The biology of the stem cell and its environment in the role of effective gene therapy.

Dando, Jonathan Samuel (2000). The biology of the stem cell and its environment in the role of effective gene therapy. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000f960

Abstract

Efficient gene delivery still represents a major hurdle for the realisation of hematopoietic stem cell gene therapy for the treatment of acquired or inherited disorders. Present gene delivery technology has centred around genetic engineering of the Moloney retrovirus to produce a recombinant retrovirus which infects a cell only once. However, the target cell has to be actively dividing at the time of virus exposure for gene transfer to be successful. Manipulation of hematopoietic stem cells is fraught with problems, which include their delicate nature unless maintained in specific culture conditions and the low cell cycle of this cell type which often results in low gene transfer and/or concommittant loss of the transplantable cell. The stromal cell microenvironment supplies a rich milieu of cytokines and growth factors to the hematopoietic cell, acting as a feeder layer which can maintain it and induce it to cycle. In this work we demonstrate that murine stromal cells can be engineered into retroviral packaging cells, which secrete a transducing retrovirus. This viral secretion is both stable and safe and produces a transducing retroviral supernatant that is superior to present murine technology. Additionally we show that an available human umbilical vascular endothelial (HUVEC) cell line has a superior supportive capacity of hematopoietic cells, as compared to other available cell lines. We also demonstrate that it can be engineered into a safe and stable retroviral packaging cell line which not only yields better gene transfer into hematopoietic cells but can also have potential use in the application of tissue specific retroviral vectors.