2008 Barcelona - European Society of Human Genetics

Concurrent Sessions
doses and long term treatment with lower doses . This was accompanied
by functional improvement and improved muscle integrity, without
any apparent toxicity. These findings are encouraging for future clinical
trials and eventual systemic application of this approach .
c15.2
Restoration of aberrant splicing and neurofibromin function
in three NF1 deep intronic mutations by antisense morpholino
oligonucleotides (AmOs)
E. Pros 1,2 , J. Fernández 1 , B. Canet 1 , L. Benito 3 , A. Benavides 4 , F. J. Ramos 5 , M.
A. López-Ariztegui 6 , G. Capellá 1 , I. Blanco 7 , E. Serra 8 , C. Lázaro 1 ;
1 Laboratori Recerca Translacional, Institut Català d’Oncologia, Hospitalet de
Llobregat, Barcelona, Spain, 2 Genetics Department, IDIBELL, L’Hospitalet
de Llobregat, Barcelona, Spain, 3 Unitat Consell Genètic, Institut Català
d’Oncologia, Hospitalet de Llobregat, Barcelona, Spain, 4 Genética, Hospital
Universitario Central de Asturias (HUCA), Oviedo, Spain, 5 Dpto. Pediatría,
Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Spain, 6 Genética,
Hospital de Cruces, Bilbao, Vizkaya, Spain, 7 Unitat de Consell Genètic, Institut
Català d’Oncologia, Hospitalet de Llobregat, Barcelona, Spain, 8 Genetics Department,
IDIBELL, Hospitalet de Llobregat, Barcelona, Spain.
Neurofibromatosis type 1 (NF1) is a common autosomal dominant
disorder caused by mutations in the NF1 gene . Aproximately 2% of
the germline mutations identified in our population consists in deep
intronic mutations . Such nucleotide changes create new splice sites
that produce the insertion of a cryptic exon in the mature mRNA . We
used antisense morpholino oligonucleotides (AMOs) to restore normal
splicing in three NF1 deep intronic mutations (c .288+2025T>G,
c .5749+332A>G and c .7908-321C>G) . All of them generate a cryptic
5’ splice donor site and result in the inclusion of a cryptic exon in the
mature RNA by the use of an existent 3’ cryptic splice site . AMOs were
designed to target the newly created 5’ splice sites in order to avoid
the incorporation of cryptic exons and promote the use of wild-type
splice sites, by the splicing machinery . Our results demonstrate that
AMOs treatment effectively restore normal NF1 splicing at the mRNA
level in primary fibroblast and lymphocyte cell lines derived from different
patients carrying the three deep intronic mutations . In addition, we
observed a decrease in the ammount of Ras-GTP (equivalent to wild
type fibroblast levels) in primary fibroblasts from patients after AMOs
treatment, consistent with the restoration of neurofibromin function.
To our knowledge this is the first time that an antisense technique is
used successfully to restore NF1 mutations, opening the possibility of
a therapeutic strategy for this type of NF1 mutations .
c15.3
Antisense therapeutics for a new deep intronic variation
identified in two Methylmalonic Acidemia patients
A. Rincón, L. R. Desviat, M. Ugarte, B. Pérez;
Centro de Biología Molecular Severo Ochoa. CIBERER, Madrid, Spain.
Isolated methylmalonic acidemia (MMA) is a life threatening organic
acidemia caused by defects in the methylmalonylCoA mutase (MCM)
or in enzymes involved in the synthesis of the active cofactor adenosylcobalamin.
In this work we describe a new point change identified
in two MMA affected patients located deep in intron 11 of the MUT
gene (IVS11-898A>G) . This change increases the splicing score of
a 5´cryptic splice site and provokes the intronic inclusion of 76 bp
(r .1957ins76) between exons 11 and 12 . Using a splicing assay we
have demonstrated that the change caused exonization of this intronic
sequence and by morpholino antisense oligonucleotide transfection
we have demonstrated that the insertion is a disease-causing mutation
in these two patients . The antisense oligonucleotide was targeted
to the 5’ cryptic splice sites to block access of the splicing machinery to
the pseudoexonic region in the pre-mRNA . After transfection of the patient’s
fibroblasts we have performed RT-PCR analysis and enzymatic
assay to determine MCM activity . Using this antisense therapeutics we
have obtained correctly spliced mRNA that was effectively translated
and methylmalonyl CoA mutase activity was rescued in patient’s fibroblasts
close to 100% of control activity . The effect of AMO is sequence
and dose dependent and was not effective in patients where the insertion
was produced by splicing background noise. These findings
add to previous results providing a new therapeutic strategy in this
genetic disorder and potentially applicable to large numbers of cases
with deep intronic changes that, at the moment, remain undetected by
standard mutation-detection techniques
c15.4
Rescue of a Lethal murine model of methylmalonic Acidemia
using AAV 8 mediated Gene therapy
R. J. Chandler, C. Venditti;
National Human Genome Research Institute, National Institutes of Health,
Bethesda, MD, United States.
Methymalonic acidemia (MMA), a severe organic acidemia, is caused
by deficient activity of the ubiquitous mitochondrial enzyme methylmalonyl-CoA
mutase (MUT) . MMA patients exhibit increased methylmalonic
acid levels in the plasma, urine and CSF and display a clinical phenotype
of lethal metabolic decompensation, growth retardation, renal
failure and metabolic strokes . To assess the potential of genetic therapy
for MUT MMA, we employed a mouse model of MMA that produces
no detectable Mut transcript or protein . AAV 8 CBA-Mut was injected
directly into the liver of newborn Mut-/- pups . Currently, 28 out of the 29
Mut-/- mice injected with 1 or 2x1011GC of AAV 8 CBA-Mut are alive
beyond DOL 90 with some treated Mut-/- mice older than 200 days .
All the untreated mutants (n=21) perished before DOL 72 . The treated
Mut-/- mice are thriving and indistinguishable from their wild-type (WT)
littermates . AAV 8 CBA-Mut treated Mut-/- mice achieved body weights
comparable to controls while untreated mutants experienced post-natal
growth retardation and reached only 40% of the weight of the WT .
Plasma methylmalonic acid levels in the treated mutant mice on an
unrestricted diet were significantly reduced compared to uncorrected
animals, indicating that substantial Mut enzymatic activity was restored
after AAV therapy . At DOL 90 the liver from a treated Mut-/- mouse had
WT levels of Mut protein by Western blot analysis . These experiments
provide the first evidence that gene therapy has clinical utility in treatment
of MMA and support the development of gene therapy for other
organic acidemias
c15.5
Evaluating suppression of nonsense mutations by
aminoglycoside antibiotics as an intervention for vision loss in
type i Usher syndrome
A. Rebibo Sabbah 1 , I. Nudelman 2 , Z. M. Ahmed 3 , T. B. Friedman 3 , T. Baasov 2 ,
T. Ben-Yosef 1 ;
1 Genetics Department, Rappaport Faculty of Medicine, Technion, Haifa, Israel,
2 Department of chemistry, Institute of Catalysis Science and Technology,
Technion, Haifa, Israel, 3 Laboratory of Molecular Genetics, National Institute
on Deafness and other Communication Disorders, NIH, Rockville, MD, United
States.
Type 1 Usher syndrome (USH1) is a recessively-inherited condition,
characterized by profound prelingual deafness, vestibular areflexia,
and prepubertal onset of retinitis pigmentosa (RP), which to date has
no effective treatment . USH1 can be caused by mutations in each of
at least six genes . While truncating mutations of these genes cause
USH1, missense mutations of some of the same genes cause nonsyndromic
deafness, suggesting that partial or low level activity of
the encoded proteins may be sufficient for normal retinal function, although
not for normal hearing . Interventions to enable at least some
translation of full-length protein, may delay the onset and/or progression
of RP in individuals with USH1 due to nonsense mutations . One
such possible therapeutic approach is suppression of nonsense mutations
by aminoglycosides . We demonstrated up to 91% suppression
of PCDH15 nonsense mutations by commercial aminoglycosides in
vitro . We also demonstrated ex vivo suppression, by the same aminoglycosides,
of the R245X mutation . We are now testing suppression of
several CDH23 nonsense mutations . In parallel, we are developing a
series of new aminoglycoside-derived compounds, which includes two
new promising derivatives, NB30 and NB54 . Based on cell toxicity assays
and on acute toxicity measurements in mice, the toxicity of both
compounds is significantly reduced, in comparison to commercially
available aminoglycosides . Based on in vitro and ex vivo experiments,
their suppressive activity is maintained . The research described here
will have important implications for development of targeted interventions
that are effective for patients with USH1 and nonsyndromic RP
caused by various nonsense mutations .