Chemical Approach to a Whole Body Imaging of Sialo-N-Linked Glycans.

PET and noninvasive fluorescence imaging of the sialo-N-linked glycan derivatives are described. To establish the efficient labeling protocol for N-glycans and/or glycoconjugates, new labeling probes of fluorescence and 68Ga-DOTA, as the positron emission nucleus for PET, through rapid 6π-azaelectrocyclization were designed and synthesized, (E)-ester aldehydes. The high reactivity of these probes enabled the labeling of lysine residues in peptides, proteins, and even amino groups on the cell surfaces at very low concentrations of the target molecules (~10⁻8 M) within a short reaction time (~5 min) to result in "selective" and "non-destructive" labeling of the more accessible amines. The first MicroPET of glycoproteins, 68Ga-DOTA-orosomucoid and asialoorosomucoid, successfully visualized the differences in the circulatory residence of glycoproteins, in the presence or absence of sialic acids. In vivo dynamics of the new N-glycoclusters, prepared by the "self-activating" Huisgen cycloaddition reaction, could also be affected significantly by their partial structures at the non-reducing end, i.e., the presence or absence of sialic acids, and/or sialoside linkages to galactose. Azaelectrocyclization chemistry is also applicable to the engineering of the proteins and/or the cell surfaces by the oligosaccharides; lymphocytes chemically engineered by sialo-N-glycan successfully target the tumor implanted in BALB/C nude mice, detected by noninvasive fluorescence imaging.

Construction and characterization of an anti-asialoglycoprotein receptor single-chain variable-fragment-targeted melittin.

Antibody-therapeutic agent conjugation to be delivered specifically to tumor cells is required for many target-based therapeutic strategies. In the present study, a recombinant immunotoxin was constructed by which melittin was fused to an anti-asialoglycoprotein receptor (ASGPR) single-chain variable fragment antibody (C1), and targeting ability and cytolytic efficacy of the fusion protein were studied. Our results suggested that the recombinant 29.4 kDa protein C1M was expressed in Escherichia coli as a soluble style. Binding of C1M to the surface of hepatocellular carcinoma (HCC) cells was confirmed by both immunohistochemistry and flow cytometry assays. C1M kept the hemolytic activity of melittin and exhibited cytolytic capacity to HepG2 cells at a concentration of 1.5 µg/mL, under which erythrocytes would not be lysed. The effects were greatly inhibited by coadministration with asialoorosomucoid, a natural ligand for ASGPR. These results suggested that C1M conferred targeting and ASGPR-specific cytotoxicity to HCC cells. This work makes it possible to further investigate its antihepatoma efficacy in vivo.

Biotin-directed assembly of targeted modular lipoplexes and their transfection of human hepatoma cells in vitro.

The asialoglycoprotein receptor, which is abundantly and near exclusively expressed on hepatocytes, has received much attention in the design of non-viral hepatotropic DNA delivery systems. Thus, asialoglycoproteins and hexopyranosyl ligands have been coupled to DNA-binding cationic polymers and liposomes in the assembly of complexes intended for uptake by liver parenchymal cells. The aim of the study was to construct a hepatocyte-targeted multimodular liposome-based transfecting complex, in which the biotin-streptavidin interaction provides the cohesive force between the ligand asialorosomucoid and the liposome bilayer, and to evaluate its transfection capabilities in the hepatocyte-derived human transformed cell line HepG2. Dibiotinylated asialoorosomucoid was attached to cationic liposomes constructed from 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T):dioleoylphosphatidylethanolamine:biotinylcholesterylformylhydrazide (MSB1) (48:50:2 mole ratio) through streptavidin interposition. Liposome-pGL3 DNA interactions were studied by gel band shift and ethidium displacement assays. The cytotoxicity of assemblies was evaluated in the HepG2 cell line and transfection capabilities determined by measuring the activity of the transgene luciferase. Binding assays showed that all DNA was liposome associated at a DNA (negative):liposome (positive) charge ratio of 1:1. Accommodation of a streptavidin dibiotinylated asialoorosomucoid assembly was achieved at a DNA:liposome:streptavidin dibiotinylated asialoorosomucoid ratio of 1:4:9 (weight basis). Complexes showed optimal transfection activity at this ratio, which was reduced 10-fold by the presence of the competing ligand asialofetuin. The streptavidin-biotin interaction has been applied for the first time to the assembly of hepatocyte-targeted lipoplexes that display asialoorosomucoid and that are well tolerated by a human hepatoma cell line in which transfection is demonstrably achieved by receptor mediation. Favorable size and charge ratio characteristics suggest that this system may be suitable for in vivo application.

Targeted delivery of macromolecular drugs: asialoglycoprotein receptor (ASGPR) expression by selected hepatoma cell lines used in antiviral drug development.

The asialoglycoprotein receptor (ASGPR), an endocytotic cell surface receptor expressed by hepatocytes, is triggered by triantennary binding to galactose residues of macromolecules such as asialoorosomucoid (ASOR). The capacity of this receptor to import large molecules across the cellular plasma membrane makes it an enticing target for receptor-mediated drug delivery to hepatocytes and hepatoma cells via ASGPR-mediated endocytosis. This study describes the preparation and characterization of (125)I-ASOR, and its utility in the assessment of ASGPR expression by HepG2, HepAD38 and Huh5-2 human hepatoma cell lines. ASOR was prepared from human orosomucoid, using acid hydrolysis to remove sialic acid residues, then radioiodinated using iodogen. (125)I-ASOR was purified by gel column chromatography and characterized by SDS-PAGE electrophoresis. The ASOR yield by acid hydrolysis was 75%, with approximately 87 % of the sialic acid residues removed. Electrophoresis and gel chromatography demonstrated substantial differences in (125)I-ASOR quality depending on the method of radioiodination. ASGPR densities per cell were estimated at 76,000 (HepG2), 17,000 (HepAD38) and 3,000 (Huh-5-2). (125)I-ASOR binding to ASGPR on HepG2 cells was confirmed through galactose- and EDTA- challenge studies. It is concluded that (125)I-ASOR is a facilely-prepared, stable assay reagent for ASGPR expression if appropriately prepared, and that HepG2 cells, but not HepAD38 or Huh-5-2 cells, are suitable for studies exploiting the endocytotic ASGPR.

Single vesicle analysis of endocytic fission on microtubules in vitro.

Following endocytosis, internalized molecules are found within intracellular vesicles and tubules that move along the cytoskeleton and undergo fission, as demonstrated here using primary cultured rat hepatocytes. Although the use of depolymerizing drugs has shown that the cytoskeleton is not required to segregate endocytic protein, many studies suggest that the cytoskeleton is involved in the segregation of protein in normal cells. To investigate whether cytoskeletal-based movement results in the segregation of protein, we tracked the contents of vesicles during in vitro microscopy assays. These studies showed that the addition of ATP causes fission of endocytic contents along microtubules, resulting in the segregation of proteins that are targeted for different cellular compartments. The plasma membrane proteins, sodium (Na+) taurocholate cotransporting polypeptide (ntcp) and transferrin receptor, segregated from asialoorosomucoid (ASOR), an endocytic ligand that is targeted for degradation. Epidermal growth factor receptor, which is degraded, and the asialoglycoprotein receptor, which remains partially bound to ASOR, segregated less efficiently from ASOR. Vesicles containing ntcp and transferrin receptor had reduced fission in the absence of ASOR, suggesting that fission is regulated to allow proteins to segregate. A single round of fission resulted in 6.5-fold purification of ntcp from ASOR, and 25% of the resulting vesicles were completely depleted of the endocytic ligand.

Development of a rapid, immunochromatographic strip test for serum asialo alpha1-acid glycoprotein in patients with hepatic disease.

Serum asialoglycoprotein (desialylated glycoproteins) concentrations have been reported to be elevated in patients with hepatic disease as compared with that of normal subjects. We recently developed a solid-phase sandwich assay for asialo alpha1-acid glycoprotein (AsAGP) as a representative of the serum asialoglycoproteins and evaluated the utility of this AsAGP as a diagnostic marker for liver cirrhosis (LC) and/or hepatocellular carcinoma (HCC). In this study, we developed a rapid, one-step immunochromatographic strip capable of specifically detecting AsAGP in serum specimens. We have produced a monoclonal antibody (mAb) to AGP, and based on ELISA and Western blot analysis, we have selected four hybridoma clones which generated mAbs to recognize AsAGP. In the immunochromatographic strip test, one mAb was used for conjugation with colloidal gold microparticles. Ricinus communis agglutinin (RCA) was immobilized onto a nitrocellulose membrane strip to form a result line in the path of chromatographic migration. Likewise, a control line was created above the result line by the immobilization of anti-mouse IgG. A serum specimen was then applied to the sample pad. The AsAGP in the sample specifically bound to the microparticles via mAb (As16.89) and co-migrated upward until the AsAGP was sandwiched with the immobilized lectin (RCA), revealing a visible result line. The colloidal gold microparticles without bound AsAGP continued to migrate, forming a visible control line. Thus, an AsAGP-positive specimen (>1.5 microg/mL) yielded a result line and a control line, whereas an AsAGP-negative specimen (<1.5 microg/mL) produced only a single control line. The entire test procedure was completed in less than 5 min. In order to examine the reliability of the testing procedures, we carried out the immunochromatographic strip test with 102 serum samples and compared the results of these tests with those obtained by ELISA. The two methods showed excellent correlation, with 83-100% above/below the cut-off value (1.5 microg/mL). Therefore, we concluded that the results of the immunochromatographic test are in excellent accordance with those of the sandwich ELISA.

Feasibility on systemic delivery of asialoorosomucoid complex to hepatic origin cells mediated by asialoglycoprotein receptor.

Receptor mediated gene delivery is a new gene transfer strategy. Asialoglycoprotein receptor (ASGP-R), the receptor of asialoorosomucoid (Asor), is specially expressed on the surface of hepatocyte. In this paper, the nuclide 131I was combined with Asor to form a kind of soluble nuclide-protein complex, which can be specifically endocytosed into hepatocyte by ASGP-R. After in travenous injection of the complex into experimental animals, the deposition of Asor in vivo and the targeting quality of hepatocyte was detected by ECT. This research testified the feasibility of targeting Asor complex delivery to hepatocyte mediated by ASGP-R in vivo, and provided foundation for the genetic diagnosis and gene therapy of hepatic cell-related diseases.

Modulation of the molecular conformation of a hepatocyte-targeting gene drug in order to improve its expression efficiency in vitro.

AIM: To construct different conformations of a plasmid DNA/vector complex (pcDNA3.1/IFN-gamma-ASOR-PLL) and transfect cells of the hepatoma cell line BEL7402 to investigate the optimal conformation of the complex for improved expression efficiency in the target cell. METHODS: Double-distilled water and adjuvant were added to the naked pcDNA3.1/IFN-gamma, target vector ASOR-PLL and the ASOR-PLL-pcDNA3.1/IFN-gamma complex to create different conformations; molecules that were transfected into BEL7402 cells and the expression efficiency was determined by measuring the IFN-g concentration in the culture supernatant by ELISA. RESULTS: Naked pcDNA3.1/IFN-gamma DNA distributed linearly in double-distilled water and condensed into a mica configuration in adjuvant; ASOR-PLL had a net-like distribution without adjuvant and a spider-like form in the adjuvant-treated group; the ASOR-PLL-pcDNA3.1/IFN-g complex had a divaricate form without adjuvant, but a bead-like or granular conformation in 0.1 and 0.2 mol/L of adjuvant, a homogeneous bacilliform or chromatoid-shaped conformation in 0.3 mol/L adjuvant, and varied shapes in 0.4 and 0.5 mol/L adjuvant. The supernatant IFN-gamma expression in the bacilliform/chromatoid conformation complex group was the highest among the different conformation groups and controls. When chloroquine was added the supernatant IFN-gamma concentration increased in the liposome group and decreased in the bacilliform/chromatoid conformation group . CONCLUSIONS: The two structural molecules and their complex, ASOR-PLL-pcDNA3.1/IFN-gamma, were adjustable in the liquid mode. The specific bacilliform/chromatoid conformation of complex was lysosome enzyme-resistant and could play an active role in improving the efficiency of gene expression. The hypothesis that a chromosome-like conformation of the target gene molecule is involved in enhancing exogenous gene expression is proposed.

Targeted gene delivery into HepG2 cells using complexes containing DNA, cationized asialoorosomucoid and activated cationic liposomes.

Unilamellar activated cationic liposomes containing 3beta[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol, dioleoyl phosphatidylethanolamine (DOPE) and the N-hydroxysuccinimide ester of cholesteryl hemisuccinate (4:5:1, molar ratio) have been prepared and their DNA-binding capacity has been assessed in a gel retardation assay. Ternary complexes composed of activated cationic liposomes, carbodiimide-cationized asialoorosomucoid (Me+AOM) and pRSVL plasmid DNA were assembled for receptor-mediated DNA delivery into cells expressing the asialoglycoprotein receptor (ASGP-R). Binding of complexes in which Me+AOM was replaced by fluoresceinated Me+AOM (FMe+AOM) to the human hepatocellular cell line HepG2 at 4 degrees C was severely reduced by co-incubation with asialoorosomucoid (AOM). Moreover, assemblies containing liposomes, pRSVL DNA and Me+AOM (8:1:4, w/w/w) promoted high levels of luciferase activity in this cell line (1.3 x 10(7) relative light units/mg soluble cell protein). Assays conducted in the presence of a hundred-fold excess of the ligand AOM afforded considerably lower levels of transfection (2.5 x 10(5) relative light units/mg soluble cell protein). In contrast, the highest level of luciferase activity achieved with liposome, pRSVL DNA, AOM complexes was only a quarter of the best levels obtained with liposome, pRSVL DNA, Me+AOM assemblies. These findings strongly support the notion that complexes gain entry into hepatocyte-derived cells by ASGP-R mediation and that they are potentially useful gene carriers to liver hepatocytes.

The efficacy of a DNA vaccine encoding herpes simplex virus type 1 (HSV-1) glycoprotein D in decreasing ocular disease severity following corneal HSV-1 challenge.

Antiviral effects of a DNA vaccine against herpes simplex virus 1 (HSV-1) glycoprotein D (gD) were evaluated in eight week-old female BALB/c mice. The nuclease-insensitive construct (gD-ASOR) consisted of an HSV-1 gD encoding plasmid coupled to asialo orosomucoid (ASOR), targeting it to cells bearing ASOR receptors. Mice were immunized on day 0 and 7 with 10 microg doses of gD-ASOR or control substances. Fourteen days later, mice were infected by the corneal route with 10(5) pfu or 10(6) pfu HSV-1, strain 17syn+. Immunized mice showed a significant decrease in ocular disease severity over a 21-day observation period following infection compared to sham-immunized mice. Acute replication kinetic assays demonstrated a 100-fold decrease in viral titers on day 6 in trigeminal ganglia from immunized BALB/c mice compared to sham-immunized mice. Immunized mice showed a significant increase in numbers of CD4(+)T cells infiltrating the trigeminal ganglia at day 6 post infection compared to sham-immunized mice. Significant differences were not seen in latent viral reservoir between immunized and unimmunized mouse groups. Immunization with gD-ASOR decreased the severity of acute ocular HSV-1 infection, induced a CD4(+) T cell response, decreased the viral load in the trigeminal ganglia, but did not diminish viral latency.

Interactions of human mesangial cells with IgA and IgA-containing immune complexes.

BACKGROUND: IgA nephropathy (IgAN) is characterized by IgA1-containing immune complexes in mesangial deposits and in the circulation. The circulating immune complexes (CIC) are composed of galactose- (Gal) deficient IgA1 and IgG or IgA1 antibodies specific for the Gal-deficient IgA1; interactions of these CIC with mesangial cells (MC) were studied. METHODS: Binding, internalization, and catabolic degradation of myeloma IgA1 protein as a standard control and the isolated CIC were studied using human MC, hepatoma cell line HepG2 expressing the asialoglycoprotein receptor (ASGP-R), and monocyte-like cell line U937 expressing the Fc(alpha)-R (CD89). Biochemical and molecular approaches were used to assess expression of CD89 and ASGP-R by MC. RESULTS: At 4 degrees C, radiolabeled IgA1 bound to MC and HepG2 cells in a dose-dependent and saturable manner. The binding was inhibited by IgA-containing CIC or excess IgA1 or its Fc fragment but not by the Fab fragment of IgA1. At 37 degrees C, the cell-bound IgA1 was internalized and catabolized. In addition to IgA1, HepG2 cells also bound (in a Ca2+-dependent manner), internalized, and catabolized asialoorosomucoid (ASOR), other asialo-(AS)-glycoproteins, and secretory component (SC). The binding by MC appeared to be restricted to IgA1 or AS-IgA1 and was not Ca2+-dependent. Furthermore, MC and HepG2 cells internalized and catabolized IgA1-containing CIC. Using RT-PCR with ASGP-R- or CD89-specific primers, mRNAs of the two respective genes were not detected in MC. CONCLUSIONS: The data showed that the ability of MC to bind IgA1 and IgA1-containing CIC in vitro was mediated by an IgA receptor that was different from CD89 or ASGP-R and had a higher affinity for IgA-CIC than for uncomplexed IgA.

H2, the minor subunit of the human asialoglycoprotein receptor, trafficks intracellularly and forms homo-oligomers, but does not bind asialo-orosomucoid.

The functional human hepatic asialoglycoprotein receptor (ASGP-R) is a hetero-oligomer composed of two subunits, designated H1 and H2, which are highly homologous. Despite their extensive homology, the major H1 subunit is stably expressed by itself, whereas in the absence of H1 most of the H2 subunits are degraded in the ER. In this study, we were able to investigate the capability of the minor ASGP-R subunit, H2, to function independently of H1, because it was apparently stabilized by fusing its NH(2) terminus with an epitope tag. We could thus create stable cell lines in hepatoma-derived SK-Hep-1 cells that expressed the H2 subunit alone. H2 was expressed on the cell surface and was internalized, predominantly through the clathrin-coated pit pathway. Since the internal pool of H2 was also able to traffick to the cell surface, we conclude that H2 recycles between the surface and intracellular compartments, similar to the constitutive recycling of hetero-oligomeric ASGP-R complexes. However, the rate of H2 recycling and internalization was approximately 25-33% that of H1. Similar to H1, the H2 polypeptides were also able to self-associate to form homo-oligomers, including trimers and tetramers. However, unlike H1, which can bind the ligand asialo-orosomucoid (ASOR) when overexpressed in COS-7 cells, H2 failed to bind or endocytose ASOR. In summary, the H2 subunit of the human ASGP-R contains functional, although weak, signal(s) for endocytosis and recycling and has the ability to oligomerize. H2 homo-oligomers, however, do not create binding sites for desialylated glycoproteins, such as ASOR, that contain tri- and tetra-antennary N-linked oligosaccharides. Nonetheless, these results raise the intriguing possibility that naturally occurring H2 homo-oligomers may exist in human hepatocytes and have an as yet undiscovered function.

Receptor-mediated gene delivery to HepG2 cells by ternary assemblies containing cationic liposomes and cationized asialoorosomucoid.

Unilamellar cationic liposomes have been prepared from an equimolar mixture of 3beta[N',N'-dimethylaminopropane)-carbomoyl] cholesterol (Chol-T), a higher homologue of 3beta[N',N'-dimethylaminoethane)-carbomoyl] cholesterol (DC-Chol), and dioleoylphosphatidyl-ethanolamine. The DNA binding capabilities of Chol-T and Chol-T/DOPE liposomes have been demonstrated in lipid impregnated paper-DNA binding assays and gel retardation experiments, respectively. These liposomes have been combined with pRSVL plasmid DNA and N-ethyl-N'-(3-trimethylpropylammonium) carbodiimide iodide modified asialoorosomucoid (Me+ CDI urea-AOM) to generate ternary electrostatic assemblies intended for selective entry into cells displaying the galactose-specific lectin. This effect has been evaluated in the human hepatocellular carcinoma cell line HepG2 in which high levels of luciferase activity were achieved (up to 1.84 x 10(7) relative light units/mg protein) after transfection with complexes containing liposomes (1-3 microg), Me+CDI urea-AOM (2 microg), and DNA (0.5 microg) in 0.5 mL culture medium. Transfections conducted in the presence of free asialoorosomucoid afforded much lower luciferase activity (up to 1.5 x 10(5) relative light units/mg protein) confirming that DNA uptake was predominantly via asialoorosomucoid receptor-mediated endocytosis. We concluded therefore that modular complexes used in our study display the carbohydrate moiety of the glycoprotein component prominently, thus permitting interaction of terminal galactose units with their cognate receptors on the cell membrane.

Effect of semisynthetic analog of alpha(1)-acid glycoprotein on immunomodulatory and antiinflammatory activity of natural glycoprotein.

Pseudo-alpha(1)-acid glycoprotein with carbohydrate chain ratio typical of native form was synthesized by a previously developed original technique of quantitative transfer of alpha(1)-acid glycoprotein carbohydrate chains to other polymeric carrier. Similarly to native glycoprotein, the semisynthetic analog inhibited lymphocyte proliferation and stimulated the production of antiinflammatory cytokines by peripheral blood mononuclear leukocytes. However, it possessed no antioxidant activity and did not inhibit complement activation by the alternative pathway. The role of carbohydrate and protein components of alpha(1)-acid glycoprotein molecule in the realization of its biological effects is discussed.

Improvement of receptor-mediated gene delivery to HepG2 cells using an amphiphilic gelling agent.

Gene transfer was performed using asialo-oroso-mucoid-polylysine (ASOR-PL) conjugates to allow targeted expression of the gene in cells of hepatic origin. In a gel-electrophoretic analysis, the ASOR-PL conjugate produced a complete DNA retardation effect at the optimal ratio of 222:1 (ASOR-PL conjugate/pCMV beta-gal plasmid). The gene-transfer efficiency of the ASOR-PL conjugate was evaluated in HepG2 cells that express asialoglycoprotein receptor and NIH 3T3 cells that do not. The expression was assayed by 5-bromo-4-chloroindol-3-yl beta-D-galactopyranoside ('X-Gal') staining and Chlorophenol Red beta-D-galactopyranoside. When an expression vector for the tumour-suppressor gene p53, pCMVp53, complexed to ASOR-PL conjugate, was transfected into HepG2 cells, the exogenously provided p53 gene was detected in the HepG2 cells by PCR. To improve the efficiency of DNA delivery and expression of the therapeutic proteins poloxamer 407, a fusogenic peptide, influenza-virus haemagglutinin HA2 and chloroquine were individually incorporated into the system. The expression level of beta-galactosidase in HepG2 cells was increased by about four times by the presence of poloxamer 407, whereas the fusogenic peptide HA2 and chloroquine had no effects. When HepG2 cells were transfected with pCMVp53 in the presence of poloxamer 407, the mRNA of transfected p53 could be detected by reverse transcriptase PCR. The current findings open the possibility that a receptor-mediated gene-delivery system for hepatic gene therapy using ASOR-PL conjugate in combination with poloxamer 407 may be developed in the future.

Human intestinal epithelial cells express a novel receptor for IgA.

Binding and transport of polymeric Igs (pIgA and IgM) across epithelia is mediated by the polymeric Ig receptor (pIgR), which is expressed on the basolateral surface of secretory epithelial cells. Although an Fc receptor for IgA (FcalphaR) has been identified on myeloid cells and some cultured mesangial cells, the expression of an FcalphaR on epithelial cells has not been described. In this study, binding of IgA to a human epithelial line, HT-29/19A, with features of differentiated colonic epithelial cells, was examined. Radiolabeled monomeric IgA (mIgA) showed a dose-dependent, saturable, and cation-independent binding to confluent monolayers of HT-29/19A cells. Excess of unlabeled mIgA, but not IgG or IgM, competed for the mIgA binding, indicating that the binding was IgA isotype-specific and was not mediated by the pIgR. The lack of competition by asialoorosomucoid and the lack of requirement for divalent cations excluded the possibility that IgA binding to HT-29/19A cells was due to the asialoglycoprotein receptor or beta-1, 4-galactosyltransferase, previously described on HT-29 cells. Moreover, the FcalphaR (CD89) protein and message were undetectable in HT-29/19A cells. FACS analysis of IgA binding demonstrated two discrete populations of HT-29/19 cells, which bound different amounts of mIgA. IgA binding to other colon carcinoma cell lines was also demonstrated by FACS analysis, suggesting that an IgA receptor, distinct from the pIgR, asialoglycoprotein receptor, galactosyltransferase, and CD89 is constitutively expressed on cultured human enterocytes. The function of this novel IgA receptor in mucosal immunity remains to be elucidated.

Murine response to DNA encoding herpes simplex virus type-1 glycoprotein D targeted to the liver.

Plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) was complexed with asialoorosomucoid conjugated to poly-L-lysine. Following its intravenous injection into BALB/c mice, this complex was targeted to the liver. Liver cells expressing gD-1 were detected immunohistochemically through day 6 post-immunization, while gD-1 DNA was detectable through 14 days post-immunization. Decline of gD-1 expression and detectable gD-1 DNA in the liver correlated with influx of T cells, predominantly CD4(+). The ASOR-poly-L-lysine DNA carrier system promotes hepatic expression of gD-1 and may be useful in vaccination against herpes simplex virus type-1.

Targeted delivery of DNA encoding herpes simplex virus type-1 glycoprotein D enhances the cellular response to primary viral challenge.

Intravenous injection of plasmid DNA encoding herpes simplex virus type-1 glycoprotein D (gD-1) complexed with asialoorosomucoid-poly-L-lysine (gD-ASOR) targets foreign DNA to the liver, leading to hepatic expression of gD-1. BALB/c mice were given two intravenous injections of gD-ASOR, pBK-ASOR (plasmid lacking the gD-1 gene but complexed with ASOR), or PBS. The skin was inoculated with 1 x 10(4) PFU of HSV-1 or sham-inoculated, and analyzed for infectious virus and cellular infiltration 1, 3, and 5 days after inoculation. Prior immunization with gD-ASOR led to significantly lower (P < 0.05) viral titers in the skin 5 days after inoculation compared with controls. Infiltration of the skin at the site of inoculation by polymorphonuclear neutrophils (PMNs), T cells, B cells, dendritic cells, and macrophages was monitored immunohistochemically. Significantly higher numbers (P < 0.05) of CD4+ and CD8+ T cells, dendritic cells, and macrophages responded to HSV-1 challenge in mice immunized with gD-ASOR than in mice immunized with pBK-ASOR or PBS. The response by PMNs and B cells was indistinguishable among the treatment groups. These results suggest that BALB/c mice sensitized to gD-1 following gD-ASOR immunization develop an enhanced T-cell response to primary HSV-1 infection.

A novel immunization method to induce cytotoxic T-lymphocyte responses (CTL) against plasmid-encoded herpes simplex virus type-1 glycoprotein D.

DNA molecules complexed with an asialoglycoprotein-polycation conjugate, consisting of asialoorosomucoid (ASOR) coupled to poly-L-lysine, can enter hepatocytes which bear receptors for ASOR. We used this receptor-mediated DNA delivery system to deliver plasmid DNA encoding glycoprotein D (gD) of herpes simplex virus type 1 to ASOR-positive cells. Maximum expression of gD protein was seen at 3 days after injection of this preparation in approximately 13% of cells from BALB/c mice [hepatocytes from mice injected intravenously (i.v.) or peritoneal exudate cells from mice injected intraperitoneally (i.p.)]. In comparison with mice injected with either the plasmid vector alone or the gD-containing plasmid uncomplexed to ASOR, mice immunized with gD-containing plasmid complexed with ASOR-poly-L-lysine induced marked antigen-specific CTL responses. BALB/c mice immunized with gD-DNA developed a T-cell-mediated CTL response against target cells expressing gD and MHC class II glycoproteins, but not against cells expressing only gD and MHC class I molecules. In C3H mice, gD-DNA induced a T-cell-mediated CTL response against target cells expressing gD and class I MHC molecules. Serum anti-gD antibody in low titers were produced in both strains of mice. DNA complexed with ASOR-poly-L-lysine induced CTL responses in mice.

Stabilization of poly-L-lysine/DNA polyplexes for in vivo gene delivery to the liver.

We are developing a self-assembling non-viral in vivo gene delivery vehicle based on poly-l-lysine and plasmid DNA. We have characterized poly-l-lysines of different chain lengths for DNA condensation and strength of DNA binding. Poly-l-lysine chains >20 residues bound DNA efficiently in physiological saline, while shorter chains did not. Attachment of asialoorosomucoid to PLL increased the PLL chain length required for efficient DNA binding in saline and for efficient DNA condensation. By electron microscopy, poly-l-lysine/DNA polyplexes appeared as toroids 25-50 nm in diameter or rods 40-80 nm long; conjugation of asialoorosomucoid to the polylysine component increased the size of resulting polyplexes to 50-90 nm. In water, poly-l-lysine and asialoorosomucoid-PLL polyplexes have effective diameters of 46 and 87.6 nm, respectively. Polyplexes containing only poly-l-lysine and DNA aggregated in physiological saline at all charge ratios and aggregated at neutral charge ratios in water. Attachment of asialoorosomucoid lessened, but did not eliminate, the aggregation of PLL polyplexes, and did not result in efficient delivery of polyplexes to hepatocytes. Conjugation of polyethylene glycol to poly-l-lysine sterically stabilized resulting polyplexes at neutral charge ratios by shielding the surfaces. For efficient in vivo gene delivery, polyplexes will need to be sterically stabilized to prevent aggregation and interaction with serum components.