Pooled Screens Identify GPR108 and TM9SF2 as Host Cell Factors Critical for AAV Transduction.

Adeno-associated virus (AAV) has been used extensively as a vector for gene therapy. Despite its widespread use, the mechanisms by which AAV enters the cell and is trafficked to the nucleus are poorly understood. In this study, we performed two pooled, genome-wide screens to identify positive and negative factors modulating AAV2 transduction. Genome-wide libraries directed against all human genes with four designs per gene or eight designs per gene were transduced into U-2 OS cells. These pools were transduced with AAV2 encoding EGFP and sorted based on the intensity of EGFP expression. Analysis of enriched and depleted barcodes in the sorted samples identified several genes that putatively decreased AAV2 transduction. A subset of screen hits was validated in flow cytometry and imaging studies. In addition to KIAA0319L (AAVR), we confirmed the role of two genes, GPR108 and TM9SF2, in mediating viral transduction in eight different AAV serotypes. Interestingly, GPR108 displayed serotype selectivity and was not required for AAV5 transduction. Follow-up studies suggested that GPR108 localized primarily to the Golgi, where it may interact with AAV and play a critical role in mediating virus escape or trafficking. Cumulatively, these results expand our understanding of the process of AAV transduction in different cell types and serotypes.

Identification of modulators of autophagic flux in an image-based high content siRNA screen.

Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. In addition to nonselective autophagic engulfment of cytoplasm, autophagosomes can recognize specific cargo by interacting with ubiquitin-binding autophagy receptors such as SQSTM1/p62 (sequestosome 1). This selective form of autophagy is important for degrading aggregation-prone proteins prominent in many neurodegenerative diseases. We carried out a high content image-based siRNA screen (4 to 8 siRNA per gene) for modulators of autophagic flux by monitoring fluorescence of GFP-SQSTM1 as well as colocalization of GFP-SQSTM1 with LAMP2 (lysosomal-associated membrane protein 2)-positive lysosomal vesicles. GFP-SQSTM1 and LAMP2 phenotypes of primary screen hits were confirmed in 2 cell types and profiled with image-based viability and MTOR signaling assays. Common seed analysis guided siRNA selection for these assays to reduce bias toward off-target effects. Confirmed hits were further validated in a live-cell assay to monitor fusion of autophagosomes with lysosomes. Knockdown of 10 targets resulted in phenotypic profiles across multiple assays that were consistent with upregulation of autophagic flux. These hits include modulators of transcription, lysine acetylation, and ubiquitination. Two targets, KAT8 (K[lysine] acetyltransferase 8) and CSNK1A1 (casein kinase 1, α 1), have been implicated in autophagic regulatory feedback loops. We confirmed that CSNK1A1 knockout (KO) cell lines have accelerated turnover of long-lived proteins labeled with (14)C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration.