Gene editing for APDS1 - a novel gene therapy approach for gain of function (GOF) mutations.

Primary immunodeficiencies (PIDs) are a group of inherited diseases that lead to a patient's immune system not working correctly. They most commonly present early in life with severe recurrent infections, which can be fatal, as well as many other problems, including severe autoimmunity, abnormal inflammation affecting joints, skin, lung and bowels and an increased risk of developing cancers. The only way to cure most PIDs is through a stem cell transplant, where a patient's blood stem cells are replaced with those from a donor. However, stem cell transplants come with significant risk with mortality of 20% or more.

APDS1 is a type of PID where a mutation in one gene leads to patients having lifelong problems with recurrent infections, lymphoma and autoimmunity. Over the last few years there have been many attempts at gene therapy to modify a cell's DNA. As most PIDs are due to a genetic problem in one gene (monogenic diseases), these have been an early target for gene therapy. An individual gene inside a cell intstructs that cell to make a specific protein. Most people have two copies of each gene in every cell (one from each parent). A mutation in one gene in APDS1 patients' immune cells causes a particular protein to become overactive, and it is this overactivity that causes the abnormal function of the immune cells. This is referred to as a gain of function mutation. This cannot be treated by previously used gene therapy methods that have 'simply' introduced a new copy of a healthy gene into affected cells (as this would lead to a great excess of overactive protein).

Our project is aiming to use new gene therapy techniques known as 'gene editing' in two ways:

(a) disrupt the mutated gene. This should prevent the manufacture of the overactive protein within the immune cells and we can see whether the immune cell can cope with just one copy of the gene and less than the normal amount of functional protein (haploinsufficiency)
(b) attempting repair of the overactive gene. This should allow the cell to make the correct amount of normally functioning protein.

We are attempting disruption and/or repair of the abnormal gene using a form of 'molecular scissors' called CRISPR/Cas9, which can cut out mutated genes and insert in normal genes. We will compare these 'in vitro' (in the lab) approaches to determine what is the most efficient way of genetic editing.

If the 'in vitro' approaches are successful we will try to do this in mice with APDS1 ('in vivo'). Our aim is that these results will help develop personalized gene therapy for patients with the disease and that we can better understand how to correct genetic mutations that result in diseases caused by overactive proteins. These results could therefore be applicable to many other serious inherited diseases.

Aim & Objectives:
This project will develop and test the functional efficiency of novel gene editing therapeutic approaches for APDS1, a model for serious diseases caused by gain of function (GOF) mutations, not amenable to conventional gene therapy approaches. In vitro experiments will compare the efficacy of targeted gene disruption with gene repair on the function of various immune cells, including cell lines and primary human cells. We will then optimise conditions for gene editing in HSCs using the most efficient and least toxic gene editing approach. Finally, we will test the efficacy of gene-corrected human HSCs (ethics in place) in an established mouse model of APDS1, which will allow us to determine the degree of correction required for clinical benefit.

This project will test the following hypotheses:

1. Targeted disruption of the GOF mutation in p110delta using WT CRISPR/Cas9 can restore functional PI3Kdelta signalling in corrected cells.

2. In vitro functional assays will demonstrate a correlation between of gene disruption and normal protein expression in edited primary human immune cells.

3. Correction of the GOF allelic mutation to wild type sequence can be achieved by:
(a). Allelic exchange (unaffected allele acts as internal template for repair); and/or
(b). Homology-directed repair (wild-type cDNA template delivered via AAV6 viral vector transduction); and/or
(c). CRISPR/Cas9 nickase mutants with HDR template.

4. Gene editing confers protection against infection in vivo by reconstituting NSG mice with human cells and by investigating chimerism in an established mouse model of APDS1.

This research project will primarily benefit scientists working in gene editing, immunology and haematology together with patients suffering from primary immunodeficiencies (PIDs).

Primary immunodeficiencies are a heterogenous group of diseases that can cause significant morbidity and mortality to those affected. Activated phosphatidylinositol-3-OH kinase delta (PI3Kd) syndrome type 1 (APDS1) is a recently identified PID, caused by monoallelic gain of function (GOF) mutations in the p110 catalytic subunit of PI3Kcd. Current conservative management for APDS1 consists of immunoglobulin replacement therapy, aggressive management of infections, immunosuppression for autoimmune complications and chemotherapy for associated lymphomas. Specific PI3Kdelta inhibitors are in use in other areas of medicine, and in trial for APDS1. While early outcome studies demonstrate good impact for lymphadenopathy, improvement in immunodeficiency and longer-term cancer risk remain to be proven. The long-term impact and or toxicity of PI3Kdelta inhibitors is unknown.

Currently, haematopoietic stem cell transplantation (HSCT) represents the only curative approach for APDS1. This has been successfully applied, particularly in children, but has a significant associated mortality and a high rate of transplant-related complications. It is not suitable for patients with significant established end-organ damage. Therefore, novel less toxic, curative approaches are required. This proposal specifically aims to address that need. Success in this approach would allow this approach to be tested for other GOF immunodeficiencies and potentially other GOF diseases such as Huntington's disease.

If I obtain funding for the project I intend to participate actively in public engagement activities in the field of gene and cellular therapy, including the British Society for Gene Therapy's annual public engagement event in Oxford. I will also highlight the work locally through the UCL Institute for Immunity & Transplantation's annual community/schools open day. I hope that this activity would benefit the wider scientific community in the UK through increasing engagement and stimulating interest in the field. As stated in my communications plan I will also get involved in a gene therapy project for sixth formers in a London state secondary school, which will give Biology A level students experience of cutting edge biomedical research. This together with more conventional public engagement may help encourage early-career scientists into the field of gene and cellular therapy.

By undertaking this PhD, I will be able to develop my skills in order to work ultimately as a clinical academic. I hope to use my skills to help develop gene therapy approaches in the UK and increase patient access to this high-tech personalised therapy. I hope that my academic and clinical interests will eventually allow me to become a future member of the community that helps maintain the UK's strong position as a leader in the development and delivery of gene and cellular therapy.

I believe the information I gain from this PhD will help drive forward research into gene therapy for immunodeficiencies. Gene therapy using Crispr/CAS9 is already in clinical trials for cancer therapeutics. I hope to demonstrate wider potential applicability in PIDs, with the long-term goal of increasing patient access to personalised therapy.