Optimizing AAV2/6 microglial targeting identified enhanced efficiency in the photoreceptor degenerative environment

Adeno-associated viruses (AAVs) are widely used to deliver genetic material in vivo to distinct cell types such as neurons or glial cells, allowing for targeted manipulation. Transduction of microglia is mostly excluded from this strategy, likely due to the cells’ heterogeneous state upon environmental changes, which makes AAV design challenging. Here, we established the retina as a model system for microglial AAV validation and optimization. First, we show that AAV2/6 transduced microglia in both synaptic layers, where layer preference corresponds to the intravitreal or subretinal delivery method. Surprisingly, we observed significantly enhanced microglial transduction during photoreceptor degeneration. Thus, we modified the AAV6 capsid to reduce heparin binding by introducing four point mutations (K531E, R576Q, K493S, and K459S), resulting in increased microglial transduction in the outer plexiform layer. Finally, to improve microglial-specific transduction, we validated a Cre-dependent transgene delivery cassette for use in combination with the Cx3cr1CreERT2 mouse line. Together, our results provide a foundation for future studies optimizing AAV-mediated microglia transduction and highlight that environmental conditions influence microglial transduction efficiency

The HDAC-Associated Sin3B Protein Represses DREAM Complex Targets and Cooperates with APC/C to Promote Quiescence

Summary: The mammalian DREAM complex is responsible for the transcriptional repression of hundreds of cell-cycle-related genes in quiescence. How the DREAM complex recruits chromatin-modifying entities to aid in its repression remains unknown. Using unbiased proteomics analysis, we have uncovered a robust association between the chromatin-associated Sin3B protein and the DREAM complex. We have determined that genetic inactivation of Sin3B results in the de-repression of DREAM target genes during quiescence but is insufficient to allow quiescent cells to resume proliferation. However, inactivation of APC/CCDH1 was sufficient for Sin3B−/− cells, but not parental cells, to re-enter the cell cycle. These studies identify Sin3B as a transcriptional corepressor associated with the DREAM complex in quiescence and reveals a functional cooperation between E2F target repression and APC/CCDH1 in the negative regulation of cell-cycle progression. : The DREAM complex associates with the co-repressor Sin3B to repress target genes during quiescence. Bainor et al. show that Sin3B inactivation is insufficient to allow S-phase re-entry but poises quiescent cells to proliferate upon inhibition of the APC/CCDH1 complex, highlighting a functional cooperation between transcriptional and post-translational cell-cycle regulation. Keywords: Sin3, DREAM, quiescence, cell cycle, E2