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Cavit Agca, Corina Kohler, Claudia Bippes, Hendrik P Scholl, Albert Neutzner; An indirect gene therapy approach for Autosomal Dominant Optic Atrophy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4092.
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Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerve. It is the most common inherited optic nerve disease with an estimated prevalence of 1 in 35.000. 70% of ADOA cases are associated with mutations in the OPA1 gene. OPA1 is a mitochondrial protein localized to the inner mitochondrial membrane and the intermembrane space and is involved in mitochondrial dynamics as a fusion promoting protein. In OPA1-related ADOA, cellular OPA1 levels are reduced due to various mutations causing haploinsufficiency, negatively impacting retinal ganglion cells eventually leading to loss of vision. The monogenic nature of OPA1-related ADOA disease lends itself to attempt gene therapeutic approaches to provide a functional cure. However, OPA1 is highly complex and multiple splicing events and subsequent proteolytic processing lead to the generation of eight different variants of OPA1 with interdependent functions. Thus, delivery of a single variant of OPA1 cDNA is unlikely to be sufficient to restore full OPA1 function. Another possible therapeutic option is an indirect gene therapy to restore OPA1 levels by boosting expression of the wt allele of OPA1 in ADOA patients.
In order to restore opa1 expression, we used Cas9-based artificial transactivators (ATAs), where a nuclease-deficient variant of Cas9 (dCas9) still possessing DNA-binding activity is fused to a transcriptional activator. By using a specific guide RNA (gRNA), dCas9-ATA is targeted to the opa1 promoter for gene activation. Using flow cytometry, Neuro2A cells expressing OPA1-targeting ATA as well as mCherry reporter were sorted to determine opa1 levels by qRT-PCR.
RT-PCR results for opa1 showed that targeting the wt allele of opa1 with an ATA successfully upregulated opa1 expression. By testing several gRNAs targeting the opa1 promoter, we were able to identify a gRNA that causes around twofold upregulation of opa1.
This study shows that it is feasible to upregulate opa1 using Cas9-based ATAs. Moreover, we have identified a gRNA that facilitates opa1 upregulation approximately twofold ideal to correct Opa1 haploinsufficiency. Therefore, indirect Opa1 gene therapy using dCas9-based ATAs is now ready for trials in animal models of ADOA. This opens up the potential to treat ADOA in humans with such novel gene expression modulators.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
ATA dependent opa1 upregulation
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