Cherie M. Walton

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.

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.

Targeted inhibition of type I procollagen synthesis by antisense DNA oligonucleotides

In hepatic fibrosis, the connective tissue biomatrix of the liver changes from the normal matrix, rich in basement membrane collagens, to a matrix enriched in interstitial fibrillar collagens. Type I collagen is the predominant component of thick fibrous bands found in matrix in advanced fibrosis. The aim of the current research was to determine whether a therapeutic approach could be developed that would specifically target collagen-producing cells to reduce the synthesis and accumulation of type I collagen. Antisense DNA oligonucleotides directed against specific sequences within α1(I) and α2(I) mRNA of type I procollagen were complexed to a cell-specific carrier, and screened for their effectiveness in reducing α1(I) and α2(I) mRNA levels. Two antisense DNA oligonucleotides delivered by the carrier were found to be most effective in reducing α1(I) and α2(I) mRNA and total collagen accumulation in the cells, but had no effect on reducing β-actin mRNA in the same cells. At similar concentrations, free antisense DNA oligonucleotides were not effective in inhibiting collagen synthesis, and/or in decreasing cellular concentrations of α1(I) or α2(I) mRNA. Collagen synthesis and mRNA levels in cells lacking receptors that recognize the carrier protein were not changed after treatment with complexed antisense DNA. The results indicate that antisense oligonucleotides can be targeted to cell types, and were effective inhibiting collagen synthesis in those cells.