Abstract
Purpose :
Test a suite of in vivo experimental tools to rapidly and quantitatively evaluate novel lead candidate post-transcriptional gene silencing (PTGS) agents for therapeutic efficacy and toxicity.
Methods :
Using a recently reported novel and refined approach to subretinal delivery of vectors using a micromanipulator-guided, trans-scleral, trans-choroidal microneedle approach (Butler and Sullivan, JoVE (141), e55894, doi:10.3791/55894 (2018)) we tested a known “model” PTGS therapeutic for preclinical efficacy and toxicity in a humanized WT RHO mouse model using OCT imaging and molecular (qRT/PCR) measures.
Results :
A recently reported shRNA agent significantly suppressed dog mutant RHO mRNA and rescued retinal degeneration in a knockdown-reconstitute strategy (Cideciyan et al., PNAS (115), No. 36, e8547, (2018)). An shRNA expression construct designed against human RHO (hRHO) strongly suppressed (>90%, p<0.05) hRHO mRNA in cultured human cells relative to a scrambled control shRNA in plasmids. These constructs were embedded into AAV delivery vectors. We injected the AAV agents (1 ul) into the nasal subretinal sector (<50% areal transduction) of mice homozygous for a WT hRHO genomic expression construct on the mouse RHO knockout background on PND 27. We observed a time-dependent thinning (to ~80%, p = <0.05) of ONL thickness with the active but not the scrambled shRNA construct, and only in the nasal retinal sector by 5 weeks post injection. There was no evidence of outer retinal toxicity with either construct or by injection. The active shRNA showed ~60% knockdown of hRHO mRNA in total eye RNA relative to scrambled shRNA construct. This is evidence of haploinsufficiency of hRHO in transduced mouse rods.
Conclusions :
Using our humanized mouse models and unique refined subretinal delivery approach we are able to rapidly evaluate retinal gene therapy agents. Strong reduction of hRHO in mouse photoreceptors leads to haploinsufficiency which must be countered with a cleavage-resistant WT reconstitution construct in a knockdown-reconstitute combined gene therapy for adRP. These approaches will be used to test our novel enhanced Facilitated-hammerhead ribozymes as a therapy for RHO adRP mutations in humanized mice. Through collaboration, these approaches can be used to rapidly test arbitrary retinal gene based therapeutics in mice.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.