Adeno-Associated Virus (AAV) as a Vector for Gene Therapy.

There has been a resurgence in gene therapy efforts that is partly fueled by the identification and understanding of new gene delivery vectors. Adeno-associated virus (AAV) is a non-enveloped virus that can be engineered to deliver DNA to target cells, and has attracted a significant amount of attention in the field, especially in clinical-stage experimental therapeutic strategies. The ability to generate recombinant AAV particles lacking any viral genes and containing DNA sequences of interest for various therapeutic applications has thus far proven to be one of the safest strategies for gene therapies. This review will provide an overview of some important factors to consider in the use of AAV as a vector for gene therapy.

Novel antibody probes for the characterization of endosialin/TEM-1.

Endosialin (Tumor Endothelial Marker-1 (TEM-1), CD248) is primarily expressed on pericytes of tumor-associated microvasculature, tumor-associated stromal cells and directly on tumors of mesenchymal origin, including sarcoma and melanoma. While the function of endosialin/TEM-1 is incompletely understood, studies have suggested a role in supporting tumor growth and invasion thus making it an attractive therapeutic target. In an effort to further understand its role in cancer, we previously developed a humanized anti-endosialin/TEM-1 monoclonal antibody (mAb), called ontuxizumab (MORAb-004) for testing in preclinical and clinical studies. We herein report on the generation of an extensive panel of recombinant endosialin/TEM-1 protein extracellular domain (ECD) fragments and novel mAbs against ECD motifs. The domain-specific epitopes were mapped against ECD sub-domains to identify those that can detect distinct structural motifs and can be potentially formatted as probes suitable for diagnostic and functional studies. A number of mAbS were shown to cross-react with the murine and human protein, potentially allowing their use in human animal models and corresponding clinical trials. In addition, pairing of several mAbs supported their use in immunoassays that can detect soluble endosialin/TEM-1 (sEND) in the serum of healthy subjects and cancer patients.

TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway.

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

Characterization of the human folate receptor alpha via novel antibody-based probes.

Folate receptor alpha (FRA) is a cell surface protein whose aberrant expression in malignant cells has resulted in its pursuit as a therapeutic target and marker for diagnosis of cancer. The development of immune-based reagents that can reproducibly detect FRA from patient tissue processed by varying methods has been difficult due to the complex post-translational structure of the protein whereby most reagents developed to date are highly structure-sensitive and have resulted in equivocal expression results across independent studies. The aim of the present study was to generate novel monoclonal antibodies (mAbs) using modified full length FRA protein as immunogen in order to develop a panel of mAbs to various, non-overlapping epitopes that may serve as diagnostic reagents able to robustly detect FRA-positive disease. Here we report the development of a panel of FRA-specific mAbs that are able to specifically detect FRA using an array of diagnostic platforms and methods. In addition, the methods used to develop these mAbs and their diverse binding properties provide additional information on the three dimensional structure of FRA in its native cell surface configuration.

Endosialin/TEM-1/CD248 regulates pericyte proliferation through PDGF receptor signaling.

Recent reports have described several cellular phenotypes that appear to be mediated by Endosialin/TEM-1/CD248 (TEM-1), including tubule formation on matrigel, migration and proliferation. It has been shown that siRNA knock-down of TEM-1 in primary human fibroblasts resulted in reduced proliferation. However, the downstream signaling events that mediate TEM-1 function(s) currently remain unknown. In this study, we demonstrate that TEM-1 mediates proliferation of primary human pericytes through a PDGF receptor signaling pathway. Normal pericytes expressing high levels of TEM-1 were able to proliferate, respond to PDGF-BB stimulation by phosphorylating both the PDGF receptor and the MAP kinase ERK-1/2, and induce the expression of the immediate early transcription factor c-Fos. However, when TEM-1 expression was knocked-down, PDGF-BB-induced proliferation, ERK-1/2 phosphorylation, and c-Fos expression were significantly impaired. Thus, our results provide evidence for a TEM-1-dependent signal pathway that controls proliferation of human pericytes and suggest targeting this pathway for future strategies aimed at mitigating tumor angiogenesis.

An arrestin-dependent multi-kinase signaling complex mediates MIP-1beta/CCL4 signaling and chemotaxis of primary human macrophages.

MIP-1beta/CCL4 is a principal regulator of macrophage migration and signals through CCR5. Several protein kinases are linked to CCR5 in macrophages including the src kinase Lyn, PI3K, focal adhesion related kinase Pyk2, and members of the MAPK family, but whether and how these kinases regulate macrophage chemotaxis are not known. To define the role of these signaling molecules, we examined the functions and interactions of endogenous proteins in primary human macrophages. Using siRNA gene silencing and pharmacologic inhibition, we show that chemotaxis in response to CCR5 stimulation by MIP-1beta requires activation of Pyk2, PI3K p85, and Lyn, as well as MAPK ERK. MIP-1beta activation of CCR5 triggered translocation of Pyk2 and PI3K p85 from the cytoplasm to colocalize with Lyn at the plasma membrane with formation of a multimolecular complex. We show further that arrestins were recruited into the complex, and arrestin down-regulation impaired complex formation and macrophage chemotaxis toward MIP-1beta. Together, these results identify a novel mechanism of chemokine receptor regulation of chemotaxis and suggest that arrestins may serve as scaffolding proteins linking CCR5 to multiple downstream signaling molecules in a biologically important primary human cell type.

Monocyte migration and LFA-1-mediated attachment to brain microvascular endothelia is regulated by SDF-1 alpha through Lyn kinase.

Infiltration of activated monocytes into the brain is a prerequisite for the development of various neurological disorders such as HIV-associated dementia, multiple sclerosis, and other inflammatory processes. In these pathologies, the chemokine SDF-1alpha (CXCL12) is over-expressed and might attract monocytes into the CNS. We demonstrate here that SDF-1alpha stimulates migration of monocytes through its receptor, CXCR4, and decreases monocyte adherence to surfaces coated with ICAM-1, a ligand for beta(2) integrins. SDF-1alpha also decreases monocyte adherence to brain microvascular endothelial cells (BMVEC) that are activated with TNF-alpha, IL-1beta, or recombinant envelope glycoprotein from HIV-1, which increase BMVEC expression of ICAM-1. The decreased adherence is linked to down-regulation on monocytes of the activation-dependent epitope of the beta(2) integrin LFA-1 by SDF-1alpha. Knockdown of Lyn in monocytes using small interfering RNA decreases SDF-1alpha-mediated migration and prevents the inhibition of monocyte attachment to ICAM-1 and activated BMVEC. Thus, in SDF-1alpha-stimulated monocytes, Lyn acts as a positive regulator of migration and a negative regulator of adhesion to BMVEC through the LFA-1 integrin. These results provide a novel Lyn-mediated signaling mechanism for the regulation of monocyte movement at the blood-brain barrier.

BCR-ABL1 alters SDF-1alpha-mediated adhesive responses through the beta2 integrin LFA-1 in leukemia cells.

Stromal-derived factor-1 (SDF-1) and its receptor, CXCR4, are essential for normal hematopoietic progenitor cell movement and adherence within the bone marrow microenvironment. In leukemia, the BCR-ABL1 oncoprotein inhibits SDF-1-dependent cell trafficking within the bone marrow through a mechanism that is not fully understood. Here, we report that BCR-ABL1 in malignant cells constitutively increases expression of activation-dependent epitopes of the beta(2) integrin LFA-1. This is associated with the complete loss of responsiveness of LFA-1 to SDF-1-induced "inside-out" signaling involving CXCR4 and Lyn, leading to aberrant adhesive responses. These data provide a novel, LFA-1-mediated mechanism whereby BCR-ABL1 inhibits SDF-1 action in malignant progenitors.

Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration.

Endosialin/TEM1 was originally discovered as a human embryonic fibroblast-specific antigen and was later found to be differentially expressed in tumor stroma and endothelium. Endosialin/TEM1 overexpression has been observed in many cancers of various tissue origin, including colon, breast, pancreatic, and lung. The knockout (KO) mouse model showed the absence of endosialin/TEM1 expression reduced growth, invasion, and metastasis of human tumor xenografts. In addition, lack of endosialin/TEM1 led to an increase in small immature blood vessels and decreased numbers of medium and large tumor vessels. This abnormal angiogenic response could be responsible for the reduced tumor growth and invasion observed in endosialin/TEM1 KO mice, suggesting a role for endosialin/TEM1 in controlling the interaction among tumor cells, endothelia, and stromal matrix. Here we report the identification of fibronectin (FN) and collagen types I and IV as specific ligands for endosialin/TEM1. More importantly, cells expressing endosialin/TEM1 exhibit enhanced adhesion to FN as well as enhanced migration through matrigel, although these properties could be blocked by a humanized antibody directed against human endosialin/TEM1. Our results pinpoint to a molecular mechanism by which expression of endosialin/TEM1 in the tumor stroma and endothelium may support tumor progression and invasion.

The Src kinase Lyn is required for CCR5 signaling in response to MIP-1beta and R5 HIV-1 gp120 in human macrophages.

CCR5 is a receptor for several beta chemokines and the entry coreceptor used by macrophage-tropic (R5) strains of HIV-1. In addition to supporting viral entry, CCR5 ligation by the HIV-1 envelope glycoprotein 120 (gp120) can activate intracellular signals in macrophages and trigger inflammatory mediator release. Using a combination of in vitro kinase assay, Western blotting for phospho-specific proteins, pharmacologic inhibition, CCR5 knockout (CCR5Delta32) cells, and kinase-specific blocking peptide, we show for the first time that signaling through CCR5 in primary human macrophages is linked to the Src kinase Lyn. Stimulation of human monocyte-derived macrophages with either HIV-1 gp120 or MIP-1beta results in the CCR5-mediated activation of Lyn and the concomitant Lyn-dependent activation of the mitogen-activated protein (MAP) kinase ERK-1/2. Furthermore, activation of the CCR5/Lyn/ERK-1/2 pathway is responsible for gp120-triggered production of TNF-alpha by macrophages, which is believed to contribute to HIV-1 pathogenesis. Thus, Lyn kinase may play an important role both in normal CCR5 function in macrophages and in AIDS pathogenesis in syndromes such as AIDS dementia where HIV-1 gp120 contributes to inappropriate macrophage activation, mediator production, and secondary injury.

Integrin inhibition through Lyn-dependent cross talk from CXCR4 chemokine receptors in normal human CD34+ marrow cells.

We studied the effects of Lyn ablation on CXCR4 receptor-mediated migration and adhesion of hematopoietic precursors. Down-regulation of Lyn expression with siRNA greatly reduced CXCR4-dependent hematopoietic cell movement, and increased cell adherence to stroma. This increase was associated with the up-regulated expression of activation-dependent epitopes of the beta(2) integrin LFA-1 and was prevented by antibodies that selectively block cell adhesion mediated by ICAM-1. Attachment to surfaces coated with ICAM-1 was also enhanced in Lyn-depleted hematopoietic cells, as compared with Lyn-expressing cells. Functional rescue experiments with Lyn siRNA targeting the 3' UTR indicated that the observed effects can be attributed directly to specific inhibition of Lyn. Our results show that in chemokine-stimulated hematopoietic cells Lyn kinase is a positive regulator of cell movement while negatively regulating adhesion to stromal cells by inhibiting the ICAM-1-binding activity of beta(2) integrins. These results provide a molecular mechanism for cross talk between the chemokine receptor CXCR4 and beta(2) integrins. This cross talk may allow chemokine receptors to modulate the arrest of rolling hematopoietic precursors on the surface of bone marrow stromal cells.

HIV-1 gp120-induced TNF-{alpha} production by primary human macrophages is mediated by phosphatidylinositol-3 (PI-3) kinase and mitogen-activated protein (MAP) kinase pathways.

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by binding of the viral envelope glycoprotein gp120 to CD4 followed by a chemokine receptor, but these interactions may also take place independently from infection. gp120 stimulation of primary human macrophages is known to trigger production of cytokines implicated in pathogenesis, particularly tumor necrosis factor alpha (TNF-alpha), but the mechanisms have not been determined. We sought to define the pathways responsible for TNF-alpha secretion by monocyte-derived macrophages (MDM) following HIV-1 gp120 stimulation. MDM exposure to recombinant macrophage-tropic (R5) gp120 led to dose- and donor-dependent release of TNF-alpha, which was cyclohexamide-sensitive and associated with up-regulated message. Pretreatment with specific inhibitors of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1/2 (ERK-1/2; PD98059, U0126) and p38 (SB202190, PD169316) inhibited the secretion of TNF-alpha. gp120-elicited TNF-alpha production was also blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors (wortmannin, LY294002). Moreover, PI-3K inhibition ablated gp120-induced phosphorylation of p38 and ERK-1/2. The response was inhibited by a CC chemokine receptor 5 (CCR5)-specific antagonist, indicating that CCR5 was in large part responsible. These results indicate that gp120-elicited TNF-alpha production by macrophages involves chemokine receptor-mediated PI-3K and MAPK activation, that PI-3K is an upstream regulator of MAPK in this pathway, and that p38 and ERK-1/2 independently regulate TNF-alpha production. These gp120-triggered signaling pathways may be responsible for inappropriate production of proinflammatory cytokines by macrophages, which are believed to play a role in immunopathogenesis and in neurological sequelae of AIDS.

Mutational analysis reveals an essential role for the LXXLL motif in the transformation function of the human herpesvirus-8 oncoprotein, kaposin.

Human herpesvirus-8 (HHV-8) is causally linked to Kaposi's sarcoma (KS). Sequence analysis of the genome and subsequent studies revealed several genes including kaposin, with transformation properties in cell culture. In this study, we have analyzed the requirement of Kaposin A for cellular transformation in an effort to understand its contribution towards KS pathogenesis. Comparative analysis of Kaposin with other proteins identified the LXXLL motif spanning from residues 31-35 (LVCLL). The observation that the LXXLL motif is present in nuclear receptor coactivators that mediate the interaction of coactivators with nuclear receptors has prompted us to investigate the relevance of this motif for Kaposin's function(s). Kaposin A coding sequences were cloned into a eukaryotic expression plasmid with the Flag (FL) epitope fused in-frame at the C-terminus (Kap-FL). To evaluate the role of leucine residues in the motif, site-directed mutagenesis was utilized, whereby alanine was substituted for the leucine residues (Kap-AXXAA-FL). Both Kap-FL and Kap- AXXAA-FL exhibited similar levels of expression in cells. Interestingly, the Kap-AXXAA-FL mutant failed to show transforming activity by two independent assays: anchorage-independent growth, and focus formation. Immunofluorescence (IFA) and FACS analysis indicated that Kap-FL was localized around the nucleus and at the cell surface, respectively. However, Kap-AXXAA-FL exhibited diffuse cytoplasmic staining as measured by IFA yet was still detectable on the cell surface by FACS. Ironically, both Kap-FL and Kap-AXXAAFL were able to activate the AP-1 promoter. These results support an important role for the LXXLL motif in the ability of Kaposin to induce transformation.

HIV-1 entry inhibitors: closing the front door.

Highly active antiretroviral therapy (HAART) has led to major declines in morbidity and mortality of HIV-1-infected individuals, but the increasing prevalence of drug-resistant viral isolates, combined with the toxicity and other limitations of current treatments, make the development of new therapies a high priority. As knowledge of viral entry has expanded, this step of the viral life cycle has become a target for novel therapeutic strategies. An emerging group of antiretrovirals, known collectively as entry inhibitors, targets several distinct steps in viral entry including CD4 binding, chemokine receptor engagement and the structural changes in the viral envelope required for fusion between viral and cellular membranes. Many entry inhibitors are in various stages of clinical development, with one already licensed for use. This review will provide an overview of the mechanisms involved in the entry process, highlight promising entry blockers under development and discuss several considerations related to treatment that are unique to this class of antiretroviral drugs.

Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signaling pathways.

Macrophages are major targets for infection by human immunodeficiency virus type 1 (HIV-1). In addition to their role as productive viral reservoirs, inappropriate activation of infected and uninfected macrophages appears to contribute to pathogenesis. HIV-1 infection requires initial interactions between the viral envelope surface glycoprotein gp120, the cell-surface protein CD4, and a chemokine receptor CCR5 or CXCR4. Besides their role in HIV-1 entry, CCR5 and CXCR4 are G protein-coupled receptors that can activate multiple intracellular signaling pathways. HIV-1 gp120 has been shown to activate signaling pathways through the chemokine receptors in several cell types including lymphocytes, neurons, and astrocytes. In some cell types, these consequences may cause cellular injury. In this review, we highlight our data demonstrating diverse signaling events that occur in primary human macrophages in response to gp120/chemokine receptor interactions. These responses include K+, Cl-, and nonselective cation currents, intracellular Ca2+ increases, and activation of several kinases including the focal adhesion-related tyrosine kinase Pyk2, mitogen-activated protein kinases (MAPK), and phosphoinositol-3 kinase. Activation of the MAPK leads to gp120-induced expression of chemokines such as monocyte chemoattractant protein-1 and macrophage-inflammatory protein-1beta and the proinflammatory cytokine tumor necrosis factor alpha. These responses establish a complex cytokine network, which may enhance or suppress HIV-1 replication. In addition, dysregulation of macrophage function by gp120/chemokine receptor signaling may contribute to local inflammation and injury and further recruit additional inflammatory and/or target cells. Targeting these cellular signaling pathways may have benefit in controlling inflammatory sequelae of HIV infection such as in neurological disease.

Role of HIV-1 Vpr in AIDS pathogenesis: relevance and implications of intravirion, intracellular and free Vpr.

Vpr, a 14-kDa, 96 amino acid protein, is conserved among the primate lentiviruses HIV-1, HIV-2 and Simian Immunodeficiency virus supporting the notion that it plays an important role in virus life cycle in vivo. Vpr appears to have several functions including cell cycle arrest at G2 stage, apoptosis, nuclear localization, nuclear import of the pre-integration complex, cation selective channel activity and transcriptionally activate HIV-1 LTR and other heterologous promoters. Over the years, we have addressed several issues pertaining to Vpr including the amount of Vpr present in the virus particles and structure-function relationship of Vpr. Here, we have reviewed the sources of Vpr that may potentially contribute to the cytopathic features observed in the context of HIV-1 infection. There are three different sources of Vpr available in the infected individuals to initiate the pathogenic effects. These include cell-associated, virion-associated (infectious, infectious-non productive, and non-infectious defective viruses) and free Vpr (cell-free and virus-free). A potential role of Vpr in neuropathogenesis of HIV infection in CNS was also suggested by early studies demonstrating neurotoxicity of recombinant Vpr protein. Interestingly, free Vpr (cell-free and virus-free) has been demonstrated in the serum of HIV-1 infected individuals and in the CSF of AIDS patients with neurological dysfunctions. Based on the toxic effects of extra-cellular Vpr on cells noted in several studies, it is likely that free Vpr could contribute to the bystander cell depletion in lymphoid tissues, peripheral blood, and the CNS. These results led us to propose a model for the role of Vpr in AIDS pathogenesis.

Human herpesvirus-8 encoded Kaposin: subcellular localization using immunofluorescence and biochemical approaches.

Human herpesvirus-8 (HHV-8) has been causally linked to the development of Kaposi's sarcoma (KS). DNA sequence analysis of the viral genome revealed a total of 81 open reading frames (ORF). Interestingly, only a small subset of these ORFs has been shown to be transcribed in cells latently infected with HHV-8 and in cells of the KS lesions. Among the genes active during latency, kaposin, is noted for its abundance and ability to transform cells in culture, thus implicating a potential role in KS pathogenesis. This has prompted us to undertake an investigation on elucidating the mechanism(s) by which Kaposin brings about transformation of cells. Towards this goal, we have generated an eukaryotic expression plasmid encoding Kaposin (Kap). As Kaposin is predicted to be a type II membrane protein, several strategies were utilized to address this, including the generation of Kaposin with the Flag (FL) epitope (DYKDDDDK) at the C-terminus of the protein (Kap-C-FL). Antibodies specific for Kaposin (kap-2), recognized both Kaposin and Kaposin-Flag, while antibodies against the Flag epitope recognized only Kaposin-Flag. Transfection of Kap and Kap-C-FL expression plasmid DNA into NIH3T3 cells resulted in cellular clones that exhibited a phenotypic property of transformation by forming large, multiclustered cells, when grown on soft agar. Because there is controversial data regarding the localization of Kaposin in cells, we examined the subcellular localization of Kaposin using confocal microscopy. We observed that Kaposin and Kaposin-Flag showed an intense staining surrounding the nucleus. Although there was no staining at the cell membrane of transfected cells, FACS analysis using kap-2 or Flag antibodies, under nonpermeable conditions, showed positivity. Cell fractionation studies further showed that the majority of Kaposin was detected in the nuclear fraction by Western blot analysis. The cytoplasmic and detergent soluble membrane fractions did not show Kaposin protein; however, a small amount was detected in the detergent insoluble membrane fraction. Taken together, these results suggest that Kaposin exhibits multicompartmental localization in cells.