Title: Next generation T cell gene therapies for children with leukaemia and immunodeficiency
Funder: Department of Health and Social Care

Abstract: Background Great Ormond Street Hospital undertakes the largest number of paediatric haematopoietic stem cell transplants (HSCT) in Europe, and is a nationally commissioned centre for children with immunodeficiencies, and also cares for children infected with HIV. This experience has brought us to the forefront of gene based therapies for conditions where it is possible to collect, engineer and return cells to patients. Paediatric disorders have been particularily suitable for these ex-vivo gene therapy approaches, partly because of their early presentation, but also because of reduced dosing requirements. I have led the translational development of T cells encoding suicide genes, anti-leukaemia receptors and anti-viral elements, and all these therapies have benefited from 'in-house' retroviral and lentiviral vector technology. However, I have recognised that existing approaches carry a number of limitations, and now propose deploying the next generation of DNA engineering techniques to address some of the key hurdles. New targeted DNA-nucleases, including transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR-Cas) nucleases, are capable of mediating highly efficient DNA scission at precisely defined genomic loci. In combination with our existing lentiviral and other vector technology, they offer burgeoning therapeutic possibilities and T cell therapies are an ideal arena for their early application. Aims My aim is to widen gene therapy applications with new reagents and to unlock approaches for otherwise chronic and disabling conditions of childhood. Three different T cell therapies have been selected, aiming to allow a broad exploration of emerging therapeutic possibilities:- Production of umblical cord blood T-cells that are available 'off the shelf' following permanent genetic disruption of their existing T-cell receptors and simultaneous engineering to express a new chimeric antigen receptor (CAR) against the B-cell leukeamia antigen, CD19.Correction of disease causing mutations in CD40 Ligand deficiency, an inherited immune disorder which results in debilitating immunodeficiency Engineering of T-cells to resist HIV by disruption of the HIV co-receptor CCR5, in combination with expression of innate, humanised antiviral restriction factors. Plan of Investigation Preclinical development (Years 1-2) of reagents and processes for ex-vivo T-cell modification using lentiviral vectors and nuclease reagents (TALENs and CRISPRs). Translational adaptation in our clean room facility will follow, ahead of submissions to the MHRA and ethics committees for clinical trials approvals. Clinical Phase testing (Years 2-5). (i) A bank of T-cells engineered to target CD19+B-cell leukaemia will be produced from healthy umbilical cord blood donations. These naive cells exhibit powerful effects in transplant patients, and sufficient T-cells could be produced from a single donation to treat over 10 children (ii) Pilot testing of CD40L gene-editing will be undertaken in children without suitable HLA-matched transplant donors, aiming to provide a pool of circulating T-cells with functional restoration of CD40L expression (iii) Clinical therapy with engineered T-cells in HIV infection will be offered to adolescents who have been on life-long antiretroviral therapy (ART) and require alternative treatment interventions. Subsequent larger phase studies (Years 5-10) will draw support from our clinical trials centre, and clinical research networks; successful approaches would then be selected for progression towards licencing and commercialisation where appropriate. Benefits to NHS This project will bring immediate access to cutting edge technology directly to NHS patients, and is designed to provide a first evaluation of a new generation of technologies. The selected treatments will, in the first instance, address areas of unmet need in children with chronic conditions. If successful, much larger numbers of patients stand to benefit from these approaches which are designed treat chronic conditions with a single intervention. This would have enormous resource saving implications for the NHS, as well as families, carers and wider society.

Lay abstract: White blood cells called T cells are a crucial part of the immune system, and circulate around the body to fight infections. T cells are long lived and can provide life long protection, dividing to provide large numbers of fighting cells when required. They can also attack and eradicate certain types of cancer, and play a leading role in bone marrow transplantation and rejection of donated organs. A critical feature of T cells is that they carry on their surface highly specialized receptors, similar to a docking arm, that can locate and latch onto infected target cells. These receptors are also capable of identifying the body's own tissues and will activate T cells to reject transplant organs, or fight against a bone marrow transplant recipient. This is why transplantation requires close matching between donor and recipient. Recently, new therapies have been developed that use T cells to mediate powerful anti-viral and anti-leukaemia effects. These approaches rely on collecting T cells either from a patient, or from a well-matched donor, and then engineering them in a special laboratory. New genes can be added into the cells using a disabled virus to introduce a new receptor against a particular cancer or virus. My laboratory has developed a number of new treatments using these methods and we have built up special expertise in our hospital for T cell therapies. These approaches overlap with other gene therapies being used to treat inherited blood and immune disorders, where an extra copy of a corrected gene is added into a patient's own cells. I now plan to exploit a toolbox of recently developed reagents that allows more sophisticated engineering of T cells, and should open the door to powerful new therapies. These reagents can be compared to very fine molecular scissors that allow highly targeted cutting of genes within cells, and this approach is known as gene-editing. Changing a gene in its normal location has important advantages over existing gene-addition approaches, where extra gene copies are randomly added into a cell. In this proposal, I plan to develop new treatments using gene-editing technology to engineer T cells for three different areas of clinical need. In view of our existing experience in delivering T cell based therapies, our centre is ideally placed to lead the first human trials of these approaches. Anti-leukaemia therapy for recurrence of leukaemia after chemotherapy. T cells will be modified to carry new receptors and will be simultaneously treated with molecular scissors to remove existing receptors that could react against the patient. This will allow the cells to be given to multiple patients, improving the cost-benefit ratio of the therapy. The therapy will use T cells collected from healthy umbilical cord blood donations because we have previously shown that these naive' cells can have especially powerful effects in transplant patients. It is possible that sufficient T cells could be produced from a single donation to treat over 10 patients as part of a clinical trial. Patients will be closely monitored for at least a year after the treatment to see how the cells grow, if they clear leukaemia or cause side effects. Some children are born with abnormal T cells that don't respond properly to infections. One of these conditions is caused by defective expression on the surface of T cells of another docking molecule called CD40Ligand (CD40L). These children usually undergo bone marrow transplantation, but an alternative approach would be to try and correct their own T cells using gene-editing. A collaboration with researchers in Seattle is being set up to develop this approach, and the molecular scissors required to cut the defective gene have been made. The next steps will be to repair the cut gene with a corrected copy. The efficiency of such manipulations has improved greatly in recent months, and if experiments are successful in the laboratory, therapy could soon be offered to affected children. When T cells are infected by HIV they stop working properly and often die. Many patients with HIV are managing to control their infection with drug therapy, but over time the virus becomes resistant and side effects from the drugs are a problem. An alternative approach may be to protect T cells from HIV infection by either removing the surface flags that the virus binds to, and by adding new anti-viral shields into the cells. This project will bring cutting edge new gene engineering reagents into the clinic for the benefit of children with chronic illness.

Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: CANCER AND NEOPLASMS;INFLAMMATORY AND IMMUNE SYSTEM