Nucleic Acids Research, Vol 25, Issue 17 3451-3458, Copyright © 1997 by Oxford University Press
G Grassi, A Forlino and JC Marini
We report here the in vitro use of hammerhead ribozymes as an approach to
the gene therapy of osteogenesis imperfecta (OI). Our strategy for the
treatment of this dominant genetic disorder is based on selective reduction
of the level of the mRNA transcripts from the mutant allele. We studied the
in vitro cleavage activity of five different hammerhead ribozymes targeted
against synthetic transcripts of two naturally occurring human collagen
mutations and against a point mutation introduced into a construct
containing a portion of the mouse COL1A1 gene. This is the first
demonstration that ribozyme cleavage is absolutely dependent on the
presence of the ribozyme cleavage site introduced by the disease-causing
mutation. Cleavage specificity and activity were unchanged when the
cleavage site was located in transcripts of progressively longer length.
Cleavage efficiency depended directly on the ratio of ribozyme/substrate,
as well as on the time and temperature of incubation. We investigated the
competitive effects of both total RNA and normal synthetic transcripts on
ribozyme cleavage activity. The ribozyme was able to localize and cleave
its specific target even in the presence of a vast excess of total RNA.
However, cleavage efficiency was linearly inhibited by the presence of a
non- cleavable competitor substrate which contained a ribozyme binding site
identical to the site present in the cleavable target. Although this
competition could be eliminated by introducing a mismatch into one ribozyme
binding arm, the presence of the mismatch decreased ribozyme cleavage
efficiency. The mutation- specificity of ribozyme cleavage demonstrated in
this work provides support for in vivo studies aimed at ribozyme
development as a treatment for dominant negative genetic disorders.
ARTICLES
Cleavage of collagen RNA transcripts by hammerhead ribozymes in vitro is mutation-specific and shows competitive binding effects
Section on Connective Tissue Disorders, Heritable Disorders Branch, National Institutes of Child Health and Human Development, Building 10, Room 9s241, 10 Center Drive, MSC 1830, Bethesda MD 20892-1830, USA.
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