| PROGRESS
REPORT OF RESEARCH BEING UNDERTAKEN march 2003
Proof-of-principle
experiments towards gene therapy for epidermolysis bullosa simplex.
Section A:
Development of an inducible
EBS mouse model expressing a mutant form of human K14 Dennis Roop, Baylor College of
Medicine, Houston.
1. Generation of
recombinant mouse embryonic stem (ES) cells
As indicated in our last progress report, we repeated the gene targeting experiment to
generate heterozygous ES cells that carry the EBS-DM allele. Approximately 25% of the ES
cell clones tested by Southern blots carried the mutant K14 allele. Six recombinant ES
cell clones were injected into C57Bl/6 blastocysts.
2. Establishing mouse lines
that carry the EBS-DM mutation
We obtained chimeric mice
from all ES cell clones that were injected. Chimeric males were intercrossed with C57Bl/6
females. Five chimeras produced agouti offspring. The coat colour marker agouti is
dominant over the black coat colour of the C57Bl/6 mice, and indicates that ES
cell-derived sperm have contributed to the offspring. Unless haplo-insufficiency occurs,
50% of the agouti offspring is expected to be heterozygous for the mutation originating
from the recombinant ES cell genome.
We have genotyped the first
litters of agouti offspring by PCR. Among these pups, we identified two animals that
carried the EBS-DM allele. As expected, the expression of the mutant allele in these mice
is suppressed by the neomycin antibiotic-resistance cassette that was placed in intron 1
of the K14 gene. Consequently, these pups are phenotypically normal.
3. Plan for the next budget
period
We are in the process of
establishing a colony of EBS-DM mutant mice. Once we have established this line, we will
intercross these mutants with transgenic mice that carry the K14.CrePR1 transgene.
Offspring that carry both the EBS-DM allele and the K14.CrePR1 transgene, will then be
treated with the inducer RU486. This treatment will result in the excision of the neomycin
resistance cassette from intron 1 and, consequently, the activation of the EBSDM allele.
Treated mice are expected to develop an EBS phenotype in RU486-treated areas.
Once activation of the
EBS-DM allele has been confirmed, these mice will be shipped to Prof. McLean. Thus, unless
something unanticipated occurs, the goals of our part of the joint application with Prof.
McLean will be completed on time.
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Section B:
Phenotypic reversion of an
inducible EBS mouse model by expression of anti-K14 ribozyme transgenes Irwin McLean,
Epithelial Genetics Group, Human Genetics Unit, University of Dundee
1. Transgenic mice
expressing Rz2(18) construct
Further to the last
progress report (June 2002), we have now generated a number of transgenic mouse lines
expressing the anti-human K14 ribozyme mini-gene, Rz2(18). This construct, which has two
18 bp targeting sequences, was found to give the most efficient cleavage of target mRNA
from in vitro experiments. We also plan to express 14 bp constructs as transgenes
later this year.
Out of 80 mice generated
from pronuclear injection experiments, 8 of these were positive for more than one PCR
specific for the transgene. Of the 8 positive founders, one mouse was a litter runt and
died for reasons apparently unconnected with transgene expression. From the remaining
7, there are mice with a
range of transgene copy numbers, estimated from ~1 copy to ~50 copies by semi-quantitative
PCR and therefore a range of ribozyme expression can be investigated for efficiency at
reversing the EBS phenotype, as outlined in the original proposal.
Currently, the founders are
being bred and all have produced litters. Therefore, there are no immediately obvious
ill-effects on the health of mice, even those carrying high numbers of the ribozyme
transgene. In view of this, we are optimistic that we will be able to progress to the next
stage of the project where these are bred with the "humanized" EBS mice being
produced by the Roop laboratory.
2. Plans for the next
budget period
We are currently breeding
the 7 Rz2(18) mouse lines to obtain sufficient mice for characterisation. These should be
ready within the next 6 weeks and will be characterised as follows: (a) RT-PCR of various
tissues, particularly skin, for the expression of the transgene and endogenous K14 levels;
(b) immunoblot characterisation of keratin expression in the epidermis, particularly K5,
K14, K15; (c) a full histological screen for any deleterious effects of transgene
expression, with particular emphasis on skin and other stratified epithelia; and (d)
ultrastructural characterisation, again with emphasis on the epidermis. In all cases,
comparisons will be made to non-transgenic littermates. Some mice will also be maintained
and characterised again as above at 6 months and 12 months of age in case there are
deleterious effects at these later time points.
During the next year, we
should also be able to import the "humanized" EBS mice from the Roop laboratory
and cross these with mice expressing various levels of the Rz2(18) transgene to see if
this can prevent the EBS phenotype and thus provide proof-of-principle for ribozyme gene
therapy in EBS and other dominant-negative genetic disorders.
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Combined Lay Summary
The two halves of the
project (in Houston and Dundee) are proceeding well and are on target for completion
within the projected period, provided no unforeseen problems arise. In Houston, mice have
been genetically engineered that carry the human K14 gene containing the most common EB
Simplex mutation. This gene exists in a silent form that can be activated by topical
application of a drug once these mice are crossed with another strain of genetically
modified mice. Crossing of these mice and testing of the drug-inducible skin blistering
system is on-going in Houston. Mice will also be characterised to show that they are a
true model of human EBS. Once this work is completed, these animals will be exported to
Dundee. These mice are the most accurate and useful animal model of EBS developed so far
and will be a test bed for a variety of therapeutic strategies.
In Dundee, a number of
lines of genetically engineered mice have been made that carry a therapeutic ribozyme gene
which should be able to prevent EBS in an animal model system.
These mice carry different doses of the ribozyme gene to allow testing over a wide range
of conditions. Currently, these mice appear healthy and so there appear to be no
immediately obvious side-effects even from large doses of the therapeutic gene. These mice
will be characterised in detail over the next year for any subtle effects. Once the
inducible EBS mice are imported from Houston, these will be crossed with the ribozyme mice
to see if the therapeutic gene really works and prevents skin blistering. |
