Standard screening methods underreport AAV-mediated transduction and gene editing

Abstract

Conventional methods to discern adeno-associated virus (AAV) vector transduction patterns are based on high, stable expression of a reporter gene. As a consequence, conventionally described tropisms omit cell types that undergo transient transduction, or have low but undetectable levels of reporter expression. This creates a blind spot for AAV-based genome editing applications because only minimal transgene expression is required for activity. Here, we use editing-reporter mice to fill this void. Our approach sensitively captures both high and low transgene expression from AAV vectors. Using AAV8 and other serotypes, we demonstrate the superiority of the approach in a side-by-side comparison with traditional methods, demonstrate numerous, previously unknown sites of AAV targeting, and better predict the gene editing footprint after AAV-CRISPR delivery. We anticipate that this system, which captures the full spectrum of transduction patterns from AAV vectors in vivo, will be foundational to current and emerging AAV technologies.

Fig. 1

Comparison of conventional and revised AAV tropism screening methods. a Following injection of AAV2/8.CMV.HI.eGFP-Cre.WPRE.SV40 (AAV8-Cre-eGFP) into C57BL/6J mice, only the subset of cells with stable, high transgene expression will show nuclear-localized eGFP. Cells with transient or low transgene expression are missed. b Injection of the same AAV8-Cre-eGFP vector into Ai14 reporter mice (containing an endogenous loxP-STOP-loxP-tdTomato locus) captures cells with stable, high transgene expression (green nuclei) as well as cells with transient or low expression of the AAV transgenes (Cre-tagged tdTomato positive cells)

Fig. 2

Ai14 mice improve the sensitivity for liver AAV transduction. a Representative photomicrographs of liver sections harvested from AAV8-Cre-eGFP treated C57BL/6J or Ai14 mice 2 weeks post IV delivery at the indicated doses (total vector genomes per mouse). eGFP (green) and tdTomato expression (red) and nuclei (blue) are evident. n = 2–3 for C57BL/6J mice, n = 3 for Ai14 mice. Scale bars = 50 µm. b Quantification of hepatocyte transduction in C57BL/6J and Ai14 at the indicated doses. Values for individual mice are shown along with the mean and standard deviation for each strain and dose group. Data were analyzed by two way-ANOVA followed by Bonferroni post hoc comparing % transduced cells per animal in BL6 (%eGFP+) versus Ai14 (%tdTomato+) mice, ****p<0.0001. c Comparison of AAV vector copies per diploid genome equivalent (dge) in liver samples. Values for individual mice are shown along with the mean and standard error for each strain and dose group. d Cre-mediated recombination in Ai14 mice at the ROSA26 locus as detected by PCR assay of liver genomic DNA. Source data are provided as a Source Data file

Fig. 3

AAV8 transduces kidney glomeruli. a Representative fluorescent photomicrographs of kidney sections from C57BL/6J and Ai14 mice 2 weeks after IV delivery of AAV8-Cre-eGFP at the indicated doses. No eGFP expression is detected in green channel in either mouse strain for all doses tested. tdTomato (red) indicates editing; nuclei are shown (blue). n = 2–3 for C57BL/6J mice, n = 3 for Ai14 mice. Scale bars = 250 µm. b Comparison of AAV vector copies per diploid genome equivalent (dge) in kidney samples from C57BL/6J and Ai14 mice 2 weeks post IV delivery. Values for individual mice are shown along with the mean and standard error for each strain and dose group. c Cre-mediated recombination at the ROSA26 locus as detected by PCR assay of kidney genomic DNA from Ai14 mice. d Immunohistochemistry for glomerular cell types in Ai14 mice 2 weeks after AAV8-Cre-eGFP delivery (3.16e11 vg). Primary antibodies labeled podocytes (anti-podoplanin), endothelial cells (anti-CD31), and mesangial cells (anti-PDGFR-β) and were detected with Alexa-488 conjugated secondary antibodies (green). Cre-tagged cells are tdTomato positive. Scale bars = 25 µm. Source data are provided as a Source Data file

Fig. 4

AAV8 tropism in mouse spleen. a Representative fluorescent photomicrographs of spleen sections from C57BL/6J and Ai14 mice 2 weeks after IV delivery of AAV8-Cre-eGFP at the indicated doses. No eGFP expression is detected in green channel in either mouse strain for all doses tested. tdTomato (red) indicates editing; nuclei are shown (blue). n = 2–3 for C57BL/6J mice, n = 3 for Ai14 mice. Scale bars = 50 µm. b Comparison of AAV vector copies per diploid genome equivalent (dge) in spleen samples from C57BL/6J and Ai14 mice 2 weeks post IV delivery. Values for individual mice are shown along with the mean and standard error for each strain and dose group. c Cre-mediated recombination at the ROSA26 locus is detected via PCR assay of spleen genomic DNA from Ai14 mice. d Flow cytometry was used to measure editing (tdTomato+) in single, live splenocytes isolated from vehicle- or AAV-injected Ai14 mice. n = 5 for both groups. e Splenocytes were co-stained for relevant cell markers and quantified via flow. Representative plots are shown from the AAV-injected group. n = 3 for each antibody co-stain. Gates for each marker were established with fluorescence minus one controls. f Plot showing the contributions of different cell types to the total tdTomato+ population. n = 3 mice for each cell type with mean and standard error shown. g Plot showing the percent of each cell type that was tdTomato+. n = 3 mice for each cell type with mean and standard error shown. Specific marker fluorochromes are indicated in Methods. Source data are provided as a Source Data file

Fig. 5

Ai14 mice capture AAV8 transgene expression across tissues. Representative photomicrographs of tissue from C57BL/6J or Ai14 mice at 2 weeks post IV delivery of 3.16e11 vg AAV8-Cre-eGFP per mouse. eGFP (green), tdTomato expression (red), and nuclei (blue) are shown. n = 3 for C57BL/6J and Ai14 mice. Insets in heart and liver highlight nuclear eGFP expression. Dashed circles highlight glomeruli. Autofluorescent material is present in testes. Scale bars = 100 µm

Fig. 6

Ai14xSpcas9 mice capture AAV8-CRISPR-Cas9 editing across tissues. a AAV8-ROSA-gRNAs vector edits the ROSA26 locus and activates tdTomato expression in Ai14xSpCas9 mice. b Representative photomicrographs of tissue from Ai14xSpCas9 mice at 2 weeks post IV delivery of 2.3e11 vg AAV8-ROSA-gRNAs per mouse. tdTomato expression (red) and nuclei (blue) are shown. n = 3 Ai14xSpCas9 mice. Scale bars = 100 µm