The overall goal of this proposal is to evaluate the ability of mobile group II introns to disrupt genes in mammalian cells focusing on HIV and CCR5 genes as targets. Group II intron mobility is mediated by a ribonucleoprotein (RNP) composed of an excised intron RNA lariat and an intron-encoded protein that has reverse transcriptase, DNA endonuclease and RNA splicing maturase activities. Mobility of the Lactococcus lactis group II intron occurs through a two-step process. First the intron catalyzes the reverse-splicing of itself into a DNA target site and then the intron RNA is reverse transcribed into DNA by the intron encoded protein. The insertion site on DNA is recognized by both the intron RNA, through about l4 base pairs, and the intron-encoded protein. Recently, we demonstrated that group II introns can be engineered to site specifically insert into targeted DNA sites in HIV- 1 pol and CCR5 genes by modifying the DNA binding sites present on the intron RNA. These retargeted introns retain the ability to insert into and disrupt their targeted DNA sites when they are transiently transfected into mammalian cells in complex with their intron-encoded protein.
The specific aims of this proposal are: 1) To continue to explore the ability of group II introns to disrupt HTV and CCR5 genes via site specific DNA insertion following transfection of reconstituted group II RNPs into mammalian cells, 2) To evaluate group II intron splicing and mobility in mammalian cells following intracellular expression of the intron RNA and the intron-encoded protein, 3) To delineate the sequence requirements for Lactococcus lactis group II intron mobility in mammalian cells. 4) To evaluate the ability of group II RNPs to inactivate HIV proviruses by site-specifically inserting into viral DNA in infected cells and to render cells resistant to HIV infection and replication. These studies will also lay the foundation for development of mobile group II introns for targeted gene disruption in mammalian cells for therapeutic arid research applications.
