The Development of Novel, Therapeutic Gene Editing Strategies for STAT1 gain-of-function Immunodeficiency.

Haematopoietic stem cell gene therapies (HSC-GT) are fast becoming a viable treatment option for many monogenic primary immunodeficiencies (PID), with several successful applications to date. However, thus far clinical HSC-GT has only targeted loss-of-function (LoF) PID where simple gene addition approaches using viral vector gene addition or CRISPR/Cas-mediated homology directed repair (HDR) suffice. Such approaches are not appropriate for heterozygous gain-of-function (GoF) mutations, which are an increasingly recognized cause of severe PID.

One such GoF PID is STAT1 GoF disease, caused by germline GoF mutations in signal transducer and activator of transcription 1 (STAT1), and characterized by overactivation of the Janus associated kinase (JAK)-STAT signaling pathway. Patients suffer from a broad clinical phenotype including Chronic Mucocutaneous Candidiasis, severe opportunistic infections, autoimmunity and malignancy amongst others. Treatment options are limited, with antimicrobial prophylaxis and prompt treatment of infections together with systemic immunosuppression for autoimmunity constituting the mainstay of conservative management. A small group of patients may undergo allogeneic haematopoietic stem cell transplantation (HSCT), although this has been associated with relatively poor outcomes (~40-50% survival rate). Very recently a small number of patients have been treated with JAK inhibitors, but no data exists on tolerability, efficacy or durability of this approach.

HSC-GT offers an attractive alternative treatment option for STAT1 GoF disease and will be explored in this PhD project. Several different gene editing approaches will be investigated including but not limited to:

- Targeted disruption of the mutant GoF STAT1 allele
- Targeted repair of STAT1 GoF mutations using HDR and/or base editing strategies
- Insertion of wild-type STAT1 cDNA under control of the endogenous STAT1 start codon

The efficacy of the above gene editing strategies in correcting immune cell function will be tested using various in-vitro and in-vivo (murine models) functional assays.

This project is relevant not only for the treatment of STAT1 GoF disease, but will be a first-of-its-kind case study for therapeutic gene editing of GoF PID. Furthermore, these gene editing strategies may also be more widely applicable to patients with other inborn errors in STAT1, including those with STAT1 deficiency.