Astrocytes are the most abundant glial cell in the central nervous system. In the brain specifically, astrocytes control or contribute to metabolism, blood flow, and water flux, as well as dynamic processes like synaptic formation, maintenance, and pruning. Astrocytes are well established as important players in understanding homeostatic brain function and disease, albeit, there are few methods to visualize or target these cells, greatly hindering our capability to fully characterize and study them. New therapeutic methods or tools to target astrocytes have the potential to combat neurodegenerative and neuropsychiatric conditions like depression, epilepsy, or Parkinson's. Each condition is characterized by either an imbalance in ion, neurotransmitter, or protein clearance— processes in the brain all modulated by astrocytes. Current methods utilizing genetic markers or fluorescent small molecules to target astrocytes suffer from a need for fixation, specificity concerns, gap junction diffusion, or an incapability for chemical modification. We have combated a few of these caveats by synthesizing an array of small molecule probes, recently demonstrating that we can deliver a variety of fluorophores to astrocytes. By leveraging organic chemistry to attach permanently positive, pyridinium‐like moieties to a variety of fluorescent small molecules, we have successfully labelled astrocytes without targeting neurons or other glia. Here, we explore the delivery of diverse small molecule cargo like transcriptional activators, calcium sensors, or drugs when modified with our astrocyte‐selective probes. Leveraging organic chemistry to attach permanently positive N‐heterocyclic amines to biologically interesting molecules will allow us to further probe astrocyte basic function and biology, all while informing the development of future tools in chemical biology. Here, we assay a modified small molecule calcium indicator and a doxycycline‐modified probe. The former will serve to mark activated astrocytes to characterize their response to various brain stimuli. Additionally, the calcium probe will help identify astrocytes that may be in direct communication with subsets of neurons. Painting a picture of the live calcium response in astrocytes could inform therapeutic development by revealing disease mechanisms in epilepsy or seizure, for example. Alternatively, the latter doxycycline probe will function as an astrocyte‐specific, transcription activator in the tetracycline‐inducible gene expression system. Despite the continued development of gene therapies, there are few that utilize regulated gene expression systems. The doxycycline probe will be used to selectively activate transcription of genes of interest in astrocytes in order to control when and how the gene product is produced in a given model. We hope to employ these, and similar molecules therapeutically where current constitutive gene therapies for Parkinson's or glioblastoma, for example, could benefit from regulated gene expression. These probes demonstrate the diver...

Astrocytes are glial cells that tile the entire central nervous system. Within the brain, they control metabolism, blood flow, and water flux, as well as dynamic processes like synaptic formation, maintenance, and pruning. Targeting astrocytes using gene therapies could be an attractive therapeutic option for seizure, epilepsy, Parkinson’s or Alzheimer’s disease, where there exists an imbalance in ion, neurotransmitter, or protein clearance— processes all modulated by astrocytes. We have recently developed chemical tags that ferry small molecule cargo into astrocytes. Here, we adapt these astrocyte targeting tags to control transcription, with the ultimate goal of using them for regulated gene therapy. Essentially, we utilize a modified transcription activator, doxycycline, to turn on the tetracycline‐inducible gene expression (Tet‐On) system— obtaining temporal control over transcription in astrocytes. Astrocyte specificity is attained by attaching a permanently positive astrocyte targeting moiety via organic chemistry to the small molecule drug doxycycline, which retains its Tet‐On activating capabilities. Our current probes include doxycycline and 9‐cyanodoxycyline variants with appended targeting moieties to improve astrocyte targeting and reduce cell permeability, decreasing off‐target affects. Our first probe, doxycycline methyl pyridinium (doxyMP) can successfully activate transcription in primary astrocytes under the strong, mammalian cytomegalovirus (CMV) promoter, eliminating the need for a cell‐specific promoter. In classical gene therapies, constitutive systems suffer from off‐target effects or variable gene expression of cell‐specific promoters. Using an astrocyte‐targeted transcription activator, we eliminate the need for these variably expressed, cell‐specific promoters, while obtaining specific and robust gene expression in astrocytes. Despite the continued development of gene therapies, there are few that utilize regulated gene expression systems. We hope to employ these molecules therapeutically where current constitutive gene therapies for Parkinson’s or glioblastoma, for example, could benefit from regulated gene expression.

Astrocytes are the most abundant type of non‐neuronal cells in the brain. In addition to maintaining blood‐brain barrier integrity and providing nutrients to the surrounding cells, they play an active role in brain function by the uptake and release of neurotransmitters through the tripartite synapse. Astrocytes play a crucial role in the maintenance of the health and function of the central nervous system and thus astrocytic dysfunction has been implicated in many neurological diseases like Parkinson's disease (PD), Alzheimer's disease (AD), Amyotrophic lateral sclerosis (ALS), glioblastoma and depression. Hence, the delivery of small molecule drugs specifically to astrocytes in coordination with gene therapy is a potential therapeutic tool to activate the expression of therapeutic genes to re‐establish healthy astrocytic functions. Recently, a targeting moiety was developed in our group that transports small molecule cargo to astrocytes. Here we adapt the astrocyte targeting moiety to deliver the drug tamoxifen, which is widely used for controlling gene expression in rodents. Since tamoxifen does not have a potential site of modification to attach the tag while retaining the activity of the molecule, we explored a traceless delivery strategy. The active metabolite 4‐hydroxytamoxifen will be caged with a linker attached to the targeting moiety, where the release of the active drug will occur in the presence of light when delivered to astrocytes. The traceless targeted delivery strategy will help us to evaluate the ability of the targeting moiety to deliver drugs to astrocytes and to explore the release of different drug candidates in the native form inside astrocytes. Additionally, it will open the door to study the controlled gene expression of specific genes in astrocytes using the tamoxifen inducible Cre‐ER/loxP system. In the future, the molecules we describe have potential use in gene therapy applications for exploring therapeutic aspects of astrocytes in various neurological diseases.