Cell-specific regulation of gene expression using splicing-dependent frameshifting

Abstract

Precise and reliable cell-specific gene delivery remains technically challenging. Here we report a splicing-based approach for controlling gene expression whereby separate translational reading frames are coupled to the inclusion or exclusion of mutated, frameshifting cell-specific alternative exons. Candidate exons are identified by analyzing thousands of publicly available RNA sequencing datasets and filtering by cell specificity, conservation, and local intron length. This method, which we denote splicing-linked expression design (SLED), can be combined in a Boolean manner with existing techniques such as minipromoters and viral capsids. SLED can use strong constitutive promoters, without sacrificing precision, by decoupling the tradeoff between promoter strength and selectivity. AAV-packaged SLED vectors can selectively deliver fluorescent reporters and calcium indicators to various neuronal subtypes in vivo. We also demonstrate gene therapy utility by creating SLED vectors that can target PRPH2 and SF3B1 mutations. The flexibility of SLED technology enables creative avenues for basic and translational research.

Magnetogenetic cell activation using endogenous ferritin

“…AAV delivery of Nb-Ft-TRPV1 Ca2+ constructs enables in vivo, remote, targeted modulation of cell types dispersed across organs, such as beta cells in pancreatic islets. The AAV-compatible magnetogenetic construct combined with advances in AAV serotypes, cell-type specific promoters, splicing and intersectional viral approaches (30)(31)(32) will greatly expand the questions that can be addressed by magnetogenetics.…”

Creation of de novo cryptic splicing for ALS/FTD precision medicine

“…In contrast, TDP-REGv2 uses synthetic splicing sensors that are embedded within the transgene sequence itself. This addresses several limitations with TDP-REGv1 and previous approaches ( 25 ): it removes the need for a lengthy upstream regulatory region, thus aiding packaging of large transgenes into AAV vectors; it prevents an upstream, unwanted peptide being expressed and released into the cell; and it is highly tunable, meaning that splicing characteristics can be optimised for the specific transgene being delivered. For example, with a gene editing enzyme one may opt for extremely tight gating to prevent the risk of DNA damage in non-target cells, whereas for a chaperone one may prefer less tight gating if it enables higher maximal expression.…”

“…Using SpliceNouveau , we successfully generated cryptic cassette exons, and also cryptic alternative splice sites, by computationally designing competitor alternative splice sites that are used preferentially when TDP-43 is present. The flexibility and high success-rate of SpliceNouveau -designed vectors therefore represents a major step forward in splicing-regulated vector technology ( 25 , 27 ).…”

Making the Next Generation of Therapeutics: mRNA Meets Synthetic Biology

“…By utilizing cell-type-specific splicing sites, SLED creates frameshifts in the coding sequence, leading to the expression of different proteins based on the specific splicing properties of the cell type. 63 Additionally, miRNAs play a crucial role in post-transcriptional gene regulation. Lockhart et al harnessed the natural function of miRNAs to control p27 encoding mRNA degradation, specifically in endothelial cells.…”

“…One application of alternative splicing is the splicing-linked expression design (SLED), which allows for a higher level of control over protein production. By utilizing cell-type-specific splicing sites, SLED creates frameshifts in the coding sequence, leading to the expression of different proteins based on the specific splicing properties of the cell type …”