Transcriptional Engineering of Hematopoietic Stem Cells using CRISPR

Project Information

HSC-CRISPR

Grant agreement ID: 101041231

DOI

10.3030/101041231

EC signature date 14 February 2022

Start date 1 September 2022

End date 31 August 2027

Funded under

• European Research Council (ERC)

Total cost

€ 1 499 923,00

EU contribution

€ 1 499 923,00

Coordinated by

AARHUS UNIVERSITET
Denmark

Periodic Reporting for period 1 - HSC-CRISPR (Transcriptional Engineering of Hematopoietic Stem Cells using CRISPR)

Reporting period: 2022-09-01 to 2025-02-28

Summary of the context and overall objectives of the project

This project revolves around the ability to modify the transcriptome of hematopoietic stem cells to enhance their therapeutic potential. These cells are used for transplantation purposes as they possess the ability to repopulate the blood and immune system for the life time of a patient. They are therefore used to treat people with genetic blood disorders or blood cancers. The challenges with these cells are mainly associated with their scarcity and the insusceptibility to precise genetic therapy in these cells. The overall objective is to use CRISPR/Cas-based tools to transcriptionally engineer these cells to enhance their properties in regards to these challenges.

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

We have performed work related to bringing RNA-based delivery approaches of CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) into clinically relevant primary cells including CD34+ hematopoietic stem cells. We have tested different transcriptional effector domains and different Cas orthologs and identified combinations that work efficiently and that enable orthogonal and multiplexed transcriptional regulation where one set of genes is upregulated and another set is downregulated following a single delivery of relevant RNA-based reagents (mRNA + sgRNAs). In addition, we have shown that we can combine this with a third Cas ortholog to enable trimodal engineering where, in addition to orthogonal transcriptional engineering, we have also included gene editing in the form of gene knockout. Additionally, we have also identified new ways of tuning transcriptional efficiency so this gene modulation effect can be tuned to a desirable level. Finally, we have identified a potential major restriction factor for AAV transduction in CD34+ hematopoietic stem cells, which we must still explore to identify its importance in genetic engineering using HDR-based CRISPR/Cas editing using AAV6 as HDR repair template.

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)

Trimodal engineering and tunable transcriptional engineering represent novel approaches to genetically manipulate cells in a sophisticated way. We believe that complex genetic engineering can find use in many avenues of cell and gene therapies. The CRISPRa and CRISPRi tools have previously not found much application in therapy, but our findings pave the way for such use. HDR-based gene correction or insertion is the most flexible way of performing gene editing in human cells but is restricted in efficiency. With the hypothesis that AAV6 HDR repair template delivery and nuclear availability is a major bottleneck in the HDR process, the identification of a major restriction factor to this might enable wide-spread therapeutic use in CD34+ hematopoietic stem cells and benefit major patient populations with diseases in the blood and immune system.