Nava Sarver

RNA enzymes (ribozymes) as antiviral therapeutic agents

Among the landmark discoveries of recent years are ribozymes, RNA molecules which possess enzymatic, self-cleaving activities. The concept of exploiting the ribozyme catalytic center for cleaving (inactivating) a specific RNA transcript is now emerging as a potential therapeutic or preventative strategy in human diseases, veterinary medicine and agriculture. Linked to the catalytic center of the ribozyme are RNA sequences which are complementary to, and thus serve to target the ribozyme to, a unique RNA sequence. Specific association of the ribozyme with its target via base pairing, cleavage of the RNA substrate and subsequent recycling of the ribozyme make these catalytic RNA molecules attractive as antiviral agents. Theoretically, ribozymes can be adapted for the destruction of any RNA species, whatever its origin.

The potential use of catalytic RNAs in therapy of HIV infection and other diseases

This article describes the applications (both real and potential) of a new antiviral strategy, based on the use of antisense, catalytic RNAs (ribozymes) as therapeutic agents. An understanding of both antisense inhibition of gene expression and RNA autocleavage reactions are essential to the use of this technology. In addition, for the successful application of this technology in clinical settings, an interdisciplinary approach involving clinicians, molecular and cellular biologists, will be necessary. The following treatise will highlight several salient features of ribozyme technology, emphasizing its application as an antiviral as well as discuss some problems and potential solutions pertinent to the clinical application of this technology.

Antisense Catalytic RNAs as Therapeutic Agents

Publisher Summary This chapter presents an overview of well-characterized ribozymes with an emphasis on the problems that remain to be overcome before ribozyme therapy can be made a useful form of medical treatment. The chapter illustrates that many techniques are currently being used to enhance ribozyme activity. Chemical modifications can be used to assess the sequence requirements for cleavage. In one case, nucleotides that may naturally inhibit the cleavage reaction have been identified. The in vitro evolution has also been used to isolate mutant forms of a group I intron, which cleave DNA up to 100-fold more efficiently than the wild-type intron. In vitro evolution takes advantage of the known ligation activities of the hairpin and group I ribozymes. Unfortunately, the hammerhead is less amenable to such in vitro selection as its ligation reaction is extremely inefficient. In conclusion, ribozymes hold considerable promise for future therapeutic applications and will play a key role in the arsenal of pharmacotherapy.

Catalytic Antisense RNA (Ribozymes): Their Potential and Use as ANTI-HIV-1 Therapeutic Agents

Catalytic RNAs were first discovered as natural processes in several biological systems 1,2,3.Their discovery is one of the landmark accomplishments of modern science since it was the first demonstration that an informational molecule could also possess enzymatic activity, and hence changed our views on the possible origins of life. The discovery and characterization of the reactions carried out by RNA molecules has facilitated the development of an entirely distinct approach to antiviral therapy, the site specific, cleavage of viral RNAs mediated by catalytic, anti-sense ribozymes. We are using ribozymes as a novel antiviral strategy. In the following paragraphs, we weill describe the development of anti-sense ribozymes with the primary emphasis being their application as anti-HIV-1 therapeutic agents.

Gene Therapy for HIV-1 Infection

The potential of gene-based therapy as a treatment of HIV/AIDS has been receiving considerable attention in recent years (for review see 1,2,3). Gene therapy is based on the notion that transfer of a therapeutic gene into target cells will render them resistant to HIV-1 replication. Infusion of cells protected by in vitro gene therapy into the patient may thus limit virus spread and delay disease progression.

Antiviral Evaluation of HIV-1 Specific Ribozyme Expressed in CD4+ HeLa Cells

Catalytic RNA, called ribozyme, has been shown to enzymatically cleave specific sites in RNA (for reviews, see Cech and Bass, 1986, and Uhlmann and Peymen, 1990). One group of ribozymes has an active site of consensus sequences forming a “hammerhead” structure. The active center of the hammerhead ribozyme consists of 11 essential bases juxtaposed near 3 target bases (Cech, 1988). The potential versatility of this system lies in the fact that simple ribozymes can be constructed which target sites in native RNA, completing the active site in a trans configuration, and inducing cleavage (Haseloff and Gerlach, 1988). Ribozymes, which have been developed to cleave human immunodeficiency virus type 1 (HIV-1) RNA, can be transcribed in both in vitro and in vivo systems (Chang et al., 1990; Sarver et al.,1990). The purpose of this report is to review the studies evaluating the effect of ribozyme on HIV-1 RNA cleavage and on the antiviral effect in mammalian cells. Using a hammerhead motif, ribozymes were constructed which targeted a gag cleavage site, were cloned into a mammalian expression vector, and used to transform HeLa-CD4+ cells. These cells expressed HIV-1-specific ribozyme and demonstrated inhibition of HIV-1 infection.