Development of Adenoviral Vectors for Studying Hematopoietic Stem Cell Function

Detta är en avhandling från Divison of Molecular Medicine and Gene Therapy

Sammanfattning: Hematopoietic stem cells (HSCs) are defined by their capacity to self-renew and differentiate into all hematopoietic cell lineages. Postnatally, HSCs reside predominantly in the bone marrow and are relatively rare, but important cells because of their therapeutic potential in bone marrow transplantation protocols and in their involvement in the origin of certain leukemias. To study how various genetic programs govern the regulation of HSCs, replication deficient viral vectors, including adenoviral vectors, have been utilized for gene transfer and expression in normal and malignant hematopoietic progenitor and stem cells. Adenoviral vectors allow high levels of transient transgene expression, however, adenoviral transduction of hematopoietic cells has been hampered by low expression of the coaxackie and adenovirus receptor (CAR), which is the primary target cell receptor for adenovirus serotype 5 (Ad5). With the aim to increase adenoviral gene transfer into hematopoietic cells, we modified the versatile Ad5 based AdEasy-1 vector system to Ad35 tropism by switching the shaft and knob domain of the fiber to that of the Ad35 species, which utilizes CD46 as a primary receptor for target cell anchoring. By using this fiber re-targeted system, termed AdEasy-1/F35, we demonstrated that primary chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML) patient cells could be efficiently transduced compared to conventional Ad5 vectors. Furthermore, we demonstrated that human candidate HSCs with NOD/SCID repopulating capacity from cord blood were efficiently transduced with the Ad35 tropism vectors. Moreover, in order to study the self-renewal process of HSCs, we utilized the Ad35 tropism vectors as reporters for monitoring human telomerase reverse transcriptase (hTERT) expression in human HSCs. Interestingly, increased hTERT expression was inversely correlated to the self-renewal capacity among primitive human hematopoietic cells and the hTERT reporter vector could be used to separate short-term from long-term repopulating human HSCs. In summary, we have developed a versatile adenoviral vector system with Ad35 tropism that can be used to transiently genetically modify normal and leukemic stem cells. This vector system can be used to study normal regulation of HSCs and has potential in the development of novel therapeutic approaches for malignant blood disorders.

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