins / Q000235:genetics / Q000493:pharmacokinetics
chemical_list: C000717249:FLG protein, human; D000091344:Filaggrin Proteins; D007381:Intermediate Filament Proteins; D011994:Recombinant Proteins