Wei-Li Di

Ex-vivo Gene Therapy Restores LEKTI Activity and Corrects the Architecture of Netherton Syndrome-derived Skin Grafts

Netherton syndrome (NS) is a debilitating congenital skin disorder caused by mutations in the SPINK5 gene encoding the lymphoepithelial Kazal-type-related inhibitor (LEKTI). It is characterized by defective keratinization, recurrent infections, and hypernatraemic dehydration with a mortality rate of about 10% in the first year of life. Currently, there are no curative treatments for NS. We have developed a HIV-1 based, self-inactivating lentiviral vector to express SPINK5 in keratinocytes as part of an ex-vivo gene therapy strategy for NS. High transduction efficiency was achieved in NS keratinocytes and reconstitution of LEKTI expression was confirmed in previously deficient cells. These genetically corrected keratinocytes were further tested in an in vitro organotypic culture (OTC) system and in vivo mouse/human skin engraftment model. Results showed correction of epidermal architecture in both OTCs and regenerated skin grafts. Importantly, the results from corrected skin grafts indicated that even where detectable LEKTI expression was restored to a limited numbers of cells, a wider bystander benefit occurred around these small populations. As LEKTI is a secreted protein, the genetically modified graft may provide not only an immediate local protective barrier, but also act as a source of secreted LEKTI providing a generalized benefit following ex-vivo gene therapy.

AKT1-mediated Lamin A/C degradation is required for nuclear degradation and normal epidermal terminal differentiation

Nuclear degradation is a key stage in keratinocyte terminal differentiation and the formation of the cornified envelope that comprises the majority of epidermal barrier function. Parakeratosis, the retention of nuclear material in the cornified layer of the epidermis, is a common histological observation in many skin diseases, notably in atopic dermatitis and psoriasis. Keratinocyte nuclear degradation is not well characterised, and it is unclear whether the retained nuclei contribute to the altered epidermal differentiation seen in eczema and psoriasis. Loss of AKT1 function strongly correlated with parakeratosis both in eczema samples and in organotypic culture models. Although levels of DNAses, including DNase1L2, were unchanged, proteomic analysis revealed an increase in Lamin A/C. AKT phosphorylates Lamin A/C, targeting it for degradation. Consistent with this, Lamin A/C degradation was inhibited and Lamin A/C was observed in the cornified layer of AKT1 knockdown organotypic cultures, surrounding retained nuclear material. Using AKT-phosphorylation-dead Lamin A constructs we show that the retention of nuclear material is sufficient to cause profound changes in epidermal terminal differentiation, specifically a reduction in Loricrin, Keratin 1, Keratin 10, and filaggrin expression. We show that preventing nuclear degradation upregulates BMP2 expression and SMAD1 signalling. Consistent with these data, we observe both parakeratosis and evidence of increased SMAD1 signalling in atopic dermatitis. We therefore present a model that, in the absence of AKT1-mediated Lamin A/C degradation, DNA degradation processes, such as those mediated by DNAse 1L2, are prevented, leading to parakeratosis and changes in epidermal differentiation.

Phase I study protocol for ex-vivo lentiviral gene therapy for the inherited skin disease, Netherton Syndrome.

Netherton syndrome (NS) is a serious inherited skin disorder caused by mutations in the serine protease inhibitor Kazal type 5 gene (SPINK5), which encodes for a serine protease inhibitor lymphoepithelial Kazal type-related inhibitor (LEKTI). Patients with NS have defective keratinization, hair shaft defects, recurrent infections, atopy, and a predisposition to skin malignancies. Historically, 1 in 10 infants has died before their first birthday. Currently, there are no proven treatments to cure this condition. A SIN-lentiviral vector encoding the codon-optimized SPINK5 gene under the control of a 572 bp element derived from the human involucrin promoter can confer compartment-specific LEKTI expression in NS keratinocytes with restoration of normal skin architecture. Here we detail a study protocol for a phase I trial for feasibility and safety evaluations of autologous epidermal sheets generated from ex vivo gene-corrected keratinocyte stem cells, which will be grafted onto patients with mutation-proven NS.

Human Involucrin Promoter Mediates Repression-Resistant and Compartment-Specific LEKTI Expression

Gene-modified skin grafts, produced through gene transfer to human keratinocyte stem cells, offer the possibility of therapeutic benefit for inherited skin diseases. We have previously described efficient lentiviral vector-mediated gene transfer to keratinocyte stem cells and the generation of human skin grafts for the inherited skin disease, Netherton syndrome, which arises due to mutations in serine protease inhibitor Kazal-type 5 (SPINK5). Vectors incorporating an internal murine retroviral-derived promoter [spleen focus-forming virus (SFFV)] in combination with a codon-optimized SPINK5 transgene supported high levels of reconstitution and robust correction of skin architecture. Subsequent longer-term experiments have uncovered unanticipated silencing phenomena, with loss of SPINK5 gene expression over time. The inadvertent introduction of CpG sites during codon optimization appears to have rendered vectors susceptible to silencing due to methylation across the promoter-transgene boundary. Substitution of the methylation-susceptible SFFV promoter with a 572-bp minimal human involucrin promoter (INVOp), which encodes very few CpG sites, prevented repression of the SPINK5 transgene and resulted in durable and highly compartment-specific reconstitution of lympho-epithelial Kazal-type-related inhibitor (LEKTI) in human skin grafted onto immunodeficient mice. We conclude that skin grafts modified with lentiviral vectors encoding INVOp offer a suitable platform for therapeutic gene therapy in Netherton syndrome, and our experience highlights unanticipated effects of transgene codon optimization.

A mechanistic target of rapamycin complex 1/2 (mTORC1)/V-Akt murine thymoma viral oncogene homolog 1 (AKT1)/cathepsin H axis controls filaggrin expression and processing in skin, a novel mechanism for skin barrier disruption in patients with atopic dermatitis

Background: Filaggrin, which is encoded by the filaggrin gene (FLG), is an important component of the skins barrier to the external environment, and genetic defects in FLG strongly associate with atopic dermatitis (AD). However, not all patients with AD have FLG mutations. Objective: We hypothesized that these patients might possess other defects in filaggrin expression and processing contributing to barrier disruption and AD, and therefore we present novel therapeutic targets for this disease. Results: We describe the relationship between the mechanistic target of rapamycin complex 1/2 protein subunit regulatory associated protein of the MTOR complex 1 (RAPTOR), the serine/threonine kinase V‐Akt murine thymoma viral oncogene homolog 1 (AKT1), and the protease cathepsin H (CTSH), for which we establish a role in filaggrin expression and processing. Increased RAPTOR levels correlated with decreased filaggrin expression in patients with AD. In keratinocyte cell cultures RAPTOR upregulation or AKT1 short hairpin RNA knockdown reduced expression of the protease CTSH. Skin of CTSH‐deficient mice and CTSH short hairpin RNA knockdown keratinocytes showed reduced filaggrin processing, and the mouse had both impaired skin barrier function and a mild proinflammatory phenotype. Conclusion: Our findings highlight a novel and potentially treatable signaling axis controlling filaggrin expression and processing that is defective in patients with AD. Graphical abstract Figure. No Caption available.

Tissue Kallikrein Inhibitors Based on the Sunflower Trypsin Inhibitor Scaffold - A Potential Therapeutic Intervention for Skin Diseases

Tissue kallikreins (KLKs), in particular KLK5, 7 and 14 are the major serine proteases in the skin responsible for skin shedding and activation of inflammatory cell signaling. In the normal skin, their activities are controlled by an endogenous protein protease inhibitor encoded by the SPINK5 gene. Loss-of-function mutations in SPINK5 leads to enhanced skin kallikrein activities and cause the skin disease Netherton Syndrome (NS). We have been developing inhibitors based on the Sunflower Trypsin Inhibitor 1 (SFTI-1) scaffold, a 14 amino acids head-to-tail bicyclic peptide with a disulfide bond. To optimize a previously reported SFTI-1 analogue (I10H), we made five analogues with additional substitutions, two of which showed improved inhibition. We then combined those substitutions and discovered a variant (Analogue 6) that displayed dual inhibition of KLK5 (tryptic) and KLK7 (chymotryptic). Analogue 6 attained a tenfold increase in KLK5 inhibition potency with an Isothermal Titration Calorimetry (ITC) Kd of 20nM. Furthermore, it selectively inhibits KLK5 and KLK14 over seven other serine proteases. Its biological function was ascertained by full suppression of KLK5-induced Protease-Activated Receptor 2 (PAR-2) dependent intracellular calcium mobilization and postponement of Interleukin-8 (IL-8) secretion in cell model. Moreover, Analogue 6 permeates through the cornified layer of in vitro organotypic skin equivalent culture and inhibits protease activities therein, providing a potential drug lead for the treatment of NS.

Persistent kallikrein 5 activation induces atopic dermatitis-like skin architecture independent of PAR2 activity

Background Upregulation of kallikreins (KLKs) including KLK5 has been reported in atopic dermatitis (AD). KLK5 has biological functions that include degrading desmosomal proteins and inducing proinflammatory cytokine secretion through protease‐activated receptor 2 (PAR2). However, due to the complex interactions between various cells in AD inflamed skin, it is difficult to dissect the precise and multiple roles of upregulated KLK5 in AD skin. Objective We investigated the effect of upregulated KLK5 on the expression of epidermal‐related proteins and cytokines in keratinocytes and on skin architecture. Methods Lesional and nonlesional AD skin biopsies were collected for analysis of morphology and protein expression. The relationship between KLK5 and barrier‐related molecules was investigated using an ex vivo dermatitis skin model with transient KLK5 expression and a cell model with persistent KLK5 expression. The influence of upregulated KLK5 on epidermal morphology was investigated using an in vivo skin graft model. Results Upregulation of KLK5 and abnormal expression of desmoglein 1 (DSG1) and filaggrin, but not PAR2 were identified in AD skin. PAR2 was increased in response to transient upregulation of KLK5, whereas persistently upregulated KLK5 did not show this effect. Persistently upregulated KLK5 degraded DSG1 and stimulated secretion of IL‐8, IL‐10, and thymic stromal lymphopoietin independent of PAR2 activity. With control of higher KLK5 activity by the inhibitor sunflower trypsin inhibitor G, restoration of DSG1 expression and a reduction in AD‐related cytokine IL‐8, thymic stromal lymphopoietin, and IL‐10 secretion were observed. Furthermore, persistently elevated KLK5 could induce AD‐like skin architecture in an in vivo skin graft model. Conclusions Persistently upregulated KLK5 resulted in AD‐like skin architecture and secretion of AD‐related cytokines from keratinocytes in a PAR2 independent manner. Inhibition of KLK5‐mediated effects may offer potential as a therapeutic approach in AD.

666. A Phase-1 Safety Study Protocol for Lentiviral-Mediated COL7A1 Gene-Modified Autologous Fibroblasts in Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating genodermatosis caused by loss-of-function mutations in COL7A1, the gene encoding type VII collagen (C7), a protein central to anchoring fibril formation at the dermal-epidermal junction (DEJ). Presently there are no curative treatments for this condition. We have been developing the gene therapy protocol for locally injection of lentiviral mediated COL7A1 gene-modified autologous fibroblasts into the patients with RDEB. Primary fibroblasts were isolated from a 6mm skin biopsy. Cells are expanded and transduced with a GMP compliant lenti-viral vector containing codon optimised COL7A1 gene cDNA under the control of the human PGK promoter. Transduced cells were further expended to reach appropriate cell numbers and cryopreserved until patients are ready. Scale up studies carried out in our GMP compliance laboratory showed C7 expression and stable integration of vector into cells. In-situ immunostaining and western blot analysis confirmed the expression of collagen VII protein (C7) not only in transduced RDEB fibroblasts but also in the culture media, indicating there is secreted C7 from transduced cells.The primary objective of our proposed Phase-I study is to determine the safety and tolerability of treatment with autologous gene modified cells administered to RDEB patients. Fibroblasts expressing codon-optimised COL7A1 will be injected intra-dermally in 5-10 adults with RDEB. The secondary objectives are to assess the efficacy of this treatment and its impact on skin and reversion of gene expression. Each study participant will receive three intradermal injections of ex-vivo transduced autologous fibroblasts expressing codon-optimised COL7A1 as the IMP and three intradermal injections of non-transduced autologous fibroblasts as control. Each injection (both IMP and control) will be administered into separate 1cm2 areas of intact skin sites. Participants will be followed up with study interventions for a total of 36-month period at various time points. If successful, a similar approach could be used to modify autologous epidermal sheets for localised treatment of blisters as well as systemic cell therapies.