Antibody targeted liposomes containing poly(rI).poly(rC) exert a specific antiviral and toxic effect on cells primed with interferons alpha/beta or gamma.

Double-stranded RNA can stimulate interferon production and mediate an antiproliferative effect on certain cell types. We evaluated the possibility of specifically targeting to cells in vitro the RNA duplex poly(rI).poly(rC) in pharmacologically active form after its encapsulation in small, unilamellar liposomes, to which was covalently coupled protein A. These liposomes became bound to and were endocytosed by murine L929 cells in the presence of protein A-binding monoclonal antibodies specific for an expressed cell surface protein, the H-2K molecule. When L929 cells were preincubated in the presence of low doses of interferon alpha/beta or gamma, they could be activated to produce interferon following exposure to either free poly(rI).poly(rC), or specifically bound liposomes poly(rI).poly(rC), but not the same liposomes in the presence of non-cell binding control antibodies. Specifically bound liposome-encapsulated poly(rI).poly(rC) was toxic to L929 cells at dose levels at least three logs lower than free poly(rI).poly(rC). This toxicity was also dependent on pre-treatment with interferon. These results indicate that liposome-encapsulated poly(rI).poly(rC) can survive endocytosis and can be released in active form to specific cell populations, at concentrations much lower than that required for pharmacologic effects of the same molecule in free form. They suggest that introduction into cells of other nucleic acids might benefit from the antibody-targeted liposome technology described here.

Regulation of transglutaminase activity in Chinese hamster ovary cells.

We have investigated the regulation of transglutaminase activity (epsilon-(gamma-glutamyl)lysine crosslinking enzyme) in Chinese hamster ovary cells in culture. We report that transglutaminase activity increases several-fold in CHO cells at maximum density in suspension culture. This increase cannot be explained by the presence of soluble regulators of the enzyme activity or the appearance of a new enzyme activity with a different affinity for substrate, but appears to be due to an increase in total enzyme activity. Treatment of CHO cells at low cell density with 8-bromo cyclic AMP results in a small increase (20--70%) in transglutaminase activity. By studying CHO mutants which have altered or absent cyclic-AMP-dependent protein kinases, we have demonstrated that the effect of cyclic AMP on transglutaminase activity at low cell density is mediated by cyclic-AMP-dependent protein kinase. However, the protein kinase mutants show normal increases in transglutaminase activity at high cell density, indicating that cyclic AMP-dependent protein kinase does not mediate density-dependent changes in transglutaminase activity.

Antiviral activity of conjugates between poly(L-lysine) and synthetic oligodeoxyribonucleotides.

Short (14 to 20-mer range) synthetic oligodeoxyribonucleotides (oligos) allow to modulate specifically viral or cellular gene expression at various stages thus providing a versatile tool for fundamental studies and a rational approach to antiviral chemotherapy. Several problems, such as metabolic stability and efficient cell internalization of oligos, still limit this approach appreciably, as briefly discussed here. We demonstrate here that the conjugation of 15-mer (beta)-anomeric oligos to poly(L-lysine) allows a specific protection of various cell lines against vesicular stomatitis virus infection at concentrations lower than 1 microM. This can be achieved with oligos complementary to the viral N-protein mRNA initiation site or to viral intergenic sequences, i.e., to untranscribed regions. No antiviral activity can be obtained with (alpha)-anomeric oligos directed against the same targets, although such analogues are much more resistant to nuclease degradation and form stable hybrids, at least in cell-free experiments.

Requirement for endocytosis of poly(rI).poly(rC) to generate toxicity on interferon-treated LM cells.

Poly(rI).poly(rC) induces a lytic reaction in interferon-treated mouse LM cells. We have attempted to determine whether the intracellular penetration of poly(rI).poly(rC) is a prerequisite for cell lysis and to gain some insight into the pathway followed. We found that poly(rI).poly(rC) coupled to Sepharose beads was unable to generate lysis of interferon-treated cells whereas the cells underwent lysis after microinjection of poly(rI).poly(rC). Some inhibitors of endocytosis were found to inhibit the development of the lytic reaction. Lysosomotropic amines or a low temperature (19 degrees C) blocked endocytosis of poly(rI).poly(rC) but did not prevent its uptake. The internalization of poly(rI).poly(rC) was energy-dependent and was blocked when sodium azide and 2-deoxyglucose were added simultaneously. We conclude that poly(rI).poly(rC) is internalized and reaches an acidic compartment before triggering the lytic reaction in the cell.

Responses to interferon of LM cells growing continuously in the absence of serum and exogenous lipids.

Mouse fibroblast LM cells growing continuously in the absence of serum and exogenous lipids are proposed as a model for studying interferon action. Several of its activities, namely the inhibition of virus and cell growth, and the increased cytotoxicity of poly(rI) X poly(rC), are shown to be potentiated in such conditions. Since cells cultured under these conditions are devoid of precursors of prostaglandin biosynthesis, the data suggest that none of the three effects of interferon depends on active prostaglandin synthesis.

Free and liposome-encapsulated double-stranded RNAs as inducers of interferon, interleukin-6, and cellular toxicity.

Poly(rI:rC) and Ampligen were entrapped in liposomes that were covalently coupled to Protein A, permitting binding to antibodies specific for the major histocompatibility complex-encoded H2K molecule of L929 cells, or to control antibodies. Free and encapsulated polynucleotides were compared for their capacity to stimulate secretion of interferon (IFN) and interleukin-6 (IL-6) and to induce cellular toxicity on L929 cells pretreated with IFN-alpha/beta. Free and encapsulated poly(rI:rC) or Ampligen (poly(rI:rC12-rU] induced similar levels of secretion of IFN over a broad dose range. The activity of the liposome-encapsulated polynucleotides was dependent on its binding to an antibody that permitted cell association and internalization; the same liposomes were inactive in the presence of control antibodies. IL-6 secretion was induced by double-stranded (ds) RNA in a dose-dependent manner, with a significantly greater effect seen for targeted, liposome-encapsulated material. The marked toxicity of targeted poly(rI:rC), as compared to free poly(rI:rC), was confirmed. Encapsulated Ampligen was less toxic than encapsulated Poly(rI:rC).

Interferon production of L929 and HeLa cells enhanced by polyriboinosinic acid-polyribocytidylic acid pH-sensitive liposomes.

The double-stranded RNA polyinosinic acid-polycytidylic acid (PolyIC) is an inducer of interferons alpha and beta (IFN) genes. With L929 and HeLa cells IFN pretreatment (priming) improves the IFN induction by PolyIC by several orders of magnitude. In the absence of the priming we demonstrate that PolyIC encapsulated into pH-sensitive liposomes (and not into pH-insensitive liposomes) enables L929 cells to secrete IFN efficiently and a low toxicity is observed; on primed cells pH-sensitive liposomes containing PolyIC trigger a high toxicity. With HeLa cells, the absence of the priming PolyIC encapsulated into pH-sensitive liposomes induces weak doses of IFN whereas free PolyIC was ineffective. Our experiments established that a pH drop (from 8 to 5.5) provoked a lipid mixing between pH-sensitive liposomes and cell membranes, likely by a fusion mechanism. Entrapment into pH-sensitive liposomes enhances the effect of PolyIC by several orders of magnitude, which might improve its therapeutic ability as an antitumor or anti-HIV agent.

Vestibular semicircular canal epithelium of the rat in culture on filter support: polarity and barrier properties.

The inner ear of mammals contains the vestibular apparatus which is involved in the maintenance of posture and balance. The tubular structure of the apparatus is bathed by the potassium-rich endolymph and sodium-rich perilymph in the luminal and abluminal compartments, respectively. The luminal compartment is lined by a continuous epithelium with islets of receptor organs, which separates the luminal from the abluminal compartment. The present work focuses on the epithelium, without the receptor organs, and shows that it can be reconstituted in culture. The epithelium from 4-day-old Wistar rats was grown on microporous membranes. High transepithelial electrical resistances (4000-6000 Omega.cm2) were achieved after 4-8 days in culture. The epithelium was characterized by the presence of cytokeratin, ZO-1 protein, occludin, and the presence of tight junctions and kinocilia. The transepithelial resistance of the cell monolayer withstood endolymph/perilymph dual bathing when the apical pole of the cells was in contact with endolymph, but collapsed in the reverse configuration. Weak but statistically highly significant basal to apical rubidium (86Rb) transport was observed. These findings show that this epithelium maintains its in vivo polarity and could enhance the potassium composition of endolymph up to maturity. This new culture model, in which dual bathing is possible, should enable further in vitro studies of the sensory vestibular epithelia.

Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide.

Recently several groups reported a dramatic improvement of reporter gene transfection efficiency using a fusogenic peptide, derived from the Influenza hemagglutinin envelop protein. This peptide changes conformation at acidic pH and destabilizes the endosomal membranes thus resulting in an increased cytoplasmic gene delivery. We describe the use of a similar fusogenic peptide in order to improve the antiviral potency of antisense oligodeoxynucleotides (anti TAT) and oligophosphorothioates (S-dC28) on de novo HIV infected CEM-SS lymphocytes in serum-free medium. We observed as 5 to 10 fold improvement of the anti HIV activities of the phosphodiester antisense oligonucleotides after chemical coupling to the peptide in a one to one ratio by a disulfide or thioether bond. No toxicities were observed at the effective doses (0.1-1 microM). No sequence specificity was obtained and the fusogenic peptide possessed some antiviral activities on its own (IC50: 6 microM). A S-dC28-peptide disulfide linked conjugate and a streptavidin-peptide-biotinylated S-dC28 adduct showed similar activities as the free S-dC28 oligonucleotide (IC50: 0.1-1 nM). As expected, all the compounds were less potent in the presence of serum but the relative contribution of peptide coupling was maintained.

Isolation and biological characterization of mouse LM fibroblast variants resistant to the cytotoxicity of polyriboinosinic polyribocytidylic acid after interferon treatment.

Mouse LM fibroblasts growing continuously in the absence of serum have an increased sensitivity to the cytotoxicity of poly(rI) X poly(rC) after interferon (IFN) exposure. This has allowed the isolation by an enrichment procedure of several independent and stable variant clones (IFN + I X C)R which are resistant to such a treatment. One of the resistant variants has been more extensively characterized as far as IFN action and IFN production are concerned. It behaves identically to the wild-type parent except for the spontaneous release of low amounts of IFN. The target of the mutation probably resides in a late step in the development of the cytotoxic response as revealed by microinjection techniques. The (IFN + I X C)R variants characterized here thus appear different from mutants in the IFN system isolated so far.