Mek1/2 gene dosage determines tissue response to oncogenic Ras signaling in the skin.

Ras genes are commonly mutated in human cancers of the skin and other tissues. Oncogenic Ras signals through multiple effector pathways, including the Erk1/2 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K) and the Ral guanine nucleotide exchange factor (RalGEF) cascades. In epidermis, the activation of oncogenic Ras induces hyperplasia and inhibits differentiation, features characteristic of squamous cell carcinoma. The downstream effector pathways required for oncogenic Ras effects in epidermis, however, are undefined. In this study, we investigated the direct contribution of Mek1 and Mek2 MAPKKs to oncogenic Ras signaling. The response of murine epidermis to conditionally active oncogenic Ras was unimpaired by deletion of either Mek1 or Mek2 MAPKKs individually. In contrast, Ras effects were entirely abolished by combined deletion of all Mek1/2 alleles, whereas epidermis retaining only one allele of either Mek1 or Mek2 showed intermediate responsiveness. Thus, the effects of oncogenic Ras on proliferation and differentiation in skin show a gene dosage-dependent requirement for the Erk1/2 MAPK cascade at the level of Mek1/2 MAPKKs.

Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer.

Sustainable correction of severe human genetic disorders of self-renewing tissues, such as the blistering skin disease junctional epidermolysis bullosa (JEB), is facilitated by stable genomic integration of therapeutic genes into somatic tissue stem cells. While integrating viral vectors can achieve this, they suffer from logistical and biosafety concerns. To circumvent these limitations, we used the Sleeping Beauty transposable element to integrate the LAMB3 cDNA into genomes of epidermal holoclones from six unrelated JEB patients. These cells regenerate human JEB skin that is normalized at the level of laminin 5 protein expression, hemidesmosome formation and blistering. Transposon-mediated gene delivery therefore affords an opportunity for stable gene delivery in JEB and other human diseases.

Peptide delivery to tissues via reversibly linked protein transduction sequences.

The development of peptide-based therapeutics has suffered from challenges associated with delivery to intact tissue. In skin, an array of protein targets resides only tens of micrometers below the surface; however, because of difficulties in traversing the cutaneous barrier, the potentialfor peptide-based therapeutics remains unrealized. We have developed a general approach for topical peptide delivery into skin using releasable protein transduction sequences to enable peptide transport across tissue boundaries. Upon entry into the cell, the disulfide linkage between the peptide transduction sequences and peptide cargo is cleaved, permitting the dissociation of the highly charged peptide transduction sequences from the active peptide. A protype cargo peptide, the hemagglutinin (HA) epitope, was conjugated to a hepta-arginine protein transduction sequence via a releasable disulfide linkage. This construct penetrated the skin to deep dermis within 1 h after topical application. Consistent with the dissociation of the protein transduction and cargo sequences, absorbed protein transduction sequences and HA peptides displayed differential intracellular localization. Reversible protein transduction sequence linkage thus represents a noninvasive platform for tissue delivery of intact peptides with no requirement for viral vectors or parenteral injection and may be of broad utility in molecular therapy.

Cutaneous gene transfer for skin and systemic diseases.

Recent progress in molecular genetics has illuminated the basis for a wide variety of inherited and acquired diseases. Gene therapy offers an attractive therapeutic approach capitalizing upon these new mechanistic insights. The skin is a uniquely attractive tissue site for development of new genetic therapeutic approaches both for its accessibility as well as for the large number of diseases that are amenable in principle to cutaneous gene transfer. Amongst these opportunities are primary monogenic skin diseases, chronic wounds and systemic disorders characterized by low or absent levels of circulating polypeptides. For cutaneous gene therapy to be effective, however, significant progress is required in a number of domains. Recent advances in vector design, administration, immune modulation, and regulation of gene expression have brought the field much nearer to clinical utility.

Sustainable systemic delivery via a single injection of lentivirus into human skin tissue.

The skin offers a tissue site accessible for delivery of gene-based therapeutics. To develop the capability for sustained systemic polypeptide delivery via cutaneous gene transfer, we generated and injected pseudotyped HIV-1 lentiviral vectors intradermally at a range of doses into human skin grafted on immune-deficient mice. Unlike Moloney murine leukemia virus (MLV)-based retrovectors, which failed to achieve detectable cutaneous gene transfer by this approach, lentivectors effectively targeted all major cell types within human skin tissue, including fibroblasts, endothelial cells, keratinocytes, and macrophages. After a single injection, lentivectors encoding human erythropoietin (EPO) produced dose-dependent increases in serum human EPO levels and hematocrit that increased rapidly within one month and remained stable subsequently. Delivered gene expression was confined locally at the injection site. Excision of engineered skin led to rapid and complete loss of human EPO in the bloodstream, confirming that systemic EPO delivery was entirely due to lentiviral targeting of cells within skin rather than via spread of the injected vector to visceral tissues. These findings indicate that the skin can sustain dosed systemic delivery of therapeutic polypeptides via direct lentivector injection and thus provide an accessible and reversible approach for gene-based delivery to the bloodstream.

Impact of laminin 5 beta3 gene versus protein replacement on gene expression patterns in junctional epidermolysis bullosa.

Molecular therapy studies to date have examined only a limited number of corrective parameters. To assess more global impacts on cellular gene expression for two major molecular therapeutic approaches, we compared gene versus protein delivery in the human genetic disease junctional epidermolysis bullosa (JEB). Both gene and protein replacement of the laminin 5 beta3 (beta3) adhesion molecule restored normal growth and adhesion to poorly viable JEB cells. Gene expression profiling was then performed using cDNA microarrays. The expression of more genes was normalized after beta3 gene transfer than after protein transfer. As anticipated for beta3 delivery, many of the genes whose expression was restored to the normal range were those encoding adhesion molecules and hemidesmosome components. Although gene transfer normalized the expression of a higher percentage of genes than did protein transfer, neither approach fully normalized expression of all genes examined. In addition, both approaches disrupted the expression of some genes, but protein transfer altered expression of a larger proportion of the genes studied. Our findings suggest that therapeutic gene and protein delivery may exert different effects on gene expression and thus may have implications for the development and analysis of molecular therapies for the treatment of genetic disorders.

In vivo restoration of laminin 5 beta 3 expression and function in junctional epidermolysis bullosa.

The blistering disorder, lethal junctional epidermolysis bullosa (JEB), can result from mutations in the LAMB3 gene, which encodes laminin 5 beta3 (beta3). Appropriate expression of LAMbeta3 in JEB skin tissue could potentially ameliorate the symptoms of the underlying disease. To explore the utility of this therapeutic approach, primary keratinocytes from six unrelated JEB patients were transduced with a retroviral vector encoding beta3 and used to regenerate human skin on severe combined immunodeficient (SCID) mice. Tissue regenerated from beta3-transduced JEB keratinocytes produced phenotypically normal skin characterized by sustained beta3 expression and the formation of hemidesmosomes. Additionally, beta3 gene transfer corrected the distribution of a number of important basement membrane zone proteins including BPAG2, integrins beta4/beta1, and laminins alpha3/gamma2. Skin produced from beta3-negative (beta3[-]) JEB cells mimicked the hallmarks of the disease state and did not exhibit any of the aforementioned traits. Therefore, by effecting therapeutic gene transfer to beta3-deficient primary keratinocytes, it is possible to produce healthy, normal skin tissue in vivo. These data support the utility of gene therapy for JEB and highlight the potential for gene delivery in the treatment of human genetic skin disease.

Genetic correction of inherited epidermal disorders.

Genetic correction of monogenic human skin disorders represents a potentially effective molecular therapy for severe diseases in which current therapy is only palliative. The stratified epithelium of the epidermis represents the tissue location with the largest number of genetic skin diseases yet characterized. Specific requirements of successful gene delivery in this setting include correct targeting within tissue, durability, and a lack of immunogenecity. Progress toward this goal has advanced from identification of disease genes to reintroduction of wild-type genes to patient cell lines and primary cells in vitro. This initial work has been extended to gene-based correction of diseased tissue regenerated in vivo in the form of human patient skin xenografts on immune-deficient mice. Efforts in this human tissue model have laid the foundation for future efforts to extend this progress toward ex vivo cutaneous gene therapy trials in humans.

Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation.

Many systemically effective drugs such as cyclosporin A are ineffective topically because of their poor penetration into skin. To surmount this problem, we conjugated a heptamer of arginine to cyclosporin A through a pH-sensitive linker to produce R7-CsA. In contrast to unmodified cyclosporin A, which fails to penetrate skin, topically applied R7-CsA was efficiently transported into cells in mouse and human skin. R7-CsA reached dermal T lymphocytes and inhibited cutaneous inflammation. These data establish a general strategy for enhancing delivery of poorly absorbed drugs across tissue barriers and provide a new topical approach to the treatment of inflammatory skin disorders.

Nuclear factor kappaB subunits induce epithelial cell growth arrest.

Nuclear factor kappaB (NF-kappaB) gene-regulatory proteins play important roles in inflammation, neoplasia, and programmed cell death. Recently, blockade of NF-kappaB function has been shown to result in epithelial hyperplasia, suggesting a potential role for NF-kappaB in negative growth regulation. We expressed active NF-kappaB subunits in normal epithelial cells and found that NF-kappaB profoundly inhibits cell cycle progression. This growth inhibition is resistant to mitogenic stimuli and is accompanied by other features of irreversible growth arrest. NF-kappaB-triggered cell cycle arrest is also associated with selective induction of the cyclin-dependent kinase inhibitor p21CiP1, with overexpression of p21(Cip1) alone inducing findings similar to those seen with NF-kappaB in vitro. An active NF-kappaB subunit expressed in the epidermis of p21(CiP1-/- mice, however, displays only partial growth-inhibitory effects, suggesting that full NF-kappaB growth inhibition is only partially p21(Cip1) dependent in this setting. These data indicate that NF-kappaB can trigger cell cycle arrest in epithelial cells in association with selective induction of a cell cycle inhibitor.

NF-kappaB determines localization and features of cell death in epidermis.

Specialized forms of physiologic cell death lacking certain characteristic morphologic features of apoptosis occur in terminally differentiating tissues, such as in the outer cell layers of epidermis. In these cell layers, NF-kappaB translocates from the cytoplasm to the nucleus and induces target gene expression. In light of its potent role in regulating apoptotic cell death in other tissues, NF-kappaB activation in these cells suggests that this transcription factor regulates cell death during terminal differentiation. Here, we show that NF-kappaB protects normal epithelial cells from apoptosis induced by both TNFalpha and Fas, whereas NF-kappaB blockade enhances susceptibility to death via both pathways. Expression of IkappaBalphaM under control of keratin promoter in transgenic mice caused a blockade of NF-kappaB function in the epidermis and provoked premature spontaneous cell death with apoptotic features. In normal tissue, expression of the known NF-kappaB-regulated antiapoptotic factors, TRAF1, TRAF2, c-IAP1, and c-IAP2, is most pronounced in outer epidermis. In transgenic mice, NF-kappaB blockade suppressed this expression, whereas NF-kappaB activation augmented it, consistent with regulation of cell death by these NF-kappaB effector proteins. These data identify a new role for NF-kappaB in preventing premature apoptosis in cells committed to undergoing physiologic cell death and indicate that, in stratified epithelium, such cell death normally proceeds via a distinct pathway that is resistant to NF-kappaB and its antiapoptotic target effector genes.

Sonic hedgehog opposes epithelial cell cycle arrest.

Stratified epithelium displays an equilibrium between proliferation and cell cycle arrest, a balance that is disrupted in basal cell carcinoma (BCC). Sonic hedgehog (Shh) pathway activation appears sufficient to induce BCC, however, the way it does so is unknown. Shh-induced epidermal hyperplasia is accompanied by continued cell proliferation in normally growth arrested suprabasal cells in vivo. Shh-expressing cells fail to exit S and G2/M phases in response to calcium-induced differentiation and also resist exhaustion of replicative growth capacity. In addition, Shh blocks p21(CIP1/WAF1)-induced growth arrest. These data indicate that Shh promotes neoplasia by opposing normal stimuli for epithelial cell cycle arrest.

Immunization via hair follicles by topical application of naked DNA to normal skin.

In order to test the immune response generated to small amounts of foreign protein in skin, we applied naked DNA in aqueous solution to untreated normal skin. Topical application of plasmid expression vectors for lacZ and the hepatitis B surface antigen (HBsAg) to intact skin induced antigen-specific immune responses that displayed TH2 features. For HBsAg, specific antibody and cellular responses were induced to the same order of magnitude as those produced by intramuscular injection of the commercially available recombinant HBsAg polypeptide vaccine. Finally, topical gene transfer was dependent on the presence of normal hair follicles.

BP180 gene delivery in junctional epidermolysis bullosa.

Epidermolysis bullosa (EB) comprises a family of inherited blistering skin diseases for which current therapy is only palliative. Junctional EB (JEB) involves dissociation of the dermal-epidermal junction and results from mutations in a number of genes that encode vital structural proteins, including BP180 (type XVII collagen/BPAG2). In order to develop a model of corrective gene delivery for JEB, we produced a retroviral expression vector for wild-type human BP180 and used it to restore BP180 protein expression to primary keratinocytes from BP180-negative patients with generalized atrophic JEB. Restoration of full-length BP180 protein expression was associated with adhesion parameter normalization of primary JEB keratinocytes in vitro. These cells were then used to regenerate human skin on immune-deficient mice. BP180 gene-transduced tissue demonstrated restoration of BP180 gene expression at the dermal-epidermal junction in vivo while untransduced regenerated JEB skin entirely lacked BP180 expression. These findings provide a basis for future efforts to achieve gene delivery in human EB skin tissue.

High-efficiency gene transfer and pharmacologic selection of genetically engineered human keratinocytes.

Low efficiencies of gene transfer to somatic cells have frustrated therapeutic gene delivery efforts in a wide array of tissues including the skin. Production of populations of keratinocytes in which all cells contain the desired therapeutic gene may be important in future genetic therapies. This may be the case in disorders such as epidermolysis bullosa and ichthyosis, where a failure to correct the vast majority of cells within tissue could perpetuate central disease features such as skin fragility and defective barrier function. We have refined retroviral gene transfer parameters to achieve significant improvements in gene delivery efficiencies to human keratinocytes compared to those previously reported. We have also generated retroviral vectors that allow rapid pharmacologic selection of human keratinocytes without interfering with the potential of these cells to regenerate epidermis in vivo--we determined that blasticidin is superior to the commonly used neomycin. The combined capabilities for efficient retroviral gene transfer and effective pharmacologic selection allow production of entirely engineered populations of human keratinocytes for use in future efforts to achieve effective cutaneous gene delivery.

Alterations in NF-kappaB function in transgenic epithelial tissue demonstrate a growth inhibitory role for NF-kappaB.

Stratified epithelium contains a mitotically active basal layer of cells that cease proliferating, then migrate outwards and undergo terminal differentiation. The control of this process, which is abnormal in cutaneous neoplasia and inflammation, is not well understood. In normal epidermis, NF-kappaB proteins were found to exist in the cytoplasm of basal cells and then to localize in the nuclei of suprabasal cells, suggesting a role for NF-kappaB in the switch from proliferation to growth arrest and differentiation. Functional blockade of NF-kappaB by expressing dominant-negative NF-kappaB inhibitory proteins in transgenic murine and human epidermis produced hyperplastic epithelium in vivo. Consistent with this, application of a pharmacologic inhibitor of NF-kappaB to intact skin induced epidermal hyperplasia. In contrast, overexpression of active p50 and p65 NF-kappaB subunits in transgenic epithelium produced hypoplasia and growth inhibition. These data suggest that spatially restricted NF-kappaB activation occurs in stratified epithelium and indicate that NF-kappaB activation in this tissue, in contrast to its role in other settings, is important for cellular growth inhibition.

Transglutaminase 1 expression in a patient with features of harlequin ichthyosis: case report.

Harlequin ichthyosis (HI) is a life-threatening disorder characterized clinically by massive generalized hyperkeratosis and ultrastructurally by an absence of lamellar bodies. However, infants who survive the perinatal period develop a phenotype resembling the nonbullous ichthyosiform erythrodermic (CIE) form of autosomal recessive ichthyosis. We studied a child with a severe hyperkeratotic skin disorder present at birth that developed into a CIE-like phenotype. Electron microscopy demonstrated an absence of lamellar bodies consistent with HI. Abnormalities of filaggrin and involucrin expression by immunostaining were evident. However, transglutaminase 1 (TGase1) was expressed in the epidermis in a pattern consistent with other diseases that involve epidermal acanthosis. Analysis of patient keratinocytes grown in vitro demonstrated expression of normal amounts of TGase1 mRNA and full length TGase1 protein, as well as normal levels of transglutaminase enzymatic activity.