An industrial standard cancer drug development platform using human induced pluripotent stem cell technology

My career intent is to become an independent research leader working on real patient problems at the interface with industry, reducing and replacing animal use through non-animal technologies and solutions. Home Office figures tell us that more than 183,000 animal procedures take place every year in cancer research in the UK. In addition, when we study figures from the Home Office and major cancer research funding charities more than 9000 mice are being used every year to study leukaemia. Mice are being used by researchers to grow patient-leukaemia cells, to study their biology and to develop better medicines. Such animal procedures can reach moderate severity; these mice often show signs of distress such as weight loss, hunched back and dishevelled fur-coat.

Cancer research and cancer drug development have been identified by the NC3Rs as a strategically important area to bring about animal replacement. Currently, children's cancer treatment comprises a 2-3 years regimen with severe side effects such as death due to infections, kidney damage, heart problems and bone destruction. Hence better medicines are urgently needed and justifiably there is a lot of research being carried out in this area. Research into childhood cancer is urgent and important and this field will continue to expand in the immediate and distant future.

Improved medicines can be developed only by researching the biology of cancer cells taken directly from patients. Such patient-derived cells faithfully replicate leukaemia biology and are indispensable in scrutinising leukaemia cell-characteristics. This in turn is essential in developing better medicines. Although leukaemia is a very aggressive disease such patient-derived cancer cells rapidly die outside the body. Hence currently these cells can be grown and studied only in mice-models. My aim is to develop a technology that will allow researchers and the pharmaceutical industry to study leukaemia cells and develop better medicines without having to use mice.

I have developed a platform whereby patient-leukemia cells can now be grown in a petri-dish with the help of leukemia-supporting human bone-marrow cells. Given these bone-marrow cells do not survive long in the petri-dish they have to be constantly sourced from different individuals. Thus they behave differently across different laboratories resulting in high variability and inconsistent drug development data. To address this problem, I have developed a novel solution by which we can now use stem cells to generate such human bone marrow cells with uniform biology. These bone-marrow stem cells will provide an environment whereby patient-leukaemia cells can be grown and their biology can be studied without having to use mice. This means that dependence on animals in children's cancer research will reduce as fewer mice will be needed to develop better cancer medicines. In addition my non-animal solution is applicable in several other types of cancer affecting children and adults. I will establish an industrial standard biological test which will have a defined workflow pipeline and quality control programmes. I have established links with Cancer Research Technologies and working with a multidisciplinary team I will ensure that my test demonstrates high consistency at all times across different laboratories nationally and globally.

In summary I will engineer a technology that will help scientists and pharmaceutical companies to test and formulate new medicines for cancer without having to heavily depend on animal-models. The defining characteristic of my platform will be setting it up at a standardised industrial level with validated protocols and authenticated quality control measures. The deliverables of this fellowship will replace and reduce animal procedures in cancer research and will also reduce drug failure rates which is currently a big problem in the pharmaceutical economy.

Although leukaemia is a very aggressive disease patient-derived cells do not survive outside the body. Hence more than 9000 mice are used annually in leukaemia research. Animals are used to expand leukaemia cells and for drug development purposes. Cell lines are used for pre-in vivo target validation of cancer therapies. Given cell lines do not represent the complexity of disease presentation this pipeline results in high in vivo drug attrition rates.

I have developed a clinically relevant ex vivo platform which expands primary leukaemia cells using human bone marrow mesenchymal stem cells (BM-MSC). However, BM-MSC can be maintained for only short periods and need to be constantly sourced from different individuals compromising reproducibility across different laboratories. The aim of this fellowship is to develop a uniform source of BM-MSC through induced pluripotent stem cell (iPSC) technology. iPSC are characterised by indefinite in vitro proliferation and ability to differentiate into a wide repertoire of cell types. Human BM-iPSC will be engineered through a virus free, integration free RNA vector using re-programming factors OCT4-SOX2-KLF4-GLIS1. Flowing deep phenotyping BM-iPSC lines will be differentiated into an array of BM-feeder cell types. The overall aim is to establish an industry standard non-animal technology with defined standard operating procedures and quality assurance programmes. Existing partnership with a multidisciplinary team in industry and academia will further ensure that rigorous standards are reached. Following this, further commercial collaborations will be sought though NC3Rs CRACKIT and Innovate UK.

Widespread endorsement will reduce in vitro to in vivo drug attrition rates in the drug development pipeline. My platform will replace and reduce animal procedures by expanding primary leukaemia cells ex vivo and by acting as a pre-in vivo screen to filter off failed candidates from going forwards with in vivo validations.

My fellowship will benefit the 3Rs, scientific, pharmaceutical and healthcare community. My 3Rs solution will reduce animal dependence in cancer research whilst developing an industry standard preclinical platform with defined standard operating procedures and rigorous quality control measures. This platform will facilitate development of improved treatments for cancer patients.

Cancer drug development is associated with the highest drug attrition rates. Thus cancer research needs preclinical models with higher in vivo and clinical predictability. 2015 Home Office annual returns state that 110,316 animal procedures were carried out in basic cancer research and 73,601 animal procedures were carried out towards translational cancer research. According to the 2015 Home Office returns and funding statistics from major cancer research funding charities more than 9000 mice are used annually in leukemia research. In addition most of these procedures can reach moderate severity and are associated with causing significant distress to the animals in form of weight loss, huddling, fur-coat ruffling and increased white cell counts.

A major difficulty is that although leukaemia is an extremely aggressive disease patient-derived cancer cells rapidly die once removed from the body. Hence the only way to study these cells is through use of animal models. Animals are routinely used to amplify patient material, investigate cancer biology and perform drug development studies.

I have engineered a novel human cell-based technology whereby primary leukaemia cells can be amplified ex vivo whilst retaining clonal abundance and leukaemia self-renewal (Pal et al, Leukemia, 2016). This preclinical tool uses human bone marrow mesenchymal stem cells (BM-MSC) as leukaemia supporting niche-feeder cells. However, the BM-MSC can only survive for short periods in culture and hence have to be constantly sourced from different individuals thereby compromising reproducibility and consistency across different laboratories. Using my PhD expertise (Pal, D., Moad, M. et al, European Urology, 2013) I will engineer a defined source of human BM-MSC through induced pluripotent stem cell (iPSC) technology. My non-animal technology will amplify primary leukaemia cells thereby reducing dependence on animal models to study leukaemia biology. In addition my 3Rs solution will derive robust single agent and drug combination indices. This strategy will replace and reduce animal procedures by filtering out unsuccessful drug candidates and allowing only optimal single agents and drug combinations to go forward with in vivo validations. The high clinical predictability of my model means that it will reduce in vivo drug attrition rates.

Existing links with the pharmaceutical sector such as Cancer Research Technology will ensure that my human cell-based preclinical technology meets industrial standards. My key objective is to engineer a well-defined biological test system using human iPSC reprogramming. In order to meet the impeccable industrial standards and promote widespread academic and industrial endorsement these human iPSC will be engineered through a state-of-the art genomic integration free, viral footprint free and animal-feeder free technology.

The human bone marrow serves to be the niche for various types of adult and childhood malignancies such as lymphomas, sarcomas, bone tumours and secondary cancers of the prostate, breast, lung and thyroid. iPSC can be differentiated into a wide variety of cancer-niche cells and a defined human iPSC-derived biological test system will thus have wide applications in cancer research. Rigorous standardisation measures will ensure consistency and reproducibility so that my non-animal technology is actively endorsed by laboratories nationally and internationally, significantly reducing and replacing animal procedures whilst addressing drug attrition rates and developing better treatments for cancer patients.