Dermal and conjunctival melanocytic proliferations in diffuse uveal melanocytic proliferation.

AIM: The goal of this case report is to describe the dermatologic and conjunctival findings in a case of bilateral diffuse uveal melanocytic proliferation (BDUMP), a paraneoplastic syndrome usually associated with gynecologic cancers. There is little information about other dermatologic melanocytic findings in these patients. METHODS: Histologic and fluorescent in situ hybridization (FISH) analysis of three separate skin biopsies, one of which was separated by 21 months from the others, were performed in a 71-year-old patient with BDUMP to assess for histologic and chromosomal abnormality. Conjunctival histologic evaluation was also done. RESULTS: Dermal melanocytic proliferation was seen in each specimen. The cells were spindle type with mitotic activity. FISH analysis showed a normal copy of chromosomes. The conjunctival sample also showed normal FISH analysis. CONCLUSION: BDUMP is associated with multifocal dermal and conjunctival melanocytic proliferation.

IL-10 delivery by AAV5 vector attenuates inflammation in mice with Pseudomonas pneumonia.

Lung infections with Pseudomonas aeruginosa and other pathogens in cystic fibrosis (CF) cause progressive airway obstruction and tissue damage, the predominant cause of morbidity and mortality in CF. We investigated whether a recombinant adeno-associated virus type 5 (AAV5) vector expressing murine interleukin (IL)-10 (AAV5.Cbeta-mIL-10), a regulatory/anti-inflammatory cytokine, could decrease airway inflammation in IL-10 knockout mice chronically infected with mucoid P. aeruginosa. Mice that received AAV5.Cbeta-mIL10 through intratracheal inoculation produced IL-10 at an average of 25 000 pg/ml in the epithelial lining fluid (ELF) and 12 000 pg/g-lung tissue 6 weeks post-vector delivery, significantly higher levels than in placebo-treated mice. At 3 days post-infection, proinflammatory cytokines (IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage inhibitory protein (MIP)-1alpha and (KC) in the ELF and lung homogenate were decreased (1-9 folds) in the AAV5.Cbeta-mIL10-treated mice accompanied by less pronounced and more localized neutrophil infiltration in lung sections, when compared with placebo-treated mice. These results suggest that AAV5.Cbeta-mIL10 induces IL-10 levels in the lungs mediating a significant anti-inflammatory response and making AAV-IL-10 gene transfer a potentially useful therapy in the treatment of CF lung disease.

In vitro and in vivo functional characterization of gutless recombinant SV40-derived CFTR vectors.

In cystic fibrosis (CF), respiratory failure caused by progressive airway obstruction and tissue damage is primarily a result of the aberrant inflammatory responses to lung infections with Pseudomonas aeruginosa. Despite considerable improvement in patient survival, conventional therapies are mainly supportive. Recent progress toward gene therapy for CF has been encouraging; however, several factors such as immune response and transduced cell turnover remain as potential limitations to CF gene therapy. As alternative gene therapy vectors for CF, we examined the feasibility of using recombinant SV40-derived vectors (rSV40s), which may circumvent some of these obstacles. To accommodate the large cystic fibrosis transmembrane conductance regulator (CFTR) cDNA, we removed not only SV40 Tag genes, but also all capsid genes. We, therefore, tested whether 'gutless' rSV40s could be packaged and were able to express a functional human CFTR cDNA. The results from our in vitro analysis determined that rSV40-CFTR was able to successfully result in the expression of CFTR protein, which localized to the plasma membrane and restored channel function to CFTR-deficient cells. Similarly, in vivo experiments delivering rSV40-CFTR to the lungs of Cftr-/- mice resulted in a reduction of the pathology associated with intra-tracheal P. aeruginosa challenge. rSV40-CFTR-treated mice had less weight loss when compared with control-treated mice as well as demonstrably reduced lung inflammation as evidence by histology and reduced inflammatory cytokines in the broncho-alveolar lavage. The reduction in inflammatory cytokine levels led to an evident decrease in neutrophil influx to the airways. These results indicate that further study of the application of rSV40-CFTR to CF gene therapy is warranted.

Melanocytic nevus-like hyperplasia and melanoma in transgenic BRAFV600E mice.

BRAF, a cellular oncogene and effector of RAS-mediated signaling, is activated by mutation in approximately 60% of melanomas. Most of these mutations consist of a V600E substitution resulting in constitutive kinase activation. Mutant BRAF thus represents an important therapeutic target in melanoma. In an effort to produce a pre-clinical model of mutant BRAF function in melanoma, we have generated a mouse expressing BRAF V600E targeted to melanocytes. We show that in these transgenic mice, widespread benign melanocytic hyperplasia with histological features of nevi occurs, with biochemical evidence of senescence. Melanocytic hyperplasia progresses to overt melanoma with an incidence dependent on BRAF expression levels. Melanomas show CDKN2A loss, and genetic disruption of the CDKN2A locus greatly enhances melanoma formation, consistent with collaboration between BRAF activation and CDKN2A loss suggested from studies of human melanoma. The development of melanoma also involves activation of the Mapk and Akt signaling pathways and loss of senescence, findings that faithfully recapitulate those seen in human melanomas. This murine model of mutant BRAF-induced melanoma formation thus provides an important tool for identifying further genetic alterations that cooperates with BRAF and that may be useful in enhancing susceptibility to BRAF-targeted therapeutics in melanoma.

Metastatic malignant melanoma.

Metastatic malignant melanoma is an incurable malignancy with extremely poor prognosis. Patients bearing this diagnosis face a median survival time of approximately 9 months with a probability of surviving 5 years after initial presentation at less than 5%. This is contrasted by the curative nature of surgical resection of early melanoma detected in the skin. To date, no systemic therapy has consistently and predictably impacted the overall survival of patients with metastatic melanoma. However, in recent years, a resurgence of innovative diagnostic and therapeutic developments have broadened our understanding of the natural history of melanoma and identified rational therapeutic targets/strategies that seem poised to significantly change the clinical outcomes in these patients. Herein we review the state-of-the-art in metastatic melanoma diagnostics and therapeutics with particular emphasis on multi-disciplinary clinical management.

The pros and cons of immunomodulatory IL-10 gene therapy with recombinant AAV in a Cftr-/- -dependent allergy mouse model.

Cystic fibrosis (CF) patients have decreased levels of lung epithelial interleukin (IL)-10 and increased levels of proinflammatory cytokines (tumor necrosis factor-alpha, IL-4, IL-8 and IL-6). This has also been documented in Cftr (cystic fibrosis transmembrane conductance regulator)-deficient mice (Cftr 489X(-/-), FABP-hCFTR(+/+)). Our laboratory has recently characterized a peculiar hyper-IgE phenotype in these mice, in response to Aspergillus fumigatus crude protein extract (Af-cpe). Thus, we hypothesized that sustained systemic circulating IL-10 levels achieved through skeletal muscle transduction with recombinant adeno-associated vectors expressing IL-10 (rAAV1-IL-10) would serve to downregulate Th1 and Th2 cytokine production. This in turn would dampen the allergic response in the Cftr(-/-)-dependent mouse model of allergic bronchopulmonary aspergillosis. After Af-cpe sensitization and airway challenge, mice treated with rAAV1-IL-10 had markedly lower IgE levels when compared to the control-treated rAAV1-GFP group. This was accompanied by a significant reduction in the levels of IL-5, IL-4 and IL-13 in the lung compartment. The lower lung cytokine profiles resulted in a near absence of eosinophil recruitment in the lung and a lower inflammatory response in the lung tissue of mice receiving rAAV1-IL-10. Unfortunately, sustained secretion of IL-10 from transduced muscle did lead to thrombocytopenia and splenomegaly in mice injected with rAAV1-IL-10. These results highlight that while IL-10 gene therapy is very effective for treating allergic responses caution must be taken with the prolonged secretion of IL-10.

Clinical gene therapy using recombinant adeno-associated virus vectors.

Recombinant adeno-associated virus (rAAV) vectors possess a number of properties that may make them suitable for clinical gene therapy, including being based upon a virus for which there is no known pathology and a natural propensity to persist in human cells. Wild-type adeno-associated viruses (AAVs) are now known to be very diverse and ubiquitous in humans and nonhuman primates, which adds to the degree of confidence one may place in the natural history of AAV, namely that it has never been associated with any human tumors or other acute pathology, other than sporadic reports of having been isolated from spontaneously aborted fetuses. On the basis of this understanding of AAV biology and a wide range of preclinical studies in mice, rabbits, dogs and nonhuman primates, a growing number of clinical trials have been undertaken with this class of vectors. Altogether, over 40 clinical trials have now been approved. Although all previous trials were undertaken using AAV serotype 2 vectors, at least two current trials utilize AAV2 vector genomes cross-packaged or pseudotyped into AAV1 capsids, which appear to mediate more efficient gene delivery to muscle. The explosion of capsid isolates available for use as vectors to over 120 has now provided the potential to broaden the application of AAV-based gene therapy to other cell types.

Progress and prospects: gene therapy clinical trials (part 1).

Over the last two decades gene therapy has moved from preclinical to clinical studies for many diseases ranging from single gene disorders such as cystic fibrosis and Duchenne muscular dystrophy, to more complex diseases such as cancer and cardiovascular disorders. Gene therapy for severe combined immunodeficiency (SCID) is the most significant success story to date, but progress in many other areas has been significant. We asked 20 leaders in the field succinctly to summarize and comment on clinical gene therapy research in their respective areas of expertise and these are published in two parts in the Progress and Prospect series.

Adeno-associated viral vector-mediated interleukin-10 prolongs allograft survival in a rat kidney transplantation model.

Interleukin-10 (IL-10) is a pleiotropic cytokine that plays a pivotal role in the regulation of immune responses. Hence, we evaluated the effects of a recombinant adeno-associated viral vector 1 (rAAV1) encoding rat IL-10 (rAAV1-IL-10) in a rat model of kidney allograft rejection. Dark Agouti rat kidneys were transplanted into Wistar-Furth (WF) rats 8 weeks following a single intramuscular administration of either rAAV1-IL-10 or rAAV1-green fluorescence protein (GFP). Isografts (WF-WF) served as an additional experimental control. Both allograft and isograft recipients received daily cyclosporine (10 mg/kg) for 14 days after transplantation. Serum IL-10 levels increased at 8, 12 and 16 weeks following vector administration in rAAV1-IL-10-treated animals, but not in rAAV1-GFP and isograft groups. rAAV1-IL-10 treatment resulted in lower BUN and creatinine levels (p<0.001), as well as increased allograft survival rates from 22% to 90%. Allograft histological abnormalities were significantly attenuated in the rAAV1-IL-10-treated rats compared with those of rAAV1-GFP controls. Serum levels of proinflammatory cytokines such as growth-related oncogene were also significantly higher in the rAAV1-GFP group than in the rAAV1-IL-10 group. These data suggest delivery of IL-10 using a rAAV1 vector improves renal function and prolongs graft survival in a rat model of kidney transplant rejection.

Recombinant adeno-associated virus-mediated alpha-1 antitrypsin gene therapy prevents type I diabetes in NOD mice.

Type I diabetes results from an autoimmune destruction of the insulin-producing pancreatic beta cells. Although the exact immunologic processes underlying this disease are unclear, increasing evidence suggests that immunosuppressive, immunoregulatory and anti-inflammatory agents can interrupt the progression of the disease. Alpha 1 antitrypsin (AAT) is a multifunctional serine proteinase inhibitor (serpin) that also displays a wide range of anti-inflammatory properties. To test the ability of AAT to modulate the development of type I diabetes, we performed a series of investigations involving recombinant adeno-associated virus vector (rAAV)-mediated gene delivery of human alpha-1 antitrypsin (hAAT) to nonobese diabetic (NOD) mice. Recombinant AAV-expressing hAAT (rAAV2-CB-AT) was administered intramuscularly to 4-week-old female NOD mice (1 x 10(10) i.u./mouse). A single injection of this vector reduced the intensity of insulitis, the levels of insulin autoantibodies, and the frequency of overt type I diabetes (30% (3/10) at 32 weeks of age versus 70% (7/10) in controls). Transgene expression at the injection sites was confirmed by immunostaining. Interestingly, antibodies against hAAT were present in a majority of the vector-injected mice and circulating hAAT was undetectable when assessed 10 weeks postinjection. This study suggests a potential therapeutic role for AAT in preventing type I diabetes as well as the ability of AAV gene therapy-based approaches to ameliorate disease effectively.

Immunity to adeno-associated virus serotype 2 delivered transgenes imparted by genetic predisposition to autoimmunity.

Adeno-associated virus (AAV) is widely considered a promising vector for therapeutic gene delivery. This promise is based on previous studies assessing AAVs safety and toxicity, ability to infect nondividing cells, elicit a limited immune response and provide long-term gene expression. However, we now find that earlier studies underappreciated the degree of AAV immunogenicity as well as the extent to which genetic background, through regulation of immune responsiveness, influences the duration of gene expression and thereby the effectiveness of AAV-mediated gene therapy. We evaluated antibody responses in 12 mouse strains to AAV serotype 2 (AAV2) and AAV2-expressed transgene products including green fluorescent protein (GFP), human alpha1-antitrypsin and murine interleukin-10. As expected, all immunocompetent mice administered AAV2 developed serologic evidence of immune responsiveness to the virus. However, a previously unidentified serologic prozone effect was observed suggesting that the concentrations of anti-AAV2 antibodies may have historically been subject to marked underestimation. Furthermore, strains with genetic predisposition to autoimmunity (eg, NOD, NZW, MRL-lpr) specifically imparted a functionally deleterious immune response to AAV-delivered transgene products. These findings suggest that more thorough studies of anti-AAV immunity should be performed, and that genetic predisposition to autoimmunity should be considered when assessing AAV efficacy and safety in humans.

Targeting recombinant adeno-associated virus vectors to enhance gene transfer to pancreatic islets and liver.

Human pancreatic islet cells and hepatocytes represent the two most likely target cells for genetic therapy of type I diabetes. However, limits to the efficiency of rAAV serotype 2 (rAAV2)-mediated gene transfer have been reported for both of these cell targets. Here we report that nonserotype 2 AAV capsids can mediate more efficient transduction of islet cells, with AAV1 being the most efficient serotype in murine islets, suggesting that receptor abundance could be limiting. In order to test this, we generated rAAV particles that display a ligand (ApoE) that targets the low-density lipoprotein receptor, which is present on both of these cell types. The rAAV/ApoE viruses greatly enhanced the efficiency of transduction of both islet cells ex vivo and murine hepatocytes in vivo when compared to native rAAV2 serotype (220- and four-fold, respectively). The use of receptor-targeted rAAV particles may circumvent the lower abundance of receptors on certain nonpermissive cell types.

Confocal examination of nonmelanoma cancers in thick skin excisions to potentially guide mohs micrographic surgery without frozen histopathology.

Precise removal of nonmelanoma cancers with minimum damage to the surrounding normal skin is guided by the histopathologic examination of each excision during Mohs micrographic surgery. The preparation of frozen histopathology sections typically requires 20-45 min per excision. Real-time confocal reflectance microscopy offers an imaging method potentially to avoid frozen histopathology and prepare noninvasive (optical) sections within 5 min. Skin excisions ( approximately 1 mm thick) from Mohs surgeries were washed with 5% acetic acid and imaged with a confocal cross-polarized microscope. The confocal images were compared with the corresponding histopathology. Acetic acid causes compaction of chromatin that increases light back-scatter and makes the nuclei bright and easily detectable. Crossed-polarization strongly enhances the contrast of the nuclei because the compacted chromatin depolarizes the illumination light whereas the surrounding cytoplasm and normal dermis does not. Fast low-resolution examination of cancer lobules in wide fields of view followed by high-resolution inspection of nuclear morphology in small fields of view is possible; this is similar to the procedure for examining histopathology sections. Both the Mohs surgeon and the patient will potentially save several hours per day in the operating room. Fast confocal reflectance microscopic examination of excisions (of any thickness) may improve the management of surgical pathology and guide microsurgery of any human tissue.

Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice.

The development of spontaneous autoimmune diabetes in nonobese diabetic (NOD) mice provides for their use as a model of human type 1 diabetes. To test the feasibility of muscle-directed gene therapy to prevent type 1 diabetes, we developed recombinant adeno-associated virus (rAAV) vectors containing murine cDNAs for immunomodulatory cytokines IL-4 or IL-10. Skeletal muscle transduction of female NOD mice with IL-10, but not IL-4, completely abrogated diabetes. rAAV-IL-10 transduction attenuated the production of insulin autoantibodies, quantitatively reduced pancreatic insulitis, maintained islet insulin content, and altered splenocyte cytokine responses to mitogenic stimulation. The beneficial effects were host specific, as adoptive transfer of splenocytes from rAAV IL-10-treated animals rapidly imparted diabetes in naive hosts, and the cells contained no protective immunomodulatory capacity, as defined through adoptive cotransfer analyses. These results indicate the utility for rAAV, a vector with advantages for therapeutic gene delivery, to transfer immunoregulatory cytokines capable of preventing type 1 diabetes. In addition, these studies provide foundational support for the concept of using immunoregulatory agents delivered by rAAV to modulate a variety of disorders associated with deleterious immune responses, including allergic reactions, transplantation rejection, immunodeficiencies, and autoimmune disorders.

Recombinant adeno-associated virus vectors for cystic fibrosis gene therapy.

Cystic fibrosis (CF) is an autosomal recessive inherited disorder that affects approximately 30,000 North Americans. Defects in the CF transmembrane conductance regulator (CFTR) gene lead to altered secretions from exocrine glands and the pulmonary airways, to a heightened susceptibility to airway infections with Pseudomonas aeruginosa, and to severe airway inflammation. Early attempts to develop a genetic therapy for CF have not met with great clinical success, but these efforts have driven the development of viral gene transfer technology for in vivo gene delivery. The recombinant adeno-associated virus (rAAV) system has proven to be safe for in vivo gene delivery in the airways of experimental animals and CF patients, although potential barriers to delivery have been identified. These barriers may limit the transduction efficiency of this vector, especially in the context of the inflamed airways of adolescent and adult CF patients. We anticipate that the use of alternative rAAV serotype capsids and other vector alterations, along with targeting the lungs of CF patients in the earlier stages of their disease, might eventually allow for these potential limitations to be overcome.

Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors.

Previous work from our group showed that recombinant adeno-associated virus (rAAV) vectors mediated long-term secretion of therapeutic serum levels of human alpha-1 antitrypsin (hAAT) after a single injection in murine muscle. We hypothesized that hepatocyte transduction could be even more efficient, since these cells represent the natural site of AAT production and secretion. To test this hypothesis, rAAV vectors containing the hAAT cDNA driven by either the human elongation factor 1 alpha promoter, the human cytomegalovirus immediate-early promoter (CMV), or the CMV-chicken beta actin hybrid (CB) promoter were injected into the portal or tail veins of adult C57Bl/6 mice. Potentially therapeutic serum levels of hAAT (600 microg/ml) were achieved after portal vein injection of doses of 4 x 10(9) infectious units (IU), a 10-fold lower dose than that required for similar levels of expression via the i.m. route. Serum levels greater than 1 mg/ml were achieved at doses of 3 x 10(10) IU. Southern blotting of liver DNA revealed the presence of circular episomal vector genomes. Immunostaining showed that transgene expression was scattered throughout the liver parenchyma. Similar results were obtained with a rAAV-CB-green fluorescent protein (GFP) vector. There was no evidence of hepatic toxicity. These data indicate that liver-directed rAAV-based gene therapy is effective in the murine model, and hence might be feasible for treatment of human AAT deficiency.