Date of Award
12-18-2024
Degree Type
Thesis
Degree Name
MPhil in Biological and Biomedical Sciences
First Advisor
Dr. Afsar Ali Mian
Second Advisor
Dr. Irfan Hussain
Third Advisor
Dr. Fawad ur Rehman
Department
Biological and Biomedical Sciences
Abstract
This study presents the development and validation of advanced in vitro disease models and targeted gene-editing approaches for β-thalassemia and SCD using prime editing systems. To model these diseases, plasmids were meticulously engineered, incorporating mutation elements for single-nucleotide mutations, including GAG→GTG at Exon 1 of the HBB gene for SCD and IVS1-5 G→C, the most prevalent β-thalassemia mutation in Pakistan. Optimized prime editing protocols were developed for K562 cells, hMSCs, and HEK293T cells. While initial trials in K562 cells and hMSCs revealed suboptimal efficiency, HEK293T cells exhibited robust GFP expression post-transfection, demonstrating effective plasmid delivery. Fluorescence-activated cell sorting and single-cell cloning allowed the isolation of targeted clones. Molecular analysis using ARMS PCR and tetra ARMS PCR confirmed successful introduction of the IVS1-5 G→C and GAG→GTG mutations, respectively. Sequencing further validated the precise genetic modifications, establishing HEK293T-based disease models for future gene-editing research. For mutation correction, plasmids targeting the IVS1-5 β-thalassemia mutation were constructed and validated using PCR and sequencing. Additionally, an RNA-based prime editing approach was employed to correct the FSC8/9 mutation in patient-derived iPSCs. Transfection into iPSCs demonstrated partial correction of the mutation, confirmed through ARMS PCR. To enhance efficiency, the pT7PEMax plasmid was linearized and transcribed into RNA for improved delivery and editing success. Furthermore, patient-derived CD34+ HSCs were isolated from the blood of β-thalassemia major patients and one healthy control. Comprehensive clinical data, including transfusion dependency and CBC parameters, provided critical insights into disease severity. Isolated HSCs demonstrated robust growth, genomic stability, and morphological integrity, as evidenced by karyotyping, DAPI staining, and Giemsa staining. These results establish the viability of HSCs for mutation correction and therapeutic studies. In conclusion, this study successfully developed disease models and optimized gene-editing strategies for β-thalassemia and SCD. The findings highlight the efficacy and adaptability of prime editing, while also identifying areas for refinement in delivery efficiency.
First Page
1
Last Page
110
Recommended Citation
Fatima, S.
(2024). Fixing B-thalassemia causing genetic variants by the aid of prime editing tools. , 1-110.
Available at:
https://ecommons.aku.edu/etd_pk_mc_mphil-bbs/33