Engineering novel synthetic factors to reprogram cell identity
Lead Research Organisation:
University of Edinburgh
Department Name: Sch of Biological Sciences
Abstract
Converting human cells such as skin to other specialised cells such as neurons, blood, heart, liver and pancreas will make it possible to regenerate any damaged or diseased tissue and develop personalised therapies. It is now possible to reprogram biopsied human cells to become induced pluripotent stem cells (iPSCs) in the laboratory. These iPSCs are pluripotent, which means they can potentially give rise to all cell types of the body. This is a major achievement as it overcomes the limitations of using of embryonic stem cells. However, the current technology uses genes called transcription factors (TFs) that are highly inefficient in reprogramming adult cells and they have been associated with tumours, making this technology quite risky and unreliable to use in human patients. Thus, we urgently need novel methods to reprogram cells in an effective and safe way to generate the quantity and the quality of cells required for therapeutic purposes.
We are building a multi-disciplinary research group, bringing together expertise from Stem Cell research, Synthetic Biology and Translational research. The goal of our research is to engineer novel reprogramming factors that can overcome these limitations. To achieve this, we plan to define which parts of natural TFs that are essential for reprogramming. Then, we will use these parts as building blocks to design novel factors that are more potent in reprogramming. We will initially focus on generating iPSCs, but we will use the same strategy to generate other cell types such as neurons. Ultimately, we aim to translate this technology to an effective method in the clinic.
It is our expectation that custom-made reprogramming factors will bring regenerative medicine one step closer to reality.
We are building a multi-disciplinary research group, bringing together expertise from Stem Cell research, Synthetic Biology and Translational research. The goal of our research is to engineer novel reprogramming factors that can overcome these limitations. To achieve this, we plan to define which parts of natural TFs that are essential for reprogramming. Then, we will use these parts as building blocks to design novel factors that are more potent in reprogramming. We will initially focus on generating iPSCs, but we will use the same strategy to generate other cell types such as neurons. Ultimately, we aim to translate this technology to an effective method in the clinic.
It is our expectation that custom-made reprogramming factors will bring regenerative medicine one step closer to reality.
Technical Summary
It is currently feasible to convert adult cells to other cell types by forcing the expression of transcription factors (TFs) in the laboratory. The key challenge is to translate this reprogramming technology to an effective method in the clinic. The prevailing strategy in the field relies on using "natural" TFs to inefficiently and partially perform this "unnatural" task. I propose that TFs have not evolved far enough to convert fully differentiated cells to other cell types.
My goal is to engineer novel reprogramming factors that overcome these limitations by exploiting the molecular features of TFs that I have previously uncovered. I will primarily use the conversion of human fibroblasts (huFibs) to induced pluripotent stem cells (iPSCs) using Oct4, Sox2, Klf4 and c-Myc (OSKM), as a paradigm for TF-mediated reprogramming.
I aim to define the molecular features that impart OSKM a pioneer activity, allowing access to silent genes within closed chromatin. To this end, I will combine a novel protein printing technology and biochemical assays with a suite of next-generation-sequencing based technologies to extract key mechanisms underlying cell fate conversion.
I aim to map the functionally-minimal OSKM subdomains that are essential in reprogramming. To achieve this, I will employ synthetic DNA assembly to dissect the minimum OSKM subdomains that are sufficient for converting huFibs to iPSCs.
I also aim to build a protein design framework to generate novel reprogramming factors. By using a novel tagging technology, I will append defined chromatin-modifying domains to the OSKM DNA-binding domains and screen for optimized reprogramming activities.
My findings will expand the reprogramming factors repertoire, which is currently limited to natural TFs, to control cell fate with improved efficiency and high fidelity. Developing this technology will lead to significant advances towards repairing tissues and organs in regenerative medicine.
My goal is to engineer novel reprogramming factors that overcome these limitations by exploiting the molecular features of TFs that I have previously uncovered. I will primarily use the conversion of human fibroblasts (huFibs) to induced pluripotent stem cells (iPSCs) using Oct4, Sox2, Klf4 and c-Myc (OSKM), as a paradigm for TF-mediated reprogramming.
I aim to define the molecular features that impart OSKM a pioneer activity, allowing access to silent genes within closed chromatin. To this end, I will combine a novel protein printing technology and biochemical assays with a suite of next-generation-sequencing based technologies to extract key mechanisms underlying cell fate conversion.
I aim to map the functionally-minimal OSKM subdomains that are essential in reprogramming. To achieve this, I will employ synthetic DNA assembly to dissect the minimum OSKM subdomains that are sufficient for converting huFibs to iPSCs.
I also aim to build a protein design framework to generate novel reprogramming factors. By using a novel tagging technology, I will append defined chromatin-modifying domains to the OSKM DNA-binding domains and screen for optimized reprogramming activities.
My findings will expand the reprogramming factors repertoire, which is currently limited to natural TFs, to control cell fate with improved efficiency and high fidelity. Developing this technology will lead to significant advances towards repairing tissues and organs in regenerative medicine.
Planned Impact
Our research will result in a significant impact on the following areas:
Academic Research: The work proposed in this research will pioneer the use of engineered factors to overcome a barrier in the inefficiency of cellular reprogramming. There is considerable research interest from investigators in stem cells, regenerative medicine and synthetic biology to use our iPCs cell lines, our library of novel reprogramming factors, and a wealth of genome wide data (see "Academic Beneficiaries"). I will disseminate our findings to a wider academic audience through publications in journals from relevant disciplines and by presenting my work to wider academic audiences.
Healthcare: Our work will help meet the worldwide need to overcome the current limitations in translating the current cellular reprogramming and iPS technology to effective methods for drug discovery and regenerative medicine. I will collaborate with clinical scientists from the closely located Royal infirmary of Edinburgh to use our approach and generate patient specific cell types for therapeutic purposes and drug discovery. These represent cost-effective methods, which will ultimately increase healthcare efficiency and decrease the burden on the National Health Service.
Technology development: The University of Edinburgh is strong in developing new technologies for high-throughput discovery platforms (Manfred Auer, Mark Bradley). In collaboration with Edinburgh Genome Foundry, we are in an excellent position to develop an automated platform to engineer novel reprogramming factors using Synthetic DNA assembly. Our work will also directly inform ongoing efforts in the UK Centre for Mammalian Synthetic Biology Research in Edinburgh (BBSRC/EPSRC/MRC) to build artificial chromosomes, mammalian parts, and artificial gene circuits for regenerative medicine and industry.
Biotech industry: Engineering novel reprogramming factors will potentially lead to patented technologies, which will be of commercial value and directly benefit the Biotech sector. I will seek the possibility of licensing any of the technologies and tools generated from this project by consulting with Edinburgh Research and Innovation (ERI). ERI is highly successful in the commercialization of the intellectual property generated from the University's research, through licensing technologies to existing companies and new University spin-outs. We will also develop protein microchips that will meet a widespread interest in diagnostics and drug discovery. I am in direct contact with Dr Lorraine Kerr and Alex Cassidy (Commercial Relations Executives), who have already helped me establish links with industry including ArrayJet to help develop this protein microchips technology. The CRM is based in the Edinburgh BioQuarter, which facilitate continuous dialogue with leaders in biotech industry.
Economy: Our work will contribute to building a skilled workforce for the future of regenerative medicine. This project will train one PDRA and one technician and will contribute to the training of undergraduate students who pass through the lab. I am also committed to training PhD students (currently I supervise one PhD student and sit on the committee of 4 PhD students from other groups).
The wider public: There is a deep-seated public interest in Stem Cell research and synthetic biology. I am committed to engage the public with my research to help inform and educate about the importance of our current research and future directions. I will also illustrate the highly rewarding aspects of research, to draw young talents to follow a career in science. In the CRM at Edinburgh, I am supported by an impressive communications and outreach team. Through this program, my work will have an ongoing impact on public understanding, engagement, and debates around synthetic biology, stem cell research, and regenerative medicine. I will use social media tools such as Twitter to reach the wider public around the globe.
Academic Research: The work proposed in this research will pioneer the use of engineered factors to overcome a barrier in the inefficiency of cellular reprogramming. There is considerable research interest from investigators in stem cells, regenerative medicine and synthetic biology to use our iPCs cell lines, our library of novel reprogramming factors, and a wealth of genome wide data (see "Academic Beneficiaries"). I will disseminate our findings to a wider academic audience through publications in journals from relevant disciplines and by presenting my work to wider academic audiences.
Healthcare: Our work will help meet the worldwide need to overcome the current limitations in translating the current cellular reprogramming and iPS technology to effective methods for drug discovery and regenerative medicine. I will collaborate with clinical scientists from the closely located Royal infirmary of Edinburgh to use our approach and generate patient specific cell types for therapeutic purposes and drug discovery. These represent cost-effective methods, which will ultimately increase healthcare efficiency and decrease the burden on the National Health Service.
Technology development: The University of Edinburgh is strong in developing new technologies for high-throughput discovery platforms (Manfred Auer, Mark Bradley). In collaboration with Edinburgh Genome Foundry, we are in an excellent position to develop an automated platform to engineer novel reprogramming factors using Synthetic DNA assembly. Our work will also directly inform ongoing efforts in the UK Centre for Mammalian Synthetic Biology Research in Edinburgh (BBSRC/EPSRC/MRC) to build artificial chromosomes, mammalian parts, and artificial gene circuits for regenerative medicine and industry.
Biotech industry: Engineering novel reprogramming factors will potentially lead to patented technologies, which will be of commercial value and directly benefit the Biotech sector. I will seek the possibility of licensing any of the technologies and tools generated from this project by consulting with Edinburgh Research and Innovation (ERI). ERI is highly successful in the commercialization of the intellectual property generated from the University's research, through licensing technologies to existing companies and new University spin-outs. We will also develop protein microchips that will meet a widespread interest in diagnostics and drug discovery. I am in direct contact with Dr Lorraine Kerr and Alex Cassidy (Commercial Relations Executives), who have already helped me establish links with industry including ArrayJet to help develop this protein microchips technology. The CRM is based in the Edinburgh BioQuarter, which facilitate continuous dialogue with leaders in biotech industry.
Economy: Our work will contribute to building a skilled workforce for the future of regenerative medicine. This project will train one PDRA and one technician and will contribute to the training of undergraduate students who pass through the lab. I am also committed to training PhD students (currently I supervise one PhD student and sit on the committee of 4 PhD students from other groups).
The wider public: There is a deep-seated public interest in Stem Cell research and synthetic biology. I am committed to engage the public with my research to help inform and educate about the importance of our current research and future directions. I will also illustrate the highly rewarding aspects of research, to draw young talents to follow a career in science. In the CRM at Edinburgh, I am supported by an impressive communications and outreach team. Through this program, my work will have an ongoing impact on public understanding, engagement, and debates around synthetic biology, stem cell research, and regenerative medicine. I will use social media tools such as Twitter to reach the wider public around the globe.
People |
ORCID iD |
Abdenour Soufi (Principal Investigator / Fellow) |
Publications
Costa A
(2022)
Repurposing the lineage-determining transcription factor Atoh1 without redistributing its genomic binding sites.
in Frontiers in cell and developmental biology
Malouf C
(2021)
miR-130b and miR-128a are essential lineage-specific codrivers of t(4;11) MLL-AF4 acute leukemia.
in Blood
Roberts GA
(2021)
Dissecting OCT4 defines the role of nucleosome binding in pluripotency.
in Nature cell biology
Soufi A
(2016)
Cycling through developmental decisions: how cell cycle dynamics control pluripotency, differentiation and reprogramming.
in Development (Cambridge, England)
Van Emmerik CL
(2020)
Ramified rolling circle amplification for synthesis of nucleosomal DNA sequences.
in Analytical biochemistry
Williams CAC
(2020)
Post-translational modification of SOX family proteins: Key biochemical targets in cancer?
in Seminars in cancer biology
Description | Early Detection Committee - CRUK-OHSU Project Award |
Amount | £250,000 (GBP) |
Funding ID | C65925/A26986 |
Organisation | Cancer Research UK |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 01/2019 |
End | 12/2021 |
Title | Cryo-EM structure of the Lin28B nucleosome core particle |
Description | Cryo-EM structure of the Lin28B nucleosome core particle |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | N/A |
URL | https://www.rcsb.org/structure/7NL0 |
Title | Dissecting OCT4 defines the role of nucleosome binding in pluripotency maintenance |
Description | Measuring Chromatin accessibility (ATAC-seq) during stem cell self-renewal, when endogenous OCT4 is knocked down and replaced with OCT4 wild-type or mutant |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | N/A |
URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE168141 |
Title | Dissecting OCT4 defines the role of nucleosome binding in reprogramming |
Description | ChIP-seq of OCT4 WT and mutants during early reprogramming |
Type Of Material | Database/Collection of data |
Year Produced | 2021 |
Provided To Others? | Yes |
Impact | N/A |
URL | https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE167632 |
Description | Molecular Dynamic modelling of nucleosome interarctions with Transcription factors |
Organisation | Royal Netherlands Academy of Arts and Sciences |
Department | Hubrecht Institute |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | By generating various mutants forms of transcription factors and measuring the biochemical affinity to nucleosomes in vitro and to chromatin in cells, we have identified important regions for nucleosome binding. |
Collaborator Contribution | Using Molecular dynamics modelling, our collaborators used our experimental data to model the three dimensional structure of transcription factors in complex with nucleosomes. |
Impact | Organized joint meeting. manuscript under preparation. |
Start Year | 2017 |
Description | Ramified Rolling Circle Amplification for nucleosomal DNA synthesis |
Organisation | Utrecht University |
Country | Netherlands |
Sector | Academic/University |
PI Contribution | We have used DNA synthesized by our collaborators to reconstitute nucleosomes in vitro |
Collaborator Contribution | To enable studies on native, genomic nucleosomes, our collaborators establshed an efficient and flexible method to produce nucleosomal DNA based on rolling circle amplification (RCA). |
Impact | manuscript under revision |
Start Year | 2017 |
Description | Reprogramming pancreatic cancer (PDAC) to discover early markers |
Organisation | Oregon Health and Science University |
Country | United States |
Sector | Academic/University |
PI Contribution | We aim to profile the chromatin landscape of PDAC during reprogramming to iPSCs using ATAC-seq and ChIP-seq for histone marks. Characterizing these chromatin-barriers would be particularly important as we reason it will reveal the essential epigenetic transition from the PanIN to PDAC stages. |
Collaborator Contribution | Our collaborator will reprogram a large number of PDAC tissue from human patients to iPSC to model cancer progression in mice. |
Impact | CRUK-OHSU project award |
Start Year | 2019 |
Description | Transcription factors lineage specificity during reprogramming |
Organisation | Hebrew University of Jerusalem |
Department | Hebrew University Hadassah Medical School |
Country | Israel |
Sector | Academic/University |
PI Contribution | We are using wide variety of genomic approaches including ChIP-seq, RNA-seq, Mnase-seq and ATAC-seq combined with bioinformatics to define how various combinations of transcription factors engage the genome and establish specific cell identity during reprogramming adult cells to embryonic stem cells and trophoblast cells |
Collaborator Contribution | Our collaborators have developed various methods of reprogramming to trophoblasts and embryonic stem cells and have carried out RNA-seq and ATAC-seq at different stages of reprogramming. |
Impact | Manuscript under preparation. two grants submitted |
Start Year | 2016 |