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Reprogramming the for a new generation of cures

The development of new and effective treatments with the potential to cure, By Dr Alex or at the very least ease patient suffering, has been the driving force in Davenport, Dr Tonya Frolov biomedical research. Although tremendous progress has been made over the and Dr Mark Kotter last few decades, cell therapies today are expensive and not particularly scalable. The development of new drugs has not kept up with the progress in biology. The majority of clinical trials fail because the drugs are either toxic or they do not work in human diseases. Human biology often proves to be distinct and sufficiently different from animal models and associated cell lines used for drug development. Widespread access to reliable human cells is needed to replace suboptimal disease and drug response models the industry currently relies on to enable a new generation of medicine. Human cells derived from induced pluripotent stem cells (iPSCs) provide an excellent platform for disease modelling and a scalable source of starting material for cell therapies. Cellular is able to overcome the traditional bottlenecks of their application: lack of consistency, purity and scalability. This synthetic biology paradigm involves activating a new cell type programme directly, skipping the usual intermediate steps that occur during development. As a result, stem cells convert directly into any desired mature cell type. We are now at a dawn of a new age in cellular biology combining in silico and techniques to revolutionise drug discovery and the development of novel cell therapies.

Drug Discovery World Spring 2020 1 Therapeutics

ver the past several decades we have control. But given the lack of reliable human cells, made tremendous progress in biomedical academics and pharmaceutical companies continue Oresearch. Scientists are now able to turn to depend on the current models due to a lack of individual genes off or on and edit them with viable alternatives. CRISPR and assess the expression of many thou- Animal models still form the backbone for the sand genes, even on a single cell level. Therapies current drug discovery workflow, despite evidence have progressed from small molecules to biolog- that their cumulative harm and cost to humans ics, with the first CAR- therapies providing a outweigh the benefits5. Apart from species differ- glimpse of a future where cancer could be eradi- ences, many diseases, such as Alzheimer’s, are lim- cated. Despite significant scientific breakthroughs ited to humans. Hence, creating disease models and technological advances, today drugs are more often involves genetic manipulation of animals or likely to fail in clinical development than 40 years the administration of toxins, with the aim of induc- ago. Inflation-adjusted Research and Develop- ing comparable disease . Instead of pre- ment (R&D) cost per drug has increased nearly dicting human response to therapies, animal mod- 100-fold between 1950 and 20101. This mismatch els can lead to misleading biomarkers and safety is concerning. Differences between human biology studies. As a consequence, otherwise effective and the models used for drug discovery are an drugs can be abandoned and resources are diverted important contributor to the problem. One of the away from more reliable models5. These issues are biggest challenges to R&D productivity remains further compounded when taking into considera- the lack of valid human screening and disease tion animal husbandry costs, staff training and models. months spent writing ethics applications and adhering to good quality regulatory frameworks. Limitations of current models in R&D Finally, given the limitations with regards to the The numbers are stark. It costs, on average, $1.78 predictability of animal models, ethical questions billion to bring a drug to market over an average have been raised. time course of 13 years with only a 3.5% probabil- ity of success2. R&D continues to rely on labora- Stem cells – introducing the human tory animals, cell- and tissue-based systems to element into drug discovery identify novel targets and to establish efficacy prior Human biology often proves to be distinct from to the design of clinical trials. Yet the majority of animal models and cell lines used for drug develop- clinical trials fail because the drugs turn out to be ment. To bridge the gap, a scalable source of con- either toxic or they do not work within human dis- sistent human cells is needed. But primary cells are eases3. difficult to source in sufficient quantities, with In vitro pre-clinical research relies predominant- some cell types proving impossible to obtain. ly on animal-derived primary cells and immor- Many in the scientific community have thus sought talised cell lines. Primary cells, usually sourced to take advantage of human stem cells. from donor tissue, are associated with high vari- Unfortunately, to date this has often been fraught ability and a lack of scalability due to often limited with problems, ranging from inconsistency, long expansion capacity. In contrast, cell lines are well and complex protocols that are difficult to repro- adapted for a culture environment, typically high- duce and lack of scalability. ly-proliferative, scalable and easy to transfect. But These can be explained by the paradigm that most cell lines have been immortalised artificially has, for the most time, been applied to the use of and propagated for decades, diversifying during stem cells. Traditional ‘’ long-term culture. As a result, they differ substan- approaches seek to replicate developmental pro- tially from the tissue of origin with regards to cesses in vitro. Consequently, the time to generate genome, and morphology. Accumulated particular mature cell types (often 50-100 days) mutations over time lead to differences in cell char- hinges on the timelines of embryonic development acteristics between laboratories and, inevitably, and depends on multiple intermediary steps. issues of reproducibility. A 2016 survey conducted Moreover, the cell fate choices that are required to by Nature found that more than two-thirds of differentiate a pluripotent stem cell to a fully dif- respondents have tried and failed to reproduce ferentiated rely on stochastic princi- someone else’s experiment, and more than half ples. The accumulation of stochastic events ulti- have failed to reproduce their own data4. mately leads to inconsistent cultures, not only Continuous propagation of cell lines can result in resulting in batch-to-batch but also intra-batch batch-to-batch variability in the absence of quality variability.

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Human glutamatergic neurons generated by Neurogenin-2- driven reprogramming of stem cells using opti-ox technology. Image courtesy of Charles River

Cellular reprogramming – using first challenged our understanding of cell types synthetic biology to induce by demonstrating that connective tissue cells cell fate changes () can be converted into skeletal mus- The initial ethical bottleneck with regards to the cle cells by the single factor use of embryonic stem cells was overcome in MyoD6. In 2010 Marius Wernig extended this 2006/07. Japanese researcher concept and demonstrated that reprogramming is discovered a way to turn adult mouse and human possible between two different germ layers, by cells into human-induced pluripotent stem cells converting fibroblasts into neurons7. These dis- (hiPSCs), which show striking similarities to coveries led to a wave of reprogramming embryonic stem cells. This can be achieved by research throughout the world that continues directly activating the pluripotency cell programme unabated to the present day. by expressing appropriate transcription factors. Cellular reprogramming is thus not limited to Known as the Yamanaka factors (Oct3/4, , generating induced stem cells from mature cells. , c-), these bypass the need for dissecting An increasing body of literature suggests that it can human , thus removing the ethical con- be applied to the specification of any cell type, be cerns associated with embryonic stem cells and it a liver cell, an immune cell, a neuronal cell or enable a scalable source of progenitor cells. hiPSCs other cell type. remain in an epigenetically native state in which progenitor cells exhibit an indefinite capacity to Next-generation proliferate while maintaining the potential to dif- cellular reprogramming ferentiate into any cell type under optimised differ- A particularly attractive starting cell for repro- entiation protocols. Yamanaka’s discovery has gramming is the iPSC: due to rapid proliferation opened the possibility to generate any human cell and their unrestricted capability to replicate, iPSCs type in unlimited supply and won him and John enable effective scale-up of cell production. Gurdon the 2012 Nobel prize for Medicine. However, until recently reprogramming of cells Yamanaka’s work built on previous findings including iPSCs has been hampered by low efficacy from Davis, Weintraub and Lassar, who in 1987 and yields. We hypothesised that these are likely

Drug Discovery World Spring 2020 3 Therapeutics

Human skeletal myocytes generated by MYOD1-driven reprogramming of stem cells using opti-ox technology

the result of evolutionary intrinsic protective mech- applied to the expression of reprogramming fac- anisms within stem cells which lead to recognition tors this results in rapid, synchronous and highly and deactivation of foreign DNA (gene silencing) defined reprogrammed cells. and thus limit reprogramming efficiency of the The first product developed using opti-ox has transcription factors introduced to stem cells. already been brought to the market: ioNEURONS/ By applying a cellular reprogramming approach glut™ human glutamatergic neurons derived from supported by a uniquely-engineered genetic switch, hiPSCs have successfully been reprogrammed using opti-ox™ (optimised inducible overexpression)8, opti-ox by forced expression of Neurogenin-2 we were able to overcome gene silencing and with (NGN2). The technology can be applied to gener- it the restrictions of inefficient cellular reprogram- ate many other mature cell types too, including ming. This proprietary technology enables tightly- human skeletal myocytes, , immune controlled expression of transcription factors and cell types and other CNS cell types. results in deterministic reprogramming of entire hiPSC cultures. To achieve this, we disassembled a Identifying human cell Tet-ON switch into its two components and gene- type programmes targeted each of them into two or more distinct These novel stem cell-derived products generated genomic safe harbour sites. The result of this is using opti-ox demonstrate that cellular repro- unprecedented homogeneous, controllable expres- gramming is not only possible, but able to achieve sion of inducible transgenes in hiPSCs. When consistency and scalability at an affordable cost.

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In order to expand the repertoire of cell types, we models of human disease and, if broadly adopted, References need to tackle the challenge of identifying the right help to overcome some of the challenges linked to 1 Scannell, JW and Bosley, J. combinations that define a the ‘reproducibility crisis’ in science4. Stem cell- When Quality Beats Quantity: Decision Theory, Drug specific cell type. A recent report identified at least derived human cells can be used in multi-lineage Discovery, and the 9 100 major clusters of human cell types . Past clas- co-cultures, , organ chips and tissue engi- Reproducibility Crisis. PLoS sifications were based on different criteria, includ- neering12. Certain human cell types, including neu- One. 2016, 11(2):e0147215. ing morphology, protein expression and func- rons and cardiomyocytes, are difficult to isolate https://journals.plos.org/ploson tion10, and recent evidence suggests that the actu- and culture in vitro; hiPSC-derived cells provide a e/article?id=10.1371/journal.po ne.0147215. al number of cell types encoded by different tran- unique opportunity for investigating pathologies 2 Paul, SM et al. How to 9 scription factors may be far greater . There are associated with these. improve R&D productivity: the approximately 20,000 genes in our DNA, of Toxicity screening currently relies on primary pharmaceutical industry’s which ~3,000 encode DNA binding proteins that hepatocytes that are difficult to culture and display grand challenge. Nature theoretically could be involved in coding cellular significant variability. Other models used are not Reviews Drug Discovery, 2010, 9(3):203-14. https://www. identity. If we postulate that cell fate is determined always predictive of human response, leading to nature.com/articles/nrd3078. by an average of three transcription factor genes, the potential of clinical failures. A robust and scal- 3 Harrison, RK. Phase II and there are 4.5 billion possible combinations to able source of stem cell-derived hepatocytes would phase III failures: 2013-2015. examine! be the ideal platform to screen candidate com- Nature Reviews Drug Advancements in molecular techniques such as pounds for toxicity. Stem cells encompassing com- Discovery, 2016, 15:817-818. https://www.nature.com/article single cell RNA sequencing and efforts such as the mon variants or with genetically-engineered muta- s/nrd.2016.184. Human Cell Atlas to develop a ‘unique ID card for tions would further optimise drug discovery by 4 Baker, M. 1,500 scientists lift each cell type’ are accelerating the discovery of ensuring that those compounds with successful the lid on reproducibility. transcription factor combinations that encode cell efficacy are personalised with regards to specific Nature, 2016, 533(7604):452-4. types11. To deal with the gigantic data sets, scien- target populations. https://www.nature.com/news/ 1-500-scientists-lift-the-lid-on- tists are applying machine learning methods to reproducibility-1.19970. identify relevant patterns. Reprogrammed cells for cell therapy 5 Akhtar, A et al. The Flaws and The use of machine learning will also help to Recent advances in CRISPR and CAR-Ts technolo- Human Harms of Animal draw connections and maps between transcrip- gies spark hopes that a new generation of therapies Experimentation. Camb Q tion factors and DNA binding proteins not yet might finally eradicate cancer. A notable example Health Ethics. 2015, 24(4): 407- 419. https://www.ncbi.nlm.nih. characterised and thus expand our ability to of the success of Chimeric Antigen Receptor (CAR) gov/pmc/articles/PMC4594046/ reprogramme cells. Bioinformatic platforms capa- technology is in the treatment of childhood Acute pdf/S0963180115000079a.pdf. ble of setting up pipelines to mine the literature Lymphoblastic Leukaemia (ALL) with Kymriah, a 6 Davis, RL, Weintraub, H and and pull out potential candidates are needed patient-specific (autologous) CAR-T cell therapy. Lassar, AB. Expression of a along with research projects to break down each This involves isolating T cells from the patient, single transfected cDNA converts fibroblasts to organ and cell type into its genetic components. genetically modifying them to express an anti-can- myoblasts. Cell, 1987, Combining bioinformatic approaches with new cer antigen receptor (in this case CD19) and then 51(6):987-1000. https://www. molecular gene editing techniques such as reinfusing them into the patient. Significant efforts cell.com/cell/pdf/0092- CRISPR has increased our understanding of cell are currently under way to develop novel cell ther- 8674(87)90585-X.pdf?_return type identity and will ultimately enable more apies. The global stem cell therapy market was val- URL=https%3A%2F%2Flinkingh ub.elsevier.com%2Fretrieve%2F accurate targeting of cellular subtypes. A newly- ued at $755.4 million in 2018 and is expected to pii%2F009286748790585X%3F 13 formed consortium between Bit Bio and the reach $11 billion in 2029 . showall%3Dtrue. London Institute of Mathematical Sciences However, cell therapy today is extremely chal- 7 Vierbuchen, T et al. Direct applies machine learning to large-scale functional lenging, causing unpredictable responses from the conversion of fibroblasts to transcription factor screens in order to drastically body’s , with many patients experi- functional neurons by defined factors. Nature, 2010, speed up the discovery process with the aim of encing neurotoxicity and release syn- 463(7284): 1035-1041. 14 developing the capability to reprogramme stem drome . Widespread access is limited due to their https://www.ncbi.nlm.nih.gov/p cells into any cell type. high price, placing economic burden on patients, mc/articles/PMC2829121/. insurance companies and governments. The cost of 8 Pawlowski, M et al. Inducible Application of reprogrammed cells treatment with Kymriah, developed by Novartis, is and Deterministic Forward Programming of Human The identification of precise transcription factor $475,000. Yescarta, developed by Kite Pharma and Pluripotent Stem Cells into combinations driving cell fate and subsequent gen- approved for large B-cell lymphoma has been Neurons, Skeletal Myocytes, and eration of high-quality human cells has the poten- priced at $373,000. . Stem Cell tial to address the translation gap and thus Harvesting patient cells and genetically modify- Reports, 2017, 8(4): 803–812. improve and lower the cost of research and drug ing them with a lentivirus or brings with https://www.ncbi.nlm.nih.gov/ pmc/articles/PMC5390118/. discovery. The precision and consistency that this it an oncogenic risk15. The cost of each batch pro- approach offers will result in more predictable duction is high, and manufacture often fails due to Continued on page ??

Drug Discovery World Spring 2020 5 Therapeutics

Continued from page ?? poor cell quality, with many patients undergoing unprecedented positive implications for academic rounds of chemotherapy treatment prior to CAR-T research, drug development and toxicity testing, 9 Han, X et al. Construction of cell therapy. A further challenge, common to adop- and regenerative medicine. Their widespread appli- a human cell landscape at single-cell level. Nature, 2020. tive T cell therapies is that activation and expan- cation has the potential to reduce failure rates in https://www.nature.com/article sion of T cells from a Naïve state to an effector clinical trials and drug development and enable a s/s41586-020-2157-4. state increases the chance of activation-induced new generation of cell therapies. DDW 10 Regev, A et al. The Human death of T cells upon transfer16. Cell Atlas. eLife, 2017, 6. pii: One approach to overcome the challenges of e27041. https://elifesciences. org/articles/27041. autologous treatments could be to use allogeneic 11 www.humancellatlas.org. donor T cells, but this comes with the risk of graft- 12 Sharma, A et al. Multi- versus-host disease. While this may improve the lineage Human iPSC-Derived quality of T cells, as healthy donors do not under- Platforms for Disease go rounds of chemotherapy, this will not solve the Modeling and Drug Discovery. Cell Stem Cell, 2020, manufacturing complications linked to activating 26(3):309-329. https://www. and rapidly expanding T cells. cell.com/cell-stem-cell/pdf/ Forward reprogramming of iPSCs to Naïve T S1934-5909(20)30064-3.pdf. cells in theory could overcome the above chal- 13 BIS Research, 2019. lenges by providing a reliable, consistent and scal- https://bisresearch.com/industr y-report/stem-cell-therapy- able platform for the manufacture of cells. The market.html. right transcription factor combination would 14 Grigor, EJM et al. Risks and enable the expansion of billions of iPSCs and upon Benefits of Chimeric Antigen activation of the transcription factor cassette, turn Receptor T-Cell (CAR-T) them into the desired T cell phenotype. Therapy in Cancer: A Systematic Review and Meta- Additionally, gene engineering the iPSCs to express Analysis. Transfusion Medicine CARs prior to T cell differentiation could improve Reviews, 2019, 33(2):98-110. therapeutic safety. https://www.sciencedirect.com With clever engineering we can also mask the /science/article/pii/S088779631 cells and avoid the host immune system, thereby 830172X?via%3Dihub. 15 Marcucci, KT et al. increasing the effectiveness of donor-derived thera- Retroviral and Lentiviral Safety pies, similar to HLA matching/masking seen in Analysis of Gene-Modified T transplantation. Cell Products and Infused HIV The use of off-the-shelf T cells will drastically and Oncology Patients. cut the cost and allow for widespread adoption of Molecular Therapy, 2018, 26(1):269-279. https://www. cell therapies in otherwise difficult-to-treat dis- cell.com/molecular-therapy- eases. Cell therapies, while currently focused on Dr Alex Davenport holds a PhD in CAR-T cell family/molecular-therapy/ the immune system and cancer, will expand to biology from the University of Melbourne and was fulltext/S1525-0016(17)30528- other indications. Indeed, immune cells are paving a postdoctoral scientist in T cell biology at the 2?_returnURL=https%3A%2F% the regulatory framework for a much larger mar- University of Cambridge before joining Bit Bio as 2Flinkinghub.elsevier.com%2Fr etrieve%2Fpii%2FS1525001617 ket of regenerative medicine, with an almost a Senior Scientist. 305282%3Fshowall%3Dtrue. unlimited number of cell types. It may become pos- 16 Gattinoni, L et al. sible to use iPSC-derived hepatocytes to regenerate Dr Tonya Frolov completed her PhD in cancer cell Acquisition of full effector damaged livers that currently require a liver trans- biology at UCL. The lack of relevant disease mod- function in vitro paradoxically plant, or to use iPSC-derived cardiomyocytes for els for her research led her to join Bit Bio as impairs the in vivo antitumor efficacy of adoptively treating heart attack patients. Both these cell types Product Specialist, with the aim of helping to transferred CD8+ T cells. are currently difficult to culture from primary sam- develop and democratise access to reliable human Journal of Clinical ples and fulfil a desperately needed gap in the mar- cell models. Investigation, 2005, 115(6): ket. Access to highly-defined, scalable human cells 1616-26. https://www.jci.org/ will enable the future of medicine. Dr Mark Kotter is a stem cell biologist and neuro- articles/view/24480. surgeon at the University of Cambridge. By com- Conclusion bining synthetic and stem cell biology, his team has Despite tremendous progress in biomedical developed a benchmark technology for the efficient research that has resulted in successful treatments and consistent production of human cells for use in for many diseases, we face significant unmet medi- research, drug development, and cell therapy. He is cal needs for individuals and for our societies. Founder & CEO of Bit Bio and co-founder of the Widespread access to human cells will have cultured meat start-up Meatable.

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