2008 Barcelona - European Society of Human Genetics

Therapy for genetic disease
P10.22
Oxidative stress in nonsyndromic sensorineural hearing loss
H. T. El-Bassyouni1 , A. M. Ashour1 , M. Afifi2 , A. Ezzat1 ;
1 2 National Research Center, Guiza, Egypt, Hearing and speech Institute, Guiza,
Egypt.
Introduction: Ototoxicity seems to result from the inhibition of cochlear
antioxidant defenses, causing an increase in the amount of reactive
oxygen species. These findings suggest a causal relationship between
the formation of reactive oxygen species, oxidative stress and functional/morphological
ear damage .
Aim: to shed more light on the link between oxidative stress and nonsyndromic
sensorineural hearing loss .
Methods: auditory function was monitored along with plasma concentrations
of copper, zinc, calcium, phosphorus, magnesium and iron .
Further more, lipid peroxidation, ß carotene, vitamin A, E and C activities
and immunoglobulin G, M and A levels were estimated .
Results: Significant decrease in calcium, phosphorus, ß carotene, and
vitamin E activities were found, as well as low levels of immunoglobulin
G and M . Increase in lipid peroxidation was observed indicating
oxidative stress which suggests a putative role of antioxidants in the
pathogenesis of sensorineural hearing loss .
Conclusion: The results support the hypothesis that dietary and immune
factors influence individual susceptibility to hearing loss. Further
studies are needed to verify whether antioxidants, correction of deficient
nutrients and/or immune modulation would improve sensorineural
hearing loss .
P10.23
Role of amniotic fluid mesenchymal stem cells in gene and cell
therapy
F. Bolda 1 , A. Bosi 2 , R. Baffelli 2 , G. Porta 3 , W. Quasim 4 , B. Gaspar 4 , F. Porta 2 , A.
Lanfranchi 2 ;
1 Stem Cell Lab, Oncohaematology and BMT Units, Ospedale dei Bambini,,
brescia, Italy, 2 Stem Cell Lab, Oncohaematology and BMT Units, Ospedale dei
Bambini, Brescia, Italy, 3 Università dell’Insubria, Varese, Italy, 4 Department of
Clinical Immunology, Great Ormond Street Hospital NHS Trust, London, United
Kingdom.
Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating
into diverse lineages: osteocytes, chondrocytes, adipocytes,
neuronal cells and cardiomyocytes. MSCs were initially identified in
adult bone marrow (BM), but cells resemblings BM-MSCs have also
been found in many other tissues: adult and foetal peripheral blood,
foetal liver, foetal spleen, placenta, umbilical cord, amniotic membrane
and synovial fluid.
Human amniotic fluid (HAF) obtained during the process of amniocentesis
is a valid source of MSCs and they could be a valid vehicles for
gene therapy .
The aim of this study was to isolate MSCs from amniotic fluid and to
test their transduction efficiency.
HAF specimens were obtained between 15 and 18 weeks’ gestation by
amniocentesis, previous written consent .
Amniotic-fluid MSCs (AFMSCs) were cultured under specific conditions
for 7 weeks and analysed by flow cytometry and quantitative real
time PCR to asses the presence and the expression levels of specific
markers . Mesenchymal markers (CD73, CD106, CD54, CD90, CD29,
CD44, CD105) present a peak of expression between 3 rd and 4 Th week
of colture .
To asses the transducibility of AFMSC we used a SIN HIV-1-based
lentiviral vector . This vector encloses SFFV-U3 promoter and eGFP . It
also encodes a mutant WPRE and cPPT .
Transduction efficiency was 60%. We demonstrate with the immunoistochemestry
and molecular techniques the presence of human multipotent
MSCs in the second-trimester amniotic fluid. We suggest that
AFMSCs may be good target for prenatal gene therapy The model
under investigation is severe combined immunodeficiency (SCID) due
to adenosine deaminase deficiency (ADA).
P10.24
BNP is a transcriptional target of the short stature homeobox
gene SHOX
A. Marchini 1 , B. Häcker 1 , T. Marttila 1 , V. Hesse 2 , J. Emons 3 , B. Weiß 1 , M. Karperien
3 , G. A. Rappold 1 ;
1 Institute of Human Genetics, Heidelberg, Germany, 2 Dept. of Pediat-
rics, Sana Klinikum Lichtenberg, Berlin, Germany, 3 Dept. of Pediatrics &
Endocrinol.&Metab. Diseases, Leiden Univ. Med. Center, Leiden, The Netherlands.
Short stature due to SHOX deficiency represents a common congenital
form of growth failure and is involved in the etiology of “idiopathic”
short stature and the growth deficits and skeletal anomalies in Léri
Weill, Langer and Turner syndrome . While much is known on the clinical
and molecular aspects of SHOX haploinsufficiency, the integration
of SHOX in the signalling pathways regulating bone growth is currently
not defined. Here we identify NPPB encoding the natriuretic peptide
BNP, a well known approved cardiac and natriuretic peptide hormone
(drug), as a transcriptional target of SHOX . The ability of SHOX to
transactivate the NPPB endogenous promoter was demonstrated in
luciferase reporter assays using serial deletions of the NPPB promoter
region . Binding of SHOX to the NPPB promoter was also demonstrated
in vivo by chromatin fixation and immunoprecipitation. We also
demonstrate the lack of promoter activation in two SHOX mutants from
patients with Léri-Weill syndrome . In addition, immunohistochemical
analysis of human growth plate sections showed for the first time a
co-expression of BNP and SHOX in late proliferative and hypertrophic
chondrocytes . Together these data strongly suggest that BNP represents
a direct target of SHOX .
One may speculate that BNP as a downstream effector of SHOX may
also open up new potential avenues for the treatment of short stature .
BNP in the systemic circulation is likely to reach growth plate chondrocytes.
It may either directly influence NPR-B signaling or indirectly increase
local CNP levels by saturating the competing receptor NPR-C .
P10.25
Designing and constructing a new package for further suicide
gene therapy
S. Hajizadeh-Sikaroodi 1 , S. Zeinali 2 , A. Kayhan 3 , M. Jafarpour 2 , L. Teimoori-
Toolabi 2 ;
1 Research and Science Azad University, Tehran, Islamic Republic of Iran,
2 Department of molecular medicine, Pasteur Institute of Iran, Tehran, Islamic
Republic of Iran, 3 Department of Hepatitis and AIDS, Pasteur Institute of Iran,
Tehran, Islamic Republic of Iran.
A commonly used strategy for gene therapy of solid tumors is suicide
gene therapy . In order to decrease the side-effects of suicide gene
therapy it would be better to express the suicide genes selectively in
cancerous tissues. Using cancer specific promoters is the most applicable
strategy to reach this aim . CD (Cytosine Deaminase) and TK
(Thymidine Kinase) are used as suicide genes, but a suitable vector is
needed for inserting any promoters upstream of them .
Firstly, the new MCS was designed by studying the sequence of our
own pUCLacZ (engineered pUC which can express LacZ in eukaryotic
cells because of its BGH-polyA downstream of LacZ) . Two partial
complementary 37 base long oligonucleotides were designed .
Cytosine deaminase and thymidine kinase genes were amplified by
PCR on saccharomyces cerevisiae and herpes simplex virus DNA,
and then were inserted downstream of MCS instead of lacZ .
Insertion of the MCS bidirectionally into pUClacZ was proved by PCR
and digestion analysis . In fact we made six constructs which are capable
of adopting suitable and specific promoters upstream of these
two suicide genes and LacZ reporter gene .
pUC-RMCS-TK
pUC-RMCS-CD
pUC-FMCS-TK
pUC-FMCS-CD
pUC-LacZ-CD
pUC-LacZ-TK
These constructs are compatible plasmids for further tumor specific
suicide gene therapy as the availability of suitable restriction sites in
MCS enables us to clone any promoter directionally upstream of reporter
and suicide genes . These reporter and suicide constructs can be
used in gene therapy of any types of cancer by the potential of accepting
any tumor specific promoter upstream of their genes.