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. 2011 Jun;19(6):1152-60.
doi: 10.1038/mt.2011.4. Epub 2011 Feb 8.

Enhancers of adeno-associated virus AAV2 transduction via high throughput siRNA screening

Alexis J Wallen  1 , Gregory A Barker, David E Fein, Huiyan Jing, Scott L Diamond
Affiliations

Enhancers of adeno-associated virus AAV2 transduction via high throughput siRNA screening

Alexis J Wallen et al. Mol Ther. 2011 Jun.

Abstract

Intracellular barriers to adeno-associated virus (AAV) transduction may limit gene delivery. We screened a short interfering RNA (siRNA) library targeting 5,520 genes to help identify pathways that modulate AAV transduction of human endothelium. In replicate screening, 50 pools (three siRNAs per gene) resulted in greater than eightfold reporter gene expression enhancement. Single siRNA confirmation tests demonstrated that at least one siRNA from each of the top 10 pools provided greater than twofold enhancement. Several siRNAs when used together resulted in additive effects and two of the most potent siRNA sequences were enhancers in cultured airway epithelium. However, enhanced transduction was not correlated with mRNA knockdown by quantitative real time PCR, indicating an off-target mechanism. In fact, four of the five most potent siRNAs contained a consensus hexamer region 5'-UGUUUC-3' at positions 2-7 of the antisense strand. The point mutation U4A within this region (but not mutations at positions 1 or 14) disrupted transduction enhancement, indicating a microRNA (miRNA)-like mechanism. Transcription profiling indicated that the hexamer also resulted in perturbation of the interferon pathway via reduced interferon-induced protein 44-like (IFI44L), interferon-inducible myxovirus resistance 1 (MX1), and interferon-induced protein with tetratricopeptide repeats (IFIT5) mRNAs. Direct interferon (α, β, and ω) receptor 2 (IFNAR2) knockdown resulted in greater than twofold transduction enhancement. In addition to providing insight into AAV biology and enhanced transduction, the results demonstrate certain beneficial siRNA off-target effects.

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Figures

Figure 1
Figure 1
Primary and secondary screening for short interfering RNA (siRNA) enhancers of adeno-associated virus (AAV) transduction. (a) Robust Z-factor for 5,520 siRNA pools (average of three replicates) examined in primary screen for enhancement of AAV transduction as detected by enhancement of the luciferase transgene. A cutoff of Robust Z-factor > 4.75 defined 50 hits (red). Data from untreated wells (average of eight replicates) is shown on the right. Robust Z-factor = 0 (median) and Robust Z = 3.0 are marked as a reference. (b) A total of 10 of the top 50 pooled screening hits were confirmed as enhancers of AAV transduction when each siRNA (sequences A, B, C) of each pool was tested individually. At least one of the three sequences tested in each pool resulted in a significant enhancement of luciferase expression. ABCA8, ATP-binding cassette sub-family A member 8; CLIC2, chloride intracellular channel 2; GART, phosphoribosylglycinamide formly transformylase, phosphoglycinamide synthetase, phosphoribosylaminoimidazole synthetase; GPR124, G-protein coupled receptor 124; KALRN, kalirin; LCK, lymphocyte-specific protein tyrosine kinase; SLC5A2, solute carrier family 5 (sodium/glucose cotransporter), member 2; SLC5A3, soluble carrier family 5 (sodium/myoinositol cotransporter), member 3; TAS2R10, taste receptor type 2 member 10.
Figure 2
Figure 2
Demonstration of off-target mechanism of action of CLIC2 short interfering RNA (siRNA). (a) Quantitative real time PCR measurement of CLIC2 mRNA knockdown in human aortic endothelial cells at 24 hour following transfection with three different siRNA sequences at concentrations of 30 nmol/l and 100 nmol/l, normalized against GAPDH mRNA signal. (b) Luciferase luminescence relative to scrambled siRNA negative control for CLIC2 siRNA sequences A, B, and C used at four concentrations of 10, 30, 50, and 100 nmol/l. Adeno-associated virus (AAV) transgene expression was uncorrelated with CLIC2 mRNA knockdown. CLIC2, chloride intracellular channel 2.
Figure 3
Figure 3
Hexamer region of CLIC(C) antisense strand mediates enhanced AAV2 transgene expression. Effect of CLIC2 short interfering RNA (siRNA) sequence C and CLIC2(C) siRNA mutants on AAV2 transduction of human aortic endothelial cells, normalized to scrambled siRNA negative control. AAV2, adeno-associated virus serotype 2; CLIC, chloride intracellular channel.
Figure 4
Figure 4
Effect of antagonism or agonism of interferon pathways on adeno-associated virus (AAV) transduction. (a) Effects of negative controls, interferon (α, β, and ω) receptor 2 (IFNAR2(A)) siRNA and IFNAR2(B) siRNA, and firefly luciferase siRNAs at 50 nmol/l on luminescence following transduction with AAV2-Luc. All results are normalized to the untreated condition. (b) Recombinant α and β interferons reduce transgene expression. siRNA; short interfering RNA.
Figure 5
Figure 5
Short interfering RNA (siRNA) mediated enhancement of AAV2 transduction of human epithelial cells. Fluorescence microscopy images and flow cytometry data for human bronchial epithelium culture treated with (a) scrambled siRNA negative control, (b) SLC5A2 sequence B, and (c) CLIC2 siRNA sequence C and then transduced with AAV2 containing enhanced green fluorescent protein. AAV2, adeno-associated virus serotype 2; CLIC2, chloride intracellular channel 2; SLC5A2, solute carrier family 5 (sodium/glucose cotransporter), member 2.
Figure 6
Figure 6
Pairwise interactions among confirmed short interfering RNA (siRNA) hits. Heat map of pairwise interactions between siRNA sequences (total siRNA concentration/well = 50 nmol/l) for the top 10 confirmed hits (a) or the three different sequences against the top three hits (b). ABCA8, ATP-binding cassette sub-family A member 8; CLIC2, chloride intracellular channel 2; GART, phosphoribosylglycinamide transformylase, phosphoriboglycinamide synthetase, phosphoribozy aminoimidazole synthetase; GPR124, G-protein coupled receptor 124; LCK, lymphocyte-specific protein tyrosine kinase; SLC5A2, solute carrier family 5 (sodium/glucose cotransporter), member 2; SLC5A3, solute carrier family 3 (sodium/myoinositol cotransporter), member 3; TAS2R10, taste receptor type 2 member 10.
Figure 7
Figure 7
Coadministration of adeno-associated virus serotype 2 and short interfering RNA (siRNA) to human aortic endothelial cells enhances transgene expression. (a) Virus was premixed with siRNA lipoplexes and then added to cells. (b) Cells were reverse transfected and virus was immediately added after adding cells to siRNA formulation. CLIC2, chloride intracellular channel 2.
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