Most current gene therapy are based on a """"""""gene addition"""""""" strategy, where a functional transgene cassette is delivered to cells and expressed from episomal or randomly integrated molecules. A potentially more desirable strategy would be to correct mutations at their normal chromosomal locations. Major advantages of this """"""""gene correction"""""""" approach include properly regulated gene expression and the removal of dominant disease- causing mutations. Gene correction is especially promising when combined with methods for the ex vivo culture and manipulation of stem cells, since even rare corrected cells could have the potential to reconstitute a diseased organ system after transplantation and in vivo proliferation. The objective of this proposal is to develop gene targeting methods that can be used to correct genes in cells capable of reconstituting the hematopoietic system. In general, the gene corruption rates that can be achieved with conventional methods have been far too low to consider therapeutic applications, and in the case of hematopoietic stem cells, gene targeting has never been demonstrated. In this proposal, a novel gene targeting method employing adeno-associated virus (AAV) vectors will be used to correct genes in stem cell populations. Gene targeting assays will be developed based on the correction or site-specific insertion of convenient reporter genes. AAV targeting vectors will be packaged in different capsid serotypes and tested for their ability to correct genes in mouse and human hematopoietic cells. Gene targeting will be demonstrated in transduced stem cells by transplantation of murine cells in myeloablated mice, or human cells in NOD/SCID mice, and assaying for expression of the corrected reporter gene in transplant recipients. Experiments will also be performed with alternative sources of hematopoietic stem cells derived from muscle and brain tissue, which have improved ex vivo culture properties and may be more susceptible to AAV-mediated gene targeting. These experiments constitute an attempt to demonstrate that gene targeting can be achieved in hematopoietic stem cells, and establish experimental conditions that may ultimately be adapted towards the treatment of blood diseases by correction.
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