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Chemical approaches towards regulated gene therapy in astrocytes

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.