Our group has a long-term interest in exploring and exploiting the process of gene targeting in human cells. Gene targeting/genome editing makes targeted changes to the genome by harnessing the cellular pathways of DNA repair. It is greatly stimulated by customised nucleases (ZFNs, TALENs, CRISPRs) that recognise the target locus. Defined genomic changes are made when an induced double-strand break (DSB) is repaired by homologous recombination (HR) with a DNA repair template co-delivered with the nuclease. Because it can be used to correct disease-causing mutations, this is an attractive approach to gene therapy for inherited genetic disorders, including haematological diseases such as b-thalassemia major (bTM) and haemophilia. Repair of DSBs by error-prone nonhomologous end-joining (NHEJ) is also used to introduce mutations in order to study gene function, and in some situations may also be used for therapeutic purposes. To promote the desired outcome in the appropriate cell type with sufficient specificity and efficiency, the key parameters in DSB repair must be identified and optimised. Such knowledge is also important for understanding factors that determine genome instability. Our projects fall into three main categories:
Investigating the underlying process of DNA DSB repair
In previous work, we have investigated the influence on gene targeting of factors such as chromosomal position, DNA delivery method and overexpressed recombination proteins such as Rad51 and Redb. We have recently developed a natural reporter system for nuclease-induced DSB repair at the X-chromosomal HPRT locus by NHEJ and HR. We are using this system to further explore parameters affecting DSB repair and gene targeting, including the nature of the host cell genotype, repair template design and DSB.
Gene disruption for functional analyses and oncogene inactivation
After pioneering homozygous gene disruption in human cells (IFI6 aka 6-16) we developed conditional gene knockouts in human cells (CDK1, TOPO II alpha), and studied their pleiotropic effects on cell cycle control, ploidy, chromosome condensation/segregation and DSB repair. More recently we have targeted the BCL6 oncogene and the NHEJ gene PRKDC in a B cell line to study their roles in cell growth and differentiation. We have also developed and tested custom endonucleases for inactivating the BCR-ABL oncogene in Chronic Myeloid Leukemia cells.
Promoting gene correction at the b-globin locus (HBB)
With the long-term goal of gene correction therapy for b-haemoglobinopathies, such as bTM, we have used a customised endonuclease to promote gene targeting at the HBB locus in cell lines. Our current goals are to explore alternative endonuclease and donor repair template designs and to investigate HBB gene correction in adult stem cells.
Dr Andy Porter
4S3, Commonwealth Building, Hammersmith Campus
Current and recent lab members
Postdocs: Olga Isman, Jeremy Brown, Max Chen, Polly Gravells, Kazunori Tomita.
PhD students: Jiaxi Zheng, Rand Al-Mudhafar, Sara Ahrabi (Oxford), Tomasso Vannocci,
MSc students: Witchayoot Huangsuranun, Nikita Dave, Liuyi He Xia, Katherine Weight, Miriam Mendez, Josh Kaplan, Jiaxi Zheng, Matthew Ellis, Tony Zheng, Yousef Alame,
BSc students: Athena Chio, Susannah Rynhart, Sonal Lakhani, Mariana Popa, Chris Chang, Kathy Wang, Katerina Constantinou.
Summer students: Liuyi He Xia, Adersh Gill, Howin Ng.
Current and recent collaborators
Internal: Tom McKinnon, Nichola Cooper, Jamshid Khorashad,
External: Tim Humphrey (Oxford), Simon Wagner (Leicester), Irene Roberts (Oxford), Christine Farr (Cambridge), Konstantin Panov (Belfast).