An antisense oligonucleotide-based strategy to ameliorate cognitive dysfunction in the 22q11.2 Deletion Syndrome

Reviewer #1 (Public review):

Summary:

This is an important and very well-presented set of experiments following up on prior work from the lab investigating knock-down (KD) of EMC10 in the restoration of neuronal and cognitive deficits in 22q11.2 Del models, including now both human iPSCs and a mouse model in vivo now with ASOs.

The valuable progress in this current manuscript is the development of ASOs, and the proof of efficacy in vivo in mice of the ASO in knock-down of EMC10 and amelioration of in vivo behavioral phenotypes.

The experiments include iPSC studies demonstrating elevations of EMC10 in a solid collection of paired iPSC lines. These studies also provide evidence of manipulation of EMC10 by overexpression and inhibition of miRNAs that exist in the 22q11 interval. The iPSC studies also nicely demonstrate the rescue of impairments with KD of EMC10 in neuronal arborization as well as KCl-induced neuronal activity. The major in vivo contributions reflect an impressive demonstration of the efficacy of two ASOs in vivo on both KD of EMC10 in vivo and through improvement in behavioral abnormalities in the 22q11 mouse in a range of different behaviors, including social behavior and learning behaviors.

Overall, there are many strengths reflected in this study, including in particular the synergy between in vitro studies in human cell models and in vivo studies in the well-characterized mouse model. The experiments are generally rigorously performed, well-powered, and nicely presented. The claims with regard to the mechanisms of EMC10 elevations and the importance of restoration of EMC10 expression to neuronal morphology and behavior are well supported by the data. The work may be further supported in future studies, by investigation of rescue by ASOs of circuit dysfunction in vivo or ex vivo through electrophysiology in the mouse model. Also, in future studies, investigation of the mechanism by which EMC10, an ER protein involved in protein processing, may function in the observed neuronal abnormalities; however, these studies are clearly for future investigations.

The potential impact of the work is found in the potential value of the ASO approach to the treatment of 22q11, or the pre-clinical evidence that knock-down of this protein may lead to some amelioration of cognitive symptoms. Overall, a very convincing and complementary set of experiments to support EMC10 KD as a therapeutic strategy.

Reviewer #2 (Public review):

Summary:

The manuscript by Thakur et. al seeks to establish a novel ASO-based approach to treat 22q11.2 deletion syndrome. Central to this thesis is that an ER membrane complex member called EMC10 is significantly increased in the disorder, which is largely attributed to the loss of miRNA-mediated repression. The authors generated three new iPSC cell lines for the disorder and showed that deletion of EMC10 rescues morphology and Ca-flux deficits. They go on to show that post-symptomatic deletion of Emc10 in mice using a conditional-off tamoxifen allele reverses social memory phenotypes. Finally, in collaboration with Ionis, they developed two new ASOs to knock down EMC10 and show that social and spatial memory phenotypes are rescued, even two months after injection.

Strengths:

In general, this represents a substantial undertaking and an impressive body of work. The experiments follow a logical progression and in most cases are well-controlled. The isolation of EMC10 effects relative to the broader miRNA disruption is viewed as impactful. The use of both genetic and ASO approaches to validate the therapeutic strategy is also viewed as highly positive. The authors' contention that EMC10 can be targeted at post-symptomatic time points to reverse 22q11.2 deletion syndrome is supported by the data. Further, they have provided a therapeutic mechanism to do so. These findings are likely to be impactful and lead to further development efforts.

Weaknesses:

The primary weaknesses of the manuscript lie in incomplete or inappropriate data analysis, as well as a failure to validate key experiments. For example, both genetic and ASO-mediated EMC10-mediated reductions are assessed at the level of mRNA, but only one experiment, in one brain region, is validated at the protein level. This brain region is the PFC, which is problematic when many of the phenotypes used have a strong hippocampal component. Likewise, the iPSC experiments make the case that excitatory neurons are central to the phenotype, but no effort is made to show that the ASOs are entering that type of neuron, or even any quantification of what percentage of cells in the target brain regions (HPC, PFC, etc.) are positive for the ASO. There is only a single image provided of staining with a phosphorothioate antibody and a claim of robust uptake, which cannot be assumed. The iPSC transcriptomics work would also benefit from a more comprehensive comparison between the EMC10 knockout lines and their parent 22q11 deletion lines. Further, there are other examples where the statistics used are either wrong (Figure 3 t-test vs ANOVA) or missing (Figure S2). These technical and analytical shortcomings make it challenging to fully interpret the data and detract from an otherwise exciting manuscript.