T-cell activation and bacterial infection in skin wounds of recessive dystrophic epidermolysis bullosa patients.

Recessive dystrophic epidermolysis bullosa (RDEB) patients develop poorly healing skin wounds that are frequently colonized with microbiota. Because T cells play an important role in clearing such pathogens, we aimed to define the status of adaptive T cell-mediated immunity in RDEB wounds. Using a non-invasive approach for sampling of wound-associated constituents, we evaluated microbial contaminants in cellular fraction and exudates obtained from RDED wounds. Infectivity and intracellular trafficking of inactivated Staphylococcus aureus was accessed in RDEB keratinocytes. S. aureus and microbial antigen-specific activation of RDEB wound-derived T cells were investigated by fluorescence-activated cell sorting-based immune-phenotyping and T-cell functional assays. We found that RDEB wounds and epithelial cells are most frequently infected with Staphylococcus sp. and Pseudomonas sp. and that S. aureus essentially infects more RDEB keratinocytes and RDEB-derived squamous cell carcinoma cells than keratinocytes from healthy donors. The RDEB wound-associated T cells contain populations of CD4+ and CD8+ peripheral memory T cells that respond to soluble microbial antigens by proliferating and secreting interferon gamma (IFNγ). Moreover, CD8+ cytotoxic T lymphocytes recognize S. aureus-infected RDEB keratinocytes and respond by producing interleukin-2 (IL-2) and IFNγ and degranulating and cytotoxically killing infected cells. Prolonged exposure of RDEB-derived T cells to microbial antigens in vitro does not trigger PD-1-mediated T-cell exhaustion but induces differentiation of the CD4high population into CD4high CD25+ FoxP3+ regulatory T cells. Our data demonstrated that adaptive T cell-mediated immunity could clear infected cells from wound sites, but these effects might be inhibited by PD-1/Treg-mediated immuno-suppression in RDEB.

Immunological mechanisms underlying progression of chronic wounds in recessive dystrophic epidermolysis bullosa.

Hereditary epidermolysis bullosa (EB) is a mechanobullous skin fragility disorder characterized by defective epithelial adhesion, leading to mechanical stress-induced skin blistering. Based on the level of tissue separation within the dermal-epidermal junction, EB is categorized into simplex (EBS), junctional (JEB), dystrophic (DEB) and Kindler syndrome. There is no cure for EB, and painful chronic cutaneous wounds are one of the major complications in recessive (RDEB) patients. Although RDEB is considered a cutaneous disease, recent data support the underlying systemic immunological defects. Furthermore, chronic wounds are often colonized with pathogenic microbiota, leading to excessive inflammation and altered wound healing. Consequently, patients with RDEB suffer from a painful sensation of chronic, cutaneous itching/burning and an endless battle with bacterial infections. To improve their quality of life and life expectancy, it is important to prevent cutaneous infections, dampen chronic inflammation and stimulate wound healing. A clear scientific understanding of the immunological events underlying the maintenance of chronic poorly healing wounds in RDEB patients is necessary to improve disease management and better understand other wound healing disorders. In this review, we summarize current knowledge of the role of professional phagocytes, such as neutrophils, macrophages and dendritic cells, the role of T-cell-mediated immunity in lymphoid organs, and the association of microbiota with poor wound healing in RDEB. We conclude that RDEB patients have an underlying immunity defect that seems to affect antibacterial immunity.

Intracellular escape strategies of Staphylococcus aureus in persistent cutaneous infections.

Pathogenic invasion of Staphylococcus aureus is a major concern in patients with chronic skin diseases like atopic dermatitis (AD), epidermolysis bullosa (EB), or chronic diabetic foot and venous leg ulcers, and can result in persistent and life-threatening chronic non-healing wounds. Staphylococcus aureus is generally recognized as extracellular pathogens. However, S. aureus can also invade, hide and persist in skin cells to contribute to wound chronicity. The intracellular life cycle of S. aureus is currently incompletely understood, although published studies indicate that its intracellular escape strategies play an important role in persistent cutaneous infections. This review provides current scientific knowledge about the intracellular life cycle of S. aureus in skin cells, which can be classified into professional and non-professional antigen-presenting cells, and its strategies to escape adaptive defense mechanisms. First, we discuss phenotypic switch of S. aureus, which affects intracellular routing and degradation. This review also evaluates potential intracellular escape mechanism of S. aureus to avoid intracellular degradation and antigen presentation, preventing an immune response. Furthermore, we discuss potential drug targets that can interfere with the intracellular life cycle of S. aureus. Taken together, this review aimed to increase scientific understanding about the intracellular life cycle of S. aureus into skin cells and its strategies to evade the host immune response, information that is crucial to reduce pathogenic invasion and life-threatening persistence of S. aureus in chronic cutaneous infections.

Fibulin-4 E57K Knock-in Mice Recapitulate Cutaneous, Vascular and Skeletal Defects of Recessive Cutis Laxa 1B with both Elastic Fiber and Collagen Fibril Abnormalities.

Fibulin-4 is an extracellular matrix protein essential for elastic fiber formation. Frameshift and missense mutations in the fibulin-4 gene (EFEMP2/FBLN4) cause autosomal recessive cutis laxa (ARCL) 1B, characterized by loose skin, aortic aneurysm, arterial tortuosity, lung emphysema, and skeletal abnormalities. Homozygous missense mutations in FBLN4 are a prevalent cause of ARCL 1B. Here we generated a knock-in mouse strain bearing a recurrent fibulin-4 E57K homozygous missense mutation. The mutant mice survived into adulthood and displayed abnormalities in multiple organ systems, including loose skin, bent forelimb, aortic aneurysm, tortuous artery, and pulmonary emphysema. Biochemical studies of dermal fibroblasts showed that fibulin-4 E57K mutant protein was produced but was prone to dimer formation and inefficiently secreted, thereby triggering an endoplasmic reticulum stress response. Immunohistochemistry detected a low level of fibulin-4 E57K protein in the knock-in skin along with altered expression of selected elastic fiber components. Processing of a precursor to mature lysyl oxidase, an enzyme involved in cross-linking of elastin and collagen, was compromised. The knock-in skin had a reduced level of desmosine, an elastin-specific cross-link compound, and ultrastructurally abnormal elastic fibers. Surprisingly, structurally aberrant collagen fibrils and altered organization into fibers were characteristics of the knock-in dermis and forelimb tendons. Type I collagen extracted from the knock-in skin had decreased amounts of covalent intermolecular cross-links, which could contribute to the collagen fibril abnormalities. Our studies provide the first evidence that fibulin-4 plays a role in regulating collagen fibril assembly and offer a preclinical platform for developing treatments for ARCL 1B.

Analysis of chemotactic molecules in bone marrow-derived mesenchymal stem cells and the skin: Ccl27-Ccr10 axis as a basis for targeting to cutaneous tissues.

BACKGROUND AIMS: Adult stem cells produce a plethora of extracellular matrix molecules and have a high potential as cell-based therapeutics for connective tissue disorders of the skin. However, the primary challenge of the stem cell-based approach is associated with the inefficient homing of systemically infused stem cells to the skin. METHODS: We examined chemotactic mechanisms that govern directional migration of mesenchymal stem cells (MSCs) into the skin by conducting a comprehensive expression analysis of chemotactic molecules in MSCs and defined cutaneous tissues from normal and hereditary epidermolysis bullosa (EB)-affected skin. RESULTS: Analysis of chemokine receptors in short-term and long-term MSC cultures showed tissue culture-dependent expression of several receptors. Assessment of epidermis-derived and dermis-derived chemokines showed that most chemotactic signals that originate from the skin preferentially recruit different sets of leukocytes rather than MSCs. Analysis of the chemotactic molecules derived from EB-affected non-blistered skin showed only minor changes in expression of selected chemokines and receptors. Nevertheless, the data allowed us to define the Ccl27-Ccr10 chemotactic axis as the most potent for the recruitment of MSCs to the skin. Our in vivo analysis demonstrated that uniform expression of Ccr10 on MSCs and alteration of Ccl27 level in the skin enhance extravasation of stem cells from circulation and facilitate their migration within cutaneous tissue. CONCLUSIONS: Collectively, our study provides a comprehensive analysis of chemotactic signals in normal and EB-affected skin and proof-of-concept data demonstrating that alteration of the chemotactic pathways can enhance skin homing of the therapeutic stem cells.

Gene expression signatures of mouse bone marrow-derived mesenchymal stem cells in the cutaneous environment and therapeutic implications for blistering skin disorder.

BACKGROUND AIMS: Multiple studies have demonstrated that mesenchymal stromal cells (MSC) can be utilized therapeutically for various congenital and acquired disorders. The involvement of MSC in the maintenance of skin homeostasis and their curative application for the treatment of skin wounds have also been documented. However, it is not known whether MSC can commit to cutaneous lineages, produce structural proteins essential for the skin integrity or be used for hereditary skin disorders. METHODS: To address these questions, we conducted a comparative expression analysis between MSC and potentially adjacent cutaneous cells, fibroblasts and keratinocytes, with specific emphasis on extracellular matrix encoding and related genes. RESULTS: Our data demonstrated that MSC share many features with cutaneous fibroblasts. We also observed that under direct influence of cutaneous fibroblasts in vitro and fibroblast-derived matrix in vivo, MSC acquired a fibroblastic phenotype, suggesting that specific cell-cell interactions play a key regulatory role in the differentiation of MSC. Additionally, the observed fibroblastic transition of MSC was underlined by a significant up-regulation of several cutaneous-specific genes encoding lumican, decorin, type VII collagen, laminin and other structural proteins. As many of the identified genes have considerable therapeutic value for dermatologic afflictions, particularly type VII collagen, we evaluated further the therapeutic potential of congenic MSC in the skin of Col7a1-null mice recapitulating human recessive dystrophic epidermolysis bullosa (RDEB). Remarkably, MSC-derived type VII collagen was sufficient for restoration of the damaged dermal-epidermal junction and partial reversal of the RDEB phenotype. CONCLUSIONS: Collectively, our results suggest that MSC may offer promising therapeutics for the treatment of RDEB and potentially other genodermatoses.

Congenital muscular dystrophy-associated inflammatory chemokines provide axes for effective recruitment of therapeutic adult stem cell into muscles.

BACKGROUND: Congenital muscular dystrophies (CMD) are a clinically and genetically heterogeneous group of neuromuscular disorders characterized by muscle weakness. The two most prevalent forms of CMD, collagen VI-related myopathies (COL6RM) and laminin α2 deficient CMD type 1A (MDC1A), are both caused by deficiency or dysfunction of extracellular matrix proteins. Previously, we showed that an intramuscular transplantation of human adipose-derived stem cells (ADSC) into the muscle of the Col6a1-/- mice results in efficient stem cell engraftment, migration, long-term survival, and continuous production of the collagen VI protein, suggesting the feasibility of the systemic cellular therapy for COL6RM. In order for this therapeutic approach to work however, stem cells must be efficiently targeted to the entire body musculature. Thus, the main goal of this study is to test whether muscle homing of systemically transplanted ADSC can be enhanced by employing muscle-specific chemotactic signals originating from CMD-affected muscle tissue. METHODS: Proteomic screens of chemotactic molecules were conducted in the skeletal muscles of COL6RM- and MDC1A-affected patients and CMD mouse models to define the inflammatory and immune activities, thus, providing potential markers of disease activity or treatment effect. Also using a pre-clinical animal model, recapitulating mild Ullrich congenital muscular dystrophy (UCMD), the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. RESULTS: Comprehensive proteomic screens evaluating relevant human and mouse skeletal muscle biopsies offered chemotactic axes to enhance directional migration of systemically transplanted cells into CMD-affected muscles, including CCL5-CCR1/3/5, CCL2-CCR2, CXCL1/2-CXCR1,2, and CXCL7-CXCR2. Also, the specific populations of ADSC selected with an affinity for the chemokines being released by damaged muscle showed efficient migration to injured site and presented their therapeutic effect. CONCLUSIONS: Collectively, identified molecules provided insight into the mechanisms governing directional migration and intramuscular trafficking of systemically infused stem cells, thus, permitting broad and effective application of the therapeutic adult stem cells for CMD treatment.

Aberrant recruitment of leukocytes defines poor wound healing in patients with recessive dystrophic epidermolysis bullosa.

BACKGROUND: Poorly healing wounds are one of the major complications in patients suffering from recessive dystrophic epidermolysis bullosa (RDEB). At present, there are no effective means to analyze changes in cellular and molecular networks occurring during RDEB wound progression to predict wound outcome and design betted wound management approaches. OBJECTIVES: To better define mechanisms influencing RDEB wound progression by evaluating changes in molecular and cellular networks. METHODS: We developed a non-invasive approach for sampling and analysis of wound-associated constituents using wound-covering bandages. Cellular and molecular components from seventy-six samples collected from early, established and chronic RDEB wounds were evaluated by FACS-based immuno-phenotyping and ELISA. RESULTS: Our cross-sectional analysis determined that progression of RDEB wounds to chronic state is associated with the accumulation (up to 90 %) of CD16+CD66b+ mature neutrophils, loss of CD11b+CD68+ macrophages, and a significant increase (up to 50 %) in a number of CD11c+CD80+CD86+ activated professional antigen presenting cells (APC). It was also marked by changes in activated T cells populations including a reduction of CD45RO+ peripheral memory T cells from 80 % to 30 % and an increase (up to 70 %) in CD45RA+ effector T cells. Significantly higher levels of MMP9, VEGF-A and cathepsin G were also associated with advancing of wounds to poorly healing state. CONCLUSIONS: Our data demonstrated that wound-covering bandages are useful for a non-invasive sampling and analysis of wound-associated constituents and that transition to poorly healing wounds in RDEB patients as associated with distinct changes in leukocytic infiltrates, matrix-remodeling enzymes and pro-angiogenic factors at wound sites.

Retroviral delivery of ECM genes.

The use of recombinant DNA has become a powerful tool in the analysis of functional and structural properties of the extracellular matrix proteins. During last decade, various procedures of plasmid DNA delivery using liposome-based or electroporation-based transfection have been developed. However, in many instances, these procedures were shown to be not effective in DNA transfer or toxic for the mammalian cells. On contrary, retrovirus-mediated infection represents a superior mode of gene delivery with a success rate and viability of the cells approaching 100% in in vitro conditions. The use of the retroviral system also allows permanent insertion of the gene of interest into the chromosome of the infected cell, resulting in efficient gene transfer in which most recipient cells will incorporate and express the transduced gene. In this chapter, we will describe several retrovirus-based systems and provide step-by-step protocols applicable for the production of the recombinant virus and efficient delivery of the ECM genes.

Characterization of the CCL21-mediated melanoma-specific immune responses and in situ melanoma eradication.

Previous studies have shown that secondary lymphoid chemokine, CCL21, can be used for modulation of tumor-specific immune responses. Here, using B16F0 melanoma cells stably expressing CCL21 under the control of cytomegalovirus and ubiquitin promoters, we showed that CCL21-activated immune responses depend on the amount of melanoma-derived chemokine, which, in turn, depends on the strength of the promoter. We showed that ubiquitin promoter-driven expression of CCL21 enabled massive infiltration of tumors with CD4(+)CD25(-), CD8(+) T lymphocytes, and CD11c(+) dendritic cells, and consequent activation of cellular and humoral immune responses sufficient for complete rejection of CCL21-positive melanomas within 3 weeks in all tumor-inoculated mice. Mice that rejected CCL21-positive tumors acquired protective immunity against melanoma, which was transferable to naive mice via splenocytes and central memory T cells. Moreover, melanoma-derived CCL21 facilitated immune-mediated remission of preestablished, distant wild-type melanomas. Overall, these results suggest that elevated levels of tumor-derived CCL21 are required for the activation of strong melanoma-specific immune responses and generation of protective immunologic memory. They also open new perspectives for the development of novel vaccination strategies against melanoma, which use intratumoral delivery of the optimized CCL21-encoding vectors in conjunction with DNA-based vaccines.

Pro-Inflammatory Chemokines and Cytokines Dominate the Blister Fluid Molecular Signature in Patients with Epidermolysis Bullosa and Affect Leukocyte and Stem Cell Migration.

Hereditary epidermolysis bullosa (EB) is associated with skin blistering and the development of chronic nonhealing wounds. Although clinical studies have shown that cell-based therapies improve wound healing, the recruitment of therapeutic cells to blistering skin and to more advanced skin lesions remains a challenge. Here, we analyzed cytokines and chemokines in blister fluids of patients affected by dystrophic, junctional, and simplex EB. Our analysis revealed high levels of CXCR1, CXCR2, CCR2, and CCR4 ligands, particularly dominant in dystrophic and junctional EB. In vitro migration assays demonstrated the preferential recruitment of CCR4+ lymphocytes and CXCR1+, CXCR2+, and CCR2+ myeloid cells toward EB-derived blister fluids. Immunophenotyping of skin-infiltrating leukocytes confirmed substantial infiltration of EB-affected skin with resting (CD45RA+) and activated (CD45RO+) T cells and CXCR2+ CD11b+ cells, many of which were identified as CD16b+ neutrophils. Our studies also showed that abundance of CXCR2 ligand in blister fluids also creates a favorable milieu for the recruitment of the CXCR2+ stem cells, as validated by in vitro and in-matrix migration assays. Collectively, this study identified several chemotactic pathways that control the recruitment of leukocytes to the EB-associated skin lesions. These chemotactic axes could be explored for the refinement of the cutaneous homing of the therapeutic stem cells.

Misbalanced CXCL12 and CCL5 Chemotactic Signals in Vitiligo Onset and Progression.

Generalized nonsegmental vitiligo is often associated with the activation of melanocyte-specific autoimmunity. Because chemokines play an important role in the maintenance of immune responses, we examined chemotactic signatures in cultured vitiligo melanocytes and skin samples of early (≤2 months) and advanced (≥6 months) vitiligo. Analysis showed that melanocytes in early lesions have altered expression of several chemotaxis-associated molecules, including elevated secretion of CXCL12 and CCL5. Higher levels of these chemokines coincided with prominent infiltration of the skin with antigen presenting cells (APCs) and T cells. Most of the intralesional APCs expressed the CD86 maturation marker and co-localized with T cells, particularly in early vitiligo lesions. These observations were confirmed by in vivo animal studies showing preferential recruitment of APCs and T cells to CXCL12- and CCL5-expressing transplanted melanocytes, immunotargeting of the chemokine-positive cells, continuous loss of the pigment-producing cells from the epidermis, and development of vitiligo-like lesions. Taken together, our studies show that melanocyte-derived CXCL12 and CCL5 support APC and T-cell recruitment, antigen acquisition, and T-cell activation in early vitiligo and reinforce the role of melanocyte-derived CXCL12 and CCL5 in activation of melanocyte-specific immunity and suggest inhibition of these chemotactic axes as a strategy for vitiligo stabilization.

Ladarixin, a dual CXCR1/2 inhibitor, attenuates experimental melanomas harboring different molecular defects by affecting malignant cells and tumor microenvironment.

CXCR1 and CXCR2 chemokine receptors and their ligands (CXCL1/2/3/7/8) play an important role in tumor progression. Tested to date CXCR1/2 antagonists and chemokine-targeted antibodies were reported to affect malignant cells in vitro and in animal models. Yet, redundancy of chemotactic signals and toxicity hinder further clinical development of these approaches. In this pre-clinical study we investigated the capacity of a novel small molecule dual CXCR1/2 inhibitor, Ladarixin (LDX), to attenuate progression of experimental human melanomas. Our data showed that LDX-mediated inhibition of CXCR1/2 abrogated motility and induced apoptosis in cultured cutaneous and uveal melanoma cells and xenografts independently of the molecular defects associated with the malignant phenotype. These effects were mediated by the inhibition of AKT and NF-kB signaling pathways. Moreover, systemic treatment of melanoma-bearing mice with LDX also polarized intratumoral macrophages to M1 phenotype, abrogated intratumoral de novo angiogenesis and inhibited melanoma self-renewal. Collectively, these studies outlined the pre-requisites of the successful CXCR1/2 inhibition on malignant cells and demonstrated multifactorial effects of Ladarixin on cutaneous and uveal melanomas, suggesting therapeutic utility of LDX in treatment of various melanoma types.

Involvement of ERCC1/XPF and XPG in oligodeoxynucleotide-directed gene modification.

Oligodeoxynucleotide (ODN)-mediated gene alteration was postulated to occur in two steps, DNA strand pairing and DNA repair. Once alignment has occurred through homologous strand pairing, a single mismatch is formed between an oligonucleotide and one of the target strands. Because of this mismatch, it has been suggested that proteins involved in a mismatch repair pathway (MMR) participate in the process. We proposed an alternative model, in which a transient assimilation of ODN to the target DNA can interrupt the trafficking of RNA polymerase, and the stalled RNA polymerase may signal for recruitment of DNA repair proteins, including transcription-coupled (TCR) DNA repair and nucleotide excision repair (NER) pathways. Recently, we found that transcription of many genes participating in NER and MMR was induced by the presence of plasmid DNA, and the extent of induction correlated with episomal gene repair rates. To investigate whether an increased level of induction of genes involved in specific DNA repair pathways has a functional role in ODN-directed gene repair, we performed episomal targeting in several cell lines with a specific defective gene in NER and MMR pathways. Comparison among several genetically related cell lines harboring a specific defective gene and complementation of missing activities showed that a primary pathway for gene correction involves some of the proteins participating in NER, primarily two endonucleases processing a DNA lesion, but not MMR.

Transcription affects formation and processing of intermediates in oligonucleotide-mediated gene alteration.

The role of transcription in oligonucleotide (ODN)-directed gene modification has been investigated in mammalian cells. The importance of transcription is demonstrated using mammalian cell lines with varying degrees of transcription of the mutant LacZ reporter gene, residing in both episome and chromosome. Gene correction occurs more efficiently when the target gene is actively transcribed and antisense ODN is more active than sense ODN. Using an approach that combines biochemical studies with a cell-based assay to measure the functional activity of intermediates it is shown that a joint molecule, consisting of supercoiled DNA and homologous ODN targeted to correct the mutated base, is a functional intermediate in the gene repair process. Furthermore, this approach showed that a resected joint molecule is a downstream intermediate of the D-loop. These results indicate that the primary reason for efficient gene repair exhibited by the antisense ODN is its increased accessibility to the non-transcribed strand, and as a consequence an increased formation of intermediate during active transcription. Moreover, the processing of intermediates was also affected by transcription, suggesting that ODN-directed gene repair may be linked to transcription-coupled repair. Thus, transcription plays an important role in ODN-directed gene repair by affecting the formation and processing of key intermediates.

Chemotaxis-driven disease-site targeting of therapeutic adult stem cells in dystrophic epidermolysis bullosa.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB), a rare genodermatosis, is characterized by the formation of intra-epidermal blistering and the development of chronic nonhealing skin wounds. Recently, attempts have been made to develop cell-based therapies for this currently intractable disorder. The molecular mechanisms that govern directional migration of the adult stem cells, allowing their efficient and controlled homing to the skin affected with DEB, are poorly understood. The key mechanism that regulates recruitment of leukocytes and progenitor stem cells to distal anatomical tissues affected with disease is chemotaxis, which depends on the signaling molecules, chemokines, and acts primarily as part of the host defense and repair mechanism. METHODS: Comprehensive proteomic screening of chemokines in the blister fluids of DEB-affected mice was conducted to define the inflammatory and immune activities, thus providing potential to examine local biological mechanisms and define the protein signature within lesional skin as a potential marker of disease activity. Also, the therapeutic relevance of identified chemotactic pathways was investigated in vivo, providing a basis for future clinical investigations. RESULTS: Assessment of blister fluid-derived chemokines showed a persistent presence of several chemotactic molecules, including CXCL1 + 2 and CXCL5. The majority of blister-originated chemotactic signals were associated with preferential recruitment of CD45(+)CXCR2(+) and CD11b(+)CXCR2(+) leukocytes. Systemic transplantation of an enriched CXCR2 population of mouse adipose-derived stem cells (mADSC) into DEB-affected mice demonstrated effective recruitment of cells to the blistering skin under the influence of blister-derived ligands and deposition of therapeutic type VII collagen. CONCLUSIONS: Collectively, these studies demonstrate that recruitment of mADSC into DEB skin is tightly controlled by disease-site chemotactic activities and suggest a potential mechanism for effective application of therapeutic stem cells for DEB.

Highly branched poly(ß-amino ester)s for skin gene therapy.

Poly(ß-amino ester)s (PAEs) have emerged as a promising class of gene delivery vectors with performances that can even be compared to viruses. However, all of the transfection studies (over 2350 PAEs) have been limited to linear poly(ß-amino ester)s (LPAEs) despite increasing evidence that polymer structure significantly affects performance. Herein, we describe the development of highly branched poly(ß-amino ester)s (HPAEs) via a new "A2+B3+C2" Michael addition approach demonstrating 2 to 126-fold higher in vitro transfection efficiencies of different cell types in comparison to their linear LPAE counterparts as well as greatly out-performing the leading transfection reagents SuperFect and the "gold-standard" polyethyleneimine (PEI) - especially on skin epidermal cells. More importantly, the ability to correct a skin genetic defect is demonstrated in vivo utilizing a recessive dystrophic epidermolysis bullosa (RDEB) knockout mouse model. Our results provide evidence that the "A2+B3+C2" approach can be controlled and offers sufficient flexibility for the synthesis of HPAEs. The branched structures can significantly improve the transfection efficiency and safety of PAEs highlighting the great promise for the successful application of non-viral gene therapy in skin disease.

Gene targeting by oligonucleotides in keratinocytes.

Oligonucleotide-directed gene alteration produces a targeted deoxyribonucleic acid (DNA) sequence change in the genome of mammalian cells at low frequency that is only detectable by highly sensitive methods. To measure the low frequency, we have established an assay using the mutant lacZ vector that contains a single point mutation in the lacZ gene, which results in a loss of enzymatic activity. When cells containing this mutant reporter gene are corrected by gene targeting, the mutant beta-galactosidase enzymatic activity is restored, and corrected cells can be visualized by histochemical staining. Using this method, we detected a low level of gene correction in the primary human keratinocytes, in spite of highly efficient nuclear uptake of oligonucleotide. Therefore, it is important to consider many other factors for successful gene repair, including DNA repair and recombination activities, status of replication and transcription, in addition to the well-known requirements like the quality and delivery of oligodeoxy nucleotides to cells. Available methods to manipulate epidermal stem cells and the accessibility of the tissue make the epidermis attractive for gene targeting. Given the low frequency, however, general selection procedures and amplification of corrected cells via epidermal stem cells are ultimately needed to make the gene repair technology practical.

Oligonucleotide-directed mutagenesis and targeted gene correction: a mechanistic point of view.

Within the last decade, a number of nucleic acid-based gene targeting strategies have been developed with the ultimate goal to cure human genetic disorders caused by mutations. Thus far, site-directed gene targeting is the only procedure that can make predefined alterations in the genome. The advantage of this approach is that expression of the corrected gene is regulated in the same way as a normal gene. In addition, targeted specific mutations can be made in the genome for functional analysis of proteins. Several approaches, including chimeric RNA-DNA oligonucleotides, short single-stranded oligonucleotides, small fragment homologous replacements, and triple-helix-forming oligonucleotides have been used for targeted modification of the genome. Due to the absence of standardized assays and mechanistic studies in the early developmental stages of oligonucleotide-directed gene alteration, it has been difficult to explain the large variations and discrepancies reported. Here, we evaluate the progress in the field, summarize the achievements in understanding the molecular mechanism, and outline the perspective for the future development. This review will emphasize the importance of reliable, sensitive and standardized assays to measure frequencies of gene repair and the use of these assays in mechanistic studies. Such studies have become critical for understanding the gene repair process and setting realistic expectations on the capability of this technology. The conventionally accepted but unproven dogmas of the mechanism of gene repair are challenged and alternative points of view are presented. Another important focus of this review is the development of general selection procedures that are required for practical application of this technology.