Λειτουργική μελέτη νέων λεντιϊκών φορέων shRNAs έναντι των ρυθμιστών της γ-σφαιρίνης BCL11A και KLF1 για τη γονιδιακή θεραπεία της β-θαλασσαιμίας

Postgraduate Thesis uoadl:1312341 298 Read counter

Unit:
Διατμηματικό / Διϊδρυτικό ΠΜΣ Μοριακή Ιατρική
Library of the School of Health Sciences
Deposit date:
2013-05-17
Year:
2013
Author:
Κοντοστάθη Γεωργία
Supervisors info:
Νικόλαος Π. Ανάγνου
Original Title:
Λειτουργική μελέτη νέων λεντιϊκών φορέων shRNAs έναντι των ρυθμιστών της γ-σφαιρίνης BCL11A και KLF1 για τη γονιδιακή θεραπεία της β-θαλασσαιμίας
Languages:
Greek
Summary:
The term thalassemia originates from the greek words «thalassa» (sea) και
«haema» (blood) and describes the previously known hemoglobinopathy
«Mediterrenean anemia». Thalassemia disorders are related to disfuctinonal
composition of α- or β chains of ΗbA (α2β2). Thalassemias are caused by more
than 200 mutations and result in reduced or no synthesis of beta globin chain.
A novel approach in the treatment of β-thalassemia includes gene therapy that
utilizes the ability of lenti-viruses to transport and insert the therapeutic
transgene (β-globin or γ-globin) in the genome of the target cell, which in
this case is ΗSCs. Transcription factors, BCL11A and KLF1 were shown to
regulate the HbF expression. They act in 2 ways in adults 1) directly: KLF1
activates β-globin and 2) indirectly: KLF1 activates BCL11A and BCL11A protein
silences γ-globin. The knockdown of the above factors utilizing shRNA
technology elevates the expression of γ-globin, providing a potential
therapeutic outcome in β-thalassemia.
Αim: Τhe improvement of CD34+ erythroid differentiation conditions ex vivo and
assessment of lentiviruses carrying shBCL11A and shKLF1 in vitro.
Conclusions: Vectors of shBCL11A and shKLF1 exhibited mean titers of 1.36X107
TU/ml and 1.42X107 TU/ml respectively and their efficiency was tested in CD34+
cord blood cells transduced at MOI (multiplicity of infection) 20. Transduced
CD34+ cells were induced towards the erythroid phenotype by the appropriate
erythroid protocol and expression of HbF was determined by flow cytometry
(FACS), for both vectors. In liquid cultures and at a clonal level there was a
trend for HbF increace. The transduction was 46.6% for shBCL11A and 45% for
shKLF1, while mean vector copy number/cell for shBCL11A and shKLF1 was 1.12 and
1.05 respectively.
Keywords:
b-thalassemia , Hemoglobin fetal, Hemoglobin A, Gene therapy, Ηaematopoietic stem cells
Index:
No
Number of index pages:
0
Contains images:
Yes
Number of pages:
108
File:
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