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A recombinant E. coli ribonuclease H (RNase H) was chemically coupled to an antisense oligodeoxynucleotide (ODN) against the 5‘-noncoding region (5‘-NCR) of the hepatitis C virus. Purity of the conjugates was confirmed by sodium deodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) as a band corresponding to approximately 23 kDa. Conjugate function was tested by the cleavage of a HCV RNA transcript including the 5‘-NCR and core region and showed HCV sequence-specific cleavage by the appearance of an expected ~1000 nt fragment of RNA. Cleavage was not seen by RNase H alone, or ODN alone. Delivery studies using ³²P- and ¹²⁵I-labeling showed that while RNAse H failed to enter cells, the conjugate was efficiently taken into the cells. To assess intracellular effects, a cell line, Huh-7/CMV−NCRCΔluc, which expresses HCV mRNA (nt 1−585) fused to a marker gene, was transfected with the conjugate. Reporter gene expression was suppressed by 51.2% with the conjugate compared to only 39.7% by ODN alone, 35.8% by a mixture of RNase H plus ODN, and not at all by RNase H alone. In conclusion, the RNase H−ODN conjugate effectively cleaved an HCV transcript in vitro and inhibited expression of an HCV-marker fusion construct in a liver-derived cell line.

Ribonuclease H (RNaseH) recognizes and efficiently cleaves the RNA strand of DNA−RNA hybrids, but has no inherent sequence selectivity. However, the formation of DNA−RNA hybrids does require specific sequence recognition. On the basis of this concept, we wondered whether antisense oligonucleotides complementary to target RNA covalently linked to RNase H could be used to direct specific cleavage events mediated by RNase H. The aim of this research was to couple a DNA oligonucleotide to RNase H to confer specificity of ribonuclease activity toward hepatitis B viral (HBV) mRNA. A modified 13-base oligonucleotide that is specific for the DR1 region of HBV mRNA was conjugated to modified E. coli RNase H using a water soluble cross-linker. A 1200 base fragment of HBV RNA including the DR1 region was synthesized as a substrate using T7 RNA polymerase. Incubation of the RNase H−oligonucleotide conjugate with the RNA transcript resulted in cleavage of the HBV mRNA transcript in a concentration dependent manner. Eighty-five percent of substrate was cleaved under optimal conditions. Controls consisting of RNase H alone, oligonucleotide alone, and incubation of the conjugate with an unrelated mRNA substrate resulted in no cleavage activity. RNase H coupled to an HBV antisense oligonucleotide can specifically cleave target HBV transcripts.

Genes can be targeted to hepatocytes in vitro and in vivo by the use of asialoorosomucoid−polylysine conjugates. After systemic application, this nonviral vector is recognized by highly selective asialoglycoprotein (AsGP) receptors on the sinusoidal liver cell membrane and is taken up via receptor-mediated endocytosis. As most of the DNA is rapidly transferred to lysosomes where it is degraded, transfection efficiency is low and gene expression transient. To address this problem, we incorporated a pH-dependent synthetic hemolytic peptide derived of the G-protein of Vesicular Stomatitis Virus (VSV) into the gene transfer system, to increase endosomal escape of internalized DNA. The multicomponent carrier binds DNA in a nondamaging way, is still recognized by the AsGP receptor, and is targeted to the liver in vivo. Injection of DNA complexes containing a luciferase marker gene resulted in luciferase expression of 29 000 pg/g liver which corresponded to an increase of a factor of 10³ overexpression after injection of DNA complexes without endosomolytic peptide. Furthermore, the amount of intact transgene within isolated liver cell nuclei was increased by a factor of 10¹−10² by the use of the multicomponent carriers. These results demonstrate that incorporation of a hemolytic peptide into a nonviral vector can greatly increase gene expression while retaining cell type targetability in vivo.

We present evidence that normal hepatocytes can be specifically protected from galactosamine toxicity in vitro by targeting an antagonist to these cells via receptor-mediated endocytosis. The strategy is based upon the following principles: 1) galactosamine is a highly selective hepatotoxin that causes a dose-dependent depletion of uridine intermediates; 2) galactosamine toxicity can be antagonized by supplemental administration of uridine; 3) normal hepatocytes possess unique cell-surface receptors that can internalize galactose terminal (asialo-)glycoproteins with subsequent degradation of the glycoprotein ligand. Based on these facts, we hypothesized that chemical coupling of a galactosamine antagonist to an asialoglycoprotein could result in cell-specific delivery and protection of normal hepatocytes by targeting the antagonist via asialoglycoprotein receptors. Using a model system consisting of freshly isolated rat hepatocytes (receptor (+)) and Morris 7777 rat hepatoma (receptor (-)) cells, sensitivity to galactosamine in vitro was determined and found to be similar for both types of cells. A targetable antagonist was synthesized by coupling uridine monophosphate to asialoorosomucoid in a molar ratio of 5 to 1. Exposure of Morris 7777 cells to the targetable antagonist in the presence of a toxic concentration of galactosamine did not protect these cells as evidenced by a steady decline in the number of viable cells in a fashion identical to cells treated with galactosamine alone. However, normal hepatocytes that received the conjugate in the presence of galactosamine were protected as their viable cell number remained the same as control (untreated) cells. Competition by an excess of asialoglycoprotein inhibited the protective effect of the conjugate, supporting the concept that the asialoglycoprotein component of the conjugate was responsible for the specific delivery of the antagonist to the target cells.

Antitumor immunotoxins were formed by covalently attaching the ribosome-inactivating protein ricin A chain (RA) to the antitumor antibodies BR96 and L6. In vitro cytotoxicity assays established that BR96-RA was cytotoxic to H2987 human lung adenocarcinoma cells (IC50 = 6 nM), while L6-RA exhibited very low levels of cytotoxic activity (18% cell kill at 67 nM). The virulence factor from the intracellular pathogen Listeria monocytogenes, listeriolysin O (LLO), was able to potentiate the cytotoxicity of BR96-RA and L6-RA by 120- and >1340-fold, respectively, resulting in IC50 values of approximately 50 pM. LLO also potentiated the cytotoxicity of the peptide anticancer drug bleomycin by a factor of >2500 but had no effect on the cytotoxic activities of the anticancer drugs cytarabine and etoposide phosphate. In addition, LLO did not potentiate the cytotoxic activity of unconjugated ricin A chain or L6-RA on H2987 cells that were saturated with L6 prior to conjugate treatment. These results are attributed to LLO-induced alteration of the intracellular trafficking of molecules that are incorporated into acidic vesicles.

A derivative of colchicine was synthesized, in a manner that preserved its important structural features, and conjugated to an asialoglycoprotein. The conjugate was characterized by ultraviolet−visible spectrophotometry and protein analysis. An average coupling ratio of 2 mol of colchicine per mole of asialoglycoprotein was achieved. The conjugate was stable to incubation in serum but was split into its separate components under chemically reducing conditions. Incubation with cells in culture revealed that the conjugate had antiproliferative activity similar to that of colchicine, but only in asialoglycoprotein receptor-containing cells. There was no effect at all on asialoglycoprotein receptor (−) cells. Furthermore, the antiproliferative effect of the conjugate on receptor (+) cells was blocked by addition of a large molar excess of free asialoglycoprotein. Immunofluorescence microscopy revealed disruption of microtubules in cell cultures that were pretreated with the conjugate. These results indicate that a colchicine conjugate that is taken up specifically into cells by asialoglycoprotein receptors and released intracellularly in a biologically active form can be prepared.

A soluble DNA carrier system was used to target a foreign gene specifically to liver in vivo via asialoglycoprotein receptors. The DNA carrier was prepared consisting of a galactose-terminal (asialo-)glycoprotein, asialoorosomucoid (AsOR), covalently linked to poly-L-lysine. The conjugate was complexed in a 2:1 molar ratio (based on AsOR content of the conjugate) to the plasmid, pSV2 CAT, containing the gene for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Intravenous injection of [32P]plasmid DNA complexed to the carrier demonstrated specific hepatic targeting with 85% of the injected counts taken up by the liver in 10 min compared to only 17% of the counts when the same amount of [32P]DNA alone was injected under identical conditions. Targeted pSV2 CAT DNA was detected at a level of 1.0 ng/g liver by hybridization of a [32P]pSV2 CAT cDNA probe to rat liver DNA extracted 24 h after intravenous injection of AsOR-poly-L-lysine-DNA complex containing 1.0 mg of DNA. Homogenates of livers taken 24 h after injection of the complex revealed that the targeted CAT gene was functional as reflected by the detection of CAT activity (approximately 4 microunits/mg protein). Livers from control animals that received individual constituents of the complex produced no CAT activity. Simultaneous injection of excess AsOR to compete with the AsOR-poly-L-lysine-DNA complex for uptake by the liver inhibited CAT gene expression. Assays for CAT activity in other organs (spleen, kidney, lungs) failed to demonstrate any activity in these organs. This new soluble DNA carrier system can permit targeted delivery of foreign genes specifically to liver with resultant foreign gene expression in vivo.

We have used a model system consisting of two human hepatoma cell lines, Hep G2, representing well differentiated normal hepatocytes, and PLC/PRF/5, representing poorly differentiated malignant hepatocytes, to demonstrate that the differential presence of asialoglycoprotein receptor activity in these cell lines can be used to influence transferrin-mediated iron uptake. We based our experiments on the following facts: Hep G2 cells possess receptors that bind, internalize, and degrade galactose-terminal (asialo-)glycoproteins; PLC/PRF/5 cells have barely detectable asialoglycoprotein receptor activity; both cell lines possess active transferrin-mediated iron uptake; transferrin releases iron during acidification of intracellular vesicular compartments; primary amines, e.g. primaquine, inhibit acidification and iron release from transferrin. When added to culture medium, [55Fe]transferrin delivered 55Fe well to both cell lines. As expected, in the presence of [55Fe]transferrin, free primaquine caused a concentration-dependent decrease in 55Fe uptake in both cell lines. To create a targetable conjugate, primaquine was covalently coupled to asialofetuin to form asialofetuin-primaquine. When PLC/PRF/5 (asialoglycoprotein receptor (-)) cells were preincubated with this conjugate, transferrin-mediated 55Fe uptake was unaffected. However, transferrin-mediated 55Fe uptake by Hep G2 (asialoglycoprotein receptor (+)) cells under identical conditions was specifically decreased by 55% compared to control cells incubated without the conjugate.

The effects of ketone bodies on the metabolism of alanine and glutamine were studied in isolated extensor digitorum communis (EDC) muscles from 24 h-fasted chicks. (1) Acetoacetate and DL-beta-hydroxybutyrate (4 mM) markedly inhibit branched-chain amino acid (BCAA) transamination and alanine formation. (2) Ketone bodies (1 and 4 mM) increase the intracellular concentration and release of glutamate and glutamine, suggesting that inhibition of BCAA transamination does not limit intracellular availability of glutamate for alanine synthesis. (3) Ketone bodies (1 and 4 mM) do not affect glucose uptake by muscles, but decrease the rate of glycolysis as well as the intracellular concentration and release of pyruvate in muscles. (4) Addition of 12 mM-glucose increases the formation of alanine in muscles incubated in the absence of ketone bodies, but has no effect in muscles incubated in the presence of 4 mM ketone bodies. (5) Addition of 5 mM-pyruvate to the media prevents the inhibiting effect of ketone bodies on BCAA transamination and alanine synthesis. These results suggest that ketone bodies decrease alanine synthesis by limiting the intracellular availability of pyruvate, owing to inhibition of glycolysis, and inhibit BCAA transamination by decreasing the intracellular concentration of amino-group acceptors such as pyruvate in EDC muscles from fasted chicks.

DNA delivered to the liver by asialoglycoprotein receptor-mediated endocytosis is degraded in lysosomes within 48 h. To test the hypothesis that microtubular disruption should promote transgene persistence by interrupting endosomal translocation to lysosomes, plasmids containing bacterial chloramphenicol acetyltransferase (pSV2-CAT) or human bilirubin-UDP-glucuronosyltransferase-1 (pSVK3-hBUGT1) genes were complexed with asialoglycoprotein-polylysine conjugates, and 1 mg of the complexed DNA was injected intravenously into bilirubin-UDP-glucuronosyltransferase-deficient Gunn rats. 30 min before DNA injection, one group received 0.75 mg of colchicine/kg of body weight intraperitoneally, which was shown by immunofluorescent confocal microscopy to disrupt the microtubular network. Control rats received normal saline. In colchicine-pretreated rats receiving pSV2-CAT, hepatic chloramphenicol acetyltransferase activity persisted for 9-14 weeks, whereas in the saline-pretreated group the activity was detectable for 48 h only. In colchicine-pretreated Gunn rats receiving pSVK3-hBUGT1, the DNA persisted in liver for 10 weeks, bilirubin glucuronides were excreted in bile, and serum bilirubin levels declined by 25-35% in 2-4 weeks and remained reduced for 8 weeks. Without colchicine pretreatment, the DNA was detectable in liver for 2 days only, and serum bilirubin levels were not reduced. Thus, microtubular disruption provides a noninvasive method for prolonging the effect of liver-targeted gene therapy.

A new method is discussed which examines trace, dried bloodstains by gel in situ hybridization using a Y-chromosome-specific deoxyribonucleic acid (DNA) probe to determine the sex of the bloodstain for forensic medicine application. The complete DNA is transferred directly by electrophoresis onto the gel intact, bypassing the possibilities of impurities contaminating the sample and of DNA degradation. The method has proven accurate for small (2.5-mm-diameter) samples aged up to eight years and is quick, simple, and easily read.

Methionine transamination is extensive in rat and chick skeletal-muscle homogenates, but is barely detectable in intact rat, but not chick, skeletal muscles. Branched-chain amino acids essentially block methionine transamination in intact muscles and homogenates from both species. The physiological significance of methionine transamination in skeletal muscle is questioned.