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Title: Advanced personalized gene therapy of B-thalassaemia
Author: Patsali, Petros
ISNI:       0000 0004 7427 9155
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2018
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Thalassaemias are amongst the commonest single‐gene disorders worldwide, but offer only limited curative treatment choices. Autologous transplantation of gene‐therapy‐corrected cells is therefore investigated by numerous groups and is already under clinical trials for β‐thalassaemia, based on gene addition by lentiviral vectors (LVs). The main aim of this project was the development of personalised gene therapy (GT) for β‐thalassaemia caused by the common and severe HBBIVSI‐110 mutation, which results in missplicing of intron 1. The resulting aberrant mRNA interferes with protein expression from normal endogenous or vector‐encoded HBB. The two therapy approaches taken were a) modification or co‐transduction of the LV ΜΑ821_ΗΒΒ (“GLOBE”) to achieve concurrent vector‐derived HBB expression and RNAi‐based suppression of aberrant RNA and b) development of designer nucleases for genome editing and functional correction of the HBBIVSI‐110 mutation. To establish proof of principle for both approaches we developed transgenic murine erythroleukaemia cell lines MEL MA821‐HBBIVSI‐110 and MEL MA821‐HBBNormal with on average two vector copies per genome (VCN) and an additional, clonal cell line of VCN=1 for MEL MA821 HBBIVSI‐110. Approach a) Depletion of aberrant HBBIVSI‐110 mRNA might enhance translation of vector‐derived or residual endogenous HBB mRNA and could be achieved by HBBIVSI‐110‐specific RNAi, mediated by expression of lentivirally delivered shRNAs. To this end, four different HBBIVSI‐110 mRNA‐specific shRNAs were cloned into the U6‐promoter‐driven pLKO.I lentiviral vector (LV). In clonal MEL HBBIVSI‐110 cells, two constructs, pLKO.I shIVSI‐110 Mid & Mid2, exhibited no discernible change of normal:aberrant HBB mRNA but a significant increase of HBB chains relative to untransduced samples. Importantly, our approach was then validated by transduction of HBB IVSI‐110‐patient‐derived hCD34+ cells, in which we observed a ~30% increase of HBB:HBA chain ratios in samples transduced with shIVSI‐110 MID, regardless of co‐transduction with other vectors. These data indicate that RNAi‐targeting of aberrant HBB mRNAs, if not therapeutic in its own right, could substantially improve efficiency of GT by HBB gene augmentation and could thus lower conditioning, VCN and gene‐expression requirements for LV‐based gene therapies. Approach b) As an alternative to gene augmentation we established a novel genome‐editing approach in which permanent functional correction the HBBIVSI‐110 mutation was achieved with the targeted disruption of the aberrant SA site and/or its sequence context. This approach, based on the efficient non‐homologous end‐joining (NHEJ) repair pathway, used HBBIVSI‐110 ‐ specific designer nucleases, transcription activator‐like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 RNA‐guided endonuclease (RGENs). Assessment of targeted disruption efficiency of our designer nucleases on gDNA or episomal reporter plasmids indicated the superiority of the HBB TALEN over RGEN for HBBIVSI‐110. Plasmid transfection of HBBIVSI‐110 specific ‐ designer nucleases into MEL MA821‐HBBIVSI‐110 transgenic cells resulted in functional correction at the RNA (RT‐qPCR) and protein level (Immunoblots). In addition, full characterisation (type, frequency and context) of induced insertions/deletions (INDELs) was achieved by T7 Endonuclease 1 assay, decomposition of sequence traces (TIDE) and Sanger sequencing of TOPO clones from edited bulk cells. Using edited MEL MA821‐HBBIVSI‐110 clones we moreover correlated specific INDELs with HBB expression at the RNA and protein level, which confirmed our hypothesis that functional correction of splicing could be achieved by disruption of upstream sequences of the aberrant SA site, leaving intact the HBBIVSI‐110 mutation. Subsequent validation of our nucleases in patient‐derived CD34+ cells by electroporation of HBB TALEN L1/R1 plasmid DNA was impaired by low targeted disruption (12%) despite high‐transfection efficiencies. Even though a marginal increase in the ΗΒΒ:ΗΒΑ chain ratio was detected, analyses of mRNA levels were inconclusive, calling for additional experimentation in CD34+ cells with more appropriate expression systems. Overall, proof of principle was established for NHEJ‐based functional repair of HBBIVSI‐110, and we suggest that the approach can be adapted for the functional correction of other of disease‐causing gain‐of‐function mutations in non‐coding regulatory regions.
Supervisor: Antoniou, Michael Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available