The proposed work is to develop a therapeutic gene targeting agent for the treatment of HIV disease. Our approach is to use triplex-forming oligonucleotides (TFOs) and peptide nucleic acids (PNAs), which bind to duplex DNA in a sequence-specific manner. Initial work has demonstrated that TFOs can stimulate recombination in mammalian cells by the ability of triple helices to provoke DNA repair and, thus, sensitize the target site to recombination. Using a series of chemical modifications, the intracellular effectiveness of TFOs has been progressively enhanced. Our company, Helix Therapeutics, Inc. (HTI), was formed to commercialize this technology for the treatment of human diseases, such as HIV/AIDS. Entry inhibitors have recently emerged as a new class of HIV therapeutics. These drugs block cell surface receptors required for HIV entry into T-cells, such as the protein encoded by the CCR5 gene. The CCR5 chemokine receptor is a major co-receptor for R5-tropic HIV-1 strains, which are responsible for most cases of initial, acute HIV infection [10]. Individuals, who possess a homozygous inactivating mutation in the CCR5 gene, are almost completely resistant to infection by R5-tropic HIV-1 strains, with no other significant adverse consequences [11]. Pharmaceutical companies are currently trying to develop entry- inhibitor drugs to block the receptor protein, although progress has been hindered by toxicity, efficacy and drug resistance. HTI is optimizing TFO-based technology as an alternative approach by targeting and inactivating the underlying gene for the receptor directly and, thereby, creating cells permanently resistant to infection by the HIV virus. These modified cells, unencumbered by HIV infection, would proliferate and, as a result, restore and maintain a patient's immune system, even in the face of HIV infection. We propose to develop our lead PNA compound to target and inactivate specifically the CCR5 gene in human cells. Experiments will be done to determine the optimal combination of PNA and donor DNA molecules, as well as the most appropriate cellular delivery methods required for maximally enhancing CCR5 gene targeting in several immune and hematopoietic model cell lines. In addition, we will test these conditions for achieving therapeutic gene modification in the intended target cells, human hematopoietic stem (CD34+) cells. Upon isolation of pure populations of cells, containing mutant CCR5 alleles, we will confirm the induced genotype(s) and phenotype(s) and will also test whether the mutant cells are resistant to HIV infection. In addition, we will test whether PNA-modified CD34+ cells engraft and properly differentiate in a mouse model of stem cell transplantation and whether the differentiated, CCR5-mutant immune cells are resistant to HIV infection. The proposed studies are critical for developing a therapeutic gene targeting agent for the treatment of HIV disease because: (1) they will establish a PNA/DNA reagent set and appropriate transfection parameters, which result in high efficiency mutation of the CCR5 gene in both model cell lines and our eventual target cells, CD34+ cells, and (2) they will serve to demonstrate that PNA-induced CCR5 gene inactivation can result in resistance to HIV infection. [unreadable] [unreadable] PUBLIC HEALTH RELEVANCE: Over 40 million people are currently living with AIDS. Helix Therapeutics, Inc. is proposing to create cells permanently resistant to infection by the HIV virus, using a therapeutic gene targeting agent. These modified cells, unencumbered by HIV infection, would proliferate, and, as a result, restore and maintain a patient's immune system, even in the face of HIV infection. [unreadable] [unreadable] [unreadable]