Enhancer-Driven Gene Expression (EDGE) Enables the Generation of Viral Vectors Specific to Neuronal Subtypes

Nair, Rajeevkumar Raveendran; Blankvoort, Stefan; Lagartos, Maria Jose; Kentros, Clifford

doi:10.2139/ssrn.3441914

Cited by 15 publications

(18 citation statements)

References 30 publications

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“…The construction of these virus, AAV-TRE-tight-GFP and AAV-TRE-tight-oChIEF-Citrine respectively, has been described in Nilssen et al (2018) Subsequently, the mCherry sequence was synthesized and cloned in an inverted orientation into EcoR1 and BamH1 sites in pAAV-CMV-FLEX-MCS-WPRE to make pAAV CMV-FLEX-mCherry-WPRE. AAV-CMV-FLEX-mCherry was recovered from pAAV CMV-FLEX-mCherry-WPRE as described elsewhere (Nair et al, 2020;Nilssen et al, 2018).…”

Section: Surgical Procedures and Virus/tracer Injectionsmentioning

confidence: 99%

Local projections of layer Vb-to-Va are more prominent in lateral than in medial entorhinal cortex

Ohara

Blankvoort

Nair

et al. 2021

eLife

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The entorhinal cortex, in particular neurons in layer V, allegedly mediate transfer of information from the hippocampus to the neocortex, underlying long-term memory. Recently, this circuit has been shown to comprise a hippocampal output recipient layer Vb and a cortical projecting layer Va. With the use of in vitro electrophysiology in transgenic mice specific for layer Vb, we assessed the presence of the thus necessary connection from layer Vb-to-Va in the functionally distinct medial (MEC) and lateral (LEC) subdivisions; MEC, particularly its dorsal part, processes allocentric spatial information, whereas the corresponding part of LEC processes information representing elements of episodes. Using identical experimental approaches, we show that connections from layer Vb-to-Va neurons are stronger in dorsal LEC compared with dorsal MEC, suggesting different operating principles in these two regions. Although further in vivo experiments are needed, our findings imply a potential difference in how LEC and MEC mediate episodic systems-consolidation.

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Section: Surgical Procedures and Virus/tracer Injectionsmentioning

confidence: 99%

Local projections of layer Vb-to-Va are more prominent in lateral than in medial entorhinal cortex

Ohara

Blankvoort

Nair

et al. 2021

eLife

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“…However, we postulated that by integrating multiple sources of data we could increase our confidence in the candidate pREs. Thus, we combined our H3K27Ac screen with previously published DLX2, LHX6 and NKX2-1 TF ChIP-seq (Lindtner et al, 2019;Sandberg et al, 2016) found that only half of the tested AAVs targeted cells in a similar pattern to the corresponding transgenic line (Nair et al, 2020). Despite improvements in computational RE prediction methods, this is likely to mean that individual candidates will continue to need empirical testing.…”

Section: Epigenetic Screen For Candidate Cin Enhancersmentioning

confidence: 99%

Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons

Rubin

Malik

Cho

et al. 2020

eNeuro

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Cortical interneuron (CIN) dysfunction is thought to play a major role in neuropsychiatric conditions like epilepsy, schizophrenia and autism. It is therefore essential to understand how the development, physiology, and functions of CINs influence cortical circuit activity and behavior in model organisms such as mice and primates. While transgenic driver lines are powerful tools for studying CINs in mice, this technology is limited in other species. An alternative approach is to use viral vectors such as AAV, which can be used in multiple species including primates and also have potential for therapeutic use in humans. Thus, we sought to discover gene regulatory enhancer elements (REs) that can be used in viral vectors to drive expression in specific cell types. The present study describes the systematic genome-wide identification of putative REs (pREs) that are preferentially active in immature CINs by histone modification chromatin immunoprecipitation and sequencing (ChIP-seq). We evaluated two novel pREs in AAV vectors, alongside the well-established Dlx I12b enhancer, and found that they drove CIN-specific reporter expression in adult mice. We also showed that the identified Arl4d pRE could drive sufficient expression of channelrhodopsin for optogenetic rescue of behavioral deficits in the Dlx5/6 +/− mouse model of fast-spiking CIN dysfunction.

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“…Identifying functional enhancers using AAV reporter vectors. To determine whether ATAC-seq peaks might provide useful enhancers for developing novel genetic tools as had been previously shown (Dimidschstein et al, 2016;Nair et al, 2020) we cloned several peaks into a reporter AAV vector backbone and packaged viral particles with the mouse blood-brain barrierpenetrant capsid PHP.eB (Fig. 3A, Chan et al, 2017).…”

Section: Concordance Of Epigenetic Marks In Human Neurons From Distinmentioning

confidence: 99%

“…AAV capsids have been identified that possess unique tropisms like retrograde transport (Tervo et al, 2016), and blood brain barrier penetrance (Deverman et al, 2016;Chan et al, 2017), and can be exploited to deliver specific transgenes in many tissues (Deverman et al, 2016;Greig et al, 2018;Song et al, 2019). Specific promoters and enhancers can be used to further control transgene expression (Nord et al, 2013;Visel et al, 2013;Silberberg et al, 2016;Dimidschstein et al, 2016;Xiong et al, 2019;Jüttner et al, 2019;Nair et al, 2020). The genome capacity of AAV is ~4.7kb, so only small gene regulatory elements (i.e.…”

Section: Introductionmentioning

confidence: 99%

Functional Enhancer Elements Drive Subclass-Selective Expression from Mouse to Primate Neocortex

et al. 2020

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Viral genetic tools to target specific brain cell types in humans and non-genetic model organisms will transform basic neuroscience and targeted gene therapy. Here we used comparative epigenetics to identify thousands of human neuronal subclass-specific putative enhancers to regulate viral tools, and 34% of these were conserved in mouse. We established an AAV platform to evaluate cellular specificity of functional enhancers by multiplexed fluorescent in situ hybridization (FISH) and single cell RNA sequencing. Initial testing in mouse neocortex yields a functional enhancer discovery success rate of over 30%. We identify enhancers with specificity for excitatory and inhibitory classes and subclasses including PVALB, LAMP5, and VIP/LAMP5 cells, some of which maintain specificity in vivo or ex vivo in monkey and human neocortex. Finally, functional enhancers can be proximal or distal to cellular marker genes, conserved or divergent across species, and could yield brain-wide specificity greater than the most selective marker genes.

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Enhancer-Driven Gene Expression (EDGE) Enables the Generation of Viral Vectors Specific to Neuronal Subtypes

Cited by 15 publications

References 30 publications

Local projections of layer Vb-to-Va are more prominent in lateral than in medial entorhinal cortex

Local projections of layer Vb-to-Va are more prominent in lateral than in medial entorhinal cortex

Regulatory Elements Inserted into AAVs Confer Preferential Activity in Cortical Interneurons

Functional Enhancer Elements Drive Subclass-Selective Expression from Mouse to Primate Neocortex

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