Project 3 will construct and produce adeno-associated virus (AAV) vectors to deliver to cells the constructs designed by investigators from Projects 1 and 2. These vectors will then be used to deliver those constructs to primary hematopoietic cells (macrophages and lymphocytes) where their antiviral effects will be evaluated. In particular the investigators will evaluate their """"""""Targeted vectors Integration"""""""" (TVI) technology for its ability to direct site-specific integration of CTVI constructs into human chromosome 19 and achieve long-term expression of these constructs in lymphoid and myeloid cells.
The specific aim of this project are to: 1) Determine whether AAV vectors can be used to deliver CTVI construct to primary hematopoietic cells and result in a antiviral effect in these cells. The efficiency of transduction of primary macrophages and lymphocytes will be determined using an AAV vector containing CTVI constructs and a marker gene (e.g., truncated rat nerve growth receptor (tNGFR). Short-term antiviral effects will be assessed in transduced primary macrophages and lymphocytes. 2) Determine whether CTVI constructs can be targeted for site specific integration into human chromosome 19, and determine the long-term antiviral effects in cloned cell lines. Cell lines will be transduced with an AAV vector containing the tNGFR marker gene and CTVI constructs and simultaneously transfected with a plasmid directing the expression of the AAV rep gene. The efficiency of long-term transduction will be determined by comparing this to the efficiency of transduction in cells transfected with a plasmid that does not contain rep. Clonal cell lines will be derived and analyzed for the site of integration and antiviral effect. tNGFR-clones will be surveyed for nonfunctional integrants. Similar experiments will also be performed with delivery of tNGFR and CTVI constructs in a plasmid containing the rep gene. The goals of this aim are to engineer a vector with the targeting capability of wild-type AAV virus and to devise a strategy for the production of this vector which eliminates or reduces the potential for wild-type AAV contamination. If successful, an AAV vector containing both the rep gene and a CTVI construct will be produced and evaluated for transduction efficiency, integration and long-term expression. An AAV vector containing the rep gene and the gene for tNGFR will be constructed, and the extent of wild- type contamination will be determined (and reduced or eliminated, if necessary). The transduction efficiency of the vector will be evaluated for cell lines and primary hematopoietic cells and the extent of integration into chromosome 19 determined. An AAV vector containing the rep gene and CTVI construct will then be produced, transduced into CD4+ primary and established hematopoietic cell lines and evaluated for integration into chromosome 19, long-term expression of the CTVI construct and antiviral effect.
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