Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

Abstract

Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors' broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency.adeno-associated virus | DNA-damage response | high-throughput screening | self-complementary vectors | RNA interference V iral vectors based on the adeno-associated virus (AAV) have received incremental attention over the past two decades as effective tools for in vivo gene transfer. The vectors' intrinsic structural simplicity, lack of pathogenicity, low immunogenicity, and ability to mediate long-term, episomal expression in nondividing cells in vitro and, most notably, postmitotic organs in vivo are desirable characteristics for several gene therapy applications (reviewed in ref. 1). Indeed, to date, over 90 clinical trials using these vectors have been carried out (www.abedia.com/wiley/index. html); the first commercial gene therapy product (Glybera) is an AAV1 vector for the treatment of familial lipoprotein lipase deficiency (2).Despite these achievements, AAV vectors still display an undeniable number of limitations in terms of restricted cellular permissivity and efficacy of transgene expression. Efficient transduction in vivo is essentially limited to postmitotic cells [in particular, cardiomyocytes, neurons, retinal cells, and skeletal muscle fibers (1)] and requires infection at a relatively high multiplicity of infection; in addition, vector-driven gene expression is achieved only after a relatively long lag period (3). These characteristics are still incompletely understood in molecular terms but certainly reside within the intrinsic biological properties of target cells. This finding is also highlighted by the peculiar biology of wild-type AAV, which, for the completion of its biological cycle, requires cellular coinfection by adenovirus or other viruses that modify the cellular environment of the target cells, rendering them permissive for viral replication (4-6).The efficient internalization of AAV vectors into different cell types is mediated by the binding of virions to ubiquitously expressed cell surface receptors (...

Gene Therapy With Angiotensin-(1-9) Preserves Left Ventricular Systolic Function After Myocardial Infarction

“…Briefly, an Ang-(1-9) expression cassette (13) was sub-cloned into plasmid adeno-associated virus-multiple cloning site (pAAV-MCS) and AAV9 vectors produced via standard protocols (16). Surgical procedures were performed in accordance with the Animals Scientific Procedures Act (1986) and approved by the University of Glasgow Animal Welfare and Ethical Review Panel and UK Home Office.…”

Effect of heart ischemia and administration route on biodistribution and transduction efficiency of AAV9 vectors

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Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

“…There have previously been two distinct approaches taken to classify the molecules mediating AAV transduction: yeast two-hybrid screens using portions of the capsid as bait 48 and siRNA library screens that assess transduction after disrupting gene expression 43 , 49 , 50 . The interactomes we identified in HEK cells (Supplemental Table 1 ) and whole mouse brain lysates (Supplemental Table 2 ) have minimal overlap with the genes recovered from the yeast two-hybrid screen using mouse liver cDNA as a library, though the authors identified nucleotide binding and intracellular trafficking as key functional categories of AAV interactors, which pathway analysis of our data also identified (Fig.…”