Gene Editing in Prkdc Severe Combined Immunodeficiency and Ataxia Telangiectasia

Versha Prakash

Research output: ThesisDoctoral Thesis

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Primary immunodeficiencies (PIDs) are inherited diseases of the immune system. A variety of molecular defects can cause PIDs, including mutations in genes PRKDC and ATM (ataxia telangiectasia-mutated), both of which in addition lead to DNA repair defects. Due to a variety of factors including gene size and sensitivity to DNA damage, these genetic defects are not easily amenable to gene addition strategies, and alternative gene therapy strategies are actively being sought. One such strategy is genome editing, the topic of the current work. PRKDC severe combined immunodeficiency (PRKDC-SCID) is a rare inherited disease caused by mutations affecting DNA-PKcs (DNA-dependent protein kinase catalytic subunit). DNA-PKcs is a key component of non-homologous end joining (NHEJ), a major defence mechanism against double-strand break DNA damage. NHEJ is also involved in V(D)J recombination, a process necessary to generate functional immunoglobulins and T cells receptors, and hence for the development of B and T lymphocytes. Consequently, defects in DNA-PKcs lead to enhanced sensitivity to DNA-damaging agents, such as ionising radiation, and severe immune defects. DNA-PKcs deficiency causes SCID in both animals (mice, dogs, horses) and humans; however, the condition is ultrarare in humans with only two patients identified so far. Like with many other PIDs, transplantation of matched bone marrow remains the only choice of effective treatment, but it is complicated by sensitivity to conditioning agents that cause DNA damage. Gene transfer to haematopoietic stem/progenitor (HSC/HSPCs) cells first shown to have major therapeutic effects in SCID-X1, continues to show promise. Advances in genome editing have further opened possible approaches to treatment by correction of genetic defects. Previous work in the laboratory using engineered zinc-finger nucleases (ZFNs) and Prkdc repair matrixes demonstrated homology-directed gene correction in SCID mouse fibroblasts and HSC/HSPCs, leading to ex vivo rescue of the SCID phenotype with the latter. The current project broadly focuses on two major goals: (i) characterisation and phenotypic analyses of ZFN-targeted SCID mouse fibroblasts, including molecular analyses of genomic correction and rescue of DNA-PK activity, and (ii) in vitro gene editing of Prkdc mutation using CRISPR-Cas9 system. In this study, we used Streptococcus pyogenes derived Cas9 system to repair the Prkdc mutation. Co-delivery of guide RNAs (gRNA) on a Cas9 plasmid along with a corrective donor template demonstrated efficient Prkdc modification in mouse fibroblasts; however, this did not restore DNA-PK activity owing to apparently error-prone DNA repair. Nonetheless, this study indicated the potential of CRISPR-Cas9 system for targeted modification of genes involved in DNA repair disorders. Based on this observation, we explored targeting of ATM, which like Prkdc, is involved in DNA repair. ATM codes for the protein ATM kinase, deficiency of which leads to Ataxia Telangiectasia (AT), a progressive neurodegenerative disease with associated immunodeficiency, radiation sensitivity and susceptibility to malignancies. Correction of mutations on ATM could rescue residual ATM kinase activity – a therapeutic approach for treating AT-related immunodeficiency. We selected and designed gRNAs for disease-causing mutations – 5762ins137, 103C>T, 2T>C and 7638del9 – and demonstrated proof-of-concept editing of ATM locus in human cells. Given the multitude of mutations on ATM, we further designed global approaches for partial ATM cDNA knock-in at endogenous N/C-terminals or full ATM cDNA knock-in at hAAVS1 locus for application in the human haematopoietic stem cells. These studies on Prkdc and ATM have shown the feasibility of CRISPR/Cas genome editing on both genes. They have also highlighted the possible enhanced risk of homology-dependent DNA repair events being associated with novel mutations at the repair site, which prevented the recovery of fully repaired fibroblast clones in the case of ZFN-treated Prkdc. This work, therefore, points to the need for extra caution in therapeutic strategies involving genome editing of DNA repair gene defects.
Original languageEnglish
Awarding Institution
  • Royal Holloway, University of London
  • Yáñez-Muñoz, Rafael J., Supervisor
  • Dickson, George, Advisor
Award date1 Jul 2017
Publication statusUnpublished - 2017


  • Gene editing
  • CRISPR-Cas
  • Radiosensitive SCID
  • Ataxia Telangiectasia

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