Critical Confirmation of Oligonucleotide-Induced Gene Repair in the rd1 Mouse

Abstract

Purpose:: To test oligonucleotide-mediated gene repair using single-stranded ONs (ssONs) in vivo in rd1 mice in independent laboratories (Atlanta and Paris) and using critical controls.

Methods:: ssONs complementary to genomic sequence subsuming the rd1 point mutation were synthesized with the WT (therapeutic) or rd mutant (control) base at the rd1 locus. Thus, the sole difference between treatment with "therapeutic ON" and "control ON" was the single base at the rd1 locus. The ONs were delivered to C3H/hen (rd1/rd1) mouse eyes by various combinations of ocular injection and transpalpebral iontophoresis at postnatal (P) days 4, 6, and 8. Mice were sacrificed at P28 and P33, stages by which rod photoreceptors are normally absent in the rd1 mouse. ON uptake and distribution was assayed for by fluorescence microscopy. DNA base conversion from mutant to WT was assayed for by allele-specific real-time PCR using genomic DNA from treated mouse retinas as template. Effects on degeneration were quantified by counting rhodopsin-immunoreactive cells in retina sections.

Results:: Coupling iontophoresis with intravitreal injection delivered ON to all nuclear layers of the retina. Treatment with therapeutic ON resulted in a 0.2% conversion from mutant to WT base at the rd1 locus. Retinas from eyes treated with therapeutic ON contained many more rhodopsin immunoreactive cells compared to retinas treated with control ON. This difference was statistically significant even with sampling at P33 (p=0.0011).

Conclusions:: Delivery of therapeutic ONs to rd1/rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence and preservation of rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ONs having the rd1 mutant sequence, the decisive control for this therapeutic approach. Importantly, critical experiments were confirmed in two separate laboratories by several different researchers using independent mouse colonies and ON preparations from independent sources.

Keywords: gene transfer/gene therapy