Targeted gene addition strategies to correct primary immunodeficiencies

Supervisors: Professor Bobby Gaspar and Dr Claire Booth

Primary immunodeficiencies (PIDs) represent a spectrum of disorders characterised by impaired development and function of the immune system with the more severe forms being lethal in infancy. Haematopoietic stem cell transplant (HSCT) is a curative treatment for many of these conditions but is limited by the availability of suitable donors and toxicities associated with cytoreductive chemotherapy and HLA disparity. Gene therapy strategies offer an alternative management option and have been developed for a number of disorders, with clinical trials underway for ADA-SCID, X-linked SCID, X-linked chronic granulomatous disease (CGD) and Wiskott Aldrich Syndrome (WAS). Initial trials using gammaretroviral vector mediated gene transfer showed impressive efficacy but were complicated by vector insertion driven leukaemia in some patients. Newer designs using modified, self-inactivating lentiviral vectors are likely to have an improved safety profile but integration of such vectors remains uncontrolled and has the potential for genomic disruption.

Designer nucleases such as Zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENS) and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas systems are homologous recombination based methods which can mediate targeted genome modification. They combine specific DNA recognition sequences with an endonuclease capable of generating a site specific double stranded break in the DNA. When a double stranded break (DSB) is induced in DNA, the break can be repaired by non-homologous end joining (NHEJ), which is an error prone mechanism, or through homologous recombination (HR) in the presence of an appropriate donor DNA sequence maintaining the integrity of the DNA. Lack of specificity and off-target effects including repair through NHEJ are however areas of concern. This technology has recently reached clinical trial with Phase II trials underway treating patients with HIV and work has also been done in primary immunodeficiencies investigating similar approaches to target SCID genes including IL2RG and RAG1 mutations. Specific sites within the genome have been investigated as ‘safe harbour sites’, where integration of a therapeutic transgene is unlikely to disrupt regulatory or coding sequences of surrounding genes. Targeted gene addition has been reported to safe sites including the IL2RG, CCR5, PP1R12C (AAVS1), DMD21 and ROSA26 loci, with the majority of human primary cell work being performed in induced pluripotent stem cells (iPS).

Our project aims to develop a reliable and efficient system to target therapeutic transgene integration into genomic safe sites in haematopoietic stem cells and T cells thus reducing the risks of uncontrolled vector integration. By providing disease specific donor DNA sequences we aim to show correction of several models of PID. We will design and optimise TALEN and CRISPR/Cas systems to target safe harbours and compare the efficiency and specificity of these approaches. The technologies developed in this project will be applicable primarily to the treatment of immune disorders but could also impact on other inherited genetic conditions which can be treated with a gene therapy approach, for example metabolic and haematological diseases, as the techniques will be readily transferable.

There is an extensive research program developing gene therapy for PID at ICH. Our facility is unique in the UK with very few other centres Worldwide. A student would benefit not only from learning the wide range of molecular biology techniques required for this project (cloning, transfection and transduction of cells, primary cell and iPS work) but working in a leading group in the field, capable of translating pre-clinical studies into clinical benefit.

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