Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

Abstract

We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach.

Clinical and Molecular Characterization ofPROM1-Related Retinal Degeneration

“…Subretinal delivery of adeno-associated viral vector–carrying PROM1 (2.5 kilobase pairs) to photoreceptors at an early stage of recessive dystrophy could replace the null protein and potentially rescue the phenotype. However, the dominant variant would need to be silenced first, eg, RNA silencing via a mirtron, 36 followed by a gene replacement therapy of the wild-type protein, ie, block-and-replace therapy. In more advanced instances of disease, where irreversible photoreceptor damage has occurred, restoring sight by optogenetic treatment 37 may be an alternative therapy.…”

Molecular Targets and Therapeutic Strategies in Spinocerebellar Ataxia Type 7

“…All these molecules act through the RISC pathway: shRNA, siRNA and mirtron lead to target mRNA degradation; sd-siRNA causes translational blockage. miRNA microRNA, mRNA messenger RNA, RISC RNAinduced silencing complex, sd-siRNA self-duplexing siRNA, shRNA short hairpin RNA, siRNA small interfering RNA, SNP single nucleotide polymorphism An alternative to allele selective targeting was recently developed using specific knockdown-replacement strategy, which combines a non-allele specific gene silencing with simultaneous delivery of a functional copy of the wild-type gene to preserve the protein physiological function [185]. In this strategy, the authors used artificial mirtrons targeting ATXN7 transcript, and a mirtron resistant ATXN7 transgene carrying silent mutations [185].…”

“…Mirtron maturation is thus independent of DROSHA and avoids overloading this processing machinery. The efficacy of the approach was significant in cells, despite potential limitation due to the splicing efficiency of mirtrons [185]. The development of knockdown-replacement strategy in vivo is challenging as both the silencing agent and the expression vector for the normal allele must be efficiently delivered, and the exogenous protein has to be expressed at an appropriate level.…”

New Perspectives of Gene Therapy on Polyglutamine Spinocerebellar Ataxias: From Molecular Targets to Novel Nanovectors

“…The non-allele-specific silencing strategy has also been applied to SCA7. Simultaneous expression of artificial mirtrons against ATXN7 mRNA and a functional mirtron-resistant ATXN7 wild-type copy was successfully accomplished in patient-derived fibroblasts [ 121 ]. Mirtrons are introns that form pre-microRNA hairpins upon splicing.…”