DEVELOPMENT OF ANTIHIV HAMMERHEAD RIBOZYMES

Project Details

Description

The goal of this program is to design a hammerhead ribozyme having
therapeutic activity against HIV-1. The present proposal, which focuses
on developing a ribozyme directed against tat mRNA, addresses these
aspects: intracellular stability of the ribozyme, target selection, and
optimized ribozyme catalytic activity. Stability in human cells will be
investigated by measuring the effects of defined terminal modifications on
the decay kinetics of an anti-tat ribozyme, and the effects of the various
modifications on ribozyme activity will be assessed in vitro and in vivo.
Target selection, which is important because there are many possible
choices that are likely to vary widely in effectiveness, will focus on
target accessibility and sensitivity of various target triplets to
cleavage. This aspect will be studied primarily in vitro, although the
conclusions will be confirmed in vivo. Improvements in anti-tat ribozyme
activity will be addressed by introducing imperfections into ribozyme-
target base pairing in order to increase the rate of cleavage. Since
there are so many possible imperfections, theoretical considerations will
be used to identify ribozymes expected to be optimally active. These will
then be examined in vitro. In addition, a bacterial system will be used
to screen for exceptionally active mutant ribozymes. Results from the
biochemical and bacterial studies of mispairing will constrain the theory,
and from the combination will emerge ribozymes to be tested in human cells
for anti-HIV activity. It is expected that the net result of the study
will be a very stable, highly active ribozyme directed at the most
accessible and cleavable site in tat RNA. This ribozyme will be tested
for its ability to block HIV-1 replication in cultured cells. Since
kinetic parameters of ribozyme-target interactions will be examined in
vitro for all of the modifications introduced, it should be possible to
attribute the effects of the modifications to particular aspects of a
proposed reaction scheme. This information may contribute to a better
understanding of the role played by particular regions of the nucleotide
sequence in ribozyme action.
StatusFinished
Effective start/end date7/1/9612/31/99

Funding

  • National Cancer Institute
  • National Cancer Institute
  • National Cancer Institute

ASJC

  • Catalysis

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