Mirtron-mediated RNA knockdown/replacement therapy for the treatment of dominant retinitis pigmentosa

Abstract

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a RhoP23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo.

VEGFA-targeting miR-agshRNAs combine efficacy with specificity and safety for retinal gene therapy

“…Furthermore, application of atypical RNAi effectors in the form of mirtrons has recently been pursued for RNA knockdown/replacement therapy. 47 Our present findings, combined with the fact that bicistronic adeno-associated viruses (AAVs) encoding RNAi effectors and proteins have been engineered, 46 suggest that application of our miR-agshRNAs in RNA replacement gene therapy might add additional advantages over current technologies, facilitating broad treatment options of dominantly inherited diseases.…”

Gene-Based Therapeutics for Inherited Retinal Diseases

“…Conveniently, mirtrons can be incorporated into polycistronic expression cassettes that contain both the mirtron and an engineered copy of the target gene that is resistant to mirtron-mediated RNAi, making them particularly useful for autosomal dominant diseases. This approach has been demonstrated in a mouse model of RHO mediated RP, where a single AAV vector containing a mirtron that targeted both the wild-type and mutant copies of RHO for RNAimediated decay, and at the same time supplied a RNAiresistant engineered copy of functional RHO to the transfected cell (Orlans et al, 2021). This mutation-agnostic approach may theoretically be applied to many dominantly inherited IRDs.…”

Benefits and Shortcomings of Laboratory Model Systems in the Development of Genetic Therapies

“…HEK293T cells were also employed to confirm targeted genome editing and genome regulation via a dual AAV split-Cas9 system [5]. Additionally, Rumachik et al found that AAV vectors produced in cell lines of human origin were significantly more potent than vectors produced in insect cell systems after testing them in HEK293T cells [17] and Orlans et al developed a novel mirtron-mediated RNA therapy for retinitis pigmentosa after evaluating the splicing efficiency and potency of artificial mirtrons in HEK293 cells [7]. Moreover, HEK cells also serve to determine the potency of the rAAV-REP1 vector used for the treatment of choroideremia.…”