UCOE-based lentiviral vectors for effective and safe gene therapy

Gene therapy holds great promise for the cure of genetically inherited diseases including disorders of the blood such as immune system defects (severe combined immune deficiency or SCID, chronic granulomatous disease or CGD), thalassaemia, sickle cell disease, and haemophilia. This has been evidenced by the recent successful treatment by gene therapy of people with SCID and CGD. However, there are still significant challenges to ensure that the treatment works safely, and also can be used for a wider rage of conditions. The way that genes are controlled has a major impact on these issues. We have shown that it may be possible to control a therapeutic gene in a safe and effective way using a newly defined 'switch' or 'regulator' that occurs naturally in human cells. For this study we want to develop the use of this switch in gene therapy, to show that it is safe, and finally to show that model diseases can be effectively treated. If this turns out to be true, then we are hopeful that it will have a broad application to the treatment of many inherited diseases for which current treatments are inadequate.

Retroviral vectors have become a promising tool for treatment of life threatening inherited diseases as exemplified by successful clinical applications targeting haematopoietic stem cells (HSC). However, two major problems are limiting their further clinical application namely (i) the significant risk of insertional mutagenesis as evidenced by four patients with SCID-X1 treated with anMLV-based vector developing clonal T-cell lymphoproliferation and (ii) susceptibility to epigenetic-mediated reduction and variegation in expression. There is a major need for vectors incorporating enhancer-less regulatory elements with markedly reduced insertional mutagenesis potential that are capable of giving rise to reproducible and stable transgene expression irrespective of tissue type or site of integration. This proposal aims to address this urgent requirement by exploiting the recently described ubiquitously-acting chromatin opening element (UCOE). UCOEs consist of methylation-free CpG islands encompassing dual, divergently transcribed promoters of housekeeping genes and confer resistance to transcriptional silencing and produce consistent and stable transgene expression. We have recently shown that the UCOE from the CBX3-HNRPA2B1 locus (A2UCOE) drives reproducible, stable and therapeutically relevant levels of transgene expression from within lentiviral vectors (LVs), in HSC in mice in vivo. Furthermore, this is achieved in the absence of classical enhancer activity and may confer a high safety profile. We now seek further funding to complete our pre-clinical evaluation of the efficacy and safety of A2UCOE-LVs. We propose two complementary parallel strands of investigation: (1) comprehensive in vitro and in vivo molecular and cell biology analyses to assess the insertional mutagenesis potential of the A2UCOE and (2) to assess novel A2UCOE-driven transcription unit designs for improved safety and increased efficiency, efficacy and range of application.