Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors

Abstract

In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or "leak" expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant "cross-over insensitive" recognition sites combined with an "ATG-out" design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.

Keywords: AAV; Cre; Flp; leak; recombinase.

Fig. 1.

Recombinase-dependent AAV constructs carrying sensitive transgenes suffer from off-target “leak” expression and both their genomes and the plasmids used to make them contain reverted transgenes. (A) Overview of recombinant AAV production from cloning to injection. The plasmid diagram shows the typical Cre-dependent DIO or FLEX recombinase-dependent design utilizing 2 overlapping anti-sense pairs of orthogonal recombinase recognition sites. (B) Examples of leak expression from Flp-dependent Cre (Left) and Cre-dependent Flp (Right) AAVs injected into V1 of reporter mice in which Flp and Cre expression, respectively, is restricted to a specific interneuron population. White arrows indicate those neurons expressing the reporter tdTomato that are not labeled by antibody for the cell type in which recombinase expression is restricted. (C) PCR of recombinase-dependent AAV genomes from different core facilities using in-sense primers spanning recombinase recognition sites produce <1-kb doublets. Sequencing of these PCR fragments reveals amplicons containing transgenes in-sense with the transcriptional motifs of the vector (I and II). (D) PCR of recombinase-dependent AAV plasmids using in-sense and anti-sense primer pairs designed to detect recombination across both 5′ and 3′ lox pairs. Colored spheres above each lane indicate primer pairs. Labeled bands in the Right lane indicate lox pairs across which recombination occurred, as found by sequencing. See also SI Appendix, Fig. S1. Acronyms: RCF, “Rosa-CAG-Frt-STOP-Frt”; nEF, hybrid HTLV/EF1α promoter.

Fig. 2.

A plasmid duplication event resulting from homologous recombination across recombinase-specific sites results in transgene reversion. (A) Design of an inverted LacZ expression plasmid based on the pUC19 expression vector. The LacZ coding sequence is inverted anti-sense to the Lac operon and start codon and bookended by 2 34-bp anti-sense recognition sites. (B) The sequences of 4 pairs of tested 34-bp “recognition sites,” both canonical and shuffled, designed to test if the mechanism of inversion was specific to the structure of site-specific recombinases or due to gross homology. Positions 1 and 2 are as denoted in A. For a full list of tested sequences and recombination rates, see SI Appendix, Fig. S3. (C) X-Gal/IPTG plate of colonies from a diluted bacterial miniprep grown from a white colony harboring the pUC19-loxp-inv_LacZ plasmid. Inset shows blue colonies among white colonies. (D) The frequency of blue colonies on X-Gal/IPTG plates grown from white colonies, from 3 minipreps grown across 3 plates. Horizontal lines signify mean value across minipreps. The average colony number per plate and the SD are shown below. (E) The relative amounts of reverted LacZ coding sequences to total LacZ copies in DNA minipreps grown from white colonies by qPCR. Horizontal lines signify the mean value across minipreps (n = 7, circles), and vertical lines are SD. (F) X-Gal/IPTG plate of colonies from a diluted bacterial miniprep grown from a blue colony found in plates shown in C from a transformation of the pUC19-loxp-inv_LacZ plasmid. Inset shows blue colonies. (G) The frequency of white colonies on X-Gal/IPTG plates grown from blue colonies, from 3 minipreps grown across 3 plates. Horizontal lines signify mean value across minipreps. The average colony number per plate and the SD are shown below. (H) The relative amounts of reverted LacZ coding sequences to total LacZ copies in DNA preparations grown from blue colonies by qPCR. Horizontal lines signify mean value across minipreps (n = 7, circles), and vertical lines are SD. (I) Schematic of proposed inter- or intraplasmid recombination events across anti-sense homologous recombinase sites at different relative positions resulting in transgene reversion and plasmid doubling. P and P′ denote mirrored plasmids. Approximate SacI site denoted by orange asterisks. For a schematic of plasmid doubling in recombinant-dependent AAVs, see SI Appendix, Fig. S4. (J) SacI restriction profile of minipreps grown from white and blue colonies from conditions in which blue colonies were present. First and last lanes are a 1-kb ladder.

Fig. 3.

Disruption of both transgene ORF and spontaneous reversion are required to abolish leak expression. (A) Schematic of model AAV plasmid constructs designed to disrupt transgene ORF (AO plasmids) or reversion by recombination (NHP plasmids). Triangles denote 34-bp shuffled loxp sites; shadings denote heterologous sequences. Asterisks denote the removal of the ATG start codon from the ORF of the transgene. (B) The HEK293T line constitutively expressing mCherry and GFP under Cre control reports Cre expression. Example images show GFP (Top row) and RFP (Bottom row) channels of images of cell monolayers transfected 48 h earlier with the plasmids listed above. Example flow cytometry results showing degree of Cre leak in HEK-mcherry- loxp-STOP-loxp-GFP cells across Cre-expressing plasmids 48 h after transfection. Controls (Left column) show example GFP signal in empty vector and iCre transfection conditions. Dotted line denotes gate bounding GFP⁺ cells without any cells falling within it in empty vector condition. Percentages at the Upper Right of the doted gate signify percent of the total shown cell population contained within the gate. (C) Summary of leak expression from iCre plasmids in HEK-mCherry-loxp-STOP-loxp-GFP cells across conditions as measured by percent of cells falling within the dotted gate. Horizontal lines signify mean value across transfections (n = 6, circles) and vertical lines are SD. The AO NHP condition is significantly different (P < 4 × 10⁻⁸, 1-way ANOVA, Tukey–Kramer post hoc multiple comparison) when compared with other modified plasmid conditions (asterisk). No significance (n.s.) was found between AO NHP condition and empty plasmid. (D) Summary of leak from EYFP plasmids in HEK293T cells across conditions as measured by percent of cells falling within the dotted gate. EYFP positive control was omitted to maintain scale. Horizontal lines signify mean value across transfections (n = 6 transfections, except for NHS in which n = 5; circles) and vertical lines are SD. The HP condition is significantly different (P < 1.5 × 10⁻⁸, 1-way ANOVA, Tukey–Kramer post hoc multiple comparison) when compared to other conditions (asterisk).

Fig. 4.

Mutated loxp sites with decreased homology mitigate spontaneous recombination and leak expression of sensitive transgenes from an ORF-disrupted AAV design. (A) Schematic of the directionally biased Cre-dependent recombination reaction when using mutant lox sites. Asterisks denote locations of mutations. (B) Candidate canonical and mutant lox sites with known compatibility and decreased homology. Nonhomologous bases within pairs are shown in red. (C) The frequency of spontaneous recombination in the pUC19-inv_LacZ test when using canonical and mutant loxp sequences as measured by qPCR. Circles indicate individual measurements, mean shown by a vertical line, and horizontal lines signify SD. n = 7. For a full list of tested sequences and recombination rates, see SI Appendix, Fig. S3. (D) Sequence homology, as measured by 1 minus the normalized Levenshtein distance between within-pair sequences, plotted against mean recombinant frequency as measured by qPCR in the pUC19_inv_LacZ test. Line of best fit was calculated using loxp derivatives (blue crosses). R² = 0.9632. NHS condition shown for comparison (pink cross). (E) Images of brain tissue slices following injections of equal quantities of AAV8 nef-DIO-FlpO (Left column) and AAV8 nef-CIAO-Flp (Right column) into primary visual cortex of RCF-tdTomato x Pvalb^Cre (Top) and RCF-tdTomato (Bottom). Asterisk in AAV model denotes removal of ATG start codon from Flp ORF. Flp-dependent tdTomato expression is in red and parvalbumin antibody in green. (Scale bars, 100 um.) (F) Quantification of total tdTomato⁺ neurons in RCF-tdTomato mice injected with AAV8 nef-DIO-FlpO (n = 5) and AAV8 nef-CIAO-Flp (n = 6). Error bars indicate SD; P = 0.0225, 2-tailed independent t test (asterisk).