GRANT=6967773;P01HL Gene therapy of sickle cell disease requires gene transfer vectors that confer table, regulated, lineage-specific expression of the gamma-globin gene at relatively high levels without the risk of cellular transformation or tumorigenesis. Our proposal to achieve this is based on two recent findings: I) adenovirus (Ad) vectors containing the Ad serotype 35 fibers (Ad5/35) particularly subsets with potential stem cell capacity and ii) incorporation of AAV ITRs into Ad vectors devoid of all viral genes (Ad.AAV) mediates efficient random vector integration and allows for stable gene transfer into human hematopoietic stem cells. To achieve adequate globin expression, we will produce helper-dependent Ad5/35 containing the 26 kb beta-globin LCR (5?HS1 to 5?HS5, beta-globin promoter, 3 HS1); whereby the LCR cassette is flanked by AAV ITRs (Ad,AAV-LCR).
In Specific Aim 1, vector production will be optimized with a recently constructed Ad.AAV-LCR vector expressing the GFP gene under the control of the beta-promoter/LCR. To minimize the risk of tumorigenesis, we will further modify Ad.AAV-LCR vectors to allow for selection against vector integration into active genes and to increase the frequency of site-specific integration by transient co-expression of AAV Rep78.
In Specific Aim 2, we will study the persistence of GFP expression and the integration pattern of our vectors in clones of transduced erythroleukemic cells.
In Specific Aim 3, expression studies and monitoring of integration sites will be performed with colonies of transduced CD34+ cells grown in semisolid cultures, followed by studies in NOD-SCID mice. In an attempt to assess vector-induced tumorigenesis, we will compare the expression profiles of the pool of transduced long-term culture initiating cells and NOD-SCID mice. In an attempt to assess vector-induced tumorigenesis, we will compare the expression profiles of the pool of transduced long-term culture initiating cells and NOD-SCID repopulating cells with that of mock-infected cells using DNA arrays. Finally, if we confirm safety and efficacy with our new vector, in Specific Aim 4, we will construct gamma-globin expressing Ad.AAV-LCR vectors and perform transduction studies in CD34+ cells derived from healthy donors and patients with sickle cell disease. Gamma-globin expression will be analyzed in progenitor assays.
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