Functional evaluation of novel lentiviral vectors for gene therapy of hemoglobinopathies

Postgraduate Thesis uoadl:2917431 15 Read counter

Κατεύθυνση Εφαρμογές της Βιολογίας στην Ιατρική
Library of the School of Science
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Tsempera Evgenia
Supervisors info:
Νικόλαος Ανάγνου, Ομότιμος Καθηγητής Ιατρικής Σχολής Πανεπιστημίου Αθηνών, ΕΚΠΑ
Διευθυντής Εργαστηρίου Κυτταρικής και Γονιδιακής Θεραπείας, ΙΙΒΕΑΑ
Original Title:
Λειτουργική αξιολόγηση νέων λεντιϊκών φορέων γονιδιακής θεραπείας αιμοσφαιρινοπαθειών
Translated title:
Functional evaluation of novel lentiviral vectors for gene therapy of hemoglobinopathies
Gene therapy for β-thalassemia has gained grounds over the last years, with the 1st successful clinical trial already being a reality and a great number of others being under way. Despite the progress however, there are still some limitations concerning safety and efficacy. The above limitations are primarily connected with suboptimal gene transfer, the need for long-term and stable tissue-specific transgene expression and designing of novel lentiviral vectors demonstrating high safety and transduction efficiency with low genotoxicity.
To this end we constructed a novel γ-globin lentiviral vector, designated GammaThal. The aforementioned vector carries a Αγ polymorphism, which gives a distinct peak in HPLC, separating the therapeutic transgene from the endogenous and thus providing means of direct measurement of the vector-producing Αγ-globin. Additionally, pseudotyping of the novel vector with the alternative envelope glycoprotein HF from the Edmonston stain of measles virus, shown to demonstrate increased tropism towards human CD34+ cells, we aimed at high transduction efficiency at low multiplicity of infection, trying thus to minimize the potential risk of insertional mutagenesis in a future clinical application.
GammaThal was assessed in vitro in CD34+ cells from normal and thalassemic donors (β+ and β0) at an MOI=25. We show that while transduction with GammaThal only leads to a trend regarding F-cells increase in normal donors, in a thalassemic background it leads to significantly increased F-cells percentage and transduction efficiency. Specifically, in thalassemic patients an average F-cell increase of 8.93% was observed (p = 0.026, n = 4) while the corresponding rate in healthy donors was 11% (p= 0.098, n=3). F-cells in thalassemic patients was associated with a mean transduction efficiency of 54.58% and an average vector copy number (VCN) per cell = 0.94. Furthermore, transduction with GammaThal improves thalassemic erythropoiesis in vitro, by reducing apoptosis (10.24%, p=0.067) and thus leading to an increase in the number of orthochromatic erythroblasts.
Overall, we have generated a new promising γ-globin lentiviral vector for the gene therapy of β-thalassemia. By conducting additional experiments both in vitro and in vivo, as well as a comprehensive integration site analysis, it will be determined if GammaThal could be an effective clinical vector that combines high level of biosafety with therapeutical improvement of β-thalassemia.
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Health Sciences
gene therapy, beta-thalassemia, lentiviral vectors, fetal hemoglobin
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