The long term objective of this proposal is to use short, cationic peptides as a efficient method of delivering biopolymers, such as intact autoantigens, immunodominant peptides, and antisense oligonucleotides into the cytoplasm of antigen presenting cells and T lymphocytes to induce antigen specific anergy and/or affect the profile of secreted cytokines as a therapy for experimental allergic encephalomyelitis (EAE). We have demonstrated that conjugation of a peptide, corresponding to nine amino acids from the HIV tat protein, to protein antigens results in their rapid uptake by a variety of cells and permits the molecules to enter both the MHC class I and ll biosynthetic pathways, which results in a dramatic increase in their antigenicity and immunogenicity. In addition, to delivering antigens to the MHC class I and ll molecules, we propose to use the tat peptide to transport PNA antisense reagents into cells to modulate cytokine production characteristic of inflammation. The studies outlined in this project seek to explore the therapeutic potential of specifically suppressing proinflammatory cytokine expression by introducing antisense PNA for the transcriptional activator NF-kappaB into murine T cell clones. If successful, these methods could be used as therapeutic strategies in mice in vivo to reduce inflammation in the CNS of mice. This project has three specific aims, 1. demonstrate that conjugation of the tat peptide to intact myelin basic protein and proteolipid protein as well as their immunodominant determinants and altered peptide ligands dramatically increases their ability to induce antigen specific tolerance and/or modify the cytokine profile of the autoantigen specific clones, 2. demonstrate that tat conjugated proteins and peptides enter antigen presenting cells, such as dendritic cells and small resting B cells, and explore whether adoptive transfer of the antigen loaded cells more efficiently induces either immune stimulation or tolerance than immunization with the peptides or proteins, and 3. demonstrate that the expression of a variety of proinflammatory proteins can be dramatically reduced with HIV-1 tat conjugated antisense PNA against the 65 kilodalton subunit of NF-kappaB in vitro and that the antisense PNA constructs can reduce inflammation when directly injected into rodents with EAE.
| Wender, P A; Mitchell, D J; Pattabiraman, K et al. (2000) The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters. Proc Natl Acad Sci U S A 97:13003-8 |
| Mitchell, D J; Kim, D T; Steinman, L et al. (2000) Polyarginine enters cells more efficiently than other polycationic homopolymers. J Pept Res 56:318-25 |
