Universal cells to overcome HLA barriers in regenerative medicine

Over 6400 people are waiting for solid organ transplants in the UK and one major issue is the need to match donors and recipients to prevent the immune system rejection. Recent developments suggest that 'genome editing' might allow us to produce 'universal' stem cells that can be used to repair or regenerated diseased organs. We are interested in applying the technology to pluripotent stem cells that could then be used to generate many different types of tissue. The process involves using tools called CRISPR-Cas9 to change the cells' DNA to make them cells invisible to the immune system. This is done by disrupting the DNA code in cells that produces surface molecules called human leukocyte antigens (HLA). These are like 'flags' that identify cells as host or 'self' and each cell can display up to 12 different very different flags from two different classes or structures.
The project will tests strategies to remove the flags. It may be neccessary to replace them with a single 'neutral' flag in case the immune system becomes alarmed that all the flags are missing. We will compare different routes to efficiently and safely manufacture 'universal cells' which will then be tested in projects already underway in the regenerative medicine hubs. In case of unwanted side effects, a safety gene will also be included to allow cells to be removed if necessary.
We will deliver the project in four stages with the bulk of the first stages to be undertaken at UCL and UoC. There is additional involvement of UKRMP hubs in Edinburgh and Cambridge in the final year. Four 'work packages are envisaged:
1. To develop and test the molecular tools that will be used to cut and edit DNA that codes for 'flags' on cells
2. Test and grow human 'stem' cells that can be later used to produce large numbers of liver cells or immune cells that help clean up cell debris. The tools developed in (1) will be used to modify these stem cells in the lab to see if they can be made 'universal'
3. Apply state of the art sequencing and computer modelling to check if the stem cells have been modified at gene sites other than those being targeted.
4. Testing the inclusion of a 'safety' switch in case cells cause side effects or get infected with viruses.
5. Testing of universal stem cells to see if they can evade immune responses in mice.
By the end of the project, we anticipate being in a position to be able to produce 'universal' pluripotent stem cells that could then be applied to various applications in early stage clinical trials.

Universal human pluripotent stem cells (HPSC) will be generated using CRISPR mediated genome modification and their stealth, or ability to evade immunological responses, investigated. The project will be delivered in four workpackages (WP) over a three year period at UCL and UoC, with additional contributions from existing UKRMP hubs.
1. Methodologies to eliminate HLA, addressing 'missing self' and safety risks.
CRISPR sgRNAs will be identified and validated for efficient HLA class I and II disruption and delivered using our hybrid lentiviral-CRISPR vector system. Transgenes encoding a replacement non-polymorphic HLA-G and/or a dimerising suicide gene will be coupled to CRISPR delivery, with Cas9 mRNA delivered transiently by electroporation. Alternative deamination base-editors will also be investigated to mitigate against the risk of translocation events in cells undergoing multiplexed editing.
2: Human pluripotent stem cell modification and differentiation
Established HPSC lines (embryonic or induced) will be evaluated for their ability to support differentiation to hepatocytes or macrophages. Lines with optimal capacity will be selected for generation of universal hepatocytes (devoid of HLA lass I) or macrophages (devoid of HLA class I & II). In vitro sensitivity to anti-HLA antibodies will be examined, as well cellular responses mediated by healthy donor mismatched T and NK cells.
3: Characterization of genotoxicity risk
Next generation sequencing will be used to quantify of on and off target activity. Sites of possible off target activity will be informed by Digenome analysis. Predicted translocations will be quantified by targeted qPCR and karyotypes assessed, including by FISH where possible.
4: Function and stealth of modified cell lines
Persistence and immune-evasion of universal HSPC derived teratomas, macrophages and hepatocytes will be examined in humanised immunodeficient mice using healthy donor T and NK cells.

This project will bring together expertise in translational aspects of regenerative medicine and genome engineered to address the immunological challenges associated with generated allogeneic, non-HLA matched, cell therapies. By working with all three of the existing UKRMP hubs, the project aims to deliver reagents to feed projects already underway, thereby maximising exploitation of previous investments while building new relationships and capacity in technologies with far reaching potential. Specifc impacts include:

i. A deeper understanding of immunological issues relevant to tissue transfer. The ability to overcome the need for HLA matching could impact broadly on transplantation medicine, the work will inform strategies evading cellular and humoral immunity. The techniques will be of particular interest to groups developing cellular immunotherapies where the role of HLA is critical.
ii. Successful generation of universal HPSC would create immediate impact in the Phase 1 setting as clinical trials are supplied with a ready source of cells. Understanding and overcoming hurdles such as the triggering of apoptosis pathways that can limit stem cell yields, or the 'shut-down' of gene expression over time are of generic interest.
iii. The application of CRISPR/Cas9 and newer base editing technologies are of interest across genetic therapies, with a number of early phase trials being planned. There will be impact not only of clinicians and researchers, but also regulatory bodies and ethics committees.
iv. Impact on industry/Pharma through proof of concept applications and policymakers/healthcare commissioner addressing unmet medical needs

A pathways to impact summary is attached.