Towards Gene Editing for Inherited Immunodeficiencies: Functional modelling & correction of novel disease-causing STAT1 variants

Signal transducer and activator of transcription 1 (STAT1) is a vital component of the Janus kinase (JAK)/STAT signalling cascade which transduces signals from diverse cell surface receptors to initiate gene transcription. STAT1 plays a particularly key role in immunity, as demonstrated by the insufficient immune responses generated by STAT1-deficient mice and by patients with STAT1 loss-of-function mutations.
In 2011, autosomal dominant STAT1 gain-of-function (GOF) mutations were first identified in humans by van de Veerdonk et al. and Liu et al. They are now known to be the main cause of chronic mucocutaneous candidiasis and are further associated with an enhanced susceptibility to bacterial and viral infections, autoimmunity, aneurysms and cancer. These outcomes owe mainly to impaired Th 17 immunity, defective natural killer cell maturation and function, B cell lymphopenia and exaggerated responsiveness to interferons.
The mechanism behind STAT1 GOF disease was previously thought to be hinderance of STAT1 dephosphorylation, leading to a build-up of phosphorylated STAT1 (pSTAT1) in the nucleus and thus overexpression of STAT1-dependent genes. However, this has been complex to decipher, and recent data suggests STAT1 GOF mutations have no effect on the dephosphorylation of STAT1, and the elevated pSTAT1 levels are explained through higher levels of total STAT1 protein.
At present, JAK inhibitors such as Ruxolitinib and Jakinibs are being used to treat STAT1 GOF manifestations but, for unknown reasons, this method proves only effective for some. Alternative approaches such as allo-HSCT have been trialled but remain difficult, with only 40% overall survival reported in 20 cases to date.
With this in mind, this project aims to utilise the novel gene-editing platform of CRISPR technology to a) correct the specific missense defects and restore wild-type STAT1 (wt-STAT1) sequence or b) knock out the overactive allele to cause haploinsufficiency as a therapeutic for STAT1 GOF disease. Initially, we will work to model the disease utilising the U3A cell line that lack endogenous STAT1 and transduce these with lentiviral constructs encoding wt-STAT1 or STAT1 GOF mutations. We will then design and screen multiple synthetic guide RNAs (sgRNAs) for their ability and specificity to guide Cas9 protein to the overactive allele for its cleavage. Having then selected the optimal sgRNAs, they will be taken forward for assessment in T cells, and used to initiate the homology-directed repair of GOF mutations via an exogenous DNA encoding wt-STAT1. If one/both of the approaches are successful, we will then explore the use of this technology in haematopoietic stem cells and potentially the practicality of the approach in vivo. Furthermore, in conjunction with the gene-editing aspect of the project, we will look to further deconstruct the pathogenesis of GOF STAT1 disease using our panel of U3A cell lines and patient primary cells, with a particular interest in GOF STAT1 disease in myeloid cells which appear to be neglected in the literature thus far.