UCL Great Ormond Street Institute of Child Health


Great Ormond Street Institute of Child Health


Gene editing in Deficiency of adenosine deaminase type 2 (DADA2)

Supervisors: Professor Despina Eleftheriou, Professor Paul Brogan, Dr Ying Hong


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 (livedo racemosa), lacunar and haemorrhagic stroke early in life, peripheral neuropathy, multi-organ failure from systemic vasculitis, and systemic inflammation which can ultimately result in death (1-3). Patients with the lowest ADA2 enzymatic activity present with severe marrow failure and immunodeficiency and die in infancy or early childhood (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). Recently, we also have discovered that development of antibodies directed against the anti-TNF therapy itself causes loss of efficacy over time (1) and this leaves patients with  no safe therapeutic alternatives because, thereafter, the only realistic therapeutic option is allogeneic haematopoietic stem cell transplant (HSCT) which itself can result in death in up to 20% (4). Moreover, HSCT may not even be possible if a well-matched donor is unavailable as exemplified by a recent paediatric case in our cohort. 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 and -Cas12a -site-specific nucleases and assess the efficacy of this approach to correct the immune defects associated with DADA2. 

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, apoptosis, interferon production and STAT signalling, interaction with endothelial cells to induce endothelial activation) 
Stage 3: Biodistribution and engraftment of gene edited HSC in NSG mice (Months 20-36). Engraftment of human leukocytes (% of CD45) and differentiation intom specific lineages will be evaluated in the haematopoietic organs and peripheral blood of NSG mice post-transplant. Chimerism will be assessed over 20 weeks as frequency and number of total human leukocytes, T, B, NK, and myeloid cells. Gene transfer efficacy (PCR, ADA2 protein expression and enzyme activity) will be established in monocytes harvested from peripheral blood and bone marrow of recipient mice.

1. Cooray S et al. Rheumatology (Oxford). 2021 Jan 9:keaa837. 
2. Lee PY et al. J Allergy Clin Immunol. 2020 Jun;145(6):1664-1672.e10. 
3.Nanthapisal S et al. Arthritis Rheumatol. 2016 Sep;68(9):2314-22. 
4. Hashem H et al. J Clin Immunol. 2021 Oct;41(7):1633-1647. 
5. Hong Y et al.  Front Immunol. 2022 Apr 22;13:852830