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Title: Using gene editing to treat a rare inflammatory disorder called Deficiency of adenosine deaminase type 2 (DADA2)
Supervisors:
Despina Eleftheriou and Paul Brogan
Project Description:
Background and aims:
Deficiency of adenosine deaminase type 2 (DADA2) is an autosomal recessive disease caused by bi-allelic loss-of-function mutations in ADA2 (1-3). The clinical features of DADA2 include skin vasculitis, stroke early in life, peripheral neuropathy, multi-organ failure, severe marrow failure and systemic inflammation which can ultimately result in death (1-3). Treatment with anti-TNF-α is effective for the autoinflammatory and vasculitic components of the disease but does not correct marrow failure or immunodeficiency (1-2). For these patients the only realistic therapeutic option is allogeneic haematopoietic stem cell transplant (HSCT) which itself can result in death in up to 20% (4). Autologous HSC gene therapy has now been successfully used to treat several immunodeficiencies and immunodysregulatory disorders, and may be a highly beneficial and long-term curative treatment for DADA2. In that context we have already demonstrated that lentiviral mediated gene addition restores ADA2 expression and function in a cell line model, macrophages and CD34 haematopoietic stem cells (HSC) from patients with DADA2 (5). However, ADA2 expression is tightly controlled, and increased ADA2 expression is seen in some cancers so concern exists that uncontrolled expression generated by lentiviral mediated gene addition could lead to further dysregulation. Gene-editing offers an alternative approach, whereby therapeutic sequences are inserted in situ, remaining under the control of the endogenous DNA regulatory mechanisms therefore providing a more optimal therapy. The aim of this PhD project is therefore to develop gene editing tools targeting the ADA2 locus, namely CRISPR-Cas9 site-specific nucleases and assess the efficacy of this approach to correct the immune defects associated with DADA2.
Timeline/methods:
Stage 1: Generation and testing of CRISPR/Cas9 reagents (Months 0-12). Initial work will focus on designing and validating CRISPR synthetic RNAs (sgRNAs) with proximity to patients with mutations causing DADA2. This work will be done in THP1 cells and monocytes/macrophages from healthy donors/patients with DADA2 where CRISPR/Cas9 reagents will be delivered as RNA complex by electroporation. Targeted breakage at the ADA2 locus will be assessed at the genomic level through established Sanger sequencing and next generation sequencing (NGS) methods.
Stage 2: Effects on ADA2 protein expression and ADA2 enzyme activity and macrophage immunophenotype (Months 12-30). Optimised reagents will be tested on macrophages derived from monocytes or CD34+HSC from patients with DADA2 and healthy controls to examine: 1) ADA2 protein expression by western blotting; 2) ADA2 enzyme activity examined by a modified ELISA assay; and 3) macrophage immunophenotype (cytokine production and gene expression, interaction with endothelial cells to induce endothelial activation)
Stage 3: Preliminary biodistribution and engraftment of gene edited HSC in NSG mice experiments (Months 20-32). Engraftment of human leukocytes and differentiation into specific lineages will be evaluated in the haematopoietic organs and peripheral blood of mice post-transplant.
Upgrade months 15-18; thesis write-up months 30-36
References:
1. Cooray S et al. Rheumatology (Oxford). 2021:9:keaa837.
2. Lee PY et al. J Allergy Clin Immunol. 2020:145:1664-1672
3.Nanthapisal S et al. Arthritis Rheumatol. 2016;68:2314-22.
4. Hashem H et al. J Clin Immunol. 2021;41:1633-1647.
5. Hong Y et al. Front Immunol. 2022;13:852830
Contact Information:
Despina Eleftheriou