Windisch, Roland (2023): From harmful to useful: exploiting a leukemia-associated transcription factor for large-scale manufacture of functional human macrophages. Dissertation, LMU München: Faculty of Medicine |
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Abstract
Expansion of hematopoietic stem and progenitor cells outside the body is hardly possible due to spontaneous differentiation. Therefore, ex vivo production of immune cells in sufficient quantities for cell therapeutic approaches is limited. Exploitation of oncogenic fusion proteins represents a new, promising possibility for ex vivo propagation of human blood progenitor cells. These chromosomal translocation-derived chimeric transcription factors are capable of driving hematopoietic progenitor cell expansion by blocking differentiation. In this context, it was investigated to what extent a controllable designed leukemia-associated fusion protein can be exploited for ex vivo proliferation of human hematopoietic progenitor cells to generate functional macrophages on a large scale after switching off the protein. As such a fusion gene, MLL-ENL was stably integrated into the genome of human hematopoietic progenitor cells via retroviral gene transfer. A destabilization domain fused to MLL-ENL was used to target protein stability using the specific small molecule ligand Shield-1. In the presence of Shield-1, late monocytic progenitor cells were expanded in a large scale in ex vivo cultures in a controlled manner. The cells exhibited an immature monocyte immunophenotype, a normal karyotype, and permanent Shield-1 dependence. Genome-wide sequencing and viral integration site analysis also confirmed the absence of copy number alterations, mutations, or integration-activated proto-oncogenes that could promote cell proliferation. Shield-1 withdrawal allowed the expanded progenitor cells to be specifically differentiated into functional macrophages with appropriate cytokines. These phagocytes expressed macrophage-associated cell surface proteins and upregulated a variety of genes that play critical roles in the innate immune response. Functionally, adhesion under shear stress, migration along a chemokine gradient, and clearance of inactivated bacteria and apoptotic cells were demonstrated. Furthermore, macrophages were able to efficiently phagocytose antibody-loaded lymphoma or leukemia cells derived from patient blood. In summary, functional human phagocytes were produced for the first time from MLL-ENL expanded monocytic progenitor cells and characterized in detail by molecular and cell biology. This successful proof of concept suggests that such acute leukemia-derived and modified transcription factors have the potential to be used as molecular tools to generate functional immune cells for cell therapy approaches.
Item Type: | Theses (Dissertation, LMU Munich) |
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Subjects: | 600 Technology, Medicine 600 Technology, Medicine > 610 Medical sciences and medicine |
Faculties: | Faculty of Medicine |
Language: | English |
Date of oral examination: | 4. May 2023 |
1. Referee: | Kellner, Christian |
MD5 Checksum of the PDF-file: | 96f890b79e31be5a3e6aa69e4cdfd125 |
Signature of the printed copy: | 0700/UMD 21143 |
ID Code: | 31829 |
Deposited On: | 02. Jun 2023 09:25 |
Last Modified: | 05. Jun 2023 10:15 |