Inhibition of CCR5-Dependent HIV-1 Infection by Hairpin Ribozyme Gene Therapy against CC-Chemokine Receptor 5

Abstract

CCR-5 is a major cellular coreceptor for R5 strains of HIV-1. Individuals carrying a homozygous 32-base-pair deletion in this gene are apparently healthy and are relatively resistant to HIV-1 infection. Since CCR5 appears to be dispensable for the host, but important for initial HIV-1 infection, CCR5 mRNA is an excellent therapeutic target for inhibiting HIV-1 replication via ribozyme knockout. We report here that hairpin ribozymes are able to reduce cellular CCR5 mRNA and cell surface CCR5 when stably introduced into PM1 cells by transduction with recombinant adenoassociated viral vector. The ribozymes effectively protect the cells from infection by R5 HIV-1 strains or non-syncytium-inducing clinical isolates commensurate with a reduction in CCR5 mRNA. These results suggest a novel gene therapy approach to preventing or slowing the disease progression of HIV-1 infection.

Identification of Cellular Cofactors for Human Immunodeficiency Virus Replication via a Ribozyme-Based Genomics Approach

“…Furthermore, it is difficult to design ribozymes that predictably and reproducibly silence gene expression. The key in vivo parameters, such as target site accessibility, on-and-off rates of the ribozymes, proximity of ribozyme and target, and local concentrations of each in a given cellular compartment, cannot be accurately predicted by computer modeling or in vitro cleavage assays (9). In practice, most ribozymes designed against a target gene down-regulate the expression of that gene but rarely knock out its expression completely.…”

Inhibition of Human Immunodeficiency Virus Type 1 Entry in Cells Expressing gp41-Derived Peptides

“…Surprisingly, very few early inhibitors for gene therapy of HIV-1 infection are available. These either have a low antiviral activity, such as the intracellular single-chain variable fragments against reverse transcriptase or integrase (17) or only act on R5-tropic virus by inhibiting CCR5 expression (7,24). We have therefore concentrated on the development of an effective and broadly active early inhibitory gene.…”

Crippling of HIV at Multiple Stages with Recombinant Adeno-Associated Viral Mediated RNA Interference

“…These second generation ribozymes are RNA molecules that cleave viral transcripts such as tat, rev, and gag at specific sequences targeting HIV-1 at critical stages, and have been shown to reduce HIV-1 levels in vitro. (20)(21)(22) RNA decoys are RNA homologues, such as TAR and RRE that bind viral proteins and compete with native ligands necessary for replication. They were also shown to inhibit HIV-1 in vitro.…”

“…Synthetic bi-specific or combinatorial constructs targeting both CXCR4 and CCR5 receptors have shown to confer resistance to HIV-1 infection much stronger than that conferred by targeting each one alone, giving a clear indication that multiple targeting is better than a single target. (22,43,61,113) …”