140. Point Mutation Correction for ADA SCID

Abstract

Adenosine deaminase (ADA) deficiency is a rare genetic disease characterised by the absence of the ADA enzyme (EC 3.5.4.4) involved in purine salvage and degradation. Patients lacking ADA build up adenosine and deoxyadenosine metabolites in the organism, something that in turn leads to an accumulation of other deoxynucleotides and gives rise to functional defects in all lymphocyte lineages and severe combined immunodeficiency (ADA SCID). This SCID phenotype can be corrected by hematopoietic stem cell transplant, enzyme replacement therapy, and integrative gene therapy protocols employing autologous CD34+ cells. ADA deficiency confers a positive selective advantage to corrected lymphocytes over deficient cells, thus offering a great system for the study of novel molecular therapies such as gene editing. The advent of the CRISPR/Cas9 system has allowed for the swift tailoring of novel gene editing strategies in laboratories without access to advanced cloning platforms. We believe this democratisation of gene editing has also made more practicable the therapeutic reversal of genetic mutations into wild type sequences, the ultimate form of genetic correction. We have designed a CRISPR/Cas9 strategy that can target the nonsense Q3X (ADA c7C>T) point mutation endemic to ADA SCID patients of Somali origin residing in the UK, as well as its equivalent site in wild type sequences. In the present work we show evidence of the Q3X site non-homologous end joining (NHEJ) gene ablation in Jurkat cells by means of DNA sequencing and ADA protein readouts (approximately 70% efficiency). Moreover, we have designed a donor for the Q3X repair step that contains an adjacent traceable silent mutation in order to track any homology directed repair (HDR) when working with wild type cells. Here we show preliminary evidence of Q3X site HDR in wild type Jurkat cells in DNA sequencing results by detection of the aforementioned traceable mutation, albeit with low efficiency (less than 3% efficiency). We are currently attempting the translation of this approach into human CD34+ cells, the ultimate target cell population for the correction of the SCID phenotype in ADA deficient patients.