Using the group II intron to repair genes in Escherichia coli

Traditional approaches to gene therapy can be categorized into DNA-based and RNA-based techniques that suffer from several problems. DNA-based approaches such as retroviral or adenoviral gene transfer, homologous recombination, chimeraplasty, triplex forming oligonucleotides and RNA-based approaches such as ribozyme-mediated RNA editing can suffer from a size limitation of therapeutic gene, safety concerns, regulation/expression limitations, as well as limited efficiency and specificity of the reactions. I wanted to investigate the feasibility of using a Group II intron to repair genes to get around some of these problems and began proof-of-principal studies in E. coli.; Group II introns define one class of catalytic RNAs that have been found in chloroplast DNAs, and higher plant and fungal mitochondrial DNAs and certain Group II introns act as mobile genetic elements and can site specifically integrate themselves into DNA. With the help from our collaborators, we found that the L1.LtrB intron from the bacterium Lactococcus Lactis can be retargeted to integrate into other (non wild type) DNA sequences, and I went on to set up a model system to study gene repair and found that a prokaryotic gene could be repaired with high efficiency when it was carried on a plasmid. In addition, I found that a mutant allele of the genomic human β-globin locus was repaired in E. coli, and the repaired locus was able to generate wild type RNA and wild type protein when transfected into mammalian cells. Finally, I was able to mobilize the Group II intron into bacterial chromosomal DNA, carry in a promoterless gene and have the endogenous promoter drive expression of the gene. These results suggest that utilizing the Group II intron to site specifically carry in promoterless repair sequences to the site of the mutation is possible and that the endogenous promoter is able to drive expression of the repair cassette and generate repaired RNA and protein. This novel approach to gene repair can alleviate some of the problems associated with DNA and RNA-based approaches to gene therapy because the endogenous promoter will drive the expression of the repair cassette circumventing regulation/expression problems as well as allow the introduction of small, specific repair sequences to the site of the DNA mutation.