An inducible CRISPR/dCAS9 strategy for directed differentiation of pluripotent stem cells

Cell therapy such as haematopoietic stem cell transplantation and red blood cell or platelet transfusion are used to treat many diseases of the blood and immune systems but these treatments are highly dependent on a limited supply of healthy donors. Also, despite extensive screening of donated blood, there is always a chance that the patient could get an infection from the donated cells. To solve these problems there has been a lot of effort to generate blood cells in the laboratory from a limitless and infection-free source. One such source is human pluripotent stem cells (hPSCs) that can be grown indefinitely in the lab as stem cells and then, under defined conditions, can be differentiated into any cells type including blood cells. However, the efficiency of this process is very low and it has not been possible to generate fully functional blood cells. Several studies have shown that the production and function of specific cell types can be improved by "directing" the cells into specific cell types by activating the expression of factors that act as molecular switches to turn on the expression of genes that are required for a particular cell function. These studies have depended on introducing transgenes into the cells using plasmid or lentiviral vectors but they result in abnormally high levels of gene expression. We propose to use a novel strategy that results in the activation of the cells' own genetic networks and results in more physiological levels of gene expression.
We will first compare the gene profile of hPSC- with adult-derived blood cells and this will identify the genetic switches required for the production of fully functional adult-like blood cells as well as novel markers that can be used to track the production process. We will then test whether activation of these genetic switches in differentiating hPSCs will result in the improved production of blood cells from hPSCs. To do this we will use a novel synthetic biology strategy whereby our chosen factors can be turned on using small molecular tools known as guide RNAs (gRNAs). gRNAs are designed to bind to the region of the genome that regulates the expression of a particular gene(s) and, together with a protein complex, known as CAS9-SAM, the expression of the gene can be activated. One of the unique aspects of our proposal is that we have designed and tested an hPSC line in which activation of the protein complex can be induced upon addition of a drug. This novel iSAM strategy represents a significant advance in the field as it makes the approach amenable to the activation of multiple factors at once which is likely to be required for the generation of fully functional blood cells. We will first use our iSAM strategy to activate single and combinations of genetic factors and monitor their effects on the production of blood cells. This will generate an experimental pipeline that will then allow us to screen all the genes in the genome in one single experiment. Cells that we produce by programming with genetic factors will be tested using methods such as flow cytometry to assess the presence of markers on the cell surface, colony-forming assays to monitor blood cell progenitors and single cell RNA sequencing to analyse the transcriptional consequences of the programming process. This project will provide a better understanding of genetic factor programming and could provide a route to producing cells for the treatment of patients with blood cell disorders. Our unique iPSC line carrying the iSAM complex will be shared with researchers studying the programming and production of other therapeutic cell types such as dopaminergic neurons to treat Parkinsons disease or pancreatic beta cells for Diabetes. Our strategy offers significant advantages over the classical transgenic technologies that are fraught with technical difficulties such as gene silencing and insertional mutagenesis that would raise significant safety concerns in the clinic.

The ultimate aim of this proposal is to develop a strategy that will allow the production of fully functional therapeutic haematopoietic stem/progenitor cells (HSPCs) from human pluripotent stem cells (hPSCs) in vitro. We will first compare the gene expression profiles of HSPCs that are generated from hPSCs in vitro with umbilical cord blood and adult HSPCs using single cell transcriptomics. This will identify genetic factors and cell surface markers that are involved in key cell fate decisions and that are unique to and/or deficient in hPSC-derived haematopoietic cells. We will drive the differentiation of hPSCs into haematopoietic lineages by manipulating the expression of critical transcription factors and we will use novel cell surface marker combinations to track the differentiation process with more precision. One of the unique aspects of our proposal is the novel technology we have developed for gene activation. We have designed and tested a DOX-inducible dCAS9/SAM-mediated gene activation strategy (iSAM) that allows for the activation of endogenous gene expression using gRNAs directed to the transcriptional start site of chosen genes. We have generated an iPSC line that carries the iSAM transcriptional activation complex and have optimized transfection protocols to deliver gRNAs to hPSCs. Integrated of iSAM into the AAVS1 locus will ensure stable expression and avoid silencing in differentiating hPSCs. Experiments using candidate genes will establish an experimental pipeline that will then be used to screen a genome-wide gRNA library. We will screen for gRNAs directed to genes associated with endothelial to haematopoietic transition (EHT), one of the most significant and tractable processes in haematopoiesis, using the SOX17-mCherry/RUNX1C-eGFP double reporter hESC line. This will be the first genome wide screen for genes associated with EHT and the strategy will be applicable to many other developmental processes.

This research has the potential for impact on the UK economy through commercial licensing of technologies to modulate gene expression and on the production of therapeutic cell types. In the longer term, there could be a societal impact by improving the lives of patients and therapeutic practice by the supply of infection-free, immunologically compatible therapeutic blood cells as well as other therapeutic cell types. The primary beneficiaries of this research in the shorter term will be commercial companies through licensed laboratory procedures which could realise the production of therapeutic blood cell types from human pluripotent stem cells (hPSCs). We have established a collaboration with an industrial partner, Plasticell and were awarded funding from Innovate UK funding (2017-2019). The aim of that project is to use our reporter hPSC lines in their unique Combicult system to simultaneous screen of large numbers of extrinsic culture conditions in the differentiation of blood cell progenitors. Complementing the current proposal, we can envisage that the identification of intrinsic genetic factors involved in the differentiation process will have significant impact on the screening of extrinsic factors and vice versa. Wider impact could also be made through researchers in associated fields by using the laboratory techniques to develop other therapeutic cell types e.g. neuronal, liver or pancreatic cells. In the mid to long term, health services, health care professionals and patients worldwide could benefit from the consistent supply of infection-free cells and patient-specific iPSCs that would be immunologically compatible.

Cell therapies such as haematopoietic stem cell (HSC) transplantation and red blood cell (RBC) or platelet transfusion are used to treat a wide range of haematological and immune disorders but these treatments are reliant on a consistent supply of high quality donor cells. Producing therapeutic blood cell types from human pluripotent stem cells (hPSCs) could provide a consistent supply of infection-free cells and patient-specific iPSCs would be immunologically compatible. If fully haematopoietic stem and progenitor cells can be produced at research grade over the next 3 years following this the processes it could be translated into the clinic to treat patients with blood cell disorders and cancer. Thus in 3-5 years, one of the most likely impacts will be through the licensing of laboratory procedures to commercial companies. Translation into the clinic would take at least a further 3 years while our research grade protocols procedures are translated into clinical-grade procedure. The ability to produce adult-like HPCs is likely to improve the production of mature blood cells such as red blood cells and platelets. Currently hPSC-derived RBCs are immature and this has been a major hurdle in the Novosang consortium's plan to take a hESC-derived RBCs to a first-in-man clinical trial (www.novosang.co.uk).The ability to produce large numbers of blood cells and their progenitors in the lab would allow other experimentation of these cells which has been limited due to the lack of numbers such as proteomic analysis and it provides a model system to study complex transcription factor networks and their regulatory element that, to date, has relied on transgenic animal models that are costly and ethically challenged. The novel gene activation technology described in this proposal will have immediate impact on researchers working in transcription factor programming of other cell lineages and in cell reprogramming. We will share our results as well as our tools and reagents with local colleagues, Profs Steve Pollard and Stuart Forbes who are planning to assess its use in the programing of cells to neuronal and liver cells, respectively. The ability to generate these cell types could have impact on the health of patients with neurodegenerative and liver diseases.