Oligonucleotide-Mediated Repair Capability of Human Retina

Abstract

Abstract: : Purpose:Several forms of retinitis pigmentosa (RP) are caused by point mutations in retina-specific genes. An approach to correct point mutations is by oligonucleotide-mediated gene repair, in which a single-stranded oligonucleotide (ON) is used to recruit endogenous DNA repair machinery to correct a mutation. We sought to determine whether human retinal cells contain DNA repair proteins required for this approach and whether repair activity can be recruited by a therapeutic oligonucleotide. Methods: An antibody specific for human MSH-2, a requisite DNA repair protein, was used to probe human retina protein extracts. DNA repair reactions consisted of a test plasmid to be repaired, an ON either specific (therapeutic) or nonspecific (negative control) to the plasmid, human retinal nuclear extract, and reaction buffer. The test plasmid contained the ampicillin (amp) resistance gene and the tetracycline (tet) resistance gene with a point mutation. Retinal extract was prepared from human cadaver tissue obtained from the Georgia Lions Eye Bank. After incubation, the plasmid was isolated, electroporated into bacterial cells, and plated onto LB agar containing amp or tet. Colony growth was monitored for several days. Results: MSH-2 immunoreactivity of correct migration appeared in western blots. In repair assays, the ratio of tet (total repaired) colonies versus amp (total transformed) colonies was used to compare experimental samples with controls. Incubation with the therapeutic ON produced a greater than 35-fold increase in growth compared to incubation with the nonspecific ON. In several experiments, incubation with the nonspecific ON produced no growth at all on tet plates. Selected tet resistant colonies were confirmed by endonuclease restriction analysis. Conclusions: The western immunoblotting results suggest that human retina contains a DNA repair pathway constituent shown by others to be requisite for ON-mediated gene therapy. Data from the cell-free in vitro assay indicate that DNA repair activity can be recruited from nuclear extracts of human retina by therapeutic ONs to correct a single base. Human retina thus may have the capability to correct DNA via ON-mediated gene repair. This raises the possibility that oligonucleotides could be used in therapies for diseases caused by short mutations, such as forms of RP.

Keywords: gene transfer/gene therapy • molecular biology • retina