Gene-replacement remains a goal for the therapy of beta-thalassemia. The main objective of this proposal is to evaluate the feasibility of utilizing a human parvovirus as a vector to transfer the normal human beta-globin gene in hematopoietic stem cells. In view of the site-specific integration of the adeno-associated virus 2 (AAV), and the erythroid cell-tropism of parvovirus B19, an AAV-B19 hybrid viral genome will be used to pursue the following: 1. Construction of recombinant parvovirus vectors containing the normal human beta-globin gene for evaluation of site-specific integration and tissue-specific expression in vitro: Recombinant clones will be constructed that contain, within the AAV inverted terminal repeats (ITRs), a genomic copy of the normal human beta-globin gene either with its own promoter or with the B19p6 promoter, and a selectable Neo marker gene. These clones will also contain the human erythroid-specific enhancer (HS-2) of the globin Locus Control Region (LCR). After encapsidation into mature AAV virions, the recombinant virus will be used to infect erythroid and non-erythroid tissue culture cells to evaluate site-specific integration as well as tissue-specific expression of the transduced beta-globin gene. 2. Definition of the ideal target-cell population for efficient infection with the recombinant parvovirus, and examination of in vitro expression of the transduced normal human beta-globin gene in clonogenic assays and in long-term bone marrow cultures: Normal human bone marrow will be fractionated to isolate cell populations that resemble the pluripotent hematopoietic stem cells (HSC). These cell populations will be evaluated for optimal growth as well as infectivity with the recombinant parvovirus by using various cytokines that promoter proliferation of these cells. In vitro colony assays will be carried out with mock-infected and recombinant parvovirus-infected HSC populations for the erythroid (BFU-E and CFU-E), and granulocyte-macrophage (CFU-GM) cell lineages. These studies will be extended to include long-term bone marrow cultures for analyses of stability as well as expression of the transduced beta-globin gene. 3. Evaluation of in vivo expression of parvovirus-mediated transduced normal human beta-globin gene following bone marrow reconstitution in mouse model systems: Normal murine bone marrow stem cells transduced with the normal beta-globin gene will be used in reconstitution experiments with lethally-irradiated mice to examine the stability and expression of the transduced gene. Similarly, bone marrow stem and progenitor cells from transgenic mice with human sickle-cell disease will be used in reconstitution experiments following transduction with the normal human beta-globin gene to evaluate the therapeutic potential of the parvovirus vectors. These studies relate to our long-term interest in the molecular correlates of parvoviruses and human disease, and may lead to the development of safe and effective vectors for gene therapy in humans.
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