Peptide and nucleic acid-directed self-assembly of cationic nanovehicles through giant unilamellar vesicle modification: Targetable nanocomplexes for in vivo nucleic acid delivery.

One of the greatest challenges for the development of genetic therapies is the efficient targeted delivery of therapeutic nucleic acids. Towards this goal, we have introduced a new engineering initiative in self-assembly of biologically safe and stable nanovesicle complexes (∼90 to 140nm) derived from giant unilamellar vesicle (GUV) precursors and comprising plasmid DNA or siRNA and targeting peptide ligands. The biological performance of the engineered nanovesicle complexes were studied both in vitro and in vivo and compared with cationic liposome-based lipopolyplexes. Compared with cationic lipopolyplexes, nanovesicle complexes did not show advantages in transfection and cell uptake. However, nanovesicle complexes neither displayed significant cytotoxicity nor activated the complement system, which are advantageous for intravenous injection and tumour therapy. On intravenous administration into a neuroblastoma xenograft mouse model, nanovesicle complexes were found to distribute throughout the tumour interstitium, thus providing an alternative safer approach for future development of tumour-specific therapeutic nucleic acid interventions. On oropharyngeal instillation, nanovesicle complexes displayed better transfection efficiency than cationic lipopolyplexes. The technological advantages of nanovesicle complexes, originating from GUVs, over traditional cationic liposome-based lipopolyplexes are discussed. STATEMENT OF SIGNIFICANCE: The efficient targeted delivery of nucleic acids in vivo provides some of the greatest challenges to the development of genetic therapies. Giant unilamellar lipid vesicles (GUVs) have been used mainly as cell and tissue mimics and are instrumental in studying lipid bilayers and interactions. Here, the GUVs have been modified into smaller nanovesicles. We have then developed novel nanovesicle complexes comprising self-assembling mixtures of the nanovesicles, plasmid DNA or siRNA, and targeting peptide ligands. Their biophysical properties were studied and their transfection efficiency was investigated. They transfected cells efficiently without any associated cytotoxicity and with targeting specificity, and in vivo they resulted in very high and tumour-specific uptake and in addition, efficiently transfected the lung. The peptide-targeted nanovesicle complexes allow for the specific targeted enhancement of nucleic acid delivery with improved biosafety over liposomal formulations and represent a promising tool to improve our arsenal of safe, non-viral vectors to deliver therapeutic cargos in a variety of disorders.

A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis.

Vascular injury is a serious complication of sepsis due to the gram-negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non-LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E-selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS-deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor-κB (NF-κB)]. In this study, we show that the particular propensity for N. meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen-activated protein (MAP) kinases, on the activity of the E-selectin promoter. Furthermore, inhibition of E-selectin expression in response to the lpxA- strain by a p38 inhibitor indicates a significant role of a p38-dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally.

Inhibition of neointimal hyperplasia in a rabbit vein graft model following non-viral transfection with human iNOS cDNA.

Vein graft failure caused by neointimal hyperplasia (IH) after coronary artery bypass grafting with saphenous veins is a major clinical problem. The lack of safe and efficient vectors for vascular gene transfer has significantly hindered progress in this field. We have developed a Receptor-Targeted Nanocomplex (RTN) vector system for this purpose and assessed its therapeutic efficacy in a rabbit vein graft model of bypass grafting. Adventitial delivery of ß-Galactosidase showed widespread transfection throughout the vein wall on day 7, estimated at about 10% of cells in the adventitia and media. Vein grafts were then transfected with a plasmid encoding inducible nitric oxide synthase (iNOS) and engrafted into the carotid artery. Fluorescent immunohistochemistry analysis of samples from rabbits killed at 7 days after surgery showed that mostly endothelial cells and macrophages were transfected. Morphometric analysis of vein graft samples from the 28-day groups showed approximately a 50% reduction of neointimal thickness and 64% reduction of neointimal area in the iNOS-treated group compared with the surgery control groups. This study demonstrates efficacy of iNOS gene delivery by the RTN formulation in reducing IH in the rabbit model of vein graft disease.

Elemental imaging of MRI contrast agents: benchmarking of LA-ICP-MS to MRI.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been used to map the spatial distribution of magnetic resonance imaging (MRI) contrast agents (Gd-based) in histological sections in order to explore synergies with in vivo MRI. Images from respective techniques are presented for two separate studies namely (1) convection enhanced delivery of a Gd nanocomplex (developmental therapeutic) into rat brain and (2) convection enhanced delivery, with co-infusion of Magnevist (commercial Gd contrast agent) and Carboplatin (chemotherapy drug), into pig brain. The LA technique was shown to be a powerful compliment to MRI not only in offering improved sensitivity, spatial resolution and signal quantitation but also in giving added value regarding the fate of administered agents (Gd and Pt agents). Furthermore simultaneous measurement of Fe enabled assignment of an anomalous contrast enhancement region in rat brain to haemorrhage at the infusion site.

Development and assessment of the constipation-related disability scale.

BACKGROUND: Chronic constipation is associated with impaired quality of life and physical discomfort. Although inability to engage in day-to-day activities has been significantly associated with psychological distress, limited research has examined this relationship in constipated samples. AIM: To develop and validate the Constipation-Related Disability Scale (CRDS), which assesses the extent of disability caused by constipation. METHODS: A total of 240 constipated participants and 103 healthy controls completed the CRDS. Reliability was measured with Cronbach's coefficient alpha and test-retest reliability was assessed with intraclass correlation coefficients. Convergent, divergent and predictive validity were assessed. RESULTS: Component and factor analyses were used to derive two factors: Work/Leisure Activities and Activities of Daily Living, as well as a total CRDS score. Good reliability was found, with alphas ≥ 0.87 and intraclass correlation coefficients ≥ 0.85. All scales were negatively correlated with the physical health subscales of the SF-36 (P < 0.001) and were not significantly correlated with the Epworth Sleepiness Scale and Social Desirability Scale, providing support for convergent and divergent validity, respectively. Evidence of predictive validity was supported by associations between the total CRDS with number of physician visits per year (P < 0.01), missed work in the last year (odds ratio [OR = 1.11, 95% confidence interval [CI] = 1.06-1.19, P < 0.001) and ER visits in the last year (OR = 1.08, 95% CI = 1.00-1.16, P < 0.05). CONCLUSIONS: The Constipation-Related Disability Scale is the first instrument that assesses the impact of constipation on daily activities. There is evidence of strong reliability and validity of the instrument.

Immunotherapy for neuroblastoma using syngeneic fibroblasts transfected with IL-2 and IL-12.

Cytokine-modified tumour cells have been used in clinical trials for immunotherapy of neuroblastoma, but primary tumour cells from surgical biopsies are difficult to culture. Autologous fibroblasts, however, are straightforward to manipulate in culture and easy to transfect using nonviral or viral vectors. Here we have compared the antitumour effect of fibroblasts and tumour cells transfected ex vivo to coexpress interleukin-2 (IL-2) and IL-12 in a syngeneic mouse model of neuroblastoma. Coinjection of cytokine-modified fibroblasts with Neuro-2A tumour cells abolished their in vivo tumorigenicity. Treatment of established tumours with three intratumoral doses of transfected fibroblasts showed a significant therapeutic effect with reduced growth or complete eradication of tumours in 90% of mice, associated with extensive leukocyte infiltration. Splenocytes recovered from vaccinated mice showed enhanced IL-2 production following Neuro-2A coculture, and increased cytotoxicity against Neuro-2A targets compared with controls. Furthermore, 100% of the tumour-free mice exhibited immune memory against tumour cells when rechallenged three months later. The potency of transfected fibroblasts was equivalent to that of tumour cells in all experiments. We conclude that syngeneic fibroblasts cotransfected with IL-2 and IL-12 mediate therapeutic effects against established disease, and are capable of generating immunological memory. Furthermore, as they are easier to recover and manipulate than autologous tumour cells, fibroblasts provide an attractive alternative immunotherapeutic strategy for the treatment of neuroblastoma.

Screening for depression among patients with multiple sclerosis: two questions may be enough.

BACKGROUND: Depression among patients with multiple sclerosis (MS) is common and has a significant impact on quality of life. As many as two-thirds of depressed MS patients receive no treatment for their depression. While guidelines for depression management suggest screening, the only validated screening tools are questionnaires, which have not been widely implemented in practice. This is the first study on the effectiveness of using two questions assessing mood and anhedonia (loss of interest or pleasure) in screening for major depressive disorder (MDD) in MS. METHODS: MS patients under the care of neurologists were recruited from a large health maintenance organization (HMO). The MDD module of the Structured Clinical Interview for the DSM-IV and screening questions was administered. RESULTS: Of the 260 participants, 26% met the criteria for MDD. Among patients with MDD, 67% received no anti-depressant medication. The MDD screen identified 99% (95% CI: 91-100%) of cases. DISCUSSION: A brief, two question screen is reliable in identifying MS patients with MDD. This suggests that asking these two brief questions could identify almost all MS patients meeting MDD criteria, with minimal numbers of false positives.

Treatment of depression for patients with multiple sclerosis in neurology clinics.

UNLABELLED: The objective of this study was to examine the adequacy of antidepressant pharmacotherapy in a sample of patients with multiple sclerosis (MS) treated by neurologists. METHODS: MS patients under the care of neurologists were recruited from a large health maintenance organization. Major depressive disorder (MDD) was diagnosed using a structured telephone interview. Antidepressant treatment data were obtained from the HMO pharmacy database. RESULTS: Study participants included 260 patients with MS treated by 35 neurologists. A total of 67 (25.8%) patients met the criteria for MDD. Among the patients with MDD, 65.6% received no antidepressant medication, 4.7% received subthreshold doses from their neurologists, 26.6% received doses at threshold, and 3.1% received doses exceeding threshold. DISCUSSION: Depression was undertreated by the neurologists treating this sample of patients with comorbid MS and MDD. Potential solutions are discussed.

Transbronchial biopsies provide longitudinal evidence for epithelial chimerism in children following sex mismatched lung transplantation.

BACKGROUND: Recent reports have shown evidence of host derived parenchymal engraftment in several human allografts including the lung, leading to speculation that stem cell therapy may be useful for lung repair in diseases such as cystic fibrosis (CF). To date, previous studies have looked at single surgical or autopsy specimens and no longitudinal studies have been reported. The aim of this study was to assess whether transbronchial biopsies could be used to study the time course of chimerism following lung transplantation. METHODS: Specimens of archived transbronchial lung biopsies from five time points taken for clinical purposes from two boys who had received a sex mismatched heart-lung transplant for end stage CF were examined. Sections were dual stained for cytokeratin (epithelium) and a mixture of leucocyte common antigen and CD68 for inflammatory cells. Co-localisation of cells containing a Y chromosome was confirmed by fluorescent in situ hybridisation. RESULTS: Evidence of chimerism was found in up to 6.6% of epithelial cells in bronchial (median 1.4% (range 0-6.6)) and alveolar (median 3.6% (range 2.3-5.5) tissue without apparent evidence of fusion. This engraftment was seen as early as 3 weeks and remained relatively constant up to 37 months. CONCLUSIONS: This study has demonstrated proof of principle for long term chimerism in lung epithelium. Transbronchial biopsies may provide a new method for studying the kinetics of stem cell engraftment in the lung.

Pulmonary gene therapy. Realistic hope for the future, or false dawn in the promised land?

Pulmonary gene therapy offers the hope of treatment for conditions such as cystic fibrosis, lung cancer, pulmonary fibrosis and acute respiratory distress syndrome for which current therapy is inadequate. Although initial clinical trials in cystic fibrosis and non-small cell lung cancer have shown promise the results have not been as good as might have been anticipated. However, clinical improvement has been demonstrated in conditions such as haemophilia [82], cardiovascular disease [83], head and neck cancer [84] and X-linked severe combined immunodeficiency disease [85]. The lack of success of pulmonary gene therapy is due, in part on the physical barriers to transfection perfected by the lung to prevent toxicity from inhaled particles, and partly due to the poor transfection efficiency of non-viral systems, and the immunogenicity of viral systems, of gene transfer. The LID vector goes some way to addressing the problems associated with current gene delivery strategies. With continued improvements in the properties of both viral and non-viral gene delivery systems leading to improved transfection efficiency with reduced toxicity, as well as the development of strategies aimed at reducing the physical barriers to pulmonary transfection, and targeting gene delivery systems to the site of injury, it is likely that pulmonary gene therapy will be used successfully to ameliorate a number of devastating pulmonary conditions.

Formation of LID vector complexes in water alters physicochemical properties and enhances pulmonary gene expression in vivo.

There is currently an urgent need to develop efficient gene-delivery systems for the lung that are free of inflammatory effects. The LID vector is a synthetic gene delivery system, comprised of lipofectin (L), an integrin-targeting peptide (I) and DNA (D) that has previously been shown to have high transfection efficiency in the lung. We have assessed the effect of alternative methods of complex preparation on structural features of the complex, levels and duration of reporter gene expression and the host response to the LID vector. We have demonstrated that making the complex in water affects the structure of the LID complexes making them smaller and more stable with a more cationic surface charge than complexes prepared in phosphate-buffered saline (PBS). When the LID vector was constituted in water and instilled intratracheally into the lungs of mice there was a 10-fold increase in luciferase activity compared with preparation in PBS. Furthermore, luciferase activity was still evident 1 week following vector instillation. This enhancement may be because of altered complex structure, although effects of the hypotonic vector solution on the lung cannot be excluded. The inflammatory effects of instilling the LID vector in water were minimal, even after three administrations of the LID vector, with only mild alterations in cytokine and broncho-alveolar lavage fluid (BALF) cell profiles. These results demonstrate that the LID vector can generate high, and prolonged, levels of gene expression in the lung from small quantities of DNA and that careful attention to synthetic polyplex structure may be important to optimize efficiency of gene expression in vivo.

Improved antitumour immunity in murine neuroblastoma using a combination of IL-2 and IL-12.

Neuroblastoma immunotherapy using cytokine-modified tumour cells has been tested in clinical trials. However, because of the complex nature of antitumour immune responses, a number of therapies may be required for complete tumour eradication and generation of systemic immunity. We report here the improved antitumour effect of two cytokines, interleukin-2 (IL-2) and interleukin-12 (IL-12), when coexpressed by neuroblastoma cell lines. Initially, transfection of human and mouse neuroblastoma cell lines resulted in high expression levels of biologically active IL-2 and IL-12 in vitro. These cytokines when expressed by transfected Neuro-2A cells completely abolished their in vivo tumorigenicity in a syngeneic neuroblastoma model. Vaccination of established tumours with IL-12-producing cells exhibited a clear effect with reduced tumour growth in the presence of IL-2. In vivo depletion studies showed that CD4(+) and CD8(+) T cells mediate the response against cytokine-producing cells. These results suggest that IL-2 and IL-12, when cotransfected in tumour cells, are effective against established disease and provide a promising immunotherapeutic approach for the treatment of neuroblastoma.

Non-viral, integrin-mediated gene transfer into fibroblasts from patients with lysosomal storage diseases.

BACKGROUND: Non-viral vectors consisting of Lipofectin/integrin-targeting peptide/DNA (LID) complexes have great potential for gene therapy, as they are safe, simple, and able to package large DNA molecules. In this study, these vectors were evaluated in vitro for the therapy of lysosomal storage disorders. METHODS: Non-viral vectors were designed to deliver therapeutic genes by integrin-mediated uptake into fibroblasts from patients with the lysosomal storage disorders fucosidosis and Fabry disease, which result from deficiencies of alpha-L-fucosidase and alpha-galactosidase A, respectively. The vectors consisted of a complex (LID) of Lipofectin and a peptide containing an integrin-targeting domain and a poly-lysine domain to which was bound plasmid DNA, containing alpha-L-fucosidase (LID-alpha-Fuc) or alpha-galactosidase A (LID-alpha-Gal). RESULTS: Patients' fibroblasts transfected with LID-alpha-Fuc and LID-alpha-Gal produced the corresponding enzyme at levels which were 10-40% of the total activity in cultures of normal fibroblasts. However, 95-98% of this activity was secreted. Transfection of endothelial cells, the main target cells in Fabry disease, with an LID-alpha-Gal produced a total alpha-galactosidase activity 65% higher than that in untransfected cultures after 6 days, 67% of the activity being secreted. Although transfection of fibroblasts with LID complexes also caused small changes in the distribution of endogenous lysosomal enzymes, it did not appear to affect the viability of the cells. CONCLUSIONS: The integrin-mediated transfer of genes encoding lysosomal enzymes into cells results in the secretion of large amounts of normal enzyme that could be taken up by other cells. This could be a useful strategy for enzyme-replacement therapy.

In vivo myocardial gene transfer: optimization, evaluation and direct comparison of gene transfer vectors.

The purpose was to determine the relative efficiency, toxicity and duration of expression following gene delivery by intramyocardial injection of naked DNA, naked DNA complexed to cationic liposomes, naked DNA complexed to cationic liposomes with integrin-targetting peptide, recombinant (E1-/E3-) adenovirus, recombinant adeno-associated virus and recombinant (ICP27-) herpes simplex virus. All vectors incorporated a LacZ reporter driven by a promoter containing the hCMV-IE promoter/enhancer. Efficiency was scored by counting positive cells in five standard microscopic sections harvested from the left ventricular apex. Rabbit hearts (n = 100) were examined from 2 to 56 days after injection. Uncomplexed and complexed naked DNA were very inefficient with less than one positive cell visible per heart. The viral vectors all resulted in robust gene expression with adenovirus being the most efficient by at least one order of magnitude before 21 days. However, despite disparate titres, the efficiency beyond 21 days of adenovirus and adeno-associated virus were comparable. In contrast to adeno-associated virus, both adenovirus and herpes-simplex virus were associated with a marked inflammatory response. Despite reporter gene activity appearing only after 21 days, adeno-associated virus shows comparative promise as a myocardial gene delivery vector.

Cyclooxygenase-2 deficiency results in a loss of the anti-proliferative response to transforming growth factor-beta in human fibrotic lung fibroblasts and promotes bleomycin-induced pulmonary fibrosis in mice.

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.

Pharmacological and antimicrobial studies on different tea-tree oils (Melaleuca alternifolia, Leptospermum scoparium or Manuka and Kunzea ericoides or Kanuka), originating in Australia and New Zealand.

Three different species of Myrtaceae growing in Australia and New Zealand are known as 'Tea-tree': the Australian Tea tree (Melaleuca alternifolia), the New Zealand Manuka (Leptospermum scoparium) and Kanuka (Kunzea ericoides). All three essential oils are used by aromatherapists, although only Melaleuca has been tested for toxicity, and its antimicrobial effects studied. The pharmacology and antimicrobial activity of the three 'tea-tree' oils was determined using guinea-pig ileum, skeletal muscle (chick biventer muscle and the rat phrenic nerve diaphragm) and also rat uterus in vitro. Differences were shown between the three essential oils in their action on smooth muscle: Manuka had a spasmolytic action, while Kanuka and Melaleuca had an initial spasmogenic action. Using the diaphragm, Manuka and Melaleuca decreased the tension and caused a delayed contracture; Kanuka had no activity at the same concentration. The action on chick biventer muscle was, however, similar for all three oils, as was the action on the uterus, where they caused a decrease in the force of the spontaneous contractions. The latter action suggests caution in the use of these essential oils during childbirth, as cessation of contractions could put the baby, and mother, at risk. The comparative antimicrobial activity showed greater differences between different samples of Manuka and Kanuka than Melaleuca samples. The antifungal activity of Kanuka was inversely proportional to its strong antibacterial activity, whilst Manuka displayed a stronger antifungal effect, though not as potent as Melaleuca. The antioxidant activity of Manuka samples was more consistent than that of Kanuka, while Melaleuca showed no activity. The variability in the Manuka and Kanuka essential oils suggests caution in their usage, as does the fact that the oils have not been tested for toxicity.

A cocaine analog, 2beta-propanoyl-3beta-(4-tolyl)-tropane (PTT), reduces tyrosine hydroxylase in the mesolimbic dopamine pathway.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis. Previously published results have established that chronic cocaine administration (30-45 mg/kg per day, 10-14 days) resulted in an upregulation of TH gene expression in dopaminergic pathways of rats. The present studies tested the effects of a tropane analog, PTT (2beta-propanoyl-3beta-(4-tolyl)-tropane), on TH expression. This drug has similar actions to cocaine, but possesses markedly different pharmacokinetics (20 times more potent at binding the dopamine transporter, markedly increased metabolic stability, and 10-20 times more potent in behavioral measures). Moreover, PTT demonstrates an increased selectivity for the dopamine (DA) and norepinephrine (NE) transporters compared with cocaine. In direct contrast to the previously reported effects of cocaine, 10 days of PTT administration (3.0 mg/kg per day, i.p.) produced a uniform downregulation of TH protein and activity gene expression. TH activity and immunoreactive protein where decreased by 54 and 69%, respectively in the nucleus accumbens. Within the ventral tegmental area, TH activity and protein were decreased by 33 and 19%, respectively. The underlying mechanisms for these fundamental differences are unclear, but likely reflect varying and selective affinities and lengths of occupancy at biogenic amine transporters.

Adhesion molecules and gene transfer.

Cell adhesion molecules are a large group of molecules involved in a variety of cell-to-cell and cell-to-extra-cellular matrix (ECM) interactions. Apart from their cellular function these molecules are exploited by a number of pathogenic micro-organisms as receptors for cell entry. Discovery of the use of adhesion molecules for binding and internalisation by naturally occurring pathogens has fuelled much research, in recent years, into the utilisation of these molecules for the targeting and uptake of both gene and drug delivery systems. This review describes the development of such systems and their potential advantages over other receptor-targeted delivery systems.

Stable integration of large (>100 kb) PAC constructs in HaCaT keratinocytes using an integrin-targeting peptide delivery system.

Transfer of large DNA constructs in gene therapy studies is being recognised for its importance in maintaining the natural genomic environment of the gene of interest and providing tissue-specific regulation and control. However, methods used to deliver such constructs have been poorly studied. We used a receptor-mediated, integrin-targeting transfection system enhanced by liposomes, to deliver a 110 kb PAC (P1-based artificial chromosome) to HaCaT keratinocytes. The PAC contained the collagen VII locus, an EGFP (enhanced green fluorescent protein) reporter gene and the puromycin resistance gene (pac) to allow selection of stably transfected cells. Analysis of puromycin resistant and EGFP-expressing colonies by Western blot showed that collagen VII production increased dramatically after transfection, indicating successful transfer of a large fully functional genomic locus. Fluorescent in situ hybridisation (FISH) and Southern blot analysis revealed that the PAC had integrated as at least one copy per cell. EGFP expression has persisted for 35 weeks, suggesting stable transgene expression. We conclude that the integrin-targeting peptide method of gene delivery is an effective means of stably delivering large DNA constructs to human keratinocytes and could be of benefit for genomic gene therapy approaches.