Integrase, LEDGF/p75 and HIV replication.

HIV integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The essential DNA cleaving and joining chemistry of integration is known, but there is less understanding of the process as it occurs in a cell, where two complex and dynamic macromolecular entities are joined: the viral pre-integration complex and chromatin. Among implicated cellular factors, much recent attention has coalesced around LEDGF/p75, a nuclear protein that may act as a chromatin docking factor or receptor for lentiviral pre-integration complexes. LEDGF/p75 tethers HIV integrase to chromatin, protects it from degradation, and strongly influences the genome-wide pattern of HIV integration. Depleting the protein from cells and/or over-expressing its integrase-binding domain blocks viral replication. Current goals are to establish the underlying mechanisms and to determine whether this knowledge can be exploited for antiviral therapy or for targeting lentiviral vector integration in human gene therapy.

Ontogeny of Phex/PHEX protein expression in mouse embryo and subcellular localization in osteoblasts.

PHEX, a phosphate-regulating gene with homologies to endopeptidases on the X chromosome, is mutated in X-linked hypophosphatemia (XLH) in humans and mice (Hyp). Although recent observations indicate that Phex protein is expressed primarily in bone and may play an important role in osteoblast function and bone mineralization, the pattern of the Phex protein expression in the developing skeleton and its subcellular localization in osteoblasts remain unknown. We examined the ontogeny of the Phex protein in the developing mouse embryo and its subcellular localization in osteoblasts using a specific antibody to the protein. Immunohistochemical staining of mouse embryos revealed expression of Phex in osteogenic precursors in developing vertebral bodies and developing long bones on day 16 postcoitum (pc) and thereafter. Calvaria from day 18 pc mice showed Phex epitopes in osteoblasts. No Phex immunoreactivity was detected in lung, heart, hepatocytes, kidney, intestine, skeletal muscle, or adipose tissue of mouse embryos. Interestingly, embryonic mouse skin showed moderate amounts of Phex immunostaining. In postnatal mice, Phex expression was observed in osteoblasts and osteocytes. Moderate expression of Phex was seen in odontoblasts and slight immunoreactivity was observed in ameloblasts. Confocal microscopy revealed the presence of immunoreactive PHEX protein in the Golgi apparatus and endoplasmic reticulum of osteoblasts from normal mice and in osteoblasts from Hyp mice transduced with a human PHEX viral expression vector. PHEX protein was not detected in untransduced Hyp osteoblasts. These data indicate that Phex protein is expressed in osteoblasts and osteocytes during the embryonic and postnatal periods and that within bone, Phex may be a unique marker for cells of the osteoblast/osteocyte lineage.

Genetic modification of human trabecular meshwork with lentiviral vectors.

Glaucoma, a group of optic neuropathies, is the leading cause of irreversible blindness. Neuronal apoptosis in glaucoma is primarily associated with high intraocular pressure caused by chronically impaired outflow of aqueous humor through the trabecular meshwork, a reticulum of mitotically inactive endothelial-like cells located in the angle of the anterior chamber. Anatomic, genetic, and expression profiling data suggest the possibility of using gene transfer to treat glaucomatous intraocular pressure dysregulation, but this approach will require stable genetic modification of the differentiated aqueous outflow tract. We injected transducing unit-normalized preparations of either of two lentiviral vectors or an oncoretroviral vector as a single bolus into the aqueous circulation of cultured human donor eyes, under perfusion conditions that mimicked natural anterior chamber flow and maintained viability ex vivo. Reporter gene expression was assessed in trabecular meshwork from 3 to 16 days after infusion of 1.0 x 10(8) transducing units of each vector. The oncoretroviral vector failed to transduce the trabecular meshwork. In contrast, feline immunodeficiency virus and human immunodeficiency virus vectors produced efficient, localized transduction of the trabecular meshwork in situ. The results demonstrate that lentiviral vectors permit efficient genetic modification of the human trabecular meshwork when delivered via the afferent aqueous circulation, a clinically accessible route. In addition, controlled comparisons in this study establish that feline and human immunodeficiency virus vectors are equivalently efficacious in delivering genes to this terminally differentiated human tissue.

A controlled, Phase 1 clinical trial to evaluate the safety and effects in HIV-1 infected humans of autologous lymphocytes transduced with a ribozyme that cleaves HIV-1 RNA.

This Phase I study, "Ribozyme Gene Therapy of HIV-1 Infection" (UCSD HSC #971072, FDA BB-IND 6405), is a prospective, open-label trial of infusion of autologous gene-altered cells into asymptomatic HIV-1 seropositive individuals. The objectives of this trial are to test the safety, feasibility, and potential efficacy of T-cell ribozyme gene therapy of HIV-1 infection. To accomplish this, autologous CD8-depleted mononuclear cells are transduced with ribozyme expressing or control murine retroviral vectors, expanded ex vivo, and and infused. Subjects are monitored intensively to determine effects of infusion on HIV burden and replication. In addition, in vivo survival of control and ribozyme transduced cells is followed in an effort to obtain evidence of proof of concept. A unique strategy of sample blinding is introduced in this protocol, wherein both subject and control specimens are supplied to the research laboratory as coded samples, spiking blood from HIV seropositive volunteers matched for CD4 lymphocyte count with known but varying numbers of cells transduced with each vector. While this study is still in progress, preliminary results indicate that infusion of gene-altered, activated T-cells in HIV infected patients is safe, and that transduced cells can persist for long intervals in HIV-infected subjects. Results also suggest ribozyme transduced cells may possess a survival advantage in vivo.

CXCR4 is required by a nonprimate lentivirus: heterologous expression of feline immunodeficiency virus in human, rodent, and feline cells.

A heterologous feline immunodeficiency virus (FIV) expression system permitted high-level expression of FIV proteins and efficient production of infectious FIV in human cells. These results identify the FIV U3 element as the sole restriction to the productive phase of replication in nonfeline cells. Heterologous FIV expression in a variety of human cell lines resulted in profuse syncytial lysis that was FIV env specific, CD4 independent, and restricted to cells that express CXCR4, the coreceptor for T-cell-line-adapted strains of human immunodeficiency virus. Stable expression of human CXCR4 in CXCR4-negative human and rodent cell lines resulted in extensive FIV Env-mediated, CXCR4-dependent cell fusion and infection. In feline cells, stable overexpression of human CXCR4 resulted in increased FIV infectivity and marked syncytium formation during FIV replication or after infection with FIV Env-expressing vectors. The use of CXCR4 is a fundamental feature of lentivirus biology independent of CD4 and a shared cellular link to infection and cytopathicity for distantly related lentiviruses that cause AIDS. Their conserved use implicates chemokine receptors as primordial lentivirus receptors.

Efficient transduction of nondividing human cells by feline immunodeficiency virus lentiviral vectors.

The molecular bases for species barriers to lentiviral replication are not well understood, but are of interest for explaining lentiviral pathogenesis, devising therapeutic strategies, and adapting lentiviruses to gene therapy. HIV-1-based lentiviral vectors efficiently transduce nondividing cells, but present complex safety concerns. Nonprimate (ungulate or feline) lentiviruses might provide safer alternatives, but these viruses display highly restricted tropisms, and their potential for adaptation as replication-defective vectors capable of transducing human cells is unknown. Feline immunodeficiency virus (FIV) does not infect humans or other non-Felidae despite prevalent natural exposure. Although long terminal repeat (LTR)-directed FIV expression was found to be negligible in human cells, promoter substitution enabled an env-deleted, three-plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells, thus bypassing the hazards of feline vector producer cells. Pseudotyped FIV vectors efficiently transduced dividing, growth-arrested, and postmitotic human targets. The experiments delineate mechanisms involved in species-restricted replication of this lentivirus and show that human cells support both productive- and infective-phase mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction. Nonprimate lentiviral vectors may offer safety advantages, and FIV vectors provide unique experimental opportunities.

A1 adenosine receptors inhibit chloride transport in the shark rectal gland. Dissociation of inhibition and cyclic AMP.

In the in vitro perfused rectal gland of the dogfish shark (Squalus acanthias), the adenosine analogue 2-chloroadenosine (2Clado) completely and reversibly inhibited forskolin-stimulated chloride secretion with an IC50 of 5 nM. Other A1 receptor agonists including cyclohexyladenosine (CHA), N-ethylcarboxamideadenosine (NECA) and R-phenylisopropyl-adenosine (R-PIA) also completely inhibited forskolin stimulated chloride secretion. The "S" stereoisomer of PIA (S-PIA) was a less potent inhibitor of forskolin stimulated chloride secretion, consistent with the affinity profile of PIA stereoisomers for an A1 receptor. The adenosine receptor antagonists 8-phenyltheophylline and 8-cyclopentyltheophylline completely blocked the effect of 2Clado to inhibit forskolin-stimulated chloride secretion. When chloride secretion and tissue cyclic (c)AMP content were determined simultaneously in perfused glands, 2Clado completely inhibited secretion but only inhibited forskolin stimulated cAMP accumulation by 34-40%, indicating that the mechanism of inhibition of secretion by 2Clado is at least partially cAMP independent. Consistent with these results, A1 receptor agonists only modestly inhibited (9-15%) forskolin stimulated adenylate cyclase activity and 2Clado markedly inhibited chloride secretion stimulated by a permeant cAMP analogue, 8-chlorophenylthio cAMP (8CPT cAMP). These findings provide the first evidence for a high affinity A1 adenosine receptor that inhibits hormone stimulated ion transport in a model epithelia. A major portion of this inhibition occurs by a mechanism that is independent of the cAMP messenger system.