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

Abstract

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.

Synthetic Biology: Emerging Concepts to Design and Advance Adeno‐Associated Viral Vectors for Gene Therapy

“…Indeed, GPR108 localizes primarily to the Golgi compartment and is expressed almost ubiquitously by most cell types. [63] Interestingly, GPR108 is not essential for AAV5, suggesting the existence of serotype-specific differences in the transduction processes. [62,63] www.advancedsciencenews.com www.advancedscience.com…”

Shiga Toxins: An Update on Host Factors and Biomedical Applications

“…In contrast with the specific requirement of LAPTM4A for Gb3 biosynthesis, TM9SF2 and TMEM165 are required for a broad range of glycosylation processes, including biosynthesis of gangliosides and heparan sulfate proteoglycan. They have been identified in a growing number of genetic screens for various toxins and viruses [ 123 , 124 , 125 , 126 , 127 ]. Mutations in TEME165 have been linked to congenital disorders of glycosylation in humans [ 128 ].…”

Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions upon Cell Entry

“…Two independent screens validated the Pillay et al screen hit GPR108 (i.e., Lung Seven Transmembrane Receptor2; LUSTR2) as an important factor for AAV transduction [ 191 , 192 ]. GPR108 is a seven-transmembrane protein with a long N-terminal lumen domain and a short C-terminal domain essential for AAV transduction [ 191 ].…”

“…AAV5 does not require GPR108 and the VP1u region of AAV2 can transfer GPR108 dependence to AAV5 [ 191 ]. Furthermore, GPR108 expression overlaps with AAVR expression in the TGN [ 191 , 192 ]. Of several possible explanations of the observations, one is that attachment of AAV2-like viruses to HSPG is followed by binding to AAVR, and eventual VP1u extrusion for a GPR108-mediated step in the TGN ( Figure 5 b) [ 191 ].…”