Gene Editing in X-Linked Agammaglobulinaemia

X-Linked Agammaglobulineamia (XLA) is an inherited immunodeficiency affecting 1 in 250,000 births. It is caused by a mutation in Bruton's Tyrosine Kinase (Btk) which is encoded in the X chromosome. This molecule is expressed in B Cell progenitors and provides survival signals to allow maturation into functional B Cells which then produce immunoglobulins which fight infections. Without this molecule patients do not have B-Cells and are unable to produce immunoglobulins. As a result they are less able to fight pathogens and therefore acquire severe infections. Conventional therapy comprises life-long infusions of immunoglobulin but the cumulative cost is high and this is not available in some developing countries. Recently a shortage of plasma donations has led to immunoglobulin supply pressures. Even with this therapy patients may still have frequent infections and these can lead to a chronic lung disease known as bronchiectasis. This risk increases when patients are poorly compliant with treatment. Complications such as inflammatory bowel disease, chronic giardiasis, chronic sinusitis and recurrent conjunctivitis are seen despite adequate Immunoglobulin replacement therapy. Currently a safe and definitive treatment is lacking.

Recent progress in gene therapy has provided proof of principle in a number of Immunedeficiencies including SCID, Wiskott Aldrich Syndrome and CGD. This process obviates the risk of graft versus host disease and probably reduces the overall risk associated with allogeneic procedures. This technology involves the use of a retroviral vector to transfer a 'functional' version of the affected gene into haematopoietic stem or progenitor cells. The gene is integrated into host DNA at a semi-random location. While effective, for conditions where accurate gene expression and regulation is desirable, it may not be fully effective, and there may be additional problems associated with dysregulated gene expression. An alternative novel approach is to use accurate gene editing technology in order to insert a therapeutic transgene in a location that enables physiological gene expression. This project will be a pre-clinical feasibility study to establish this technique for the treatment of XLA.

At first, immortalised cell lines will be edited but as the technique is refined, human stem cells will be used from normal donors and XLA patients. Specialised enzymes will transferred into cells to cut the DNA at an area in the BTK gene. Subsequently a correct version of the gene will be delivered into cells using a viral vector and this will be transferred into the area of the cut.

. The edited cells will then have their DNA checked to show that the changes have been made in the correct place and not elsewhere (so called "off target" effects). Subsequently, the edited cells will be tested to ensure they can differentiate into B-Cells which produce Immunoglobulin. We aim to then test these cells in immune deficient mice and observe whether the mice start to make B Cells and Immunoglobulins.

In recent years the cost of whole genome sequencing has fallen greatly and now it is easier to elicit the genetic cause of diseases including Primary Immunodeficiencies. Gene editing has the potential to reverse these mutations. If applied to embryos it could stop hereditary disease being inherited. However, this application is controversial and is unlikely to be trialled in the near future. Attempting gene-editing in adults with monogenic immunodeficiencies is a more realistic first step to bring this technology to clinical practice. If this project is successful then it can be adapted for other immunological and haematological conditions.

X-linked agammaglobulinemia (XLA) is caused by a mutation in Bruton's Tyrosine Kinase (Btk) which is encoded at the Xq22.1 locus. When developing lymphocytes reach the pre B-Cell phase they express a pre B Cell receptor and after cell surface expression it dimerises sending survival signals via Btk. Mutations of this molecule lead to the cessation of B-cell development. Patients therefore have absent B Cells and no detectable Immunoglobulins.

This project will assess the CRISPR-Cas system of gene editing to correct this mutation. This involves nucleofection of cells with the Cas 9 protein complexed with a guide RNA complementary to our chosen target. This leads to a double stranded DNA break (DSB) at the Btk gene locus. Subsequently a Adeno Associated Virus 6 vector will be used to introduce a normal Btk minigene gene into the locus. Following optimisation of the technique in a Btk knockout DG75 cell line, we will trial it in CD34+ cell from healthy donors and then XLA patients. CD34+ cells will be separated from blood using Magnetic-Activated Cell Sorting. Editing efficiency will be determined by Digital Droplet PCR. Subsequently, we will perform in vitro comparisons will assess the function of gene edited patient cells.
To assess for off target effects we will use GUIDE-sec (Genome-wide, unbiased identification of DSBs enabled by sequencing). This method involves tagging any DSBs in the genome with a blunt double-stranded oligodeoxynucleotide (dsODN). Integration points are then identified using PCR amplification and sequencing of products.

We finally aim to test gene edited cells in a NSG mice model. Primary and secondary transplantation will be performed and each experiment will consist of 3 groups (negative control, gene edited group and positive control). To assess restoration of the humoral immune system we will perform flow cytometry to determine B Cell numbers, test vaccination, Immunoglobulin quantification and cell proliferation assays.

The main aim of this project is to deliver a gene editing technique to be used in patients with X-linked Agammaglobulinemia (XLA). If successful this will lead to clinical trials of a gene-edited cell therapy which will be potentially curative.

The primary beneficiaries will therefore be XLA patients who can suffer a great deal from severe and chronic infections despite adequate Immunoglobulin replacement therapy. Our 90% of patients develop bronchiectasis and suffer from frequent respiratory infections. The potential improvements in morbidity and mortality deliverable from a gene-editing therapy are therefore great.

Although a gene-editing therapy would have a high one-off cost, it is likely to be much cheaper than the current alternative of lifelong immunoglobulin treatment. For example, a 30 gram infusion of Privigen costs the NHS £1377 and if given every 3 weeks this will amount to between £1.5 million and £2 million over a lifetime. Importantly, due to the increase of secondary antibody deficiency the demand for immunoglobulin is increasing and this is likely to lead to price rises. Taking all these costs into account it is clear that a curative gene-edited cell therapy will result in a substantial cost saving for the NHS and allow resources to be used for other priorities.

Regular Immunoglobulin Infusions also affect a patient's quality of life and careers. A survey carried out by patient support group PID UK in 2016 showed a loss of wages averaging £7,143 per annum. Respondents also felt that regular infusions impacted their ability to travel abroad, socialise and manage household activities. Freeing up XLA patients from regular Immunoglobulin Infusions will therefore have a positive impact on the economy and allow them a live a life as close to normal as possible.

In addition, the techniques developed during this project can be applied to treat other diseases including other monogenic primary immunodeficiencies as well as monogenic haemophilias which affect many more people.

During the course of the project I will be completing experiments that involved disrupting the endogenous Btk Locus. As a result, we may learn more about the gene itself along with its regulation. For example, a previous study involving gene-editing in cells from Chronic Granulomatous Disease patients revealed restoration of gp91 expression after insertion of a CYBB minigene into exon 2, but not exon 1 of the endogenous CYBB gene[1]. This therefore highlighted a potential role of Exon 1/Intron 1 in gene expression and regulation.

The project will involve engagement with patient groups including UKPIPS as well as the wider public through UCL's Public Engagement Unit. By raising the profile of the research in this way we will act as advocates for those with rare diseases such as XLA, who often feel that their condition does not receive an appropriate level of research funding.

As an Immunology trainee, I am required to gain experience with running a laboratory. This includes setting up and validating assays as well as troubleshooting when equipment does not perform as expected. This project will allow me to gain a great deal of experience in this area and these skills would be relevant to my future work as an Immunology Consultant.

I hope to become an academic Immunologist and aim to bring cell based gene therapy into clinical use. Therefore this project will be vital for acquiring the necessary skills and knowledge to do this. I aim to lead my own research group in the future and would like to train future students. I would also like to take part in University level teaching. This will expand the field and ultimately improve the care of patients with immunodeficiencies.

1. Santilli G, Thrasher AJ. A New Chapter on Targeted Gene Insertion for X-CGD: Do Not Skip the Intro(n). Mol Ther. 2017;25(2):307-309.