A programmable dual-targeting siRNA scaffold supports potent two-gene modulation in the central nervous system.

Divalent short-interfering RNA (siRNA) holds promise as a therapeutic approach allowing for the sequence-specific modulation of a target gene within the central nervous system (CNS). However, an siRNA modality capable of simultaneously modulating gene pairs would be invaluable for treating complex neurodegenerative disorders, where more than one pathway contributes to pathogenesis. Currently, the parameters and scaffold considerations for multi-targeting nucleic acid modalities in the CNS are undefined. Here, we propose a framework for designing unimolecular 'dual-targeting' divalent siRNAs capable of co-silencing two genes in the CNS. We systematically adjusted the original CNS-active divalent siRNA and identified that connecting two sense strands 3' and 5' through an intra-strand linker enabled a functional dual-targeting scaffold, greatly simplifying the synthetic process. Our findings demonstrate that the dual-targeting siRNA supports at least two months of maximal distribution and target silencing in the mouse CNS. The dual-targeting divalent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g. Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting divalent siRNAs against each gene. This work enhances the potential for CNS modulation of disease-related gene pairs using a unimolecular siRNA.

Di-valent siRNA-mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington's disease.

Huntington's disease (HD) is a severe neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeat tract in the huntingtin gene. Inheritance of expanded CAG repeats is needed for HD manifestation, but further somatic expansion of the repeat tract in non-dividing cells, particularly striatal neurons, hastens disease onset. Called somatic repeat expansion, this process is mediated by the mismatch repair (MMR) pathway. Among MMR components identified as modifiers of HD onset, MutS homolog 3 (MSH3) has emerged as a potentially safe and effective target for therapeutic intervention. Here, we identify a fully chemically modified short interfering RNA (siRNA) that robustly silences Msh3 in vitro and in vivo. When synthesized in a di-valent scaffold, siRNA-mediated silencing of Msh3 effectively blocked CAG-repeat expansion in the striatum of two HD mouse models without affecting tumor-associated microsatellite instability or mRNA expression of other MMR genes. Our findings establish a promising treatment approach for patients with HD and other repeat expansion diseases.

A programmable dual-targeting di-valent siRNA scaffold supports potent multi-gene modulation in the central nervous system.

Di-valent short interfering RNA (siRNA) is a promising therapeutic modality that enables sequence-specific modulation of a single target gene in the central nervous system (CNS). To treat complex neurodegenerative disorders, where pathogenesis is driven by multiple genes or pathways, di-valent siRNA must be able to silence multiple target genes simultaneously. Here we present a framework for designing unimolecular "dual-targeting" di-valent siRNAs capable of co-silencing two genes in the CNS. We reconfigured di-valent siRNA - in which two identical, linked siRNAs are made concurrently - to create linear di-valent siRNA - where two siRNAs are made sequentially attached by a covalent linker. This linear configuration, synthesized using commercially available reagents, enables incorporation of two different siRNAs to silence two different targets. We demonstrate that this dual-targeting di-valent siRNA is fully functional in the CNS of mice, supporting at least two months of maximal target silencing. Dual-targeting di-valent siRNA is highly programmable, enabling simultaneous modulation of two different disease-relevant gene pairs (e.g., Huntington's disease: MSH3 and HTT; Alzheimer's disease: APOE and JAK1) with similar potency to a mixture of single-targeting di-valent siRNAs against each gene. This work potentiates CNS modulation of virtually any pair of disease-related targets using a simple unimolecular siRNA.

Investigation of Neurodevelopmental Deficits of 22 q11.2 Deletion Syndrome with a Patient-iPSC-Derived Blood-Brain Barrier Model.

The blood-brain barrier (BBB) is important in the normal functioning of the central nervous system. An altered BBB has been described in various neuropsychiatric disorders, including schizophrenia. However, the cellular and molecular mechanisms of such alterations remain unclear. Here, we investigate if BBB integrity is compromised in 22q11.2 deletion syndrome (also called DiGeorge syndrome), which is one of the validated genetic risk factors for schizophrenia. We utilized a set of human brain microvascular endothelial cells (HBMECs) derived from the induced pluripotent stem cell (iPSC) lines of patients with 22q11.2-deletion-syndrome-associated schizophrenia. We found that the solute permeability of the BBB formed from patient HBMECs increases by ~1.3-1.4-fold, while the trans-endothelial electrical resistance decreases to ~62% of the control values. Correspondingly, tight junction proteins and the endothelial glycocalyx that determine the integrity of the BBB are significantly disrupted. A transcriptome study also suggests that the transcriptional network related to the cell-cell junctions in the compromised BBB is substantially altered. An enrichment analysis further suggests that the genes within the altered gene expression network also contribute to neurodevelopmental disorders. Our findings suggest that neurovascular coupling can be targeted in developing novel therapeutical strategies for the treatment of 22q11.2 deletion syndrome.

In Vivo Modulation of the Blood-Brain Barrier Permeability by Transcranial Direct Current Stimulation (tDCS).

tDCS has been used to treat various brain disorders and its mechanism of action (MoA) was found to be neuronal polarization. Since the blood-brain barrier (BBB) tightly regulates the neuronal microenvironment, we hypothesized that another MoA of tDCS is direct vascular activation by modulating the BBB structures to increase its permeability (P). To test this hypothesis, we used high resolution multiphoton microscopy to determine P of the cerebral microvessels in rat brain. We found that 20 min 0.1-1 mA tDCS transiently increases P to a small solute, sodium fluorescein (MW 376) and to a large solute, Dextran-70k, with a much higher increase in P to the large solute. By pretreating the vessel with a nitric oxide synthase inhibitor, we revealed that the tDCS-induced increase in P is NO dependent. A transport model for the BBB was further employed to predict the structural changes by the tDCS. Comparing model predictions with the measured data suggests that tDCS increases P by temporarily disrupting the structural components forming the paracellular pathway of the BBB. That the transient and reversible increase in the BBB permeability also suggests new applications of tDCS such as a non-invasive approach for brain drug delivery through the BBB.

What is the Role of Hyaluronic Acid Ester in Myringoplasty? Systematic Review and Meta-Analysis.

OBJECTIVE: To reveal odds of tympanic membrane closure and postoperative hearing outcomes for myringoplasty utilizing hyaluronic acid ester via systematic review and meta-analysis. DATA SOURCES: 1) Search of English articles in PubMed/Medline, Embase, and Cochrane databases published between January 1, 1998 and March 31, 2018. STUDY SELECTION: Inclusion criteria: 1) English language; 2) clinical studies; 3) reported posttreatment perforation status, hearing outcomes, or complications. EXCLUSION CRITERIA: hyaluronic acid used for middle ear packing or topical application of hyaluronic acid solution. DATA EXTRACTION: Number of patients, surgical technique, mean age, overall rate of tympanic membrane closure, success rate based on size of perforation, mean air-bone gap improvement, and postoperative speech scores and complications. DATA SYNTHESIS: Ten studies encompassing 531 patients met criteria. Reported success rates for closure of chronic perforation ranged from 70.0 to 92.7% (mean, 85.21%). Smaller perforation predicted success in complete closure. Mean air-bone gap closure was 10.6 dB (4-24 dB). There were five complications reported. Meta-analysis was performed on five studies. No difference was noted in the success rates between hyaluronic acid ester myringoplasty and conventional tympanoplasty using fascia or perichondrium, with an overall closure rates of 89.8 and 89.4%, respectively (odds ratio [OR] 1.04, 95% confidence interval [CI] 0.59-1.82, p = 0.896). A higher closure rate was seen in hyaluronic acid ester myringoplasty (87.9%) when compared with fat graft myringoplasty (70.8%), (OR 3.01, 95% CI 1.42-6.35, p = 0.004). CONCLUSIONS: Hyaluronic acid (HA) ester myringoplasty appears to be safe and effective at attaining complete closure of tympanic membrane perforation, although there exists significant selection bias and inconsistent reporting among existing papers.