Site-directed RNA repair of endogenous Mecp2 RNA in neurons

Abstract

Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G> A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.

In vivo repair of a protein underlying a neurological disorder by programmable RNA editing

“…Six copies of a Mecp2 targeting guide or a non-targeting guide ( Supplementary Table S1) were cloned downstream of the U6 small nuclear RNA polymerase III promoter and upstream of the recombinant phage λN-hADAR2 deaminase domain protein (Editase). Because the target adenosine in Mecp2 317G>A is not in an ideal sequence context for ADAR2 editing [38], we utilized a version of Editase that contains a mutation, E488Q, resulting in hyperactivity [19,39]. The non-targeting guide lacks both the BoxB RNA hairpin recognized by the λN RNA binding domain in Editase as well as any Mecp2 sequences.…”

“…Several pioneering studies have paved the way for programmable Ato-I editing of exogenous RNAs by harnessing the deaminase domains of ADAR1 or 2 [13][14][15]. Many of these studies utilized fusions of the ADAR deaminase domain with a heterologous RNA binding protein, for example Cas13 or the bacteriophage λN peptide, with a RNA guide specific for the target RNA substrate [13,[15][16][17][18][19][20][21]. Endogenous RNAs have been shown to be repaired by programmable RNA editing in cell lines and primary cells [17][18][19]22].…”

“…Many of these studies utilized fusions of the ADAR deaminase domain with a heterologous RNA binding protein, for example Cas13 or the bacteriophage λN peptide, with a RNA guide specific for the target RNA substrate [13,[15][16][17][18][19][20][21]. Endogenous RNAs have been shown to be repaired by programmable RNA editing in cell lines and primary cells [17][18][19]22]. A recent study demonstrated repair in mouse models of Duchenne muscular dystrophy and ornithine transcarbamylase deficiency [21], establishing the utility of this approach for both muscle and liver.…”

“…Importantly, a database of mutations causing Rett syndrome [35] indicates that 36% are caused by G>A mutations or C>T mutations that create opal stop codons, raising the possibility that adenosine deamination in these contexts may repair MeCP2 protein function. We previously generated a mouse line in which the mouse Mecp2 gene contained the human patient mutation, MECP2 317G>A (R106Q) [19]. This mutation, resulting in classic Rett syndrome, is located in the DNA binding domain.…”

“…Consequently, MeCP2 protein becomes unstable and has greatly reduced ability to bind to chromatin [19,36,37], easily quantifiable metrics for recovery of protein function. Indeed, by expressing the ADAR2 deaminase domain fused to the bacteriophage λN peptide (Editase) [15], together with a mouse Mecp2 guide containing the RNA sequence recognized by the λN peptide, endogenous Mecp2 317G>A RNA in cultured neurons was repaired efficiently [19]. However, whether we could virally deliver sufficient amounts of Editase in vivo, whether different neuronal types would be equally accessible to directed RNA editing, and the extent of off-target editing, remained open questions.…”