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. 2022 Dec 27;6(3):e202201592. doi: 10.26508/lsa.202201592

Figure 1. Overview of therapeutic outline, in vitro knockdown efficiency of miTmc1D11, and gene therapy constructs.

Figure 1.

(A) Illustration of mutation-specific RNAi, in which a targeted RNAi construct suppresses a mutant allele. (B) Illustration of mutation-agnostic RNAi, wherein the vector delivers an RNAi construct to suppress endogenous (WT and mutant) transcripts of the gene, and a knockdown-resistant exogenous WT allele, which is engineered to have synonymous variants at the miRNA-targeting site. (C) qRT-PCR analysis isolated from COS-7 cells co-transfected with WT Tmc1 and either miTmc1D02-D11 or empty vector. Knockdown efficiency of miTmc1D11 was >80%. Data are the mean ± SEM, and each point represents a single biological replicate, the average of three technical triplicates. (D) Outline of transgene control and gene therapy constructs. The mU6 promoter drives miSafe or miTmc1D11 expression, and CMV promoter drives expression of eGFP or engineered Tmc1 cDNA ± WPRE. (E) miTmc1D11 sequence and diagram of knockdown-resistant exogenous Tmc1. Exogenous WT mouse Tmc1 has been engineered to have six synonymous variants in exon 17 to escape RNAi suppression.