Research Project
6016619
Recent advances in molecular biology have increased the scientific understanding of the genetic basis for disease and have provided the tools for novel advances in gene therapy. A major obstacle to the realization of the full potential of gene therapy is the development of effective means for delivering these therapeutic nucleotide sequences to the interior of primary cells in a form capable of stabily integrating into the target cell s chromosome. While various means and vehicles for gene transfer are under development and have been applied successfully in some systems, there is still room for improvement in areas such as practicality, safety, efficiency, target cell types transduced, and ex vivo and in vivo applicability. The long-term goals of this research project are to develop vehicles that can safely and efficiently mediate stable gene transfer into human hematopoietic stem cells, while maintaining the pluripotent nature of these cells. This proposal combines a vector delivery system capable of delivering genetic materials to the interior of a cell, with the molecules necessary for the integration of such genetic materials into the genome of the target cell. Specifically, recombinant rep 68 and rep 78 proteins of the adeno-associated virus type II (AAV) will be formulated into cochleate delivery vehicles, with plasmids containing marker genes flanked by the iterated terminal repeat (ITR) sequences from the AAV genome. The overall goals of these studies are to determine whether rep protein DNA cochleate complexes have the structural and ex vivo gene transfer capabilities in human hematopoietic cells to warrant further scientific investigation or commercial development. This will be accomplished through the following Specific Aims: 1. Characterize and optimize the integrative protein DNA cochleate vehicles. 2. Understand the structure- function relationship between the components of the integrative protein DNA cochleate vehicles and the efficiency of their ability to mediate stable gene expression in human hematopoietic cells with and without selective pressure. 3. Determine whether the integrative protein DNA cochleate vehicles mediate site specific integration of plasmid DNA into chromosome 19, analogous to adeno associated virus (AAV). 4. Partially characterize the phenotype of cells transiently and stabily transduced, as an indication of whether the multilineage and repopulating capacity (pluripotency) of hematopoietic cells in maintained. Proof of this would await studies of in vivo repopulation capacity proposed for future studies. PROPOSED COMMERCIAL APPLICATION The wide spread commercialization of human gene therapy requires the development of a non-toxic, non-replicating, non-immunogenic, and effective gene delivery system. Cochleate vehicles could function in this capacity.
