Gene, Stem and Cellular Therapies Funding Summary

Norrie disease is a rare genetic condition resulting from mutations in the NDP gene. It affects mainly boys, who are born blind and from adolescence develop progressive hearing loss. This dual sensory deprivation severely restricts patients’ social interactions and independence. Currently there is no treatment for the hearing loss other than cochlear implants, which are ineffective for most patients. Our research aims to slow or prevent the hearing loss by delivering a new copy of the NDP gene. This treatment may also help preserve some vision in individuals with Norrie disease, if treated as newborns.

We’ve proved our concept using mice with Norrie disease. When administered sufficiently early, NDP gene replacement therapy is able to rescue abnormalities in the cochlea (inner ear) and the eye. However, in these experiments we used a gene delivery method that expressed NDP throughout the body. Therefore, although it successfully treated the eye and ear it also had neurological side effects due to mislocalised expression of NDP.

In order to avoid these side effects and deliver the gene precisely, we aim to pinpoint the normal pattern of expression of NDP and its targets in the developing human eye and ear. We will then compare means of delivery (serotypes of adeno-associated viral vectors, AAV) along with the “on switch” (promoter) of the NDP gene to achieve expression of NDP in only the specific cell types of the eye or cochlea. Combinations of the promoter and viral serotypes will be tested in vitro in cell cultures or retinal and cochlear organoids that mimic the structure of these organs.

Based on the results of this project, the new gene therapy constructs will be tested in future pre-clinical studies for the ability to rescue their vision and hearing.

For patients diagnosed with severe immunodeficiencies, treatment options are limited. Bone marrow transplant, where bone marrow stem cells from a healthy donor are transplanted into patients, is usually the only curative therapy. However, poor outcomes are seen if the donor is not well matched to the patient, driving the search for safer and effective alternative therapeutic approaches. For several diseases, there has been significant success in clinical trials using modified viruses to deliver a corrective copy of the defective gene into the patient’s own stem cells, termed gene therapy.

Gene corrected stem cells and the cells that develop from them constantly produce large amounts of the protein missing in the patients' own cells. However, some immunodeficiencies are caused by genes that aren’t produced in all cells all the time, or indeed in the stem cells themselves. Continuous expression could be detrimental to immune system cells and even lead to an increased risk of cancer. For these diseases, gene editing technologies may offer a better approach by correcting a gene at its place in the genome rather than adding a new gene, using the gene's usual regulatory signals contained in the surrounding DNA to replicate the normal expression pattern.

Currently gene editing requires the use of an electric shock to the cells and a virus to deliver all the necessary components, compromising the health of the cells and making it challenging to move to the clinic. We want to test a novel gold nanoparticle technology, aiming to overcome barriers and bring curative, safe, gene edited stem cell therapies closer to the clinic. Initially, we will investigate these techniques in the context of X-linked lymphoproliferative disease (XLP), a severe disease with an unmet clinical need. However, there are many inherited diseases which could also benefit from this technology and could be the focus of further studies.

Gene therapy is a branch of medicine that involves replacing non-functional genes that cause inherited diseases with functioning ones. Primary immunodeficiencies are diseases in which the non-functional gene causes serious problems in the immune system – they are a good target for gene therapies because immune stem cells can be easily isolated from the blood and given back to patients after being modified, allowing them to replace the non-functioning immune system without any risk of incompatibility. To do this, it is necessary to introduce the functional gene into the patient’s cells, get it to remain there stably, and ensure that the cell reads it correctly so as to produce functional protein in the right quantity and context.

One way to ensure the corrected gene is read appropriately is to put it directly into the patient’s own DNA, overwriting the faulty gene. This requires a targeted cut to be made at the faulty gene, and DNA of the correct sequence to be provided separately to be inserted by the cell’s repair mechanisms. This project involves two potential improvements to the viruses used to provide the corrected DNA sequence. The first involves completely removing the genome of a virus and inserting a fully synthetic replacement sequence, to maximise safety. The second involves a different type of virus (AAV), which works well but has a limit to the length of DNA it can carry. The DNA can be split over two AAVs that co-infect the target cell, but this is inefficient. We will physically link the two partner AAVs so that both viruses always co-infect the same cell, so that delivery is more efficient. The technologies will improve the effectiveness and feasibility of replacing genes in human immune stem cells to treat XLA, a B cell disorder and our disease model in this application.

Patients with acute myeloid leukaemia (AML) are faced with low survival rates and high relapse rates (can get sick again). Much of this is due to AML cancer stem cells which lead to disease relapse after chemotherapy. Eliminating and targeting AML stem cells is challenging as they are a rare population arising from many stages of cell development and their cell surface markers may be different to the bulk AML tumour population. As such, focusing and targeting a single AML cancer marker may be insufficient to destroy all AML cancer cells. Development of therapies targeting multiple cancer markers may be more robust.

The work in this project looks into the development of multiplex chimeric antigen receptor (CAR) T-cell therapy, whereby the patient’s own immune system cells are engineered to fight cancer cells by targeting 3 AML cancer markers “turned on” in AML stem cells – CD33, CD123, and CLL1. To date, our multiplex CARs can destroy AML cells through each of the cancer markers in a test tube and in living cancer models in the lab, equally as well as CAR’s that can only recognise one cancer marker and show protection even when cancer cells stop expressing cancer markers. Before treating patients, we would like to further inspect our multiplex CARs to find versions that are more able and better in targeting and killing AML cancer cells in order to support funding applications to launch a clinical study. The overall aim of the project is to improve outcomes for paediatric and adult patients with high-risk relapsed AML.