Hammerhead ribozymes as a candidate gene-based therapy for autosomal dominant retinitis pigmentosa due to rhodopsin mutations
Kolniak, Tiffany A.
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Post-transcriptional gene silencing ( PTGS ) holds vast potential as a method of treatment for autosomal dominant diseases. Significant bottlenecks exist in the development of gene therapeutics, including the identification of regions of target messenger mRNA ( mRNA ) accessibility, colocalization of the PTGS agent with the target mRNA in the cellular environment, and stable expression of the PTGS agent in the target cells. Not only do these factors need to be taken into consideration in the development of gene therapies, but the lack of toxic effects induced by the PTGS agent needs to be investigated as well in a rapid, inexpensive manner. A technology platform examining PTGS safety determinations in cultured human cells that are expected to simulate the major common cellular housekeeping microenvironment has been developed. A high throughput screening ( HTS ) cytotoxicity assay in 96-well plate format has been generated. The assay is based around the SYTOX Green dye which is excluded from healthy viable cells and becomes substantially fluorescent only after entering cells and binding to nuclear DNA. In this format a number of PTGS agents can be tested for cellular toxicity relative to control elements. A HTS 96-well plate assay that can be used to assess the impact of any given PTGS agent on stimulating a variety of common cellular signaling pathways (e.g. CRE, SRE, AP-1, NFκB, Myc, and NFAT) that could indicate possible deleterious effects of PTGS agents either independent or dependent upon base pair complementarity with target mRNAs has also been developed exploiting the secreted alkaline phosphatase ( SEAP ) Pathway Profiling System where the expression of a secreted reporter protein is coupled to transcriptional activation of a variety of promoter elements involved in common cell signaling pathways. These assays allow for an initial rapid, inexpensive determination of potential cellular toxicity of PTGS agents. The development of gene therapeutics also involves design of a PTGS agent that is not only relatively nontoxic but also provides efficient reduction of target mRNA. PTGS agents are likely to have value as therapeutics for the treatment of many eye diseases, such as Retinitis Pigmentosa ( RP ). To date over 150 mutations in human rhodopsin ( RHO ) have been linked to autosomal dominant Retinitis Pigmentosa ( adRP ). As such, PTGS holds vast potential as a means to treat the disease by reducing the amount of toxic mutated RHO mRNA and protein that is expressed. By designing PTGS agents using a mutation independent approach a single agent can be used to combat all or most disease causing mutations in RHO. An efficacious hammerhead ribozyme (hhRz) has been developed which significantly reduces the amount of RHO mRNA and protein when evaluated in cell culture studies. Once a PTGS agent has been designed and deemed efficacious in cell culture studies, it needs to be further evaluated by testing in an animal model. An appropriate animal model for testing is one in which the target mRNA of choice is expressed. While the mouse models of adRP that currently exist have been useful in elucidating the mechanisms by which mutation in RHO cause disease, most are not ideal in that either a mutated mouse RHO transgene is expressed and targeted for reduction or a mutated human RHO transgene is expressed but on the background of wild type (WT) mouse RHO. Targeting mutated RHO from a species other than human is not conducive to the development of PTGS agents to treat human disease as there are sequence variations that may cause differences in secondary structures between species leading to a change in efficacy of the PTGS agent. In order to relieve this bottleneck in the evaluation of PTGS agents, a novel mouse model of adRP has been generated that strictly expresses a single copy of the human P347S RHO transgene and one copy of the WT human RHO transgene on the mouse RHO knockout background. This model also exhibits a slow rate of retinal degeneration allowing time to institute the PTGS therapy before significant photoreceptor loss has occurred indicating the model is amenable to investigation into the ability of PTGS agents to prevent retinal degeneration.