Abstract

RNA molecules with enzymatic activity (ribozymes) targeted to the genome of HIV-1 were developed and demonstrated to mediate site-specific cleavage of the HIV-1 RNA in vitro and to inhibit viral proliferation intracellularly. Two classes of ribozymes that incorporate a "hammerhead" catalytic center were utilized, eotide elements in the viral sequence to provide half of the The first class used nuclcatalytic center. A complementary strand harboring the remaining active site nucleotides was supplied in trans and directed the site-specific cleavage of the HIV-1 RNA target. A second type of ribozyme incorporated the entire catalytic center, minus the site of cleavage, within a single RNA molecule. Ribozymes of this design were targeted to two different sites in the HIV-1 gag gene and a single site in the viral 5* LTR region. Efficient catalysis of each target was obtained at a physiological temperature. One of the ribozymes was expressed in CD4+ HeLa cells and was shown to be functionally active against an HIV-1 gag RNA target in a cell-free assay. When challenged with HIV-1, the CD4+ HeLa cells expressing the ribozyme produced 10-fold less gag-p24 antigen compared to CD4+ Hela parental cells. Polymerase chain reaction analysis of the total RNA from infected cells supported the hypothesis that ribozymes mediated the sitespecific cleavage of HIV-1 RNAs intracellularly, resulting in lowered susceptibility to HIV-1 infection.

The possibility of delivering preformed ribozymes into the cells was also explored. Preliminary data suggests that a sufficient quantity of preformed ribozymes was taken up by H9 lymphocytes to render them more resistant to HIV-1 infection than control cells. The amount of delivered, preformed ribozyme can be increased by a lipid-delivery system (liposome). Experiments utilizing a liposome delivery system into human H9 lymphocytes are described.

Finally, this study describes the use of the yeast Saccharomyces cerevisiae as eukaryotic model for studying ribozyme expression and function. Several ribozyme expressing constructs have been transformed into yeast, setting the stage for future studies in this organism.

Catalytic RNAs such as those described in this thesis should prove to be effective therapeutic agents against HIV-1.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

John J. Rossi

Second Advisor

E. Clifford Herrmann

Third Advisor

Thomas A. Linkhart

Fourth Advisor

Donna D. Strong

Fifth Advisor

R. Bruce Wilcox

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1990

Date (Title Page)

8-1990

Language

English

Subject - Local

HIV-1 -- drug effects; Antiviral Agents -- therapeutic use; RNA, Catalytic -- therapeutic use

Type

Dissertation

Page Count

ix; 155

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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