Enhancer-AAVs allow genetic access to oligodendrocytes and diverse populations of astrocytes across species

Reviewer #1 (Public review):

The goal of this study was to generate a library of new enhancer-driven AAVs in order to selectively and efficiently target astrocytes and oligodendrocytes in rodents. The implied criteria are that such viral vectors should have high specificity for the intended cell type and effectively express in all astrocytes/oligos in the brain or, alternatively, be specific for defined brain regions, layers, or subtypes of astrocytes/oligos. In addition, they should be compatible with intravenous retro-orbital delivery to facilitate experimentation and brain-wide targeting (i.e., show organ specificity and high efficiency in the brain). Ideally, these new AAVs would also maintain their characteristics across disease contexts and show applicability in non-human primates. Tools with such characteristics are generally lacking in studying glial cells and would be extremely useful to scale up and accelerate glial research, allowing targeting of astrocytes/oligos with distinct molecular identity and intersectional strategies.

At present, however, none of the enhancer-AAVs presented in the study seems to meet this combination of criteria, at least not with the level of stringency typically expected in the field. The main reason is that, in its current form, the study does not present one candidate AAV iteratively improved to meet all these criteria; instead, it presents a catalogue of new AAVs with various degrees of specificity, completeness, and mixed characteristics. Therefore, their utility should be interpreted cautiously. Moreover, the way specificity and completeness are intermixed in the analysis makes it difficult to evaluate the actual utility of any given AAV. The study might have been strengthened by focusing on a small set of the most promising candidates (i.e., AiE0890m_3x2C) and validating them thoroughly for expression specificity, completeness, effective cargo expression, ability to allow specific pan-astrocyte or astrocyte-subtype targeting in vivo, and preserved properties in NHPs and in disease, as this would encourage their adoption by the community. Currently, too many AAVs are assessed inconsistently against the desired criteria, with none being evaluated through and through.

The impact of the catalogue is also greatly diminished by the fact that a suite of AAVs with outstanding specificity and efficiency is already available for the study of astrocytes (e.g., 4x6T AAVs) and was not utilized as a standard to benchmark the new library, making it difficult to appreciate the relative benefits of the new AAVs. The inclusion of expression data in NHPs is very significant, but benchmarking against established AAVs would also be needed to fully appreciate their value.

Importantly, readers should also be aware that the study seems noticeably limited in its literacy with glial biology. The introduction and discussion frame the field in a way that seems outdated, creating the impression that the diverse roles of glia in health and disease have not yet been studied, which may inadvertently be perceived as dismissive and stigmatizing.

In summary, the paper introduces potentially useful viral tools and lays the foundations for future multiplexed targeting of distinct glial cell subpopulations in rodents and in non-human primates, which are extremely important directions. Some of the regionally restricted or even sparsely expressed AAVs may prove valuable in enabling subpopulation-specific targeting or molecular profiling strategies, but currently lack full benchmarking. At present, the promises over the utility of the new tools seem overstated, and the library may not yet represent an actionable resource for targeting astrocytes and oligodendrocytes.

Reviewer #2 (Public review):

Enhancer elements are regulatory DNA sequences that are capable of driving specific expression patterns. As these elements are generally short and context-independent, enhancers can be used in expression vectors (e.g., packaged in an adeno-associated virus, AAV) to limit expression to target cell populations. This approach was identified as a major strategy for cell-type-specific manipulation in the brain and has been pursued by both standard research studies as well as large-scale efforts led by the BRAIN Initiative. This manuscript describes a major effort to generate enhancer-AAVs targeting astrocytes and oligodendrocytes orchestrated by a large research team led by the Allen Institute for Brain Science. This manuscript parallels other recent publications describing sets of enhancer-AAVs, following rigorous, similar methods with relatively broad testing and application.

To identify and screen candidate enhancers, the scientists prioritized candidates via analysis of single-nucleus accessibility and methylation datasets (i.e., snATAC-seq) and tested them in mice. The scientists prioritized candidate enhancers that exhibited specificity of accessibility in the target cell type. Following selection, the scientists cloned the candidate sequences into AAV vectors with a minimal promoter and reporter gene, packaged the virus, delivered it to the mouse brain, and screened for activity based on reporter expression. Candidates that passed initial screening were further characterized via imaging and sorting, followed by single-cell RNA-seq. This process had around a 50% success rate and yielded 25 astrocyte and 21 oligodendrocyte enhancer-AAVs with the targeted cell-type-specific expression patterns.

The scientists went on to test for subtype-specific activity patterns, finding wide diversity in astrocyte activities across sub-populations and conversely, homogenous oligodendrocyte activation. They optimized a few of these via concatenating the enhancer core sequence to increase expression levels of the reporter gene and showed strong specificity and completeness of cell targeting for a set of these enhancer-AAVs. Following characterization and validation, they then deployed these enhancer-AAVs in a number of demonstration applications to show the utility for basic and translational science. All the constructs developed here are available for public use via Addgene, ensuring that these new tools can be used by other researchers.

There really are no obvious weaknesses in the work presented here, from the generation of the enhancer-AAVs to use in sophisticated validation studies. The enhancer-AAV testing is rigorous and provides critical information necessary for other scientists to select and use these constructs. The applications demonstrate the power of enhancer-AAV approaches. The toolbox presented here may not enable specific targeting of all relevant cellular subtypes or activity states for astrocytes and oligodendrocytes, and future work will be needed to fully understand the activity of the enhancers, identity of the target cell types, and context-dependent utility of these constructs. However, the set of enhancer-AAVs developed here should be transformative for researchers working on accessing and manipulating these cell types and have a major impact on the field.