This NCDG proposal represents an integrated series of preclinical studies designed to develop an effective treatment for HIV patients via transplantation of genetically modified, viral resistant, stem cells. The three research projects of this program represent a systematic and thorough approach to address the current limitations of this emerging technology and utilize novel strategies that have been developed in the laboratories of the principal investigators. Project I will develop potent RNA-based inhibitors with primary emphasis on the use of ribozymes. However, unlike the current studies which employ the use of trans-cleaving ribozymes, Project I will develop the use of trans-splicing ribozymes which offer critical advantages over conventional ribozymes: the ability to follow ribozyme activity in the cell and hence systematically improve ribozyme function in vivo, and the ability to generate trans-dominant inhibitors which should further enhance their antiviral potency. Project II will assess the function of HIV inhibitors generated in Project I in monocyte/macrophage and CD4+ T cells derived from transduced stem cells, and will develop methods to improve retroviral delivery of HIV inhibitors into hemopoietic stem cells, with the emphasis on identifying conditions which preserve the undifferentiated phenotype of the vector transduced cells. A novel and simple paradigm using multiparameter FACS analysis developed by the P.I. of Project II, is a critical component in the proposed studies. In Project III, novel methods will be developed incorporating a new proprietary technology (termed Strand Displacement Amplification) developed at Becton Dickinson by the PI of Project III to measure in situ the presence of vector and HIV sequences. Coupled with phenotypic and functional characteristics of the genetically modified cells, it will provide sensitive, accurate, and quantitative information about the effectiveness of HIV gene therapy strategies in clinical settings. This information will be critical for the rational design of improved vectors, inhibitors, transduction protocols, and the design of new clinical studies. This NCDG program is characterized by extensive and close interactions between investigators with complementary and unique expertise at Duke University, the University of Virginia, and Becton Dickinson Immunocytometry Division. This interaction provides a critical mass of diverse scientific and medical talent collectively focused on developing effective gene therapy based treatments for HIV infection and AIDS.
