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UNEXPECTED Report Summary

Project ID: 648894
Funded under: H2020-EU.1.1.

Periodic Reporting for period 2 - UNEXPECTED (Uncovering targets for ex vivo expansion of hematopoietic stem cells to enhance cell therapies of blood disorders)

Reporting period: 2017-02-01 to 2018-07-31

Summary of the context and overall objectives of the project

While stem cells with high regenerative potential exist in many organs, their clinical use in tissue regeneration has been limited as retrieving and successfully delivering the stem cells poses a great challenge in most tissue types. The inherent ability of intravenously infused hematopoietic stem cells (HSCs) to seed and engraft their tissue, the bone marrow, is an exception in this regard and forms the basis for a more than four decades long clinical application of bone marrow transplantation (BMT). BMT is a conceptual and elegant example of stem cell therapy, used successfully in life-saving treatment of malignant and inherited hematological disorders. However, a significant proportion of patients who would benefit from BMT cannot receive treatment due to insufficient numbers of stem cells for so called autologous transfers when the patients own cells are used, or a lack of suitable matched donors for so called allogeneic transplantation (where donor cells are required). Umbilical cord blood is (UCB) a very promising stem cell resource, which can be easily and safely harvested from the umbilical cord of newborn babies. Initiatives have been taken all over the world to bank large quantities of cord blood, providing a rich repertoire of donor cells for transplantation. However, the stem cell numbers in UCB harvests are typically too low for efficient transplantation in adult patients. Insufficient stem cell numbers are, therefore, significant constraints in settings of both autologous and allogeneic transplantation. To tackle this problem, intense efforts are being made to find strategies that would enable the expansion and amplification of the stem cells outside the body (i.e. ex vivo), prior to transplantation but to date these efforts have been met with very limited success. Ex vivo stem cell expansion is therefore one of the most desired, yet elusive goals in experimental hematology and transplantation medicine. Successful development of strategies for expansion of HSCs from cord blood samples would implement cord blood as an accessible and sufficient source of HSCs for large numbers of patients with hematological malignancies currently non-eligible for potentially curable allogeneic transplantation therapy.

Our work is focused on understanding the genes and pathways that regulate blood stem cells in order to be able to develop novel strategies for ex vivo stem cell expansion. We are particl´ularly interested in the basic mechanisms that are responsible for the ability of stem cells to self-renew, i.e. to make new copies of themselves. If we were able to mimic the process of stem cell self-renewal that normally takes place in the bone marrow environment, in a culture dish, we should also be able to amplify the number of stem cells prior to transplantation. My research team has developed several new approaches to study hematopoiesis and stem cells from human sources. Rather than studying one gene or one factor at the time we have developed methods that allow the screening of thousands of genes in parallel for their ability to functionally influence the stem cells. This is achieved by specially designed viruses that can enter into the stem cells and deliver molecules (RNAi or CRISPR/Cas9) that specifically silence any given gene. Using these screens, our aim is to identify the most significant regulators that could be relevant to target in order to achieve stem cell expansion.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the project we have used very large numbers of human HSCs derived from umbilical cord blood we to screen a large fraction of all genes in our genome by applying RNA interference. Among our top-ranked genes (top 0.5%), we found no less than 4 out of 5 components of the so called cohesin protein complex. Our findings implicate a direct role for the cohesin complex in balancing the critical decision of whether a stem cell should self-renew and multiply, or differentiate into mature blood cells (Galeev et al, Cell Reports 2016). Understanding the precise details of how this decision is made may hold the key to successful expansion of HSCs. Another gene, CYTH1, that we recently identified in our screens represents a new molecular target that may be considered in therapeutic strategies to influence the localization of HSCs following transplantation (Rak et al, Blood 2017).
We have further used our new knowledge from the screens to systematically develop better culture conditions for expansion. Indeed, our first screens have identified several highly promising ”druggable” candidates such as the NFkB signaling pathway. We have now found that NFkB inhibition by specific drugs, dramatically enhances the stem cell numbers of cultured cord blood HSCs, compared to our previous “best” culture conditions (submitted).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

A major focus in my laboratory is now to further assess these novel findings to develop expansion conditions that will be suitable in clinical settings. We will identify suitable patients and prepare for clinical trials using expanded stem cells with the aim of treating the first patients with expanded stem cells during the next 5 years. In parallel we will extend and further develop our screens using the recently discovered gene editing technology (the CRISPR/Cas9 system) that allows for precise silencing or activation of genes. In this manner we hope to identify other, and more potent stem cell regulators.

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