Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons

Rubin, Anna Noren; Malik, Ruchi; Cho, Kathleen K.A.; Lim, Kenneth J.; Lindtner, Susan; Schwartz, Sarah E. Robinson; Vogt, Daniel; Rubenstein, John L.R.

doi:10.1523/eneuro.0211-20.2020

Cited by 14 publications

(12 citation statements)

References 85 publications

(176 reference statements)

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“…Targeted delivery of transgenes, encoding fluorescent reporters, functional sensors/effectors, supplemental proteins to rescue imbalanced cell functions or genome editors, to specific cell populations enables us to untangle complex biological circuits and offers promise as a therapeutic approach [1][2][3][4] . Targeting strategies include transgenic animal models, engineered viral vectors with biased tropisms [5][6][7][8][9][10][11][12][13] , gene regulatory elements (for example, promoters [14][15][16][17][18] , enhancers [19][20][21][22][23][24][25] and/ or microRNA target sites [26][27][28] ) and various delivery routes. These methods, often used in combination 2,27 , have granted selective access to a few cell types but are still far from covering most molecularly distinct cell populations.…”

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confidence: 99%

Spatial transcriptomics for profiling the tropism of viral vectors in tissues

et al. 2023

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A barrier to advancing engineered adeno-associated viral vectors (AAVs) for precision access to cell subtypes is a lack of high-throughput, high-resolution assays to characterize in vivo transduction profiles. In this study, we developed an ultrasensitive, sequential fluorescence in situ hybridization (USeqFISH) method for spatial transcriptomic profiling of endogenous and viral RNA with a short barcode in intact tissue volumes by integrating hydrogel-based tissue clearing, enhanced signal amplification and multiplexing using sequential labeling. Using USeqFISH, we investigated the transduction and cell subtype tropisms across mouse brain regions of six systemic AAVs, including AAV-PHP.AX, a new variant that transduces robustly and efficiently across neurons and astrocytes. Here we reveal distinct cell subtype biases of each AAV variant, including a bias of AAV-PHP.N toward excitatory neurons. USeqFISH also enables profiling of pooled regulatory cargos, as we show for a 13-variant pool of microRNA target sites in AAV genomes. Lastly, we demonstrate potential applications of USeqFISH for in situ AAV profiling and multimodal single-cell analysis in non-human primates.

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confidence: 99%

Spatial transcriptomics for profiling the tropism of viral vectors in tissues

et al. 2023

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“…So far, not much is known about its function, but it has been shown that Arl4D is involved in neurite growth [ 70 ], adipogenesis [ 71 ] and actin remodeling [ 72 ]. In adult mice, Arl4d is expressed in neocortical layer 1 and hippocampus, mostly in cortical interneurons (CIN), whose loss or alteration have been related to neurological disorders such as autism, schizophrenia, and epilepsy [ 73 ]. Interestingly, both DMRs are directly or indirectly related to neurological disorders such as ASD.…”

Section: Discussionmentioning

confidence: 99%

Maternal–fetal stress and DNA methylation signatures in neonatal saliva: an epigenome-wide association study

et al. 2022

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Background Maternal stress before, during and after pregnancy has profound effects on the development and lifelong function of the infant’s neurocognitive development. We hypothesized that the programming of the central nervous system (CNS), hypothalamic–pituitary–adrenal (HPA) axis and autonomic nervous system (ANS) induced by prenatal stress (PS) is reflected in electrophysiological and epigenetic biomarkers. In this study, we aimed to find noninvasive epigenetic biomarkers of PS in the newborn salivary DNA. Results A total of 728 pregnant women were screened for stress exposure using Cohen Perceived Stress Scale (PSS), 164 women were enrolled, and 114 dyads were analyzed. Prenatal Distress Questionnaire (PDQ) was also administered to assess specific pregnancy worries. Transabdominal fetal electrocardiograms (taECG) were recorded to derive coupling between maternal and fetal heart rates resulting in a ‘Fetal Stress Index’ (FSI). Upon delivery, we collected maternal hair strands for cortisol measurements and newborn’s saliva for epigenetic analyses. DNA was extracted from saliva samples, and DNA methylation was measured using EPIC BeadChip array (850 k CpG sites). Linear regression was used to identify associations between PSS/PDQ/FSI/Cortisol and DNA methylation. We found epigenome-wide significant associations for 5 CpG with PDQ and cortisol at FDR < 5%. Three CpGs were annotated to genes (Illumina Gene annotation file): YAP1, TOMM20 and CSMD1, and two CpGs were located approximately lay at 50 kb from SSBP4 and SCAMP1. In addition, two differentiated methylation regions (DMR) related to maternal stress measures PDQ and cortisol were found: DAXX and ARL4D. Conclusions Genes annotated to these CpGs were found to be involved in secretion and transportation, nuclear signaling, Hippo signaling pathways, apoptosis, intracellular trafficking and neuronal signaling. Moreover, some CpGs are annotated to genes related to autism, post-traumatic stress disorder (PTSD) and schizophrenia. However, our results should be viewed as hypothesis generating until replicated in a larger sample. Early assessment of such noninvasive PS biomarkers will allow timelier detection of babies at risk and a more effective allocation of resources for early intervention programs to improve child development. A biomarker-guided early intervention strategy is the first step in the prevention of future health problems, reducing their personal and societal impact.

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“…AAV is gaining traction as a powerful method for enhancer-driven, cell-type-specific manipulations 15,16,66 .…”

Section: Discussionmentioning

confidence: 99%

Parallel functional testing identifies enhancers active in early postnatal mouse brain

Lambert

Su-Feher

Cichewicz

et al. 2021

eLife

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Enhancers are cis-regulatory elements that play critical regulatory roles in modulating developmental transcription programs and driving cell-type specific and context-dependent gene expression in the brain. The development of massively parallel reporter assays (MPRAs) has enabled high-throughput functional screening of candidate DNA sequences for enhancer activity. Tissue-specific screening of in vivo enhancer function at scale has the potential to greatly expand our understanding of the role of non-coding sequences in development, evolution, and disease. Here, we adapted a self-transcribing regulatory element MPRA strategy for delivery to early postnatal mouse brain via recombinant adeno-associated virus (rAAV). We identified and validated putative enhancers capable of driving reporter gene expression in mouse forebrain, including regulatory elements within an intronic CACNA1C linkage disequilibrium block associated with risk in neuropsychiatric disorder genetic studies. Paired screening and single enhancer in vivo functional testing, as we show here, represents a powerful approach towards characterizing regulatory activity of enhancers and understanding how enhancer sequences organize gene expression in the brain.

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Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons

Cited by 14 publications

References 85 publications

Spatial transcriptomics for profiling the tropism of viral vectors in tissues

Spatial transcriptomics for profiling the tropism of viral vectors in tissues

Maternal–fetal stress and DNA methylation signatures in neonatal saliva: an epigenome-wide association study

Parallel functional testing identifies enhancers active in early postnatal mouse brain

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