Abstract
Purpose: :
To develop RNA knockdown strategies as part of an RNA replacement strategy for the treatment of autosomal dominant retinitis pigmentosa (ADRP).
Methods: :
Ribozymes and siRNAs specific for mouse rhodopsin (RHO) mRNA were designed and tested in vitro. Ribozymes (Rz) were analyzed first in cell free reactions to identify those with the fastest cleavage kinetics. Both siRNAs and ribozymes were then tested in co–transfection experiments in tissue culture to verify that they could reduce levels of wild–type rhodopsin mRNA. Ribozyme coding sequences and DNA segments coding RNA hairpin precursors of the siRNA were cloned in Adeno associated virus (AAV) and packaged in AAV serotype 5 capsids. They were injected subretinally in the right eyes of rhodopsin hemizygote (RHO+/–) mice (at postnatal day 6) and of P23H line 3 transgenic rats (at P 16). Left eyes were sham injected or received AAV–GFP. Impact of the AAV injections was monitored at regular intervals by scotopic electroretinography (ERG) and, eventually, by histology. Reduction of rhodopsin mRNA was measured by RT–PCR and of rhodopsin protein by immunoblots.
Results: :
In tissue culture, ribozymes and siRNA led to a 60 –80% reduction in rhodopsin mRNA. In wild type mice, Rz397 led to a 50% reduction in ERG b–wave amplitude and an 80% reduction in rhodopsin protein content in RHO+/– mice. The same AAV–ribozyme had little impact on the ERG response of wild–type mice, despite a 50% reduction in rhodopsin levels. AAV–siRNA led to a similar reduction in ERG response in RHO+/– hemizygous mice and a 70% reduction in rhodopsin protein. Rz525 was tested in P23H rats because it cleaves mouse but not rat rhodopsin mRNA. Injection of AAV expressing this ribozyme led to an increase in scotopic ERG response relative to the control eyes and a reduction in the level of the mutant transgene mRNA.
Conclusions: :
AAV delivered ribozymes and siRNA lead to significant reductions of rhodopsin mRNA in rodents sufficient to impact rhodopsin content and ERG response. When coupled with ribozyme– or siRNA–resistant RHO mRNA, these tools should permit replacement of endogenous RHO mRNA with wild–type RHO and alter the balance between mutant and normal rhodopsin in a treatment for ADRP.
Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • gene/expression