No abstract

Inherited metabolic diseases impose a significant worldwide burden both financially and socially. Although there have been advances in treating these disorders, alternative therapeutic approaches are being developed to correct the responsible genetic defects. Gene augmentation using viral vectors and transgene expression to replace an absent or defective protein has shown limited success in treating a variety of genetic disorders. In fact, the mechanisms by which cells and organisms protect against viral infections have presented significant barriers for long-term stable expression of the introduced transgene. In part, due to the disadvantages of using biological vectors, a variety of different approaches for nonviral gene delivery have been developed. 1 In particular, targeting of nonviral delivery systems to specific tissues by receptor/ligand interactions has been exploited for in vivo gene transfer. 2 By targeting the asialoglycoprotein receptor (ASGPR), gene delivery to hepatocytes has been significantly improved both in vitro 3 and in vivo. 4,5 Thus, we have focused on using nonviral approaches to correct the genetic defect in hepatocytes using galactosylated compounds attached directly to polycations, or incorporated in liposomes. 6 The strategy of in situ genomic repair using chimeric RNA/DNA molecules (chimeraplasts) developed from studies investigating molecular aspects of DNA repair. It became apparent that there was a significant increase in pairing efficiency between an oligonucleotide Յ50 bases and a genomic DNA target if RNA replaced DNA in a portion of the targeting oligonucleotide. 7,8 The original chimeric 68-mer design incorporated 10 2'-O-methylated RNA residues flanking each side of a 5 bp stretch of DNA, poly-T hairpin loops, a 3' GC clamp and a complementary all-DNA strand resulting in a stable, nucleaseresistant duplex molecule. The 25 bp homology segment between the chimeraplast and its genomic target is constructed such that it includes one mismatch with the DNA sequence of the gene. This engineered mismatch permits the directed alteration of the genomic target using the chimeraplast as a template for the intended correction of target sequence. It is postulated that the 'mismatched' chimeraplast complexes with the genomic DNA and creates the illusion of a genetic mutation leading to the recruitment of endogenous repair functions. 9,10 This novel approach of gene repair, based on in vitro experimental evidence, was shown by Yoon et al 11 and Cole-Strauss et al 12 to be capable of introducing targeted single base conversions in episomal and genomic DNA in cultured cells. We then reported that the chimeraplast