Overcoming delivery challenges in stem cell biology with nanotechnology

Sammanfattning: The discovery of the CRISPR-Cas9 system has revolutionized several areas of biological research by making genome engineering easy, affordable, fast, accessible and scalable. One field that has not yet benefited from this technological advance is that of mitochondrial DNA research. Study I addresses the challenges associated with adapting CRISPR-Cas9 to the mitochondrial environment, and identifies the lack of reliable methods to deliver RNA to mitochondria as the major limiting factor that continues to prevent site-specific Cas9-based modification of mitochondrial DNA.Study II aimed to overcome a long-standing challenge in stem cell biology. For both research-related and clinical applications, being able to deliver biomolecules to cells is essential. Traditional methods for delivering different payloads to stem cells can be highly efficient and suitable for many applications, but they negatively affect cell viability and function. Study II establishes nanostraws as a gentle alternative delivery method that enables efficient transfection of hematopoietic stem cells without the negative side-effects that accompany traditional methods.Study III and IV both involve the generation of Cas9-mediated gene knockouts in primary human hematopoietic stem and progenitor cells (HSPCs). Study III confirmed that the combined loss of STAG2 and STAG1 is lethal to both HSPCs and primary AML cells, while the loss of either one of these genes is well tolerated. This finding might be relevant to the development of future treatments against STAG2-negative AML. In Study IV, RPS19 was knocked out in HSPCs to generate a disease model that mimics the genotype of Diamond-Blackfan anemia (DBA). RPS19 knockout HSPCs exhibited the expected DBA-associated erythroid differentiation block. These cells could be used to validate the efficacy of an RPS19-encoding lentiviral vector, which was shown to ameliorate the differentiation block in treated cells.Generation of STAG2 KO cells was possible by delivering STAG2-targeting Cas9 to HSPCs by electroporation. When RPS19 was targeted with the same approach, a near complete loss of cell viability occurred, since loss of RPS19 sensitizes cells to stressful events, such as electroporation. Using nanostraws to delivery RPS19-targeting Cas9 removed the electroporation-associated stress from the equation and enabled recovery of sufficient numbers of viable RPS19 KO HSPCs, further highlighting the utility of the findings from Study II.In Study V, a small molecule screen was performed to identify new drug candidates for AML differentiation therapy. The molecule H4 was found to be a potent inducer of differentiation in primary FLT3-wt AML samples that acts through activation of the PKC pathway. Its effect could be further potentiated by inhibition of the BET pathway.