Over 34 million people are currently living with HIV/AIDS. Combination antiviral therapies have significantly reduced the morbidity and mortality of the disease; however, there is currently no cure for the disease. Discovery that a naturally occurring variant of CCR5, the delta32 mutation, renders cells resistant to HIV infection has led to efforts by several academic laboratories and biotech companies to find gene therapy approaches to mimic the delta32 mutation. This project will demonstrate the feasibility of using peptide nucleic acids (PNAs) as a therapeutic to genetically truncate the CCR5 protein in CD34+ stem cells and block HIV cell entry. Preliminary experiments that modified the CCR5 gene used a targeting PNA with a donor DNA sequence to induce a stop codon near the delta32 mutation. In this Phase I project, we will develop a novel strategy for PNA-induced CCR5 gene modification to increase the CD34+ mutation frequency to clinically useful levels. Specific Aim 1 experiments will test the feasibility of a PNA targeting strategy where we will conjugate DNA strand breaking molecules directly to the PNA to induce site specific breaks in the CCR5 gene. Mutagenic repair of these breaks via the error-prone non-homologous end joining pathway will lead to frameshift mutations and a truncated CCR5 protein. This repair mechanism is expected to generate a significant increase in CCR5 frameshift mutations. In Specific Aim 2 experiments, we will compare clinically relevant PNA delivery methods for modification of the CCR5 gene in human CD34+ stem cells. A successful Phase I project will identify PNA conjugates and delivery methods suitable for a Phase II project that will use our novel strategy to establish HIV-resistant T cells in the humanized mouse model of HIV infection. Our long- term goal is to develop a highly specific clinical PNA conjugate CCR5 gene modification protocol that will create HIV-resistant immune cells in HIV patients and reduce their need for long-term antiretroviral therapy.