Novel extended-release transdermal formulations of the psychedelic N,N-dimethyltryptamine (DMT).

There is considerable evidence from the literature that psychedelics, such as N,N-dimethyltryptamine (DMT), are safe and effective treatments for depression. However, clinical administration to induce psychedelic effects and expensive psychotherapy-assisted treatments likely limit accessibility to the average patient. There is emerging evidence that DMT promotes positive behavioral changes in vivo at sub-hallucinogenic dosages, and depending on the target indication, subjecting patients to high, bolus dosages may not be necessary. Due to rapid metabolic degradation, achieving target levels of DMT in subjects is difficult, requiring IV administration, which poses risks to patients during the intense hallucinogenic and subjective drug effects. The chemical and physical properties of DMT make it an excellent candidate for non-invasive, transdermal delivery platforms. This paper outlines the formulation development, in vitro, and in vivo testing of transdermal drug-in-adhesive DMT patches using various adhesives and permeation enhancers. In vivo behavioral and pharmacokinetic studies were performed with lead patch formulation (F5) in male and female Swiss Webster mice, and resulting DMT levels in plasma and brain samples were quantified using LC/MS/MS. Notable differences were seen in female versus male mice during IV administration; however, transdermal administration provided consistent, extended drug release at a non-hallucinogenic dose. The IV half-life of DMT was extended by 20-fold with administration of the transdermal delivery system at sub-hallucinogenic plasma concentrations not exceeding 60 ng/mL. Results of a translational head twitch assay (a surrogate for hallucinogenic effects in non-human organisms) were consistent with absence of hallucinations at low plasma levels achieved with our TDDS. Despite the reported low bioavailability of DMT, the non-invasive transdermal DMT patch F5 afforded an impressive 77 % bioavailability compared to IV at two dosages. This unique transdermal delivery option has the potential to provide an out-patient treatment option for ailments not requiring higher, bolus doses and is especially intriguing for therapeutic indications requiring non-hallucinogenic alternatives.

Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries.

New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.

Convergent coexpression of autism-associated genes suggests some novel risk genes may not be detectable in large-scale genetic studies.

Autism spectrum disorder (ASD) is a heritable neurodevelopmental disorder characterized by deficits in social interactions and communication. Protein-altering variants in many genes have been shown to contribute to ASD; however, understanding the convergence across many genes remains a challenge. We demonstrate that coexpression patterns from 993 human postmortem brains are significantly correlated with the transcriptional consequences of CRISPR perturbations in human neurons. Across 71 ASD risk genes, there was significant tissue-specific convergence implicating synaptic pathways. Tissue-specific convergence was further demonstrated across schizophrenia and atrial fibrillation risk genes. The degree of ASD convergence was significantly correlated with ASD association from rare variation and differential expression in ASD brains. Positively convergent genes showed intolerance to functional mutations and had shorter coding lengths than known risk genes even after removing association with ASD. These results indicate that convergent coexpression can identify potentially novel genes that are unlikely to be discovered by sequencing studies.

Transcriptional and functional consequences of alterations to MEF2C and its topological organization in neuronal models.

Point mutations and structural variants that directly disrupt the coding sequence of MEF2C have been associated with a spectrum of neurodevelopmental disorders (NDDs). However, the impact of MEF2C haploinsufficiency on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern its regulation. To explore the functional changes associated with structural variants that alter MEF2C expression and/or regulation, we generated an allelic series of 204 isogenic human induced pluripotent stem cell (hiPSC)-derived neural stem cells and glutamatergic induced neurons. These neuronal models harbored CRISPR-engineered mutations that involved direct deletion of MEF2C or deletion of the boundary points for topologically associating domains (TADs) and chromatin loops encompassing MEF2C. Systematic profiling of mutation-specific alterations, contrasted to unedited controls that were exposed to the same guide RNAs for each edit, revealed that deletion of MEF2C caused differential expression of genes associated with neurodevelopmental pathways and synaptic function. We also discovered significant reduction in synaptic activity measured by multielectrode arrays (MEAs) in neuronal cells. By contrast, we observed robust buffering against MEF2C regulatory disruption following deletion of a distal 5q14.3 TAD and loop boundary, whereas homozygous loss of a proximal loop boundary resulted in down-regulation of MEF2C expression and reduced electrophysiological activity on MEA that was comparable to direct gene disruption. Collectively, these studies highlight the considerable functional impact of MEF2C deletion in neuronal cells and systematically characterize the complex interactions that challenge a priori predictions of regulatory consequences from structural variants that disrupt three-dimensional genome organization.

Role of angiotensin II type 1 (AT1) and type 2 (AT2) receptors in airway reactivity and inflammation in an allergic mouse model of asthma.

Objective: Angiotensin II (Ang II) exerts its effects through two G-protein coupled receptors: angiotensin II type 1 receptors (AT1) and type 2 receptors (AT2). Both these receptor subtypes are poorly understood in asthma. In this study, we investigated effects of AT1 receptor antagonist losartan, novel AT2 receptor agonist novokinin and AT2 receptor antagonist PD 123319 in a mouse model of asthma. Methods: Mice were divided into control (CON) and allergen sensitized (SEN) groups. SEN was sensitized with ovalbumin (OVA) on days 1 and 6 (30 µg; i.p.), followed by 5% OVA aerosol challenge (days 11-13). Treatments included (a) losartan (SEN + LOS; 20 mg/kg i.p., day 14), (b) novokinin (SEN + NOV; 0.3 mg/kg i.p., day 14), and (c) PD 123319 (SEN + PD; 5 mg/kg i.p., day 14). Experiments for airway responsiveness, bronchoalveolar lavage, and tracheal ring reactivity using isolated organ bath were performed. Results: Airway responsiveness to methacholine (MCh) (48 mg/mL) was significantly higher in SEN (563.71 ± 40% vs. 294.3 ± 123.84 in CON). This response was potentiated in SEN + PD group (757 ± 30%; p < .05 compared to SEN). SEN + LOS (247.61 ± 86.85%) and SEN + NOV (352 ± 11%) had significantly lower response compared to SEN. SEN + LOS (26.22 ± 0.29%) and SEN + NOV (46.20 ± 0.76%) treatment significantly (p < .001) attenuated total cell count and eosinophils compared to SEN group (69.38 ± 1.5%), while SEN + PD (73.04 ± 0.69%) had highest number of eosinophils. Tracheal response to MCh was significantly higher in SEN group compared to controls, and this response was significantly lowered with the losartan and novokinin treatments. Conclusions: These data suggest that AT1 and AT2 receptors have opposite effects in modulating airway hyperresponsiveness and inflammation in asthma.