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Transglutaminase 1 Replacement Therapy Successfully Mitigates the Autosomal Recessive Congenital Ichthyosis Phenotype in Full-Thickness Skin Disease Equivalents

Open ArchivePublished:November 15, 2018DOI:https://doi.org/10.1016/j.jid.2018.11.002

      Abbreviations:

      ARCI (autosomal recessive congenital ichthyosis), TG1 (transglutaminase 1), tNG (thermoresponsive nanogels)
      To the Editor
      Autosomal recessive congenital ichthyosis (ARCI) disrupts normal keratinization, resulting in generalized scaling of the skin. There are presently no curative therapies available (
      • Fleckman P.
      • Newell B.D.
      • Van Steensel M.A.
      • Yan A.C.
      Topical treatment of ichthyoses.
      ). Local protein replacement is, therefore, an encouraging approach for a more specific treatment.
      ARCI refers to a heterogeneous group of rare skin keratinization disorders with an estimated prevalence of 1 in 50,000–200,000 (
      • Dreyfus I.
      • Bourrat E.
      • Maruani A.
      • Bessis D.
      • Chiaverini C.
      • Vabres P.
      • et al.
      Factors associated with impaired quality of life in adult patients suffering from ichthyosis.
      ). The disease is characterized by notable impairments to the skin’s barrier function, resulting in frequent infections and increased transepidermal water loss. ARCI is caused by mutations in 1 of 12 identified genes involved in epidermal differentiation. The most common of these are loss of function mutations in TGM1, affecting approximately 30% of patients (
      • Rodriguez-Pazos L.
      • Ginarte M.
      • Vega A.
      • Toribio J.
      Autosomal recessive congenital ichthyosis.
      ). TGM1 encodes transglutaminase 1 (TG1), a protein that plays an essential role in the formation of the cornified envelope (
      • Eckert R.L.
      • Sturniolo M.T.
      • Broome A.M.
      • Ruse M.
      • Rorke E.A.
      Transglutaminase function in epidermis.
      ). Because animal models of severe keratinization disorders such as ARCI are not viable and animal skin poorly represents human skin (
      • Gerber P.A.
      • Buhren B.A.
      • Schrumpf H.
      • Homey B.
      • Zlotnik A.
      • Hevezi P.
      The top skin-associated genes: a comparative analysis of human and mouse skin transcriptomes.
      ), the use of organotypic skin equivalents has emerged as a valid tool to investigate ARCI.
      In the present study, full-thickness skin equivalents generated from the fibroblasts and keratinocytes of ARCI patients with mutations in TGM1 were treated topically with TG1. Because biomacromolecules do not normally overcome the skin barrier, owing to their high molecular weight, protein delivery was mediated by use of thermoresponsive nanogels (tNG) (
      • Cuggino J.C.
      • Alvarez I.C.I.
      • Strumia M.C.
      • Welker P.
      • Licha K.
      • Steinhilber D.
      • et al.
      Thermosensitive nanogels based on dendritic polyglycerol and N-isopropylacrylamide for biomedical applications.
      ). Proteins as large as 150 kDa have been encapsulated within tNGs and subsequently released above a thermal trigger point (
      • Giulbudagian M.
      • Yealland G.
      • Hönzke S.
      • Geisendörfer B.
      • Kleuser B.
      • Hedtrich S.
      • et al.
      Breaking the barrier—potent anti-inflammatory activity following efficient topical delivery of etanercept using thermoresponsive nanogels.
      ,
      • Witting M.
      • Molina M.
      • Obst K.
      • Plank R.
      • Eckl K.M.
      • Hennies H.C.
      • et al.
      Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules.
      ). Our groups previously reported the epidermal delivery of functional TG1 using topically applied tNGs and rescue of barrier defects in TGM1 knockdown skin equivalents (
      • Witting M.
      • Molina M.
      • Obst K.
      • Plank R.
      • Eckl K.M.
      • Hennies H.C.
      • et al.
      Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules.
      ). However, whether TG1-loaded tNGs are an effective topical treatment for ARCI skin with TGM1 mutations, rather than transiently induced TGM1 knockdowns, was still unclear.
      The study was approved by the Ethics Committee of the Medical University of Innsbruck, Austria, and samples were taken after obtaining written informed consent of the probands. Full-thickness skin equivalents were generated from fibroblasts plus normal keratinocytes, keratinocytes with transient TGM1 knockdowns, or keratinocytes from ARCI patients with TGM1 mutations (Figure 1). In comparison to normal equivalents, TGM1 knockdown and patient equivalents both demonstrated slightly thinned stratum corneum and epidermis, with reduced cell number within the granular layer. The epidermal differentiation markers keratin 14 and 10 were distributed appropriately. TG1 activity was present in normal skin equivalents but not in those generated from patient cells or TGM1 knockdown keratinocytes, in line with the inactivating mutations found in patient 1, and the absence of persistent TG1 expression in patient 2 and knockdown equivalents. Notably, knockdown equivalents demonstrated increasing TGM1 transcript levels over time (>50% after 10 days cultivation), indicating a loss of effective repression (Supplementary Figure S1 online).
      Figure thumbnail gr1
      Figure 1Characterization of full-thickness skin equivalents: Cryosections of (a) normal, (b) TGM1 knockdown, (c) ARCI patient 1, and (d) ARCI patient 2 skin equivalents. From left to right, images show hematoxylin and eosin, keratin 14 (green), keratin 10 (green), TG1 (green), and TG1 activity staining (counter-staining with DAPI in blue). Scale bars = 50 μm. The dashed, yellow line indicates the epidermal–dermal junction. The viability of normal human keratinocytes derived from healthy subject (black), patient 1 (light gray), or patient 2 (dark gray) following (e) 24-hour or (f) 48-hour incubation with TG1, tNGs, and TG1-loaded tNGs as assessed by MTT assay. (g) Viability of normal skin equivalents following full treatment regimen with TG1-loaded tNGs (5 μg/cm2 TG1, 500 μg/cm2 tNG; applied four times over 8 days) as assessed by MTT assay; results are expressed as % untreated control. Statistical differences were assessed by one-way analysis of variance with Dunnett’s correction for multiple comparisons (n = 3). SDS served as positive control. ARCI, autosomal recessive congenital ichthyosis; H&E, hematoxylin and eosin; tNG, thermoresponsive nanogel; TG1, transglutaminase 1.
      To assess their biocompatibility, TG1-loaded tNGs were incubated with normal, patient 1, and patient 2 keratinocytes, as well as fibroblasts for up to 48 hours, resulting in no significant cytotoxicity at any of the tested concentrations (Figure 1b, Supplementary Figures S2 and S3 online). Concordantly, no significant cytotoxicity was observed following the application of tNGs onto skin equivalents (Figure 1g). Additionally, the ability of TG1, alone or loaded in tNGs, to enter keratinocytes was assessed by confocal microscopy. In both cases, TG1 entered the cytoplasm in a time-dependent manner (Supplementary Figure S4 online). Notably, tNGs entered more rapidly than the TG1, which, with their lack of clear intracellular co-localization, would suggest that the tNGs and TG1 enter keratinocytes separately, concordant with the relatively quick release of protein at temperatures ≥35°C. It should be noted, however, that previous evidence indicates tNGs are largely unable to overcome the stratum corneum of even barrier-deficient skin, suggesting that, in most cases, little or no contact will occur between them and viable epidermal cells (
      • Giulbudagian M.
      • Hönzke S.
      • Bergueiro J.
      • Işik D.
      • Schumacher F.
      • Saeidpour S.
      • et al.
      Enhanced topical delivery of dexamethasone by β-cyclodextrin decorated thermoresponsive nanogels.
      ).
      Finally, patient 1 skin equivalents were topically treated with TG1, either in solution or loaded in tNGs, four times over 8 days. Untreated patient 1 equivalents demonstrated decreased barrier function, shown by the significant increases in their apparent permeabilities to testosterone compared to normal equivalents (Figure 2a). Following full treatment regimens with TG1-loaded tNGs, significant decreases in apparent permeabilities—indicating improved barrier function—correlating to TG1 dose were seen (Figure 2a, 2d, Supplementary Figure S5 online). Importantly, permeation was almost unaffected by the application of unloaded tNG or TG1 dissolved in phosphate buffered saline only (Figure 2b, 2c). Activity staining confirmed the delivery of functional TG1 into viable epidermal layers (Figure 2e), and the distribution of activity was comparable to normal equivalents. Improvement of barrier activity was further confirmed by permeability tests with Lucifer yellow (Figure 2f) and N-hydroxy-sulfosuccinimide-LC-biotin (Supplementary Figure S6 online). Compared to equivalents with normal keratinocytes, a 59-fold increase was seen in the amount of Lucifer yellow fully passing through patient 1 equivalents. Similarly, 39-fold and 43-fold increases were respectively seen in patient 1 equivalents treated with unloaded tNG and TG1 dissolved in phosphate buffered saline. However, following treatment with TG1-loaded tNGs, full Lucifer yellow penetration was only 1.2-fold that of the control, clearly corroborating the role of TG1-loaded tNGs in the reconstitution of patient equivalent barrier function. It is highly likely that the majority of TG1 penetrating into the viable epidermis did so independently of the tNGs because they do not overcome the stratum corneum (
      • Giulbudagian M.
      • Hönzke S.
      • Bergueiro J.
      • Işik D.
      • Schumacher F.
      • Saeidpour S.
      • et al.
      Enhanced topical delivery of dexamethasone by β-cyclodextrin decorated thermoresponsive nanogels.
      ).
      Figure thumbnail gr2
      Figure 2Skin-barrier function and TG1 activity of skin equivalents following TG1-loaded tNG treatment. Apparent permeabilities of normal or patient 1 skin equivalents, untreated (white) or treated with either unloaded tNGs (dashed white bars), TG1 in PBS (gray), or TG1 loaded in tNGs (dashed gray bars). (a) The addition of unloaded tNGs did not alter the significant difference in Papp values between normal (wt) and patient 1 (TG1Δ) skin equivalents. (b) A significant difference is seen between TG1Δ equivalents treated with the vehicle control and 10 μg/cm2 TG1 in the tNG treatment group, but not the PBS treatment group (n = 3, error bars = standard error of the mean, P ≤ 0.05, ∗∗P ≤ 0.01). Corresponding permeation profiles of skin equivalents following treatment with (c) unloaded tNGs, (d) TG1 in PBS, or (e) TG1-loaded tNGs (UTs are identical in all panels). TG1 staining and activity in (f) patient 1 equivalents treated with TG1/PBS (control), (g) normal equivalents treated with TG1/tNG, (h) TG1 knockdown equivalent treated with TG1/tNG and (i) patient 1 equivalent treated with TG1/tNG (blue = DAPI, green = TG1 and biotinylated-cadaverine staining, respectively). Scale bars = 50 μm. Lucifer yellow permeation into (j) normal skin equivalent, (k) patient 1 skin equivalent, (l) patient 1 skin equivalent treated with unloaded tNGs, (m) patient 1 equivalent treated with TG1 in PBS (10 μg/cm2 TG1), and (n) patient 1 equivalent treated with TG1/tNG (10 μg/cm2 TG1 and 500 μg /cm2 tNG). Dashed, yellow line indicates the epidermal–dermal junction (blue = DAPI, green = Lucifer yellow). Scale bars = 75 μm. Note that due to limited availability of patient keratinocytes, the control experiments shown here (l, m) were performed once and still require confirmation by further independent experiments. Papp, apparent permeabilities; PBS, phosphate buffered saline; tNG, thermoresponsive nanogel; TG1, transglutaminase 1; UT, untreated control.
      This study aimed to further characterize the therapeutic potential of TG1-loaded tNGs in ARCI skin, as well as to better understand their mechanism of action, based on a previous proof-of-principle study demonstrating epidermal delivery of TG1 following topical application of TG1-loaded tNGs (
      • Witting M.
      • Molina M.
      • Obst K.
      • Plank R.
      • Eckl K.M.
      • Hennies H.C.
      • et al.
      Thermosensitive dendritic polyglycerol-based nanogels for cutaneous delivery of biomacromolecules.
      ). Overall, these data verify that topical protein substitution could mitigate or even reverse the ARCI disease phenotype. Notably,
      • Aufenvenne K.
      • Larcher F.
      • Hausser I.
      • Duarte B.
      • Oji V.
      • Nikolenko H.
      • et al.
      Topical enzyme-replacement therapy restores transglutaminase 1 activity and corrects architecture of transglutaminase-1-deficient skin grafts.
      previously demonstrated that topical applications of TG1 mixed with cationic liposomes successfully delivered the functional protein to skin equivalents, formed from TGM1 mutant ARCI patient cells, grafted onto humanized mice. In contrast to our system, no changes to barrier function were observed upon treatment, likely a result of their model; unlike the typical ARCI phenotype, the grafted animals demonstrated compact hyperkeratosis and transepidermal water loss levels close to non-ARCI controls.
      In summary, topical TG1 replacement therapy is a highly promising therapeutic avenue for ARCI patients with disease-causing TGM1 mutations. The work here indicates TG1 delivery to the intercellular spaces between keratinocytes, and possibly their intracellular environments, can produce therapeutic improvements to the skin-barrier function of the ARCI phenotype. It is hypothesized that increasing the concentration or enzymatic activity of TG1 within the tNG will result in improved therapeutic efficacy and is the likely starting point for future development. The ability of tNGs to encapsulate a wide variety of proteins and deliver these past the stratum corneum of barrier-deficient skin makes them a promising platform technology to treat a range of inflammatory and monogenic skin diseases.

      ORCIDs

      Hans Christian Hennies: http://orcid.org/0000-0001-7210-2389

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      The authors would like to thank Katja Fuchs and Maria Molina for their scientific support, Christian Ploner for providing skin samples, and fu:stat for excellent help with the statistical analysis of the data.
      Funding from the German Research Foundation (HE7440/2-1) and the Berlin-Brandenburg Research Platform BB3R to SH and the German Research Foundation (HE3119/9-1), the Austrian Science Fund (FWF, I2259-B26), the German Federal Ministry for Education and Research (E-Rare-2 01GM1201), and the Cologne Fortune Program of the Faculty of Medicine, University of Cologne, to HCH is greatly acknowledged.

      Supplementary Material

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