Titolo della tesi: Gene Therapy For Hemoglobinopathies Enhancing Lentiviral Vector Therapeutic Efficacy
Background: Thalassemia and sickle cell disease demonstrate the highly common form of hereditary hemoglobinopathies. Β-thalassaemia corresponds to an imbalance in globin chain level due to haemoglobin beta total absence or underproduction. Sickle cell disease corresponds to aberrations in structure of the β-globin gene. Gene therapy has posed a promising future, with the integration of hematopiotic stem cell (HSC) gene therapy being developed that incorporates Lentiviral vector correction for the β- globinopathies.
Primary Objective: Enhancement of β-globin containing LV designs to increase HBB expression for increased efficacy of gene therapy followed by Functional analysis of novel β-globin containing LV designs to increase HBB expression for increased efficacy of gene therapy. The HBB transcription unit within the prototypical GLOBE LV, was modified to enhance either transcription (by HBB promoter extension) or pre-mRNA processing (by addition of the HBB full-length second intron and/or the HBB transcription termination region). HBB was also modified to contain three amino acid substitutions enhancing its anti-sickling properties.
Secondary Objective: Compare the function and efficacy of our developed LVs with the BlueBird vector currently in the market.
Methods: Synthesis and development of the 2 lentiviral vectors to contain CTCF binding sites, as well as synthesis of Bluebird vector. Follows is the production of lentivirus containing the vectors of interest transduction of LVs of different cell lines including human primary CD34+ haematopoietic stem and progenitor cells (HSPCs), then the average vector copy number per cell was quantified and transgene expression of lentiviruses was assessed by qPCR and results were interrelated with respect to VCN.
Results: The inclusion of the CTCF in the MA1221 and MA1222 LV resulted in a 2-2.5-fold increase in HBB-AS3 expression per vector copy in differentiated murine erythroleukaemia (MEL) cells achieved with three different. Human primary CD34+ derived erythroid cells isolated from healthy donor and β-thalassaemia patients gave a 1.18- and an average 2.11-fold increase in HBB transgene mRNA level per vector copy. Elements that can provide higher functional efficiency of the GLOBE vectors such as CTCF binding sites, could be introduced in LV designs for increased transgene expression following thorough evaluation aimed towards a more efficacious correction of β-globinopathies by gene therapy