Macdonald, Catherine; (1998) A study of the expression and role of the amphotropic retrovirus receptor in human haemopoietic cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Retroviral vectors based on Amphotropic Murine Leukaemia Virus (A-MLV) are widely used for gene transfer into human haemopoietic stem cells (HSCs) as they can stably integrate the gene of interest into the genome of the target cell, resulting in transfer of the gene to all the progeny of that cell. Unfortunately, the efficiency of gene transfer (assessed by long term reconstitution with transduced cells) has been very low - typically <1%. The cell-surface receptor, Glvr2, is an essential requirement for the binding and entry of A-MLV, and previous studies suggest that it may have an important role in determining transduction efficiency. In order to test the hypothesis that Glvr2 expression is a key limiting factor in the transduction of human HSCs with A-MLV-based vectors, it was necessary to set up a number of assays and experimental systems. A virus binding assay, which gave an indirect measure of receptor number, showed that the level of binding to CD34+ cells was low and difficult to upregulate.We then developed a system in Chinese Hamster Ovary cells in which receptor number could be manipulated independently of other factors. Transduction efficiency proved to be dependent on receptor number and comparison of the level of virus binding to the CHO system clones and peripheral blood-derived CD34+ cells allowed us to infer that a viral vector with a titre of 10^5 infectious units per ml would, when used to transduce CD34+ cells, have an effective titre of 10^3 infectious units per ml. The conclusion that Glvr2 expression is likely to be a major limiting factor in this context has important implications for the design of future gene therapy protocols. This limitation could be circumvented by improved vector design either through increasing vector titre or by engineering vectors to make use of cell-surface receptors other than Glvr2.

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