Catherine H. Wu

Delivery systems for gene therapy

Introduction of foreign genes into mammalian cellsin vitro has been accomplished previously by a variety of methods. The few techniques that have been developed for transfection of mammalian cellsin vivo, are technically difficult or lack cell specificity.We have developed a soluble, targetable DNA carrier system consisting of an asialoglycoprotein covalently coupled to a polycation. The strategy was based on: 1) the presence of unique receptors on hepatocytes which internalize galactose-terminal (asialo-)glycoproteins; 2) polycations can bind DNA in a non-covalent, non-damaging interaction. Using chloramphenicol acetyltransferase (CAT) as a marker gene, specific delivery and expression of CAT was demonstratedin vitro using asialoglycoprotein receptor ( +) and (-) cell lines.Intravenous injection of conjugate-DNA complexes in rats resulted in detection of CAT DNA sequences in liver 10 min later by dot blots with a CAT cDNA probe. CAT enzyme activity 24 hrs later was found specifically in liver but no other tissues or control livers. Targeted hepatic CAT expression was transient, maximal at 24 hrs but declined to barely detectable levels by 96 hrs. Persistent foreign gene expression was achieved by injection of DNA complex followed by 67% partial hepatectomy. High levels of hepatic CAT activity were detected through 11 weeks post-hepatectomy.The data indicate that a targetable gene delivery system can permitin vivo expression of an exogenous gene after simple intravenous injection. The foreign gene expression can be enhanced and made to persist by induction of hepatocyte replication.

In vitro and in vivo comparative study of chimeric liver-specific promoters

Targeting therapeutic genes to the liver is essential to improve gene therapy protocols of hepatic diseases and of some hereditary disorders. Transcriptional targeting can be achieved using liver-specific promoters. In this study we have made chimeric constructs combining promoter and enhancer regions of the albumin, alpha 1-antitrypsin, hepatitis B virus core protein, and hemopexin genes. Tissue specificity, activity, and length of gene expression driven from these chimeric regulatory sequences have been analyzed in cultured cells from hepatic and nonhepatic origin as well as in mice livers and other organs. We have identified a collection of liver-specific promoters whose activities range from twofold to less than 1% of the CMV promoter in human hepatoma cells. We found that the best liver specificity was attained when both enhancer and promoter sequences of hepatic genes were combined. In vivo studies were performed to analyze promoter function during a period of 50 days after gene transfer to the mouse liver. We found that among the various chimeric constructs tested in this work, the alpha1-antitrypsin promoter alone or linked to the albumin or hepatitis B enhancers is the most potent in directing stable gene expression in liver cells.

Receptor-mediated delivery of foreign genes to hepatocytes

Naturally existing cell surface receptors provide convenient systems for transporting substances from the extracellular environment to the interior of cells. When such receptors are highly selective for certain cell types, the possibility exists for targeting biological agents of interest to specific tissues. In this review, the concepts, progress and problems in targeting genes and their subsequent expression in hepatocytes based on the asialoglycoprotein receptor in vitro and in vivo will be discussed. The use of protein carriers for hepatocyte specific delivery of double stranded DNA to introduce novel gene expression novel, as well as, delivery of single stranded DNA for inhibition of endogenous genes with examples of each are reviewed.

Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3′-Terminal Sequences

ABSTRACT The 3′-terminal sequences of hepatitis C virus (HCV) positive- and negative-strand RNAs contribute cis-acting functions essential for viral replication. The secondary structure and protein-binding properties of these highly conserved regions are of interest not only for the further elucidation of HCV molecular biology, but also for the design of antisense therapeutic constructs. The RNA structure of the positive-strand 3′ untranslated region has been shown previously to influence binding by various host and viral proteins and is thus thought to promote HCV RNA synthesis and genome stability. Recent studies have attributed analogous functions to the negative-strand 3′ terminus. We evaluated the HCV negative-strand secondary structure by enzymatic probing with single-strand-specific RNases and thermodynamic modeling of RNA folding. The accessibility of both 3′-terminal sequences to hybridization by antisense constructs was evaluated by RNase H cleavage mapping in the presence of combinatorial oligodeoxynucleotide libraries. The mapping results facilitated identification of antisense oligodeoxynucleotides and a 10-23 deoxyribozyme active against the positive-strand 3′-X region RNA in vitro.

Targeting of genes to the liver with glycoprotein carriers

Abstract Several techniques are available to deliver foreign genes to cells in vitro. These techniques have offered valuable insights into gene regulation and expression. Many of these methods, however, have limited in vivo potential for clinical gene therapy because they compromise cell viability or cannot be targeted to desired cell types. Strategies with high potential for use in gene therapy need to satisfy several criteria: (1) use of a stable vector for the foreign DNA, (2) efficient expression of foreign DNA in host cells, and (3) a means to target a desired cell type or organ. Hepatocytes are attractive targets for gene therapy because of their large number, rich blood supply and important role in intermediary metabolism. Hepatocytes have also been found to have specific cell surface receptors that can be utilized for targeted delivery of foreign DNA. This article discusses in vitro and in vivo methods of targeted delivery and expression of foreign genes in hepatocytes with emphasis on those techniques with potential for clinical gene therapy. Methods to enhance and prolong foreign gene expression such as the use of the lysosomotropic agent chloroquine, endosomal disruption by adenovirus and influenza virus hemagglutinin HA-2 subunit, and partial hepatectomy, which has been found to augment foreign gene expression, are covered. Finally, the use of ligand-based systems to target sense and antisense DNA in gene therapy to correct metabolic disorders and the targeted delivery of protective agents to the liver are addressed.

siRNA‐resistance in treated HCV replicon cells is correlated with the development of specific HCV mutations

Summary.  RNA interference (RNAi) has been extremely effective against hepatitis C viral (HCV) gene expression in short‐term cell culture. Our aim was to determine whether long‐term RNAi might result in HCV‐resistant mutants. Huh7 HCV subgenomic replicon cells were transfected with short interfering RNAs (siRNAs). HCV‐RNA was quantified by real‐time RT‐PCR, and HCV NS5A levels were assayed by Western blots using specific antibody. Treatment with HCV‐siRNA resulted in a 50% inhibition of HCV‐RNA levels compared with pretreatment levels after 4 weeks (P < 0.05). HCV‐RNA returned to 85% of pretreatment levels after cessation of HCV‐siRNA treatment. Sequencing of the HCV‐siRNA target and upstream region was performed on 10 colonies from subcloning using PCR products, each before, during and after siRNA treatment. All colonies except one from HCV‐siRNA‐treated cells during and after treatment had mutations. There were no mutations in the HCV‐siRNA target region following control HBV‐siRNA treatment. Subcloned replicon cells containing the point mutations in the target region were found to be resistant to HCV‐siRNA inhibitory effects. In conclusion, even after 4 weeks of treatment of replicon cells with HCV‐siRNA, HCV‐RNA and HCV‐NS5A protein expression could not be completely eliminated. HCV replicons isolated during or after treatment were associated with mutations in the siRNA target region, while controls were not.

Targeted delivery of antisense DNA in woodchuck hepatitis virus‐infected woodchucks

Summary. An asialoglycoprotein‐based DNA delivery system containing an antisense oligo DNA against the polyadenylation region and adjacent upstream sequences of woodchuck hepatitis virus (WHV) was prepared. Experimental woodchucks were inoculated neonatally with the woodchuck virus 23 weeks before initiating the study, and all animals subsequently developed hepatitis as evidenced by the presence of measurable levels of circulating viral DNA. Animals were injected intravenously (i.v.) with asialoorosomucoid (AsOR)‐poly‐l‐lysine complexes containing 0.1 mg kg‐1 antisense DNA for five consecutive days. Levels of surface antigen did not differ substantially between treated and control animals. However, intravenous administration of complexed antisense DNA significantly decreased viraemia, as shown by a five‐to 10‐fold decrease in circulating viral DNA 25 days post treatment. The decline lasted for at least 2 weeks, after which there was a gradual increase in DNA levels. Antisense DNA alone or a complex containing a random oligo DNA of the same size and linkage failed to have any significant effect on viral DNA levels. We conclude that antisense oligo DNA can be targeted to the liver in vivo, resulting in a substantial and prolonged decrease in viral DNA levels in WHV‐infected woodchucks.

Granulomatous liver diseases: A review

Granulomas that consist of focal accumulations of macrophages are commonly found in the liver due to stimulation of the immune system by a number of agents. Manifestations are variable depending on whether the underlying cause is a systemic disease or a primary hepatic granulomatous reaction. This article describes the common causes, presentation, histopathology, and manifestations of granulomatous diseases as well as various diagnostic and management strategies.

Human hepatocytes transplanted into genetically immunocompetent rats are susceptible to infection by hepatitis B virus in situ

Immune tolerance of human cells without generalized immunosuppression was created in groups of normal fetal rats at 17 days of gestation by inoculation ip with primary human hepatocytes in utero. One day after birth, suspensions of human hepatocytes were transplanted via intrasplenic injection and one week later groups of rats were inoculated with hepatitis B virus (HBV). Tolerized rats that were transplanted with human hepatocytes and subsequently infected with HBV produced hepatitis B surface antigen (HBsAg) in serum beginning on day 3. Levels rose fivefold and remained stable at 0.75 pg/ml through at least 60 days. Of cells that stained positive for human serum albumin, approximately 30% were found to be also positive for HBsAg by immunohistochemistry. Serum HBV DNA was detectable from 1 to 15 weeks postinfection. Finally, covalently closed circular DNA, reflecting HBV replication, was found in liver and serum. Controls that were tolerized and not transplanted, but inoculated with HBV, as well as untreated controls, had no evidence of HBV gene expression or replication under identical conditions. The data support the conclusion that primary human hepatocytes transplanted into genetically immunocompetent rodent hosts, survive and maintain sufficient differentiation to produce human serum albumin and be infected by HBV.

Liver-directed gene delivery

Abstract Cell surface receptors can provide a convenient natural mechanism for delivery and internalization of substances to cells. We have utilized asialoglycoprotein receptors on hepatocytes to target DNA specifically to these cells. Studies initially began with marker genes in cell culture studies. More recently, normal genes in the form of targetable protein-DNA complexes have been introduced into animals possessing inherited metabolic disorders. Partial, transient corrections of these diseases have been achieved by intravenous injection of the complexed genes.