Development of enhanced gene specific technology for the isolation of proteins binding at a single locus in vivo.

Lead Research Organisation: King's College London
Department Name: Cancer Studies

Abstract

The identification of proteins that bind to and regulate gene expression within the cell is an important, ongoing but time consuming process. Current methodologies, such as chromatin immunoprecipitation (ChIP) require foreknowledge that a particular protein may be binding to a particular region of DNA in the cell. This protein is then used as a 'handle' to isolate all the DNA in the cell to which that protein might be binding. (This is generally achieved by using antibodies specific for the particular protein). Then an assessment of this DNA is carried out to determine whether the original DNA of interest is enriched relative to other sequences. The method can be extended somewhat by using the protein 'handle' as a means to isolate other additional proteins that might be interacting with the target protein, thus some kind of picture can be laboriously assembled as to the general protein occupancy of a given promoter. The methodology however suffers from several flaws:- 1) one needs to identify a specific protein as a starting point, 2) one needs to have access to high quality antibodies specific for the target protein, 3) such antibodies differ in their affinity/specificity between individual batches and between protein targets, leading to potentially large variations in results between experiments, 4) ALL the DNA regions that are bound by the target protein throughout the cell will be isolated, along with a majority of the other proteins that it interacts with at these other DNA loci. Thus identifying DNA region specific binding proteins via follow on experiments such as mass spectrometry is impossible.

One clever approach to circumvent these problems has been to use small DNA molecules (oligonucleotides) complementary to the DNA region of interest. These have a single chemical handle attached (des-thiobiotin), so that when these oligonucleotides bind to their target DNA sequence they can be retrieved from the cell, along with those proteins that have remained bound to the target DNA. So far this method has only been used to examine proteins bound to DNA that is repetitive, thus artificially boosting the sensitivity of the approach. Also, one of the key steps in this method is denaturing (melting) the cell's DNA to allow the oligonucleotide to bind it. This represents a possible downside, in that some of the DNA:protein interactions we were hoping to capture may be lost during the process.

We propose to develop a method that circumvents these issues, delivering a reagent that will have applications for scientists throughout the world, in identifying in one go, all of the proteins that are associated with a specific, individual gene. Our approach is to use zinc finger proteins (or similar acting TALE proteins), that can be designed to bind virtually any DNA sequence, linked to multiple small tags (biotin acceptor peptides) that become modified in vivo, and that will provide an incredibly strong 'handle' for DNA isolation. We suggest that this approach, which does not require any DNA denaturation and can have multiple 'handles' to increase sensitivity, will provide a versatile and highly sensitive route for the isolation of individual DNA regions throughout the genome and the proteins that bind them. Additional routes will involve enhancing the 'handle' efficacy of these enzymes within the cell via increasing their degree of modification in vivo.

The ultimate aim will be to enable scientists to rapidly and confidently build up a picture of events in the cell as they happen at specific regulatory regions of DNA, during processes such as cell division and differentiation, stem cell renewal and disease onset and progression.

Technical Summary

Eukaryotic promoters represent loading points for a diverse range of proteins who's complement fluctuates during the course of e.g. cell cycle progression, DNA repair, cellular differentiation, induced de-differentiation (e.g. IPSCs) and programmed gene transcription and repression. The identification of a promoter's protein complement in any given scenario is currently a laborious task, usually ChIP based and requiring some pre-knowledge or guess work about which proteins might be present at the promoter in question. Such methods do not provide complete descriptions of composition at the promoter. While DNA capture based methods are available for the purification of proteins associated with specific genomic loci, they are perhaps not widely applicable to non-specialist labs and also may suffer from certain flaws inherent in the nature of the capture devices used in these instances, for example the use of des-thiobiotin and the requirement for a genomic DNA denaturation step prior to promoter targeting. We propose the development and characterisation of a more user friendly tool and protocol for the capture of proteins associated with specific promoter sequences. The essence is to combine/compare zinc finger or TALE based promoter specific targeting components, with peptide sequences that are readily biotinylated in vivo, in the form of gene specific fusion proteins that can be expressed in the cell of interest. Targeting of multple biotin acceptor sites in vivo to the promoter of interest will allow affinity capture of complexes mediated through the powerful streptavidin:biotin interaction. Further enhancements will include specific protease release of streptavidin bead-immobilised capture protein:DNA complexes. Our successful targeting of DNA methylation to the CDKN2B promoter using zinc finger fusion proteins, suggests this region as a suitable test case for the development of this methodology.

Planned Impact

The work contained in this application on the development of methods for the purification of proteins associated with single gene loci will improve the UK's profile and competitiveness in the following ways:

-Commerce: Gene expression mechanisms are an emerging area for pharmaceutical intervention. Drugs such as the DNA methyltransferase inhibitors 5-Azacytidine (Vidaza) & Decitabine and histone deacetylase inhibitors that target epigenetic mechanisms are in clinical use at King's College Hospital as well as elsewhere. It is envisaged that more drugs will be needed in the future and small companies as well as big pharma have interests in identifying and exploiting such targets. The market for such drugs was estimated to reach USD 4.1 billion by 2012 and the UK needs to have a presence in this important emerging area. The identification of total protein components associated with individual gene regulatory regions for example is therefore a requirement for further assessment and drug discovery processes. The ability in the longer term to identify the protein make-up associated with each gene promoter in the genome, and how these change with different cellular processes or states, will ultimately have an enormous impact on our understanding of virtually all aspects of cell biology. Such a program would ultimately benefit from the inclusion of Pharma companies such as Sangamo or Cellectis Biosciences, which have both proven track records in gene targeting as well as extensive libraries of gene specific reagents already in place.

-Health and wellbeing: Abnormalities in transcriptional programs for example, occur in all cancers and other diseases. These recruit epigenetic modifyers including DNMT1 and HDACs. We and others have used DNMT inhibitors to treat patients with MDS and combination drug trials are in progress. There is a need to understand how transcriptional/epigenetic mechanisms affect the expression of important regulatory genes in order to define more precisely which abnormalities are relevant in patients and how such abnormalities can be exploited by the development of new therapies. Complete characterisation of proteins associated with candidate disease-specific promoters would considerably enhance our understanding of all the key players involved in both the transcriptional and epigenetic fingerprints associated with the diseased versus normal state. Importantly, the UK is a leading innovator in drug trials, particularly in leukaemias. New, emerging epigenetics drugs are likely to be effective in treating many patients with a variety of diseases, with the aim of providing therapies that minimise side-effects. Intellectual property arising from this project will be identified, protected and rapidly exploited in consultation with KCL Business.

-Teaching. King's is a major UK research and teaching institution and has a strong history in the provision of high quality teaching to undergraduates and in post-graduate courses. Research-active staff, such as Drs Ford and Darling and Prof. Farzaneh, bring a wealth of knowledge and understanding of cutting-edge science to lectures at all levels. This is particularly important for lectures to specialist audiences in transcription, epigenetics, haematology and immunology. In addition, non-academic audiences at all levels, such as charity workers and fundraisers, as well as opinion and policy makers benefit from the ability to distil and explain exciting new research.

-Research. The development of state of the art research in such an exciting area and its publication in high impact journals has a huge impact on the profile of KCL and the UK's competitive position. This is evidenced by the willingness of leading international researchers to collaborate with this department and with the viral and immune gene therapy groups in particular.
 
Description The central objective of this study was development of site directed endonucleases. These studies and the expertise gained in site directed endonuclease activity, has underpinned our current work in collaboration with Cellectis Pharmaceuticals (France) for the production of allogeneic T cells expressing Chimeric Antigen Receptors (UniCAR T cells). This specific aspect of our work, in combination with work supported by BB/D014301/1 and BB/E005896/1) has contributed to a new contract with Cellectis (2016-2019) at a value of £11 million pounds, plus additional smaller contracts with Autolus UK, Ltd, and with Cell and Gene Therapy Catapult.
Exploitation Route Current collaborations with the Pharmaceutical industry are being actively pursued. These collaboration are likely to result in further projects with Autolus, a new biotech company set up by Syncona (Wellcome Trust). Further collaborations are being negotiated with another recent start up biotech LiFT and with two major pharmaceutical companies: Pfizer and Servier.
Sectors Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Extensive collaborations with the Pharmaceutical industry and inward investment from France into UK
First Year Of Impact 2013
Sector Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Societal,Economic

 
Description Cellectis: Production of viral vectors (primarily lentivirus), and gene modified cells, for clinical applications of cell and gene therapy 
Organisation Cellectis
Country France 
Sector Private 
PI Contribution Development and production of multiple lentivirus and retrovirus vectors for a range of clinical studies in collaboration with both academic and industry partners, the largest of which is the collaboration with Cellectis culminating in over £1.6 million of funding todate, plus a new contract for £10.1 million over the next 3 years.
Collaborator Contribution Provision of funding and know-how in specific areas (e.g. site directed endonuclease mediated inhibition of endogenous T cell receptors, in order to allow the generation of allogeneic (off-the-shelf) Chimeric Antigen Receptor (CAR) T cells for the treatment of malignant disease. This project is directly supported by BB/N003853/1 and assisted by the outputs from our previous BBSRC grants: BB/E005896/1, BB/D014301/1 and BB/K013785/1.
Impact The development of allogeneic CAR-T cells (referred to as UniCAR-T) for the treatment of malignant disease. There has also been substantial inward investment (over £11,000,000 between 2016 and 2019 from Cellectis alone) underpining further developments that we expect to culminate in substantially larger collaborations with other pharmaceuticaal companies (active discussions in progress with Cell Therapy Catapult, Pfizer and Servier). This collaboration has also resulted in a separate collaboration with a UK based start-up company - Autolus (reported as a separate collaboration).
Start Year 2015
 
Title Development of GMP compliant manufacturing strategies for the production of clinical grade viral vectors 
Description The production of viral vectors, in particular lentivirus and gamma-retrovirus in sufficient quantities and able to meet the regulatory standards of quality is particularly challenging. Using the technologies that were developed as part of our BBSRC supported projects, we have established a range of manufacturing, purification and concentration strategies that have enabled us to manufacture the largest number (academia or industry) of retroviral and lentivirus vectors for regulatory approved clinical trials in Europe. This extensive research and development programme has now culminated in over £15 million pound of income (2012 to 2019) for King's College London from overseas based companies. 
IP Reference  
Protection Protection not required
Year Protection Granted 2016
Licensed Yes
Impact The background manufacturing IP and know-how is licensed (non-exclusive) to Cellectis and to Cell Therapy Catapult. Discussions are in progress with other organisations in taking similar non-exclusive licenses.
 
Title The processes developed in the course of this study have directly contributed to the success of subsequent contracts with the Industry, including Autolus and Cellectis (biotech and pharmaceutical companies. 
Description We have developed procedures for the fast manufacture of retrovirus and lentivirus vectors in compliance with the regulatory requirements for clinical use (GMP compliant procedures). These highly optimised procedures have enabled the production of high titre vectors (about 50,000 million infectious units of vector) from relatively small scale cultures (circa 10 litres), with greater than 50% recovery (frequently in excess of 70%) and minimal quantities of contaminating proteins and nucleic acids. This knowhow has recently been licensed on non-exclusive deals to the industry (Cellectis) in contracts producing in excess of £15 million pounds of income over the next 3 years. 
IP Reference  
Protection Protection not required
Year Protection Granted 2016
Licensed Yes
Impact We have produced, for regulatory approved clinical trials, the largest number of lenti- and retroviral vectors in Europe. Each of the 4 BBSRC supported projects have contributed to this outcome. We are now extending this expertise with a view to similarly innovative manufacture of Adeno Associated Virus (AAV) manufacture for clinical use.