Alternatively Spliced Genes
161
from patient samples can be technically
challenging. The most frequently used
methods include RT-PCR and analyses
of defective protein products. In general,
these methods are still limited to research
studies. Systematic approaches to detect-
ing disease-causing splicing mutations are
yet to be developed for diagnostic applica-
tions in clinics.
4.2
Potential Therapeutic Approaches
Molecular studies and mechanistic char-
acterization
of
splicing
mutations
that
cause human disease have led to the
development of a number of potential
therapeutic approaches. They can be clas-
siFed into four types, oligonucleotide-,
ribozyme-, trans-splicing- and chemical
compound-based approaches. These ap-
proaches are still at the stage of research
and development.
4.2.1
Oligonucleotide-based Approaches:
Antisense, RNAi, and Chimeric Molecules
Several groups have tested oligonucleotide-
based methods for designing therapies
to treat diseases caused by aberrant pre-
mRNA splicing. These approaches include
using modiFed antisense oligonucleotides
or RNA interference (RNAi). Antisense
oligonucleotides could be used to restore
the proper function of mRNAs that are dis-
rupted by splicing mutations. Antisense
oligonucleotides designed to block cryp-
tic sites in the mutated globin gene in
β
-thalassemia were reported to increase
wild-type mRNA and decrease aberrant
mRNA. A lentiviral vector-based system
using a modiFed U7snRNA containing
a sequence blocking the aberrant splice
sites, reduced aberrant pre-mRNAs and
increased levels of the correctly spliced
β
-
globin mRNA and protein. The production
of stress-induced aberrant AChE-R mRNA
was reduced by antisense oligonucleotides.
Another study using the antisense oligonu-
cleotide approach reported the reversal of
the aberrant splicing pattern caused by
mutations associated with ±TDP-17 in the
human
tau
gene. On the other hand, anti-
sense oligonucleotides have been used to
increase the inclusion of exon 7 of SMN2
in order to develop therapeutic agents for
spinal muscular atrophy. 2
0
-O-Methylated
antisense oligoribonucleotides were used
to modify the splicing pattern of the dys-
trophin pre-mRNA in the mdx mouse
model of Duchenne muscular dystrophy.
Recently, RNAi was used to selectively de-
grade alternatively spliced mRNA isoforms
in
Drosophila
by treating cultured cells with
dsRNA corresponding to an alternatively
spliced exon.
One of most common splicing defects
associated with human diseases is exon
skipping. Because an RS domain can act
as a splicing activator, small chimeric
molecules containing a minimal synthetic
RS domain covalently linked to an anti-
sense moiety have been tested to target
defective
BRCA1
or
SMN2
pre-mRNA
transcripts and shown to restore splicing.
These oligonucleotide-based approaches
have shown feasibility in correcting splic-
ing defects and treating the corresponding
splicing diseases.
4.2.2
Ribozymes
Ribozymes are RNAs that catalyze bio-
chemical
reactions
in
cells,
especially
cleavage of other nucleic acids. Efforts
have been made in developing derivatives
from small naturally occurring RNAs, in-
cluding the hammerhead, the hairpin, the
tRNA processing ribonuclease P (RNase
P), and group I and group II ribozymes
as
therapeutic
agents.
Ribozymes
can
be used to reduce aberrant or defective
previous page 161 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online next page 163 Encyclopedia of Molecular Cell Biology and Molecular Medicine read online Home Toggle text on/off