Production of HLA-null universal platelets from GMP banked human pluripotent stem cell lines

Each year 280,000 bags of platelets are given to patients in the UK. Platelets promote blood clotting after injury. Cancer therapy or heavy bleeding due to trauma or extensive surgery can reduce the number of platelets in the blood. Patients with a low number of platelets have an increased risk of bleeding (including in the bowels and brain). To prevent bleeding, platelets are given that are derived from blood donations.
Platelets cannot be stored in the fridge. Instead they are kept at room temperature, which means they can only be kept for 7 days. This makes stock management a challenge. Also, as with all donor-derived products (blood, organs etc) there is a small risk of transmission of infections from donors to patients. To minimize this risk, blood donations are screened for infections such as HIV or hepatitis. Finally, some patients develop an immune reaction against the "self" signature of platelets that would result in them destroying platelets from "random" donors. These patients are often women who have had several children or patients who repeatedly receive platelets (such as those undergoing chemotherapy). For these patients we use in England 15,000 bags of platelets per year that are specially matched. These bags are 3 times more expensive than unmatched bags. . The production of matched bags requires donors with the same "self" signature on the surface of their platelets. The "self" signature is called HLA Class I. In only 1/3 of cases a perfect match can be found and for 2/3 the platelets are at best a "near match". When "not perfectly matched" platelets are given they last shorter than "perfectly matched" ones which means they need to be given more often. Also, with not perfectly matched platelets it is more likely that patients will bleed and need to stay in hospital longer.
The option of producing platelets from banks of stem cells in the laboratory would allow for guaranteed supply, remove the risk of infection that comes with donor-derived platelets and would allow for better matching of platelets to patients.
Platelets are produced from megakaryocytes (MKs) in the bone marrow. We have in the last 4 years had a breakthrough in developing an efficient method to produce MKs from stem cells in the laboratory. In addition, we have made a devise that allows us to harvest platelets from those manufactured MKs. Our technology can be adapted to make platelets in such a way that they are safe to give to patients. Finally, we are able to delete the "self" signature from the surface of the platelets by modifying the DNA of the stem cells. These "anonymous" platelets would not be recognized by the immune system and would represent a "perfect" match for patients who cannot receive platelets from "random" donors. Crucially, they would not trigger an immune reaction in anyone.
We plan to use our technology to produce platelets for a first trial in humans in the next 5 years. We will use stem cells that can be used to produce products to give to humans. We call these cells "clinical-grade". We will identify which of these "clinical grade" stem cells are very efficient at producing platelets. Subsequently, we will remove the "self" signature from these clinical-grade stem cells to make them a "universal" match. We have already done this for stem cells that are not "clinical-grade" and have shown that taking away the "self" signature does not impact on the quality of the platelets the stem cells make. We now need to confirm this in "clinical-grade" stem cells before we can start producing "clinical-grade" "universal" platelets for a human trial.

WP1. Generation of HLA Class I null lines
Three hPSC lines with the best MK output will be transfected with each of the paired guide RNAs and Cas9 nickase cloned into 2 vectors containing either GFP or dTomato. Double-positive single cells will be sorted and subsequent colonies will be screened for HLA Class I expression by flow cytometry. Pluripotency will be confirmed in standard assays. This method has been validated on research hPSC lines in our lab.
WP2. Determine off-target rate.
The whole genome sequence of each HLA null line will be compared to the parent line to ascertain the off-target rate. This will be supported by the HipSCi initiative.
WP3. FoP MK production and phenotyping.
Using validated batches of lentiviral vectors from the CiC project we will FoP parent and HLA null lines to compare the dynamics of MK differentiation and proliferation, the potential to freeze-thaw the MKs and their ability to form proplatelets. RNA sequencing will be used to confirm whole genome expression levels are the same in null MKs compared to HLA expressing ones.
WP4. Platelet output and functionality.
We will seed mature FoP MKs from the parent and null lines in our bioreactor to compare the efficiency of platelet production. Platelet functionality will be assessed in a range of assays including adhesion and spreading, aggregation, thrombus formation in collagen-coated flow chambers and ultrastructure analysis by electron microscopy.
WP5. In vivo platelet survival studies.
NSG mice will be injected with 40-240x10^6 (equivalent to a therapeutic dose of platelets [3x10^11] in adult patients) in vitro-produced "null" or "wild-type" platelets or donor-derived platelets. The survival in circulation will be monitored by flow cytometry Subsequently, the IgG fraction of patient plasma samples containing polyclonal HLA antibodies will be injected into NSG mice and platelet survival studies repeated to demonstrate a survival advantage of "null" over "wild-type" platelets.

The impact of the proposed research project will be on 3 main groups: 1. NHS and patients, 2. Academic beneficiaries and 3. Commercial partners and UK economy.
1. NHS and patients.
The potential advantages of in vitro- over donor-derived platelets are numerous: no risk of transmission of donor-acquired infections, a continuous supply and in particular, better matching. The R&D directorate of the NHSBT (the likely future end user/distributor of in vitro-produced blood cells in England) holds an annual stakeholder meeting that comprises patient and religious groups, industry representatives and blood product prescribers. The programme of research proposed in this application has received strong support at the 2013 and 2015 meetings. This clearly indicates that stakeholders see benefit in in vitro-produced blood products. A market research and customer survey commissioned by CGT identified improved safety, supply and matching as the main drivers for the potential purchase of in vitro-derived platelets by health providers. This engagement with both patients and end users will continue during the lifetime of this grant and will be key to the future promotion of the product and commercialization. The NHSBT will provide practical and financial support for these meetings and events.
The creation of a "universal" platelet product will have a major impact on the provision of platelets to refractory alloimmunised patients who face longer hospital stays (associated with higher costs to the NHS), an increased risk of bleeding and decreased survival. It will also benefit patients who are likely to need repeated prophylactic platelet transfusions such as patients with malignancies or haematological disorders who receive about 50% of all platelet transfusions.
2. Academic beneficiaries.
The main impact of the scientific strategy used in the current proposal is likely to be in the field of regenerative medicine. A nationalnetwork of key hubs has been created through the UK Regenerative Medicine Platform initiative. Dr Ghevaert has an ongoing collaboration with two key hubs in this field, namely, the Biochemistry of the Niche Hub (Prof S Forbes, Edinburgh) and the Pluripotent Stem Cell Hub (Profs P Andrews and A Smith, Sheffield/Cambridge). Through contact with these hubs, dissemination of the knowledge gained within this project can be carried out effectively and we would therefore envisage that this project will have an impact beyond the production of platelets in vitro.
3. Commercial partners and UK economy.
A robust manufacturing protocol for the generation of platelets from hPSCs has a global market of $300m. Our long-term intention is to translate the 3 key technologies of FoP MKs, bioreactor with 3D functionalized scaffolds and HLA null lines into a manufacturing process for human use. Our product is expected to obtain a large share of the market due to advantages in providing consistent cell supply from a validated, pathogen-free source and the generation of "universal" cells. Based on figures from other hPSC regenerative medicine licensing deals, the ROI is expected to be at least 10-fold. The novel platelet production process will therefore have wider benefits, such as providing job opportunities and revenue to the UK economy.